WO2020057561A1 - 一种由淀粉制备d-阿洛酮糖的方法 - Google Patents

一种由淀粉制备d-阿洛酮糖的方法 Download PDF

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WO2020057561A1
WO2020057561A1 PCT/CN2019/106498 CN2019106498W WO2020057561A1 WO 2020057561 A1 WO2020057561 A1 WO 2020057561A1 CN 2019106498 W CN2019106498 W CN 2019106498W WO 2020057561 A1 WO2020057561 A1 WO 2020057561A1
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psicose
mixture
starch
column
glucose
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PCT/CN2019/106498
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English (en)
French (fr)
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任世阔
牛志国
韩子明
赵红兵
吴会广
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上海立足生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Definitions

  • the invention relates to the field of functional foods, in particular to a method for preparing D-psicose from starch.
  • D-psicose is a rare sugar that exists in nature. According to research reports, D-psicose is a new type of functional monosaccharide. Compared with sucrose, it has low calories and pure taste, and will not be affected by the human body. Metabolism can well control blood sugar. In 2011, the FDA of the United States has approved D-psicose as a food ingredient and considered it to be a safe food; therefore, D-psicose is expected to become a substitute for sucrose, as a food or food additive, widely used in diet, health care and Medicine and other fields.
  • Another method is to separate D-psicose from the existing syrup (if glucose syrup) containing D-psicose.
  • D-psicose is still low in purity (about 10% or less based on the total amount of all sugars) and still contains many other monosaccharides (especially fructose).
  • D-psicose As a new type of sweetener, D-psicose has to be widely popularized and used. It is also necessary to further reduce the production cost while preparing high-purity D-psicose.
  • the purpose of the present invention is to provide a low-cost, high-efficiency, method suitable for industrialized preparation of high-purity psicose products.
  • a first aspect of the invention provides a method for producing D-psicose, comprising the steps:
  • steps (a) and (b) do not include a step of separating sugars.
  • the starch is selected from the group consisting of corn starch, potato starch, or a combination thereof.
  • the starch raw material liquid is a raw material liquid in which starch is liquefied.
  • step (a1) is further included before step (a): liquefying the starch to form a starch raw material liquid.
  • the starch is liquefied in the presence of amylase.
  • the raw starch is mixed with water to form a starch water mixture before liquefaction.
  • the mass ratio of starch to the starch-water mixture is 25-35: 100.
  • the liquefaction treatment has one or more of the following characteristics:
  • the added amount of amylase is 10-50u / g starch
  • pH is 5.5 ⁇ 6.5;
  • the temperature of the liquefaction treatment is 100-130 ° C; and / or
  • the liquefaction time is 30-60min.
  • amylase is added in an amount of 10-25u / g starch.
  • step (a) the starch raw material liquid is subjected to saccharification treatment in the presence of a saccharifying enzyme to form a first mixture.
  • step (a) the saccharification process has one or more of the following characteristics:
  • the added amount of saccharifying enzyme is 10-200u / g starch
  • the pH of saccharification is 4.0 ⁇ 5.5;
  • Saccharification time is 20-40h; and / or
  • the temperature of the saccharification treatment is 35 to 70 ° C.
  • the pH of the saccharification treatment is 4.3 to 4.8.
  • the saccharifying enzyme is added in an amount of 50 to 100 u / g of starch.
  • the processing temperature of the saccharification treatment is 45 to 65 ° C; more preferably, it is 55 to 60 ° C.
  • step (a) further includes a step of inactivating the saccharifying enzyme and the liquefying enzyme in the first mixture at 90 to 110 ° C. to obtain a first mixture in which the enzyme has been inactivated.
  • step (a) further includes the step of adding activated carbon to the first mixture and performing a decoloring treatment to obtain a first mixture subjected to decolorization treatment.
  • the step (a) includes or does not include a step of removing inactivated saccharifying enzyme and liquefying enzyme by filtration.
  • the content of glucose is ⁇ 70%, preferably ⁇ 80%, more preferably ⁇ 90%, most preferably ⁇ 95%, according to the dry matter in the first mixture Total mass meter.
  • the first mixture contains 90-99 wt% glucose, preferably 95-99 wt% glucose, based on the total dry matter mass in the first mixture.
  • the Y value is ⁇ 80%
  • C1 is the concentration of glucose
  • C2 is the concentration of maltose
  • C3 is the concentration of isomalt
  • C4 is the concentration of maltotriose
  • C5 is the concentration of maltotetraose.
  • step (a) further includes the step of: performing a concentration treatment on the first mixture to form a concentrated first mixture.
