CN109022521A

CN109022521A - A method of D-Psicose is prepared by starch

Info

Publication number: CN109022521A
Application number: CN201811087533.2A
Authority: CN
Inventors: 任世阔; 牛志国; 韩子明; 赵红兵; 吴会广
Original assignee: SHANGHAI LIZU BIOTECHNOLOGY Co Ltd
Current assignee: SHANGHAI LIZU BIOTECHNOLOGY Co Ltd
Priority date: 2018-09-18
Filing date: 2018-09-18
Publication date: 2018-12-18
Anticipated expiration: 2038-09-18
Also published as: WO2020057561A1; CN109022521B

Abstract

The present invention provides a kind of methods for preparing D-Psicose by starch, and specifically, the present invention is made from starch, and handle by saccharification processing, isomerization, and concentration and chromatographic isolation processing, can get the D-Psicose of up to 98-99% purity.Method of the invention can prepare D-Psicose efficiently at low cost.

Description

A method of D-Psicose is prepared by starch

Technical field

The present invention relates to functional food fields, and in particular, to a method of D-Psicose is prepared by starch.

Background technique

D-Psicose is a kind of rare sugar existing for nature, and according to research reports, D-Psicose is a kind of novel function Can property monosaccharide compared with sucrose, heat is low, pure taste, will not can be very good control blood glucose by body metabolism.2011 Year, U.S. FDA approved D-Psicose is considered safety food as raw-food material；So D-Psicose is expected into Diet, health care and medicine and other fields are widely used in as food or food additives for the substitute of sucrose.

Currently, in order to obtain the D-Psicose of higher degree, a kind of to use method be to directly adopt fructose as raw material D-Psicose is prepared, however this has the disadvantage that the fructose of high-purity improves production cost, and compared with the fructose meeting of low-purity Introduce other impurity (including other monosaccharide or disaccharides).

Another method is the separation D-Psicose from the existing syrup (such as fructose syrup) containing D-Psicose, however Although the concentration through D-Psicose in separation product increases, D-Psicose purity is still lower (with all sugars Total amount meter is about 10% or lower), and still contain many other monosaccharide (especially fructose).

Therefore, the production cost of D-Psicose is also relatively high at present.D-Psicose as a kind of novel sweetener, Universal popularization and use is obtained, need to also further decrease production cost while preparing high-purity D-Psicose.

Therefore, there is an urgent need in the art to develop a kind of low cost, it is efficient, be suitble to preparation of industrialization high-purity A Luo ketone The method of sugar product.

Summary of the invention

The purpose of the present invention is to provide a kind of low cost, efficient, suitable preparation of industrialization high-purity psicoses The method of product.

The first aspect of the present invention provides a kind of method for producing D-Psicose, comprising steps of

(a) saccharification processing is carried out to starch raw material liquid, to form the first mixture containing saccharification product, the saccharification Product includes: glucose, maltose, isomaltose, maltotriose and maltotetraose；

(b) isomerization processing is carried out to first mixture, glucose isomerase is turned into fructose and then is isomerized to D- Psicose, to form the second mixture containing D-Psicose；With

(c) D-Psicose is separated from second mixture, to obtain the separation product containing D-Psicose.

In another preferred example, do not include the steps that separating sugar in step (a) and (b).

In another preferred example, the starch is selected from the group: cornstarch, potato starch or combinations thereof.

In another preferred example, the starch raw material liquid is material liquid of the starch through liquefaction processing.

In another preferred example, further include step (a1) before step (a): liquefaction processing being carried out to starch, thus shape At starch raw material liquid.

In another preferred example, liquefaction processing is carried out to starch in the presence of amylase.

In another preferred example, before liquefaction, raw starch and water are mixed to form starch water.

In another preferred example, starch and the mass ratio of the starch water are 25~35:100.

In another preferred example, liquefaction processing has following one or more features:

The additional amount of amylase is 10-50u/g starch；

PH is 5.5~6.5；

The temperature of the liquefaction processing is 100-130 DEG C；And/or

Liquefying time is 30-60min.

In another preferred example, the additional amount of amylase is 10-25u/g starch.

In another preferred example, in step (a), in the presence of carbohydrase, saccharification processing is carried out to starch raw material liquid, from And form the first mixture.

In another preferred example, in step (a), the saccharification processing has following one or more features:

The additional amount of carbohydrase is 10-200u/g starch；

The pH of saccharification processing is 4.0~5.5；

Saccharificatinn period is 20-40h；And/or

The treatment temperature of saccharification processing is 35~70 DEG C.

In another preferred example, in step (a), the pH for the processing that is saccharified is 4.3~4.8.

In another preferred example, in step (a), the additional amount of carbohydrase is 50~100u/g starch.

In another preferred example, in step (a), the treatment temperature for the processing that is saccharified is 45~65 DEG C；It is highly preferred that for 55~ 60℃。

In another preferred example, step (a) further comprises the steps of: at 90~110 DEG C to the sugar in first mixture Change enzyme and α-amylase carries out inactivation treatment, obtains the first mixture that enzyme has inactivated.

In another preferred example, step (a), which is further comprised the steps of:, is added active carbon into first mixture, is taken off Color processing, obtains the first mixture through decolorization.

In another preferred example, include in step (a) or do not include being filtered to remove the step for having inactivated carbohydrase and α-amylase Suddenly.

In another preferred example, in the first mixture, content >=70% of glucose, preferably >=80%, more preferably >=90%, most preferably >=95%, based on the gross mass of dry matter in first mixture.

In another preferred example, first mixture includes the glucose of 90-99wt%, it is preferable that includes 95- The glucose of 99wt%, on the basis of dry matter gross mass in the first mixture.

In another preferred example, in first mixture, Y value >=80%

Y=C1/ (C1+C2+C3+C4+C5)

In formula,

C1 is the concentration of glucose；

C2 is the concentration of maltose；

C3 is the concentration of isomaltose；

C4 is the concentration of maltotriose；

C5 is the concentration of maltotetraose.

