CN101575629A - Method for producing isomaltulose without purification step - Google Patents
Method for producing isomaltulose without purification step Download PDFInfo
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- CN101575629A CN101575629A CNA2009100120036A CN200910012003A CN101575629A CN 101575629 A CN101575629 A CN 101575629A CN A2009100120036 A CNA2009100120036 A CN A2009100120036A CN 200910012003 A CN200910012003 A CN 200910012003A CN 101575629 A CN101575629 A CN 101575629A
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- sucrose
- palatinose
- isomaltulose
- isomerase
- crystallization
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Images
Abstract
The invention provides a method for directly producing isomaltulose by condensation crystallization without a separating purification step. The method is characterized in that: in the process of preparing the isomaltulose, the complex processes of separating and purifying the isomaltulose by ion exchange resin and the like are omitted in industrialized production, and the vacuum condensation and crystallization of conversion solution of sucrose are directly performed to obtain the purified isomaltulose. In the method, cell debris containing activity of sucrose isomerase is firstly prepared into a biocatalyst, the sucrose is subjected to biological conversion by catalysis to generate a product of the isomaltulose not containing byproducts such as glucose, fructose and the like, and the conversion solution of sucrose is directly subjected to vacuum condensation, crystallization and drying to produce the crystallized product of the isomaltulose. The method has the advantages of greatly lowering production cost, saving a large amount of process water and simplifying the production process.
Description
Technical field
The present invention relates to a kind of is the production technology of feedstock production Palatinose with sucrose, and the Palatinose production technique that need not to separate byproducts such as removing glucose and fructose in the Palatinose production process.
Background technology
The chemical name of Palatinose is α-D-glucopyranoside-1, and the 6-D-fructofuranose is found in honey the earliest.Because its content is trace very, therefore do not cause people's attention always.Up to nineteen fifty-seven, in the beet sugar manufacture process, find and crystallization goes out that this " newly " disaccharide compound---behind the Palatinose, the importance of Palatinose just is subject to people's attention gradually.Subsequently, some bacterium can become sucrose inversion the discovery of Palatinose, has promoted the big quantity research of the consumption and the production possibility of Palatinose.Should not take in sugar for those and need the special population of careful selection sweeting agent, the discovery of Palatinose is undoubtedly a kind of stem-winding happy message.
As a kind of functional natural sweet taste sugar, Palatinose has and sweet taste characteristic like the sucrose, and it is faster than sucrose to the initial stimulation rate of taste bud, and the strongest sweet stimulus is the same with sucrose, and sweet stimulus at the end is then than a little less than the sucrose.Palatinose does not have any peculiar smell, and its sugariness is 50% of a sucrose, and does not vary with temperature and change.Palatinose is applied in candy and the chocolate-like food, does not find to have evident difference between it and sucrose.Palatinose not only has the non-carious tooth that causes, but also can stop the generation of insoluble glucan, forms with prevention of dental caries.Because human body itself can't digest Palatinose, have only and just can be entered blood after slowly being decomposed by enteric microorganism and be absorbed by the body, therefore, edible back discharges monose in blood speed stimulates insulin secretion slowly and not than sucrose, thereby is of value to the control of diabetes and can prevents fatty too much accumulation.The all unfermentable Palatinose that utilizes of most of bacteriums and yeast, when Palatinose was applied to leavened food and drink production, its antimicrobial property made the sweet taste of product be easy to keep.