  • the concentration of the dry matter in the first mixture without concentration is 10-70 wt%, preferably 20-65 wt%, more preferably 30-60 wt%, and the concentration is the total
  • concentration of the dry matter is based on the total mass of the first mixture.
  • the dry matter concentration in the concentrated first mixture is 45-55% by weight, based on the total mass of the first mixture.
  • the concentration treatment is performed using an MVR evaporator.
  • step (b) includes:
  • the method before step (b1), further includes the step of: adding a metal ion to the first mixture; preferably, the metal ion is selected from the group consisting of: Mn 2+ , Co 2+, or a combination thereof.
  • the final concentrations of the metal ions are each independently 1-10 mM.
  • steps (b1) and (b2) are performed at 40-80 ° C; preferably, they are performed at 50-70 ° C.
  • steps (b1) and (b2) are performed simultaneously or sequentially.
  • step (b) or the steps (b1) and (b2) are performed in the same reactor.
  • step (b) glucose isomerase and C-3 isomerase are added to the first mixture, and a reaction is performed to obtain the second mixture.
  • the first mixture flows through a reaction column having a glucose isomerase immobilized to obtain a fructose-containing mixture; and / or
  • step (b2) the fructose-containing mixture is passed through a reaction column to which a C-3 isomerase is immobilized to obtain the second mixture.
  • the first mixture flows through a reaction column having a glucose isomerase immobilized and a reaction column having a C-3 isomerase, thereby obtaining the first Two mixtures.
  • the total concentration of the saccharides in the second mixture is 30-68 wt% (preferably, 50-65 wt%), based on the total mass of the second mixture.
  • the purity of the allosene in the second mixture is greater than 5 wt%, preferably greater than 11 wt%; more preferably, greater than 14 wt%, based on the total dry matter mass in the second mixture.
  • the second mixture includes: ptoseose, glucose, fructose, and oligosaccharides (including disaccharides).
  • the oligosaccharide is selected from the group consisting of maltose, isomaltose, maltotriose, maltotetraose, or a combination thereof.
  • the content of the oligosaccharides (including disaccharides) in the second mixture is 2-10% by weight, based on the total dry matter mass in the second mixture.
  • the content of the glucose in the second mixture is 5-55 wt%, preferably 10-40 wt%, more preferably 20-30 wt%, based on the dry matter in the second mixture.
  • the total mass is the benchmark.
  • the content of the fructose in the second mixture is 1-50 wt%, preferably 5-30 wt%, more preferably 10-25 wt%, based on the dry matter in the second mixture.
  • the total mass is the benchmark.
  • step (c) a chromatographic separation method is used to separate the second mixture to obtain an isolated product containing D-psicose.
  • the chromatographic separation method is a simulated moving bed chromatography method.
  • the chromatographic separation method includes steps
  • the chromatographic separation includes:
  • Elution step the eluent D is passed into a chromatographic column for elution, and the eluent D is water;
  • a discharging step collecting a discharging liquid, wherein the discharging liquid comprises a D-psicose separated product;
  • the chromatographic separation device includes 2-20 of the chromatographic column and / or column segment, and the filler of the chromatographic column and / or column segment is a cationic resin, and each of the chromatographic column and / or chromatographic column is The column segments are connected in series.
  • step (2.1) and step (2.2) are performed intermittently; and / or step (2.3) is performed continuously.
  • the material-water ratio is 1: (0.5-3.0); wherein the material-water ratio is the mass ratio of feed F: eluent D; preferably, the material-water ratio is 1: (0.8 -2.5); preferably, 1: (1.0-2.0).
  • the mass ratio of the separated product of D-psicose to the total amount of feed F is (0.9-1.5): 1; preferably, (1.0-1.3): 1.
  • the feed flow rate of the method is 0.002-0.150 BV (bed volume) / h; preferably, the feed flow rate of the method is 0.005-0.10 BV / h; more preferably, 0.01-0.05 BV / h.
  • the flow rate of the eluent is 0.005-0.375 BV (bed volume) / h; preferably, the flow rate of the feed of the method is 0.0125-0.10 BV / h; More preferably, it is 0.025-0.125 BV / h.
  • step (2.3) the flow rate of the discharge liquid is 0.002-0.150 BV (bed volume) / h.
  • the column temperature of the chromatographic column and / or column segment of the method is 20-80 ° C; preferably, 30-70 ° C; more preferably, 50-65 ° C.
  • the particle size of the cationic resin is: 50-500um.
  • the density of the cationic resin filled in a single column and / or column segment is 0.85 to 0.95 g / cm 3 .