In another preferred example, step (a), which is further comprised the steps of:, carries out concentration to first mixture, thus shape At the first concentrated mixture.

In another preferred example, the concentration of dry matter is 10-70wt% in not concentrated first mixture, preferably Ground, 20-65wt%, more preferably, 30-60wt%, the concentration is the concentration of total solids, with the first mixture gross mass On the basis of.

In another preferred example, dry matter concentration is 45-55wt% in the first concentrated mixture, with described first On the basis of mixture gross mass.

In another preferred example, the concentration is carried out using MVR evaporator.

In another preferred example, the step (b) includes:

(b1) the first enzymatic reaction is carried out to first mixture with glucose isomer enzyme, to obtain containing fructose Mixture；With

(b2) the second enzymatic reaction is carried out to the mixture containing fructose with C-3 allomerase, to obtain described Second mixture.

In another preferred example, it is further comprised the steps of: before step (b1) and metal ion is added into first mixture； Preferably, the metal ion is selected from: Mn²⁺、Co²⁺Or combinations thereof.

In another preferred example, the final concentration of the metal ion respectively stands alone as 1-10mM.

In another preferred example, step (b1) and step (b2) carry out at 40-80 DEG C；Preferably, at 50-70 DEG C into Row.

In another preferred example, the step (b1), (b2) are carried out at the same time or are successively carried out.

In another preferred example, the step (b) or the step (b1) and (b2) are carried out in same reactor.

In another preferred example, in the step (b), into first mixture be added glucose isomer enzyme and C-3 allomerase, and reacted to obtain second mixture.

In another preferred example, in the step (b1), first mixture flow is through being fixed with glucose isomerization The reaction column of enzyme reacts to obtain the mixture containing fructose；And/or

In step (b2), the mixture flow containing fructose obtains institute through being fixed with the reaction column of C-3 allomerase State the second mixture.

In another preferred example, in the step (b), first mixture flow is through being fixed with glucose isomer enzyme Reaction column and C-3 allomerase reaction column, to obtain second mixture.

In another preferred example, the total concentration of the glucide of second mixture is that 30-68wt% (preferably, is 50-65wt%), on the basis of the gross mass of the second mixture.

In another preferred example, psicose purity is greater than 5wt% in second mixture, it is preferable that is greater than 11wt%；It is more preferably, greater than 14wt%, on the basis of dry matter gross mass in the second mixture.

In another preferred example, second mixture include: psicose, glucose, fructose and oligosaccharide (including Disaccharides).

In another preferred example, the oligosaccharide is selected from the group: maltose, isomaltose, maltotriose, maltotetraose, Or combinations thereof.

In another preferred example, in second mixture, the content of the oligosaccharide (including disaccharides) is 2~10w%, On the basis of dry matter gross mass in the second mixture.

In another preferred example, in second mixture, the content of the glucose is 5-55wt%, preferably, 10- 40wt%, more preferably, 20-30wt%, on the basis of dry matter gross mass in the second mixture.

In another preferred example, in second mixture, the content of the fructose is 1-50wt%, preferably, 5- 30wt%, more preferably, 10-25wt%, on the basis of dry matter gross mass in the second mixture.

In another preferred example, in step (c), by chromatography, contain from second mixture is isolated The separation product of D-Psicose.

In another preferred example, the chromatography is Simulated Moving Bed Chromatography partition method.

In another preferred example, the chromatography includes step

(1) the second mixture is provided as charging F；And

(2) chromatographic isolation: being separated to described containing sugared mixed liquor by the chromatographic isolation equipment based on mobile bed chromatic, Obtain the separation product containing D-Psicose；

The chromatographic isolation includes:

(2.1) feed step: will be in the chromatographic column that be passed through containing sugared mixed liquor；

(2.2) elution step: eluent D being passed through in chromatographic column and is eluted, and the eluent D is water；

(2.3) discharge step: collecting out-feed liquid, wherein the out-feed liquid includes the separation product of D-Psicose；

Wherein, the chromatographic isolation equipment includes 2-20 chromatographic columns and/or chromatography shell of column, and the chromatographic column And/or the filler of chromatography shell of column is resin cation, each chromatographic column and/or chromatography shell of column are connected in series.

In another preferred example, step (2.1) and step (2.2) interval carry out；And/or step (2.3) is carried out continuously.

In another preferred example, material-water ratio 1:(0.5-3.0)；Wherein, the material-water ratio is charging F: the matter of eluent D Amount ratio；Preferably, material-water ratio 1:(0.8-2.5)；It preferably, is 1:(1.0-2.0).

In another preferred example, the separation product of D-Psicose and the mass ratio of charging F total amount are (0.9~1.5): 1； Preferably, (1.0~1.3): 1.

In another preferred example, in step (2.1), the flow of the charging of the method is 0.002-0.150BV (bed body Product)/h；Preferably, the flow of the charging of the method is 0.005-0.10BV/h；It is highly preferred that being 0.01-0.05BV/h.

In another preferred example, in step (2.2), the flow of eluent is 0.005-0.375BV (bed volume)/h,；It is excellent Selection of land, the flow of the charging of the method are 0.0125-0.10BV/h；It is highly preferred that being 0.025-0.125BV/h.

In another preferred example, in step (2.3), the flow of the out-feed liquid is 0.002-0.150BV (bed volume)/h.

In another preferred example, the column temperature of the chromatographic column of the method and/or chromatography shell of column is 20-80 DEG C；Preferably, it is 30-70℃；It is highly preferred that being 50-65 DEG C.

In another preferred example, the partial size of the resin cation are as follows: 50-500um.

In another preferred example, the single chromatographic column and/or the density of the resin cation of chromatography shell of column filling For 0.85~0.95g/cm³。

The single-column of the chromatographic column and/or the single long length of chromatography shell of column are 50-200cm.

In another preferred example, the single hop diameter height ratio 1/20-1/0.4 of the single-column of the chromatographic column and/or chromatography shell of column；It is excellent Selection of land is 1/15-1/1.