Palatinose is to be raw material with sucrose, and (alpha-glucosyl transferring enzyme, EC.5.4.99.11) catalyzed reaction turn to Palatinose with the sucrose isomery, by the catalytic sucrose isomerization reaction of sucrose isomerase as shown in Figure 1 through sucrose isomerase.In this isomerization process, α in the sucrose molecules-1, the 2-glycosidic link at first is hydrolyzed in the sucrose isomerase active centre and disconnects and transposition, glucose monomer forms α-1 with the fructose monomer more subsequently, and 6-or α-1, the 1-glycosidic link, and from the enzyme molecule, come off and produce two kinds of sucrose isomer, simultaneously, the α of disconnection-1,2-glycosidic link also can form glucose and fructose with water molecule reaction.Therefore, in the sucrose isomerization process, except generating α-1, outside the Palatinose that 6-connects, also form α-1, the cell wall sugar that 1-connects also can form a part disconnected glucose and fructose simultaneously.At present, the sucrose isomerase that is used for Palatinose production derives from various microorganisms, as (Protaminobacter) such as serratia (Serratia), Klebsiella (Klebsiella), Rhodopseudomonas (Psedumonas), erwinia (Erwinia), Agrobacterium (Agrobacterium) and Protaminobacters.When United States Patent (USP) 4670387 uses the immobilized cell of Erwinia rhapontici, Protaminobacter rubrum and Serratia plymuthica to produce Palatinose, approximately can transform 70～95% sucrose.When United States Patent (USP) 5229276 usefulness immobilization Agrobacteriumradiobacter bacterium produced Palatinose, the ratio of Palatinose only had 10%.Though the microbial bacterial that has been found that or use can the isomerization of catalysis sucrose produce Palatinose, but output is all very unstable, and transformation efficiency is generally 8～86%, and, in the product except Palatinose, the formation that also to be attended by three kinds of byproducts be cell wall sugar, glucose and fructose.The ratio difference of the various products such as Palatinose that the sucrose isomerase catalysis of different sources generates, wherein cell wall sugar is the same with Palatinose, be a kind of non-decayed tooth sugar that causes, the cell wall sugar in the product does not influence the special trait of Palatinose, need not in the production process to consider to separate to reject.But, also contain by products such as 2～7% glucose and fructose in the converted product of these sucrose isomerases, have a strong impact on the quality of Palatinose product, in the leaching process of back, must separate and remove.Must adopt the separation and purification process of complexity such as ion exchange resin to remove these impurity in the production process, cause production cost significantly to improve, be a considerable industrial problems.We find that by the comparative analysis of sucrose isomerase catalytic reaction process the existence form of sucrose isomerase influences the ratio of sucrose isomerization product.
Summary of the invention
The purpose of this invention is to provide a kind of effective technology, provide a cover to need not the simplification production technique of separation and purification process direct production Palatinose simultaneously with easy steps and device fabrication high purity Palatinose.Compare with other disclosed patents, the sucrose inversion rate reaches 99.8%, and Palatinose content is up to 88%, and glucose and fructose content are lower than 0.1%.According to the present invention, can realize the suitability for industrialized production of Palatinose at lower cost.
Concrete operations step of the present invention is as follows:
The first step, the preparation of sucrose isomerase active cells fragment: klebsiella LSI bacterium is inoculated in 100 milliliters of sucrose medium by preserving the inclined-plane, under 30 ℃, 150 rev/mins conditions, shaking culture is after 6～8 hours in shaking table, transfer in fresh sucrose medium by 5% inoculum size, cultivated 10～12 hours in 30 ℃ of vibrations (150 rev/mins), under 6000 rev/mins, 25 ℃ conditions centrifugal 10 minutes, with distilled water wash somatic cells 3 times, again be suspended in the 25mM phosphoric acid-citric acid solution of original volume, and separated into two parts.A part is used for enzyme activity determination as intact cell, and another part ultrasonic disruption is made sucrose isomerase active cells fragment.Wherein, contain 2% sucrose in the sucrose medium prescription, 0.1% yeast powder, 0.1% peptone, 0.05% sal epsom, 0.1%NaH
2PO
4, 0.1%KNO
3, pH6.6.
Centrifugal 10 minutes of 14000 rev/mins of cell debriss that obtains are washed 3 times, and are suspended in again in the above-mentioned buffered soln of original volume, can be used for the enzymic catalytic reaction analysis.
Gene order synthetic primer according to sucrose isomerase, genomic dna with klebsiella LSI bacterium is a template, adopt round pcr amplification sucrose isomerase gene, and at GST expression system (GST GeneFusion System, Pharmacia) express sucrose isomerase in, merge sucrose isomerase with special-purpose affinity chromatography column separating purification sucrose isomerase and GST.
Second step, sucrose inversion: the active cells fragment is mixed in conversion tank with 1: 10 ratio with 10～20% sucrose solutions, under the condition that keeps malleation, in 30 ℃ of insulations 6～15 hours, is Palatinose with sucrose inversion, obtains sucrose inversion liquid;
The 3rd step, condensing crystal: under 65 ℃ of conditions, the sucrose inversion liquid that above-mentioned steps is obtained carries out vacuum concentration, treat that sugared concentration reaches at 60～75% o'clock, add 0.1～1% Palatinose crystal seed, agitation condition is slowly cooling down, the Palatinose crystallization is separated out, obtain small-particle crystallization Palatinose;
The 4th step, drying: with after the crystalline Palatinose drip washing decolouring, be dried to water content 7～8% o'clock, obtain the Palatinose crystalline product.
The steric effect of sucrose isomerase existence influences the kind and the ratio of enzymatic conversion product.It is 10～20% sucrose solution with 1: 10 mixed with concentration respectively that the sucrose isomerase of the intact cell that obtains in the first step, cell debris suspension, purifying and GST are merged sucrose isomerase, condition according to second step, the sucrose inversion rate is reached more than 99.8%, obtain the Palatinose conversion fluid.