  • a single column and / or a single segment length of the chromatographic column is 50-200 cm.
  • the diameter ratio of a single column and / or a single segment of the chromatographic column is 1 / 20-1 / 0.4; preferably, it is 1 / 15-1 / 1.
  • the moving bed chromatography includes 4-12 (preferably 5-10) chromatographic columns and / or column segments.
  • the switching time of the chromatographic column and / or the chromatographic column segment of the method is 3-15 minutes; preferably, it is 5-8 minutes.
  • the cationic resin is selected from the group consisting of: calcium-type cationic resin, sodium-type cationic resin, potassium-type cationic resin, magnesium-type cationic resin, lithium-type cationic resin, or a combination thereof; preferably, calcium-type resin A cationic resin, a magnesium-type cationic resin, or a combination thereof.
  • step (c) before the chromatographic separation, the method further includes: desalting the second mixture to obtain a desalted second mixture.
  • the desalination treatment includes desalination by an ion exchange column.
  • the conductivity of the desalted second mixture is ⁇ 20 ⁇ s.
  • the purity of D-psicose is ⁇ 95%, based on the total mass of dry matter; preferably, the purity is 96-99 %; More preferably, the purity is 98-99%.
  • step (c) the method further includes a step of separating and recovering fructose and glucose from the second mixture.
  • fructose and glucose are recovered as a solution containing "fructose + glucose".
  • the total concentration of glucose and fructose is 15-40 wt%, preferably 20-35 wt%, based on the total solution mass.
  • the "fructose + glucose” solution includes: 40-70 wt% glucose and 30-55 wt% fructose, preferably, 50-60 wt% glucose and 40-50 wt% fructose, with The total mass of dry matter is the benchmark.
  • the D-psicose-containing isolated product includes a liquid product and a solid product.
  • the method further includes the steps:
  • the D-psicose-containing isolated product is concentrated and crystallized (preferably, crystallized by cooling) to obtain solid D-psicose.
  • said D-psicose-containing isolated product is concentrated by an MVR evaporator.
  • step (d) the concentration is performed under reduced pressure (preferably, the degree of vacuum is -0.07 to -0.1 MPa).
  • step (d) concentration is performed at 74 to 76 ° C.
  • the one-way conversion rate of allosexose is 4.5-14.5 g of allosexose per 100 g of starch.
  • the amount of the starch raw material liquid is 1 to 5 tons; preferably, 1 to 3 tons; more preferably, 2 tons.
  • the D-psicose yield of the method is 100-1250 kg / day; preferably, 100-750 kg / day; more preferably, 200-500 kg / day.
  • step (b2) the C-3 isomerase is D-psicose-3-epimerase.
  • the glucose isomerase and / or C-3 epimerase is selected from the group consisting of an enzyme solution, an enzyme dry powder, an immobilized enzyme, or a combination thereof.
  • the D-psicose-3-epimase is a D-psicose-3-epimase of Paenibacillus senegalensis.
  • D-psicose-3-epimerase is selected from the following group:
  • the amino acid sequence has a homology of ⁇ 70% (preferably ⁇ 80%, more preferably ⁇ 90%) with the amino acid sequence shown in SEQ ID NO :: 1, and has a derivative that catalyzes the production of psicose Peptide.
  • FIG. 1 is a schematic diagram of a continuous chromatography separation process of the present invention.
  • the inventor unexpectedly developed a method for producing D-psicose based on starch raw materials for the first time.
  • the method of the present invention through continuous and specially optimized saccharification, isomerization and separation processes, not only can D-psicose be continuously industrially produced, but high purity (up to 98-99%) D-psicose, and can significantly reduce the production cost of D-psicose.
  • the present inventors have completed the present invention.
  • dry matter refers to the sum of all substances except water.
  • concentration refers to the weight of a particular substance as a percentage of the total weight of the solution, for example, the concentration of psicose is the weight of psicose / total solution * 100%.
  • the term "purity" refers to the weight of a specific substance as a percentage of the total weight of substances other than water.
  • the purity of psicose is the weight of psicose / the weight of dry matter in the solution * 100%.
  • saccharide substance is a molecular compound composed of three elements of C, H, and O.
  • the saccharide substance is ptoseose, fructose, glucose, and the like.
  • weight ratio is the average weight ratio between materials over a period of time or when a certain amount of material is separated.
  • enzyme activity refers to the amount of enzyme that is converted to 1 umol corresponding product per minute, which is 1 u.
  • dry matter refers to the components of the mixture other than water, and in the present invention are primarily sugars.
  • one-pass conversion refers to the amount of psulose produced per unit mass of starch without recycling the solution obtained from "fructose + glucose” when separating psicose.