In another preferred example, the mobile bed chromatic includes 4-12 (preferably 5-10) chromatographic columns and/or color Compose shell of column.

In another preferred example, the switching time of the chromatographic column of the method and/or chromatography shell of column is 3~15min；It is preferred that Ground is 5-8min.

In another preferred example, the resin cation is selected from: calcium cation resin, sodium form resin cation, potassium Type resin cation, magnesium types resin cation, lithium type resin cation, or combinations thereof；Be preferably, calcium cation resin, Magnesium types resin cation, or combinations thereof.

In another preferred example, in step (c), before the chromatographic isolation further include: carried out to second mixture Desalting processing, to obtain the second mixture through desalination.

In another preferred example, the desalting processing includes ion exchange column desalination.

In another preferred example, the conductivity of the second mixture through desalination≤20 μ s.

In another preferred example, in the separation product containing D-Psicose, purity >=95% of D-Psicose, On the basis of dry matter gross mass；Preferably, purity 96-99%；It is highly preferred that purity is 98-99%.

In another preferred example, in step (c), further comprise the steps of: from second mixture separation and recovery fructose and Glucose.

In another preferred example, in the separation and recovery, with containing " fructose+glucose " solution form recycling fructose and Glucose.

In another preferred example, in " fructose+glucose " solution, the total concentration of glucose and fructose is 15- 40wt%, it is preferable that be 20-35wt%, on the basis of total solution quality.

In another preferred example, described " fructose+glucose " solution includes: the glucose and 30- of glucose 40-70wt% The fructose of 55wt%, it is preferable that the glucose of 50-60wt% and the fructose of 40-50wt%, on the basis of dry matter gross mass.

In another preferred example, the separation product containing D-Psicose includes liquid form product and solid product.

In another preferred example, the method further comprises the steps of:

(d) the separation product to described containing D-Psicose is concentrated and is crystallized (preferably, decrease temperature crystalline), thus Obtain solid D-Psicose.

In another preferred example, the separation product to described containing D-Psicose carries out being concentrated by MVR evaporation Device carries out.

In another preferred example, described to carry out concentration (preferably, vacuum degree is -0.07 to be depressurized in step (d) ~-0.1MPa) concentration.

In another preferred example, it in step (d), at 74~76 DEG C, is concentrated.

In another preferred example, in the method, the conversion per pass of psicose be 4.5-14.5g psicose/ 100g starch.

In another preferred example, in step (a), the amount of the starch raw material liquid is 1~5 ton；It preferably, is 1~3 Ton；It is highly preferred that being 2 tons.

In another preferred example, the raw amount of the D-Psicose of the method is 100~1250kg/ days；Preferably, it is 100~750kg/ days；It is highly preferred that being 200~500kg/ days.

In another preferred example, in step (b2), the C-3 allomerase is D-Psicose -3- epimerase.

In another preferred example, the glucose isomerase and/or C-3 epimerase are selected from the group: enzyme solution, enzyme are dry Powder, immobilised enzymes, or combinations thereof.

In another preferred example, the D-Psicose -3- epimerase is series bacillus (Paenibacillussenegalensis) D-Psicose -3- epimerase.

In another preferred example, the D-Psicose -3- epimerase is selected from the group:

(i) amino acid sequence polypeptide as shown in SEQ ID NO.:1；

(ii) polypeptide of amino acid sequence shown in SEQ ID NO.:1 is (preferable by one or more amino acid residues Ground, 1-50, more preferably, 1-30, more preferably, 1-10, most preferably, 1-6) replace, miss or add and formed, Or addition signal peptide sequence after formed and have catalysis generate the active derived peptides of psicose；

(iii) contain the derived peptides that amino acid sequence is pressed described in (i) or (ii) in sequence；

(iv) amino acid sequence shown in amino acid sequence and SEQ ID NO.:1 homology >=70% (preferably >= 80%, more preferably >=90%), and there is catalysis to generate the active derived peptides of psicose.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 is continuous chromatography separation process schematic diagram of the invention.

Specific embodiment

After extensive and in-depth study, by the screening of a large amount of process routes, the present inventor unexpectedly develops for the first time A method of D-Psicose is produced based on starch raw material.In the methods of the invention, pass through sugar that is continuous and especially optimizing Change processing, isomerization processing and separating treatment, not only can be with continuous industrial production D-Psicose, but also can obtain high-purity The D-Psicose of (up to 98-99%) is spent, and the production cost of D-Psicose can be significantly reduced.On this basis, The present inventor completes the present invention.

Term

As used herein, term " dry matter " refers to the summation for removing all substances other than water.

As used herein, term " concentration " refers to that predetermined substance weight accounts for the percentage of total solution weight, for example, A Luo ketone The concentration of sugar is psicose weight/total solution weight * 100%.

As used herein, term " purity " refers to that predetermined substance weight accounts for the percentage of substance total weight than water, example Such as, the purity of psicose is the weight * 100% of dry matter in psicose weight/solution.

As used herein, the molecular compound that term " glucide " is made of tri- kinds of elements of C, H, O, in this application institute Stating glucide is psicose, fructose, glucose etc..

As used herein, term " weight ratio " is within a certain period of time or when separating a certain amount of material between each substance Average weight ratio.

As used herein, term " enzyme activity " refers to the enzyme amount for being converted into the corresponding product of 1umol per minute, is 1u.

As used herein, term " dry matter " refers to the component in mixture than water, predominantly sugared in the present invention Substance.

As used herein, term " conversion per pass " refers to, obtains " fructose+Portugal when not recycling separation psicose When the solution of grape sugar ", the amount for the psicose that the starch of unit mass is prepared.

C-3 allomerase

As used herein, term " C-3 allomerase ", " C-3 fructose difference phase isomerase " can be mutual with " C-3 difference phase isomerase " Change use, refer to can efficient catalytic generate psicose enzyme.