As shown in Figure 2, compare with the product of cell walls enzyme, in the converted product of pure enzyme, the glucose ratio obviously increases, near other with zymoid ratio (12.5～13%), cell wall sugar is then little increase.When in that the N-of sucrose isomerase is terminal when connecting a gst fusion protein, may be because the proteic spatial obstacle effect of GST, the glucose ratio in the sucrose inversion product decrease (7%).This shows that sucrose isomerase is during with cell walls enzyme state catalysis sucrose inversion, product purity is the highest.
Immobilized cell is produced Palatinose:
The klebsiella LSI cell suspending liquid of adding 15% in 2.5% sodium alginate soln of the bacterium of going out after mixing, is added drop-wise to the CaCl of 0.65% sterilization under aseptic condition
2In the solution, carry out fixed-typely, and in this solution, placed 4 hours, to strengthen calcium-alginate-immobilized cell.Immobilized cell changed over to contain 0.1%CaCl
2Sucrose medium (2% sucrose, 0.1% yeast powder, 0.1% peptone, 0.05% sal epsom, 0.1%NaH
2PO
4, 0.1%KNO
3, pH6.6) in, 30 ℃ of vibrations (150 rev/mins) were cultivated 16 hours, made the immobilized cell activation.Immobilized cell after will activating then fills in the column reactor, flow velocity Continuous Flow by 0.5～5 ml/min adds 10～20% sucrose solution, under 30 ℃ of conditions, carry out enzymatic conversion reaction, by controlling flow velocity to adjust the residence time of sucrose solution in reactor, sucrose is transformed fully, collect effluent liquid, measure the concentration of Palatinose.
Sucrose isomerase vigor detection method is as follows among the present invention:
The active cells fragment suspension (0.1 milliliter) that obtains in the first step is mixed with 0.4 milliliter of 2% (w/v) sucrose solution that is dissolved in 25mM phosphoric acid-citric acid solution (pH6.6), reaction is after 10 minutes in 30 ℃ of water-baths, add 1.5 milliliter 3 immediately, the 5-edlefsen's reagent stops enzyme reaction, and in boiling water bath, heat 5 minutes with colour developing, detect absorbancy at 520 nano wave length places, determine reducing sugar content by the Palatinose typical curve.A sucrose isomerase unit of activity is defined as: under above-mentioned reaction conditions, the isomerization of per minute catalysis sucrose forms the needed enzyme amount of 1 μ mol Palatinose.
The detection of Palatinose can be adopted thin-layer chromatography (TLC) or high performance liquid chromatography (HPLC) method among the present invention.
Thin-layer chromatography (TLC) method detection Palatinose and sugar component measuring method are as follows:
With the sucrose inversion supernatant liquor behind point sample on the silica gel thin-layer chromatography plate, in separation chamber, launch the back colour developing, solvent systems is: the volume ratio of ethyl acetate-acetate-water 4: 3: 0.8, developer is the volume ratio 5: 5: 1 of this amine (4%)-two amine (4%)-phosphoric acid (85%), colour temp is 80 ℃, developing time is 5 minutes, and Palatinose forms typical yellow-green colour spot.
Ratio for various sugar components in the quantitative analysis conversion fluid, behind the TLC chromatography, downcut the component that contains Palatinose, cell wall sugar and glucose in the silica-gel plate respectively, and use dissolved in distilled water, adopt 3,5-dinitrosalicylic acid method is measured sugared concentration, calculates the ratio of Palatinose, cell wall sugar and glucose.
High performance liquid chromatography (HPLC) detection Palatinose and proportion of products method are as follows:
Adopt Waters 2690 HPLC of system to separate the Palatinose product, chromatography column is Waters glycan analysis dedicated columns (4.6 * 250 millimeters), detector is Waters 2410 refractive index detectors, sample is with 70% second cyanogen aqueous solution wash-out, eluent flow rate is 1 ml/min, by containing HPLC chromatogram that the active cell debris catalysis of sucrose isomerase sucrose inversion forms product as shown in Figure 3, can calculate sugared content and proportion of products according to peak area.
Description of drawings
The synoptic diagram of Fig. 1, the isomerization reaction of sucrose isomerase catalysis sucrose.
The thin layer chromatogram of Fig. 2, different existence form sucrose isomerase catalysis sucrose isomerization products.
The HPLC of Fig. 3, sucrose isomerase catalysis sucrose inversion product analyzes collection of illustrative plates.