  • C-3 isomerase As used herein, the terms “C-3 isomerase”, “C-3 fructose isomerase” and “C-3 isomerase” are used interchangeably and refer to the high-efficiency catalysis of the production of poloxone Sugar enzymes.
  • a typical C-3 isomerase is D-psicose-3-epimerase.
  • the typical D-psicose-3-epimerase is the protein shown in SEQ ID No.:1 (i.e., wild-type D-psicose-3-epimer Conformational enzyme) or its derived protein (for example, the mutant D-psicose-3-epimerase shown in SEQ ID No .: 2), the D-psicose-3-epimerase Isomerases are derived from Paenibacillus senegalensis.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in the natural state of living cells are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state.
  • isolated D-psicose-3-epimerase as used herein means that the protein is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • D-psicose-3-epimase of the present invention can purify using standard protein purification techniques. Substantially pure protein produces a single band on a non-reducing polyacrylamide gel.
  • D-psicose-3-epimerase should also include variant forms of the protein, which variant forms have It has the same or similar function as the "D-psicose-3-epimase of the present invention", but its amino acid sequence is the same as that shown by the wild-type D-psicose-3-epimase There are minor differences in the sequence.
  • variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, more preferably such as 1-8, 1-6) amino acid deletions, insertions and / or substitutions, and addition of one or more (usually within 20, preferably within 10, more It is preferably within 6 amino acids.
  • substitution of amino acids with similar or similar properties usually does not change the function of the protein.
  • adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of D-psicose-3-epimerase protein.
  • Polypeptide variants include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, which can interact with "yellow D-psicose of the invention” under conditions of high or low stringency.
  • "-3-Epimerase” encodes a protein encoded by DNA that hybridizes to DNA.
  • the invention also includes other polypeptides, such as fusion proteins comprising "D-psicose-3-epimase of the invention” or fragments thereof.
  • the present invention should also include the active fragment of "D-psicose-3-epimase of the present invention".
  • the fragment has at least about 10 consecutive amino acids, typically at least about 30 consecutive amino acids, and preferably at least about 50 consecutive amino acids of the "D-psicose-3-epimase of the invention" Contiguous amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
  • the invention also provides analogs of "D-psicose-3-epimase".
  • the difference between these analogs and the natural "D-psicose-3-epimerase of the present invention” may be a difference in the amino acid sequence, a difference in a modified form that does not affect the sequence, or both There are.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.
  • Analogs also include analogs with residues (such as D-amino acids) that are different from natural L-amino acids, and analogs with non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • Modified (usually unchanged primary structure) forms include chemically derived forms of the polypeptide, such as acetylated or carboxylated, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize their lytic properties.
  • the conservative variant polypeptide of "D-psicose-3-epimerase” refers to the amino acid sequence compared with the wild-type D-psicose-3-epimase There are at most 20, preferably at most 10, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide, but the conservative variant polypeptide still has The amino acid sequence is the same or similar activity as the protein shown in SEQ ID NO: 1, that is, the activity of catalyzing the production of psicose.
  • substitution residues Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys
  • the protein of the present invention may be a recombinant protein, a natural protein, or a synthetic protein, and is preferably a recombinant protein.
  • the protein of the present invention may be a naturally purified product or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • a prokaryotic or eukaryotic host eg, bacteria, yeast, higher plants, insects, and mammalian cells
  • the proteins of the invention may be glycosylated or may be non-glycosylated.
  • the proteins of the invention may also include or exclude initial methionine residues.
  • D-psicose-3-epimase of the present invention also includes fragments, derivatives and similar of "D-psicose-3-epimase” Thing.
  • fragment refers to a biological function or activity that substantially maintains the same "D-psicose-3-epimerase” of the invention Peptide.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that extends the half-life of a polypeptide, such as (Polyethylene glycol), a polypeptide formed by fusion, or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify the polypeptide or a protein sequence, or a fusion) protein).
  • conservative or non-conservative amino acid residues preferably conservative amino acid residues
  • substituted amino acid residues It may or may not be encoded by the genetic code, or (ii)
  • any biologically active fragment of "D-psicose-3-epimase” can be applied to the present invention.
  • the biologically active fragment of "D-psicose-3-epimerase” refers to the fragment of "D-psicose-3-epimase", but it can still be maintained Full or partial function of the full-length "D-psicose-3-epimerase”.
  • the biologically active fragment retains at least 50% of the activity of the full-length "D-psicose-3-epimerase". Under more preferred conditions, the active fragment is able to maintain 60%, 70%, 80%, 90%, 95%, 99%, Or 100% active.