In the present invention, a kind of typical C-3 allomerase is D-Psicose -3- epimerase.In the present invention, Typical D-Psicose -3- the epimerase is albumen (i.e. wild type D-Psicose-shown in SEQ ID NO.:1 3- epimerase) or its derived protein (for example, saltant type D-Psicose -3- epimerism shown in SEQ ID NO.:2 Enzyme), the D-Psicose -3- epimerase comes from series bacillus (Paenibacillus senegalensis).

The term as used herein " separation " refers to that substance is separated from its primal environment (if it is natural object Matter, primal environment are natural surroundings).As under the native state in active somatic cell polynucleotide and polypeptide be not separate Purifying, but same polynucleotide or polypeptide in native state in other substances with existing for such as from separating, then is separation Purifying.Therefore, the term as used herein " isolated D-Psicose -3- epimerase " refers to the albumen substantially not Containing natural relative other albumen, lipid, carbohydrate or other materials.Those skilled in the art can use the protein of standard Purification techniques D-Psicose -3- epimerase of the invention.Substantially pure albumen is in non-reducing polyacrylamide Single band can be generated on gel.However, in view of the teachings of the present invention and the prior art, those skilled in the art also Ying Ming White " D-Psicose -3- epimerase " should also include the variant form of the albumen, and the variant form has and " this hair The same or similar function of bright D-Psicose -3- epimerase ", but its amino acid sequence and wild type D- A Luo ketone Amino acid sequence shown in sugar -3- epimerase has a small amount of difference.These variant forms include but is not limited to: one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, also more preferably such as 1-8,1-6) amino Missing, insertion and/or the substitution of acid, and C-terminal and/or N-terminal addition it is one or more (usually within 20, compared with Being goodly is more preferably within 6 within 10) amino acid.For example, those skilled in the art are known, with similar performance or phase As amino acid when being replaced, do not usually change the function of protein.For another example, in C-terminal and/or N-terminal addition one A or several amino acid will not generally also change the function of protein.The term further includes D-Psicose -3- epimerase The active fragment and reactive derivative of albumen.

The variant form of polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation, induces and dash forward Variant, can be miscellaneous with the coding DNA of " yellow D-Psicose -3- epimerase of the invention " under high or low stringency The encoded albumen of the DNA of friendship.The invention also includes other polypeptides, such as comprising " D-Psicose -3- epimerism of the invention The fusion protein of enzyme " or its segment.Other than the almost polypeptide of overall length, the present invention should also include " D-Psicose-of the invention The active fragment of 3- epimerase ".In general, the segment has the ammonia of " D-Psicose -3- epimerase of the invention " At least about 10 continuous amino acids of base acid sequence, typically at least about 30 continuous amino acids, preferably at least about 50 it is continuous Amino acid, more preferably at least about 80 continuous amino acids, most preferably at least about 100 continuous amino acids.

The present invention also provides the analogs of " D-Psicose -3- epimerase ".These analogs and the natural " present invention D-Psicose -3- epimerase " difference can be the difference on amino acid sequence, be also possible to not influence sequence Difference on modified forms, or have both at the same time.These polypeptides include natural or induction genetic variant.Induce variant can To be obtained by various technologies, random mutagenesis such as is generated by radiating or being exposed to mutagens, can also pass through site-directed mutagenesis Or the technology of other known molecular biology.Analog further includes with residue (such as D- amino different from natural L-amino acids Acid) analog, and with it is non-naturally occurring or synthesis amino acid (such as β, gamma-amino acid) analog.It should be understood that Albumen of the invention is not limited to enumerated representative albumen.

Modification (not changing primary structure usually) form includes: the chemical derivative form such as acetyl of internal or external polypeptide Change or carboxylated.Modification further includes glycosylation.Modified forms further include with phosphorylated amino acid residue (such as phosphotyrosine, Phosphoserine, phosphothreonine) sequence.It further include being modified to improve its anti-proteolytic properties or optimize molten Solve the albumen of performance.

In the present invention, the conservative variation's polypeptides of " D-Psicose -3- epimerase " refer to and wild type D- A Luo Amino acid sequence shown in ketose -3- epimerase is compared, and has at most 20, and preferably at most 10, more preferably at most 5, Most preferably at most 3 amino acid are replaced by amino acid with similar or analogous properties and form polypeptide, but the conservative variation Polypeptide still has and amino acid sequence the same or similar activity of albumen as shown in SEQ ID NO:1, that is, catalysis generates A Luo The activity of ketose.

Therefore, in view of the teachings of the present invention and the prior art, those skilled in the art can basis, such as carried out shown in following table Amino acid substitution and the mutant for generating conservative variation.

Therefore, " containing " used herein, " having " or " comprising " include "comprising", " mainly by ... constitute ", " base On this by ... constitute " and " by ... constitute "；" mainly by ... constitute ", " substantially by ... constitute " and " by ... structure At " belong to the subordinate concept of " containing ", " having " or " comprising ".

Albumen of the invention can be recombinant protein, native protein, synthetic proteins, preferably recombinant protein.Egg of the invention It is white to can be native purified product or chemically synthesized product, or use recombinant technique from protokaryon or eucaryon host (example Such as, bacterium, yeast, higher plant, insect and mammalian cell) in generate.According to host used in recombinant production scheme, originally The albumen of invention can be glycosylated, or can be nonglycosylated.Albumen of the invention may also include or not include starting Methionine residues.

It will be understood by those skilled in the art that " D-Psicose -3- epimerase " of the invention further includes " D- A Luo ketone Segment, the derivative and analogue of sugar -3- epimerase ".As used herein, term " segment ", " derivative " with it is " similar Object ", which refers to, is kept substantially " D-Psicose -3- epimerase " identical biological function or active more of the invention Peptide.Polypeptide fragment of the invention, derivative or the like, which can be (i), has one or more conservative or non-conservative amino acids residual Base (preferably conservative amino acid) substituted polypeptide, and such substituted amino acid residue can be and may not be By genetic code encoding, or (ii) mature with the polypeptide of substituent group, or (iii) in one or more amino acid residues Polypeptide and another compound (for example extending the compound of polypeptide half-life period, such as polyethylene glycol) fusion are formed by polypeptide, Or (iv) additional amino acid sequence be fused to this polypeptide sequence and the polypeptide that is formed (such as leader sequence or secretion sequence are used to Purify the sequence or proprotein sequence or fusion protein of this polypeptide).According to the definition of this paper these segments, derivative and similar Object belongs to scope known to those skilled in the art.