Embodiment
Embodiment 1, conversion tank transform the production Palatinose in batches
Klebsiella LSI bacterium from keeping on the inclined-plane, is seeded to sucrose medium (2% sucrose, 0.1% yeast powder, 0.1% peptone, 0.05% sal epsom, 0.1%NaH are housed
2PO
4, 0.1%KNO
3PH6.6) activation culture 8 hours (30 ℃, 150 rev/mins) changes in the fermentor tank that fresh sucrose medium is housed by 5% inoculum size in the triangular flask, ventilates to cultivating after 12 hours, in fermentor tank under aseptic condition the ultrasonic disruption cell, make active cells fragment suspension.In 10 tons of conversion tank, prepare 5%, 10%, 15% and 20% sucrose solution respectively, 95 ℃ of sterilizations were cooled to 30 ℃ after 20 minutes.In 1: 10 ratio above-mentioned active cells fragment suspension is changed in 10 tons of conversion tank, 30 ℃ of insulations, logical aseptic wind is to keep the barotropic state in the conversion tank.When sucrose concentration was 5% and 10%, sucrose 100% transformation time was no more than 6 hours, and during 15% and 20% sucrose concentration, required time is no more than 9 hours when reaching sucrose and all transforming.
Embodiment 2, immobilized cell are produced Palatinose
Behind per 5 milliliters of klebsiella LSI inoculums and the 100 milliliter of 2% sodium alginate soln thorough mixing, be added drop-wise to 0.65% (w/v) CaCl with syringe
2In the solution, and in solution, under 4 ℃ of conditions, placed 4 hours, to strengthen immobilization particle.Then immobilized cell is changed over to and contain 0.1%CaCl
2Sucrose medium (2% sucrose, 0.1% yeast powder, 0.1% peptone, 0.05% sal epsom, 0.1%NaH
2PO
4, 0.1%KNO
3, pH6.6) in, 30 ℃ of vibrations (150 rev/mins) overnight incubation, with the activation immobilized cell.Immobilized cell is packed in 500 milliliters of filling type reactors, under 30 ℃ of conditions, add 20% sucrose solution, and detect the concentration of Palatinose in the effluent liquid by different in flow rate stream.When the sucrose flow acceleration was 4 ml/min, the sucrose inversion rate was 99.9%, and the concentration of Palatinose and cell wall sugar and glucose is respectively 88.0%, 11.9%, 0.1% in the effluent liquid.When continuing to improve the sucrose flow acceleration, the sucrose inversion rate reduces gradually, but the ratio of various sugar components does not obviously change in the product.When flow acceleration was 8 ml/min, the sucrose inversion rate had been reduced to 75.3%.
Claims (4)
1, a kind of method for producing isomaltulose that does not have purification step is characterized in that may further comprise the steps:
The first step, the preparation of sucrose isomerase active cells fragment: klebsiella LSI bacterium is inoculated in 100 milliliters of sucrose medium by keeping the inclined-plane, under 30 ℃, 150 rev/mins conditions, shaking culture is 6～8 hours in shaking table, transfer in fresh sucrose medium by 5% inoculum size, in 30 ℃, 150 rev/mins shaking culture 10～12 hours, ultrasonic disruption is made sucrose isomerase active cells fragment suspension; Wherein, contain 2% sucrose in the sucrose medium prescription, 0.1% yeast powder, 0.1% peptone, 0.05% sal epsom, 0.1%NaH
2PO
4, 0.1%KNO
3, pH6.6;
Second step, sucrose inversion: the active cells fragment is mixed in conversion tank in the ratio of 1:10 with 10～20% sucrose solutions, under the condition that keeps malleation, in 30 ℃ of insulations 6～15 hours, is Palatinose with sucrose inversion, obtains sucrose inversion liquid;
The 3rd step, condensing crystal: under 65 ℃ of conditions, the sucrose inversion liquid that above-mentioned steps is obtained carries out vacuum concentration, treat that sugared concentration reaches at 60～75% o'clock, add 0.1～1% Palatinose crystal seed, agitation condition is slowly cooling down, the Palatinose crystallization is separated out, obtain small-particle crystallization Palatinose;
The 4th step, drying: with after the crystalline Palatinose drip washing decolouring, be dried to water content 7～8% o'clock, obtain the Palatinose crystalline product.
2, a kind of method for producing isomaltulose that does not have purification step according to claim 1 is characterized in that the described sucrose isomerase of the first step is to contain the active cell debris of sucrose isomerase.
3, a kind of method for producing isomaltulose that does not have purification step according to claim 1 is characterized in that not containing by products such as glucose and fructose in the described sucrose inversion liquid of second step.
4, a kind of method for producing isomaltulose that does not have purification step according to claim 1 is characterized in that not containing the Palatinose purification procedures in the described condensing crystal operation of the 3rd step.
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