  • D-psicose-3-epimerase can be made into other utilization forms such as immobilized enzyme.
  • amylase refers to an enzyme (e.g., alpha-starch) capable of hydrolyzing O-glucose bonds in starch, glycogen, and related polysaccharides, which acts on starch in a random manner to cut off alpha- 1,4 glycosidic bonds.
  • sacharifying enzyme also known as glucoamylase [Glucoamylase, EC.3.2.1.3.] This enzyme can hydrolyze starch from a non-reducing end to a-1.4 glucosidic bonds to produce glucose, and can also slowly An enzyme that hydrolyzes a-1.6 glucosidic bonds and converts them to glucose. It can also hydrolyze dextrin and release ⁇ -D-glucose at the non-reducing end of glycogen.
  • fast component refers to a component with a shorter retention time, first-in (first-out chromatographic separation).
  • slow component refers to a component with a longer retention time and a later-out (slower leaving the chromatographic separation).
  • a method for preparing D-psicose comprising the steps:
  • the method for preparing D-psicose is as follows:
  • step (a) starch is liquefied and saccharified by amylase to form a starch saccharified solution (the starch saccharified solution is the first mixture in step (a)).
  • the starch saccharification solution contains a certain concentration (such as ⁇ 70%, ⁇ 80%, ⁇ 90%, or ⁇ 95%) of glucose and the balance of other sugars (including maltose, isomalt) , Maltotriose, etc.) (for example, the components of starch saccharification solution are: about 95.4% glucose, 1.9% maltose, 0.9% isomaltose, 0.5% maltotriose, 1.4% polysaccharides above maltotetraose, based on the total mass of dry matter ).
  • the starch saccharification solution isomerized by glucose isomerase and C-3 phase isomerase.
  • the saccharification solution may not be separated, and the starch saccharification solution may be appropriately concentrated and directly isomerized with glucose isomerase and C-3 phase isomerase to form a mixed solution.
  • step (a) the starch saccharification solution is heat-treated (temperature 80-100 ° C.) to inactivate the amylase and saccharification enzyme therein.
  • amylase liquefaction and saccharification enzyme inactivation after heat treatment in the starch saccharification solution are removed by filtration.
  • the components of the starch saccharified solution after glucose isomerization and C-3 epimerization are:
  • the concentration is 30-60% (for example, 50%), based on the total mass of the mixed liquid
  • the isomerization is by directly adding glucose isomerase and C-3 isomerase to the starch saccharification solution, or passing the starch saccharification solution through the glucose isomerization enzyme immobilized. And C-3 isomerase reaction column.
  • the isomerization is an inactivation treatment that can be used in step (a) without separating glucose isomerase and C-3 isomerase directly into the starch saccharification solution. Enzyme.
  • the composition of the psicose solution is as follows:
  • Partial solution containing psicose Psicose has a purity of 97% to 99%, an isolation yield of more than 95%, and a concentration of 4 to 7.2%. (The concentration is processed into a ptoseose product through processes such as concentration and crystallization.)
  • "Fructose + glucose” solution dry matter concentration of 25 to 35%, the purity of each component in the "fructose + glucose” solution is 30 to 55% fructose, 40 to 70% glucose, 0.6 to 1.0% maltose, Isomaltose 0.3 to 0.5%, maltotriose 0.13 to 0.3%, maltotetraose or higher polysaccharide 0.4 to 0.7%, and psicose 0.05 to 0.3%.
  • the method shown includes the following steps:
  • starch and water concentration 25-35%, adding amylase 10-50u / g starch, adjusting pH), liquefaction (liquefaction at 100-130 ° C and maintaining for 30-60 minutes), neutralization (adjusting pH), saccharification ( Adding saccharifying enzyme 10-200u / g starch, saccharification at 58-62 ° C) to obtain the starch saccharification solution (the components of the starch saccharification solution are about 95.4% glucose, 1.9% maltose, 0.9% isomaltose, 0.5% maltotriose, Polysaccharides above maltotetraose are 1.4%, based on the total mass of dry matter); preferably, the starch saccharification solution is heated to inactivate the enzyme, activated carbon is added to decolorize and filtered.
  • the starch is adjusted to a 25-35 wt% (e.g., 30 wt%) emulsion with purified water, the pH is adjusted to 6.2-6.4 (such as mediated with sodium carbonate), and amylase (20- 50u / g starch), uniformly prepared, spray liquefaction at 110 ⁇ 5 ° C, and the liquefaction DE value is 15-20%. Cool the liquefied liquid to 55-60 ° C, adjust the pH to 4.3-4.8, add 0.05% saccharifying enzyme (50-100u / g starch) by weight of starch, start saccharification at 55-60 ° C, to DE> 98%, warm up Inactivate to 100 ⁇ 10 °C for 2-3min.