In view of state of the art and the teachings of the present invention, those skilled in the art are not difficult to obtain D- A Luo ketone of the present invention The active fragment of sugar -3- epimerase.Therefore, the bioactivity piece of any " D-Psicose -3- epimerase " Section can be applied to the present invention.Herein, the bioactive fragment of " D-Psicose -3- epimerase " refers to " D- The segment of psicose -3- epimerase ", but it is still able to maintain the complete of overall length " D-Psicose -3- epimerase " Portion or partial function.Under normal conditions, the bioactive fragment at least keeps overall length " D-Psicose -3- epimerism 50% activity of enzyme ".Under still more preferential conditions, the active fragment is able to maintain overall length " D-Psicose -3- difference is to different 60%, 70%, 80%, 90%, 95%, 99% or 100% activity of structure enzyme ".

Based on the teachings of the present invention and the prior art, those skilled in the art are to be further understood that can be by D-Psicose- The other utilizations form such as immobilised enzymes is made in 3- epimerase.

" amylase ", which refers to, as used herein can hydrolyze starch, glycogen and the enzyme (example in relation to the O- glucose key in polysaccharide Such as, alphalise starch), cut off α-Isosorbide-5-Nitrae glycosidic bond when acting on starch in a random way inside starch molecule.

Term " carbohydrase " as used herein, also known as glucoamylase [Glucoamylase, EC.3.2.1.3.], this Starch can be generated glucose from irreducibility end hydrolysis a-1.4 glucoside bond by kind enzyme, also can slowly hydrolyze a-1.6 grape Glycosidic bond is converted into the enzyme of glucose.Simultaneously also can hydrolyse dextrin, the non-reducing end of glycogen discharges β-D-Glucose.

The method for preparing D-Psicose

In the present invention, a kind of method for preparing D-Psicose is additionally provided, comprising steps of

(a) saccharification processing is carried out to starch, obtains the first mixture；

(b) isomerization processing is carried out to first mixture, obtains the second mixture；With

(c) second mixture is post-processed, obtains D-Psicose.

In a preferred embodiment, the method for preparing D-Psicose is as follows:

In step (a), starch liquefies by amylase and saccharification enzymatic conversion, forms starch saccharificating liquid (the starch saccharification The first mixture in liquid, that is, step (a)).

In the present invention, in the starch saccharificating liquid, containing a certain concentration (as >=70%, >=80%, >=90% or >= 95%) other glucides (including maltose, isomaltose, maltotriose etc.) of glucose and surplus are (for example, starch Saccharified liquid ingredient are as follows: glucose about 95.4%, maltose 1.9%, isomaltose 0.9%, maltotriose 0.5%, maltotetraose The above polysaccharide 1.4%, on the basis of dry matter gross mass).

The starch saccharificating liquid passes through glucose isomerase and C-3 difference phase isomerase isomerization.

In a preferred embodiment, the saccharified liquid can not be separated, starch saccharificating liquid is directly over by appropriate concentration Glucose isomerase and C-3 difference phase isomerase isomerization form mixed liquor

In another preferred example, in step (a), the starch saccharificating liquid makes by heat treatment (80-100 DEG C of temperature) Amylase and carbohydrase inactivation therein.

In another preferred example, be filtered to remove in the starch saccharificating liquid it is heat-treated after inactivate amylase liquefaction and Be saccharified enzymatic conversion.

Starch saccharificating liquid mixed liquor (mixed liquor, that is, step (b) second after glucose isomerase and C-3 epimerism Mixed liquor) ingredient are as follows:

1) concentration 30-60% (for example, 50%), on the basis of the gross mass of mixed liquor；

2) component: psicose 5.5~7.5%, fructose 18.6~20.5%, glucose 20.5~25.9%, maltose 1.9%, the above polysaccharide 1.4% of isomaltose 0.9%, maltotriose 0.5%, maltotetraose, using the gross mass of mixed liquor as base It is quasi-；

3) pass through desalination conductivity≤20 μ s (neutrality).

In another preferred example, described to be isomerized to by directly adding glucose isomer enzyme into starch saccharificating liquid With C-3 allomerase, or flows through starch saccharificating liquid and be fixed with glucose isomer enzyme and C-3 allomerase reaction column.

In another preferred example, described to be isomerized to by directly adding glucose isomer enzyme into starch saccharificating liquid With C-3 allomerase can not use in separating step (a) through inactivating treatment enzyme.

Into chromatographic isolation, two parts solution is obtained after separation, wherein psicose solution composition is as follows:

1) part solution containing psicose: the purity of psicose reaches 97%~99%, and 95% or more separation yield is dense Degree 4~7.2%.It is (described to be processed into psicose product by techniques such as concentration, crystallizations.)

2) contain the solution of " fructose+glucose ": dry matter concentration 25~35%, each component in the solution of " fructose+glucose " Purity be fructose 30~55%, glucose 40~70%, maltose 0.6~1.0%, isomaltose 0.3~0.5%, malt The above polysaccharide 0.4~0.7% of trisaccharide 0.13~0.3%, maltotetraose, psicose 0.05~0.3%.

In another preferred example, shown method includes the following steps:

1) starch liquefacation and saccharification prepare starch saccharificating liquid:

Mixing (concentration 25-35%, starch and water are added amylase 10-50u/g starch, adjust pH), (100-130 DEG C of liquefaction Liquefy and maintain 30-60 minutes), neutralize (adjust pH), saccharification (carbohydrase 10-200u/g starch, 58-62 DEG C of saccharification is added) obtains The starch saccharificating liquid (the starch saccharificating liquid ingredient be glucose about 95.4%, maltose 1.9%, isomaltose 0.9%, The above polysaccharide 1.4% of maltotriose 0.5%, maltotetraose, on the basis of dry matter gross mass)；Preferably, the starch is heated Saccharified liquid inactivates enzyme, and active carbon decoloring is added and filters.

In another preferred example, starch is tuned into the lotion of 25-35wt% (such as 30wt%) with purified water, adjust pH to 6.2-6.4 (is such as reconciled with sodium carbonate), and the amylase (20-50u/g starch) of starch weight 0.05% is added, and allotment is uniform, 110 ± 5 DEG C of injection liquefaction, liquefaction DE value are 15-20%.The liquefier is cooled to 55-60 DEG C, adjusting pH is 4.3-4.8, is added The carbohydrase (50-100u/g starch) for entering starch weight 0.05%, starts to be saccharified, until DE > 98%, is warming up to 100 at 55-60 DEG C ± 10 DEG C of inactivation 2-3min.It is cooled to 50 DEG C, is added the active carbon of starch weight 0.5~1.5% (such as 1.0%), stirring 15~ 45min (such as 30min) decoloration, overanxious removing insoluble impurities and zymoprotein, obtain dilute saccharified liquid, are dehydrated by being concentrated under reduced pressure, Obtain the starch saccharificating liquid of high concentration.

In another preferred example, the glucose containing 50 ± 2wt% is prepared (with starch saccharificating liquid total weight in 1kg starch On the basis of) 1.5~2.5kg starch saccharificating liquid.

2) above-mentioned starch saccharificating liquid (for example, using MVR or other evaporators) is concentrated to dry matter concentration 30-60wt% (preferably, 45-55wt%), in terms of gross mass；And metal ion is added.Pass through according to certain flow rate and " glucose isomerase is housed The enzyme immobilization pillar of enzyme " and " C-3 fructose difference phase isomerase ".Cross the mixed liquor that column obtains (the i.e. step (b) of mixed liquor Second mixture) ingredient are as follows: psicose 5.5~7.5%, fructose 18.6~20.5%, glucose 20.5~29%, maltose 1.9%, the above polysaccharide 1.4% of isomaltose 0.9%, maltotriose 0.5%, maltotetraose, using the gross mass of mixed liquor as base It is quasi-.

3) desalination: the ion exchange column desalination of calcium type is used, subsequently into chromatographic isolation equipment.

4) chromatographic isolation: the pillar that dedicated chromatography separation resin is housed and has been regenerated with calcium chloride using 4-8, process is such as Shown in Fig. 1, by taking 4 columns as an example.

As shown in Figure 1, discharging is carried out continuously, intermittent charging (charging interval time such as 30-60min, with resin is carried out Amount and packed density etc. it is related) and elution (elution interval time such as 30-60min has with the amount of resin and packed density etc. Close), collect respectively fast component (main component is psicose, i.e. the separation product of D-Psicose) and slow component (mainly at Dividing is glucose and fructose).

Specifically, 4 Coupled columns connections, mobile phase flow successively through (flow velocity 0.002-0.150BV/h, wherein 4 Whether flow velocity is identical or different in a pillar) this 4 chromatographic columns, while chromatographic column can be mobile to mobile phase flow direction.

(feed rate 0.002-0.150BV/h) is fed first, feeds the laggard water elution of 0.01-0.02BV, and pass through shifting Dynamic chromatographic column makes the water entry position of eluant, eluent (be added) between fast component unloading position and slow component unloading position, color The switching time t for composing column is 5-8min.Wherein, the switching time refers to, chromatographic column is in a certain position, in the t time, chromatography Column starts that next position on mobile phase flow direction is moved and be moved to along mobile phase flow direction；For example, as shown in Figure 1, After a certain chromatographic column is moved to 1 position of chromatographic column, after the t time, which is moved quickly by 1 position of chromatographic column The position of chromatographic column 2 in figure.

In another preferred example, the material-water ratio (quality of material: the quality of eluting water) of the chromatography separating method is 1: (1.5~3), fast component/slow component (psicose solution) are (2~3): 1.

For example, chromatographic condition can be with are as follows:

Feed F=0.4Kg/h (related with amount of resin), eluant, eluent D=0.6Kg/h, discharging AD (slow component)=0.33Kg/ H, BD (fast component)=0.66Kg/h, treating capacity 0.03Kg feed liquid/Kg resin, material-water ratio 1:1.5, BD/AD=2；

Momentary operation condition: F → B=33ml/min (fast component discharges when charging), D → A (slow component discharges when elution) =33ml/min, internal circulating load (are inversely proportional) R=33ml/min, D → B (when elution fast component discharge)=33ml/ with switching time min。

As a result:

(1) psicose solution purity 98-99% is obtained, (on the basis of dry matter gross mass)；Psicose yield 95% or more；

(2) " fructose+glucose " mixed liquor, dry substance concentration 25-35wt% are obtained.

In dry matter the content of fructose be 30-55%, glucose content 40-70%.

(3) " maltose+isomaltose+maltotriose+polysaccharide+psicose " mixed liquor, dry matter concentration 5- are obtained 20%.

Wherein: maltose 3-10wt%, isomaltose 3-10wt%, maltotriose 1-5wt%, polysaccharide 0.5-5wt%, Ah Lip river ketose 1-6wt% (on the basis of the quality of " maltose+isomaltose+maltotriose+polysaccharide+psicose " mixed liquor).

Main advantages of the present invention include:

(1) present invention is made from starch for the first time, by treatment processes such as saccharification, isomerization, concentration, chromatographic isolations, is obtained The D-Psicose of 98-99% is up to purity.

(2) present invention uses special C-3 allomerase for the first time, it is from series bacillus (Paenibacillus Senegalensis D-Psicose -3- epimerase), can efficient catalytic generation D-Psicose.

(3) D-Psicose is produced with method of the invention, can significantly reducing the production cost of D-Psicose, (cost can Decline is 30%).