  • 1.5 kg to 2.5 kg of starch saccharification solution containing 50 ⁇ 2 wt% of glucose (based on the total weight of starch saccharification solution) is prepared from 1 kg of starch.
  • starch saccharified solution for example, using MVR or other evaporator
  • a dry matter concentration of 30-60 wt% preferably, 45-55% by weight
  • metal ions are added.
  • the components of the mixed solution obtained after passing through the column are: 5.5-7.5% of ptoseose, 18.6% -20.5% of fructose, 20.5-29% of glucose, 1.9% of maltose, isopropyl Maltose 0.9%, maltotriose 0.5%, and maltotetraose or higher polysaccharide 1.4%, based on the total mass of the mixed liquid.
  • Desalination Use a calcium ion exchange column to desalinate, and then enter the chromatographic separation equipment.
  • Chromatographic separation 4-8 columns equipped with special chromatographic separation resin and regenerated with calcium chloride are used. The process is shown in Figure 1, with 4 columns as an example.
  • the discharging is continuously performed, and intermittent feeding (such as 30-60min for feeding interval, which is related to the amount of resin and packing density) and elution (such as 30-60min for eluting interval, and The amount of resin is related to the packing density, etc., and the slow component (the main component is D-psicose), and the fast component (the main components are glucose and fructose) are collected.
  • intermittent feeding such as 30-60min for feeding interval, which is related to the amount of resin and packing density
  • elution such as 30-60min for eluting interval
  • the amount of resin is related to the packing density, etc.
  • the slow component the main component is D-psicose
  • the fast component the main components are glucose and fructose
  • chromatographic columns are connected in series, and the mobile phase flows in sequence (the flow rate is 0.002-0.150 BV / h, in which the flow rates in the four columns are the same or different).
  • the four chromatographic columns can flow to the same time. Phase flow direction moves.
  • First feed feed flow 0.002-0.150BV / h
  • elute with water and move the chromatographic column so that the water inlet point (the position where the eluent is added) is located at the fast component.
  • the column switching time t is 5-8min.
  • the switching time means that the chromatographic column is at a certain position, and at time t, the chromatographic column starts to move in the opposite direction of the mobile phase flow direction and moves to the next position in the opposite direction of the mobile phase flow direction; for example, As shown in FIG. 1, after a chromatographic column is moved to the position of chromatographic column 1, and after time t has elapsed, the chromatographic column is rapidly moved from the position of chromatographic column 1 to the position of chromatographic column 4 in the figure.
  • the material-water ratio (material quality: mass of elution water) of the chromatographic separation method is 1: (1.5 to 3), and the fast component / slow component (psicose solution) is (2 to 3): 1.
  • the chromatographic conditions can be:
  • the purity of the prosulose solution is 98-99% (based on the total mass of the dry matter); the yield of the prosulose solution is more than 95%;
  • the content of fructose in dry matter is 30-55%, and the content of glucose is 40-70%.
  • the present invention uses starch as a raw material for the first time, and through the processes of saccharification, isomerization, concentration, and chromatographic separation, D-psicose having a purity of 98-99% is obtained.
  • the present invention uses a special C-3 isomerase for the first time. It is a D-psicose-3-epimerase from Paenibacillus senegalensis, which can efficiently catalyze the production of D- Puloseose.
  • the method of the present invention can also recycle the raw materials (glucose, fructose and metal ions), which greatly reduces the cost.
  • the method of the present invention is suitable for industrial production.
  • the glucose isomerase, amylase and saccharifying enzyme used in the examples were purchased from Novozymes.
  • C-3 epimerase ie, C-3 isomerase
  • starch saccharification solution about 95.4% of glucose, 1.9% of maltose, 0.9% of isomaltose, 0.5% of maltotriose, and 1.4% of maltotetraose or higher polysaccharides (based on dry matter).
  • reaction product two enzymes (glucose isomerase and C-3 epimerase) were removed by membrane filtration.
  • a chromatographic separation was performed using a simulated moving bed to separate and obtain an aqueous solution containing mainly ptoseose and a mixed solution containing mainly fructose and glucose ("fructose + glucose” solution).
  • the obtained "fructose + glucose” solution is recovered and recycled, that is, after being concentrated, it is converted again with glucose isomerase and C-3 epimerase.
  • the effluent was measured, and 14.3% glucose in the effluent was converted to D-psicose.