(4) raw material (glucose, fructose and metal ion) also can be recycled in method of the invention, greatly reduces into This.

(5) the method for the present invention is suitable for industrial production.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and Number is weight percent and parts by weight.

Glucose isomerase used in embodiment, amylase and carbohydrase are believed purchased from Novi.

C-3 epimerase used in embodiment (i.e. C-3 allomerase), sequence is as shown in SEQ ID No.:2.

If not otherwise specified, material used in embodiment and reagent are commercial product.

Embodiment 1

Starch saccharificating liquid preparation

First 100Kg starch is tuned into 30% lotion with purified water, adjusts pH to 6.36.2-6.4 with sodium carbonate, be added and form sediment The amylase (20u/g starch) of powder weight 0.05%, allotment uniformly, are liquefied in 110 degree of injections, and liquefaction DE value is 18%.It should Liquefier is cooled to 55 degree, and adjusting pH is 4.5, and the carbohydrase (100u/g starch) of starch weight 0.05% is added, opens at 55 degree Begin to be saccharified, until DE > 98%, is warming up to 100 degree of inactivation 3min, is cooled to 50 degree, the active carbon of starch weight 1.0% is added, stirs 30min decoloration, overanxious removing insoluble impurities and zymoprotein are mixed, dilute starch saccharificating liquid is obtained, is dehydrated, obtains by being concentrated under reduced pressure The starch saccharificating liquid of the high concentration of 200Kg glucose content 50%, starch saccharificating liquid ingredient: glucose about 95.4%, maltose 1.9%, the above polysaccharide 1.4% of isomaltose 0.9%, maltotriose 0.5%, maltotetraose (on the basis of dry matter).

Embodiment 2

The preparation of D-Psicose

2.1 isomerization

In the reaction kettle of 5L, 3.0Kg starch saccharificating liquid is added and (is prepared in embodiment 1, glucose content 50%, about It is made by 1.5Kg starch), 1.0mM MnSO₄.7H₂O, glucose isomer enzyme and C-3 epimerase, total enzyme activity are respectively 50000u and 20000u is reacted at 60 DEG C, and the conversion of about 3h glucose reaches balance.Obtain reaction product.

The reaction product is measured, the results showed that about 14.6% glucose is converted into D-Psicose.

The removal of 2.2 enzymes

To the reaction product, two enzymes (glucose isomer enzyme and C-3 epimerase) is filtered to remove by film.

2.3 separation D-Psicoses

Through calcium cation resin treatment, manganese ion is removed from reaction product, and recycles solution with manganese ions.

Then, carry out chromatographic isolation with Simulation moving bed, thus separate acquisition mainly the aqueous solution containing psicose and Mixed liquor (" fructose+glucose " solution) mainly containing fructose and glucose.Wherein gained " fructose+glucose " solution is recovered It recycles, i.e., after it being concentrated, is converted again with glucose isomer enzyme and C-3 epimerase.

As a result

(a) " fructose+glucose " mixed liquor 6.0Kg is obtained, the content of dry is 31%.In dry, fructose contains Amount is 46%, glucose content 52%, by dry matter total weight.

(b) the aqueous solution 3.0Kg of psicose is obtained, wherein the content of D-Psicose is 7%, D-Psicose Purity is 98.7%；The content < 1.3% of impurity (including fructose, glucose and other sugars), by dry matter total weight.

Gross production rate: 0.140kg psicose/kg starch.

Embodiment 3

The preparation of D-Psicose

3.1 isomerization

In the reaction kettle of 5L, it is added 3Kg starch saccharificating liquid (preparation of embodiment 1), 1.0mMMnSO₄.7H₂O is preheated to 60 Degree is flowed through the immobilized reactant of (flow velocity for crossing column is 1Kg/h) glucose isomer enzyme and C-3 epimerase by pump conveying Column is converted at 60 DEG C by biological enzyme, and efflux is reaction product.

The efflux is measured, has 14.3% glucose to be converted into D-Psicose in efflux.

3.2 separation D-Psicoses

As a result

(a) " fructose+glucose " mixed liquor 6.0Kg has been obtained, the content of dry is 30%.The fructose in dry matter Content is 46%, glucose content 52%；

(b) the aqueous solution 3.0Kg of psicose has been obtained, wherein the content (dry substance concentration) of D-Psicose is 6.7%, D-Psicose purity is 98.1%, and the content < 1.9% of impurity (including fructose, glucose and other sugars) is pressed The total weight of dry matter.

Gross production rate: 0.134kg psicose/kg starch.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>Biotechnology Co., Ltd is based oneself upon in Shanghai

<120>a kind of method that D-Psicose is prepared by starch

<130> P2016-1406

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 292

<212> PRT

<213>series bacillus (Paenibacillus senegalensis)