  • a chromatographic separation was performed using a simulated moving bed to separate and obtain an aqueous solution containing mainly ptoseose and a mixed solution containing mainly fructose and glucose ("fructose + glucose” solution).
  • the obtained "fructose + glucose” solution is recovered and recycled, that is, after being concentrated, it is converted again with glucose isomerase and C-3 epimerase.

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Abstract

一种制备D-阿洛酮糖的方法，以淀粉为原料，经过糖化处理、异构化处理，通过浓缩和色谱分离处理，制得D-阿洛酮糖，纯度为98-99％。

Description

一种由淀粉制备D-阿洛酮糖的方法 技术领域
本发明涉及功能食品领域，具体地，涉及一种由淀粉制备D-阿洛酮糖的方法。
背景技术
D-阿洛酮糖是自然界存在的一种稀有糖，据研究报道，D-阿洛酮糖是一种新型功能性单糖，和蔗糖相比，其热量低、口味纯正，不会被人体代谢，可以很好的控制血糖。2011年，美国FDA已批准D-阿洛酮糖作为食品原料，并认为是安全食品；所以D-阿洛酮糖有望成为蔗糖的替代品，作为食品或食品添加剂，广泛用于饮食、保健和医药等领域。
目前，为了获得较高纯度的D-阿洛酮糖，一种采用方法是直接采用果糖为原料来制备D-阿洛酮糖，然而这存在缺点：高纯度的果糖提高了生产成本，而较低纯度的果糖又会引入其它杂质(包括其它单糖或二糖)。
另一种方法是从含D-阿洛酮糖的现有糖浆(如果葡糖浆)中分离D-阿洛酮糖，然而经分离产品中虽然D-阿洛酮糖的浓度有所提高，但D-阿洛酮糖纯度仍较低(以所有糖分的总量计，为约10％或更低)，并且仍含有许多其它单糖(尤其是果糖)。
因此，目前D-阿洛酮糖的生产成本还相对较高。D-阿洛酮糖作为一种新型甜味剂，要得到普遍的推广和使用，还需在制备高纯度D-阿洛酮糖的同时进一步降低生产成本。
因此，本领域迫切需要开发一种低成本的、高效的、适合工业化制备高纯度阿洛酮糖产品的方法。
发明内容
本发明的目的在于提供一种低成本的、高效的、适合工业化制备高纯度阿洛酮糖产品的方法。
本发明的第一方面提供了一种生产D-阿洛酮糖的方法，包括步骤：
(a)对淀粉原料液进行糖化处理，从而形成含有糖化产物的第一混合物，所述糖化产物包括：葡萄糖、麦芽糖、异麦芽糖、麦芽三糖及麦芽四糖；
(b)对所述第一混合物进行异构化处理，将葡萄糖异构化为果糖进而异构化为D-阿洛酮糖，从而形成含D-阿洛酮糖的第二混合物；和
(c)从所述第二混合物分离D-阿洛酮糖，从而得到含D-阿洛酮糖的分离产品。
在另一优选例中，在步骤(a)和(b)中不包括对糖分进行分离的步骤。
在另一优选例中，所述的淀粉选自下组：玉米淀粉、土豆淀粉或其组合。
在另一优选例中，所述的淀粉原料液是淀粉经液化处理的原料液。
在另一优选例中，在步骤(a)之前还包括步骤(a1)：对淀粉进行液化处理，从而形成淀粉原料液。
在另一优选例中，在淀粉酶存在下对淀粉进行液化处理。
在另一优选例中，在液化前，将原料淀粉与水混合形成淀粉水混合物。
在另一优选例中，淀粉与所述淀粉水混合物的质量比为25～35:100。
在另一优选例中，所述的液化处理具有下述一个或多个特征：
淀粉酶的加入量为10-50u/g淀粉；
pH为5.5～6.5；
所述液化处理的温度为100-130℃；和/或
液化时间为30-60min。
在另一优选例中，淀粉酶的加入量为10-25u/g淀粉。
在另一优选例中，在步骤(a)中，在糖化酶存在下，对淀粉原料液进行糖化处理，从而形成第一混合物。
在另一优选例中，在步骤(a)中，所述糖化处理具有下述一个或多个特征：
糖化酶的加入量为10-200u/g淀粉；