<400> 1

Met Lys Phe Gly Thr Tyr Phe Ala Tyr Trp Glu Gln Ser Trp Asp Thr

1 5 10 15

Asp Tyr Leu Lys Tyr Val Lys Lys Val Ala Asp Leu Gly Phe Asp Val

20 25 30

Leu Glu Val Gly Ala Ala Gly Ile Val Asn Met Ser Asp Asp Ala Leu

35 40 45

Ser Ala Leu Lys Ser Glu Ala Glu Asn Tyr Ala Ile Thr Leu Thr Ala

50 55 60

Gly Ile Gly Leu Pro Lys Gln Phe Asp Val Ser Ser Glu Asn Glu Ser

65 70 75 80

Val Arg Gln Asp Gly Ile Ala Phe Met Lys Lys Ile Leu Asp Ala Leu

85 90 95

His Lys Ala Gly Ile Lys Ala Ile Gly Gly Thr Ile Tyr Ser Tyr Trp

100 105 110

Pro Val Asp Tyr Ser Ala Pro Ile Asn Lys Pro Ala Val Arg Lys Gln

115 120 125

Ser Ile Lys Ser Met Gln Glu Leu Ala Asp Tyr Ala Ala Gln Tyr Asp

130 135 140

Ile Thr Leu Leu Val Glu Ser Leu Asn Arg Phe Glu Gln Phe Leu Val

145 150 155 160

Asn Asp Ala Lys Glu Ala Val Asp Tyr Val Lys Ala Val Asn Lys Pro

165 170 175

Asn Val Lys Val Met Leu Asp Ser Phe His Met Asn Ile Glu Glu Asp

180 185 190

Tyr Leu Gly Asp Ala Ile Arg Tyr Thr Gly Asp Tyr Leu Gly His Phe

195 200 205

His Ile Gly Glu Cys Asn Arg Lys Val Pro Gly Lys Gly His Met Pro

210 215 220

Trp Ser Glu Ile Gly Gln Ala Leu Arg Asp Ile Gln Tyr Asp Gly Cys

225 230 235 240

Val Val Met Glu Pro Phe Val Arg Pro Gly Gly Ile Val Gly Ser Asp

245 250 255

Ile Lys Val Trp Arg Asp Leu Ser Asp Asn Ala Asp Glu Ala Lys Leu

260 265 270

Asp Ala Asp Ile Lys Glu Ser Leu Glu Phe Val Lys Gln Thr Phe Leu

275 280 285

Lys Ser Thr Pro

290

<210> 2

<211> 292

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 2

Met Lys Phe Gly Thr Tyr Phe Ala Tyr Trp Glu Gln Ser Trp Asp Thr

1 5 10 15

Asp Tyr Leu Lys Tyr Val Lys Lys Val Ala Asp Leu Gly Phe Asp Val

20 25 30

Leu Glu Val Gly Ala Ala Gly Ile Val Asn Met Ser Asp Asp Ala Leu

35 40 45

Ser Ala Leu Lys Ser Glu Ala Glu Asn Tyr Ala Ile Thr Leu Thr Ala

50 55 60

Gly Ile Gly Leu Pro Lys Gln Phe Asp Val Ser Ser Glu Asn Glu Ser

65 70 75 80

Val Arg Gln Asp Gly Ile Ala Phe Met Lys Lys Ile Leu Asp Ala Leu

85 90 95

His Lys Ala Gly Ile Lys Ala Ile Gly Gly Thr Ile Tyr Ser Tyr Trp

100 105 110

Pro Val Asp Tyr Ser Ala Pro Ile Asn Ala Pro Ala Val Arg Lys Gln

115 120 125

Ser Ile Lys Ser Met Gln Glu Leu Ala Asp Tyr Ala Ala Gln Tyr Asp

130 135 140

Ile Thr Leu Leu Val Glu Ser Leu Asn Arg Phe Glu Gln Phe Leu Val

145 150 155 160

Asn Asp Ala Lys Glu Ala Val Asp Tyr Val Lys Ala Val Asn Lys Pro

165 170 175

Asn Val Lys Val Met Leu Asp Ser Phe His Met Asn Ile Glu Glu Asp

180 185 190

Tyr Leu Pro Asp Ala Ile Arg Tyr Thr Gly Asp Tyr Leu Gly His Phe

195 200 205

His Ile Gly Glu Cys Asn Arg Lys Val Pro Gly Lys Gly His Met Pro

210 215 220

Trp Ser Glu Ile Gly Gln Ala Leu Arg Asp Ile Gln Tyr Asp Gly Cys

225 230 235 240

Val Val Met Glu Pro Phe Val Arg Pro Gly Gly Ile Val Gly Ser Asp

245 250 255

Ile Lys Val Trp Arg Asp Leu Ser Asp Asn Ala Asp Glu Ala Lys Leu

260 265 270

Asp Ala Asp Ile Lys Glu Ser Leu Glu Phe Val Lys Gln Thr Phe Leu

275 280 285

Lys Ser Thr Pro

290

Claims

1. a kind of method for producing D-Psicose, which is characterized in that comprising steps of

(a) saccharification processing is carried out to starch raw material liquid, to form the first mixture containing saccharification product, the saccharification product It include: glucose, maltose, isomaltose, maltotriose and maltotetraose；

(b) isomerization processing is carried out to first mixture, glucose isomerase is turned into fructose and then is isomerized to D- A Luo Ketose, to form the second mixture containing D-Psicose；With

2. the method as described in claim 1, which is characterized in that before the step (a) further include step (a1): being carried out to starch Liquefaction processing, to form starch raw material liquid.

3. method according to claim 2, which is characterized in that carry out liquefaction processing to starch in the presence of amylase.

4. method as claimed in claim 3, which is characterized in that the liquefaction processing has following one or more features:

The additional amount of amylase is 10-50u/g starch；

PH is 5.5~6.5；

The temperature of the liquefaction processing is 100-130 DEG C；And/or

Liquefying time is 30-60min.

5. the method as described in claim 1, which is characterized in that in step (a), in the presence of carbohydrase, to starch raw material Liquid carries out saccharification processing, to form the first mixture.

6. method as claimed in claim 5, which is characterized in that in step (a), saccharification processing have following one or Multiple features:

The additional amount of carbohydrase is 10-200u/g starch；

The pH of saccharification processing is 4.0~5.5；

Saccharificatinn period is 20-40h；And/or

The treatment temperature of saccharification processing is 35~70 DEG C.

7. the method as described in claim 1, which is characterized in that the step (b) includes:

(b1) the first enzymatic reaction is carried out to first mixture with glucose isomer enzyme, to obtain mixed containing fructose Close object；With

8. the method for claim 7, which is characterized in that further comprised the steps of: before step (b1) to first mixing Metal ion is added in object；Preferably, the metal ion is selected from: Mn²⁺、Co²⁺、Mg²⁺、Fe²⁺Or combinations thereof.

9. the method as described in claim 1, which is characterized in that in step (c), by chromatography, from described second The isolated separation product containing D-Psicose of mixture.

10. the method as described in claim 1, which is characterized in that in the method, the conversion per pass of psicose is 4.5- 14.5g psicose/100g starch.