糖化处理的pH为4.0～5.5；
糖化时间为20-40h；和/或
糖化处理的处理温度为35～70℃。
在另一优选例中，步骤(a)中，糖化处理的pH为4.3～4.8。
在另一优选例中，步骤(a)中，糖化酶的加入量为50～100u/g淀粉。
在另一优选例中，步骤(a)中，糖化处理的处理温度为45～65℃；更优选地，为55～60℃。
在另一优选例中，步骤(a)还包括步骤：在90～110℃下对所述第一混合物中的糖化酶及液化酶进行灭活处理，得到酶已失活的第一混合物。
在另一优选例中，步骤(a)还包括步骤：向所述第一混合物中加入活性炭，进行脱色处理，得到经脱色处理的第一混合物。
在另一优选例中，步骤(a)中包括或不包括过滤除去已失活糖化酶及液化酶的步骤。
在另一优选例中，在第一混合物中，葡萄糖的含量≥70％，较佳地≥80％，更佳地≥90％，最佳地≥95％，按所述第一混合物中干物质的总质量计。
在另一优选例中，所述第一混合物包含90-99wt％的葡萄糖，优选地，包含95-99wt％的葡萄糖，以第一混合物中干物质总质量为基准。
在另一优选例中，所述第一混合物中，Y值≥80％
Y＝C1/(C1+C2+C3+C4+C5)
式中，
C1为葡萄糖的浓度；
C2为麦芽糖的浓度；
C3为异麦芽糖的浓度；
C4为麦芽三糖的浓度；
C5为麦芽四糖的浓度。
在另一优选例中，步骤(a)还包括步骤：对所述第一混合物进行浓缩处理，从而形成经浓缩的第一混合物。
在另一优选例中，未经浓缩的所述第一混合物中干物质的浓度为10-70wt％，较佳地，20-65wt％，更佳地，30-60wt％，所述浓度为总干物质的浓度，以所述第一混合物总质量为基准。
在另一优选例中，所述经浓缩的第一混合物中干物浓度为45-55wt％，以所述第一混合物总质量为基准。
在另一优选例中，所述浓缩处理，采用MVR蒸发器进行。
在另一优选例中，所述步骤(b)包括：
(b1)用葡萄糖异构化酶对所述第一混合物进行第一酶促反应，从而得到含有果糖的混合物；和
(b2)用C-3异构化酶对所述含有果糖的混合物进行第二酶促反应，从而得到所述第二混合物。
在另一优选例中，在步骤(b1)前还包括步骤：向所述第一混合物中加入金属离子；优选地，所述金属离子选自：Mn 2+、Co 2+或其组合。
在另一优选例中，所述金属离子的终浓度各自独立为1-10mM。
在另一优选例中，步骤(b1)及步骤(b2)在40-80℃下进行；优选地，在50-70℃下进行。
在另一优选例中，所述步骤(b1)、(b2)在同时进行或者依次进行。
在另一优选例中，所述的步骤(b)或所述步骤(b1)和(b2)在同一反应器中进行。
在另一优选例中，在所述步骤(b)中，向所述第一混合物中加入葡萄糖异构化酶及C-3异构化酶，并进行反应得到所述第二混合物。
在另一优选例中，在所述步骤(b1)中，所述第一混合物流经固定有葡萄糖异构化酶的反应柱反应得到含有果糖的混合物；和/或
在步骤(b2)中，所述含有果糖的混合物流经固定有C-3异构化酶的反应柱得到所述第二混合物。
在另一优选例中，在所述步骤(b)中，所述第一混合物流经固定有葡萄糖异构化酶的反应柱及C-3异构化酶的反应柱,从而得到所述第二混合物。
在另一优选例中，所述第二混合物的糖类物质的总浓度为30-68wt％(优选地，为50-65wt％)，以第二混合物的总质量为基准。
在另一优选例中，所述第二混合物中阿洛酮糖纯度大于5wt％，优选地，大于11wt％；更优选地，大于14wt％，以第二混合物中干物质总质量为基准。
在另一优选例中，所述第二混合物包括：阿洛酮糖、葡萄糖、果糖、及低聚糖(包括二糖)。
在另一优选例中，所述低聚糖选自下组：麦芽糖、异麦芽糖、麦芽三糖、麦芽四糖、或其组合。
在另一优选例中，所述第二混合物中，所述低聚糖(包括二糖)的含量为2～10w％，以第二混合物中干物质总质量为基准。
在另一优选例中，所述第二混合物中，所述葡萄糖的含量为5-55wt％，较佳地，10-40wt％，更佳地，20-30wt％，以第二混合物中干物质总质量为基准。
在另一优选例中，所述第二混合物中，所述果糖的含量为1-50wt％，较佳地，5-30wt％，更佳地，10-25wt％，以第二混合物中干物质总质量为基准。
在另一优选例中，在步骤(c)中，通过色谱分离法，从所述第二混合物分离得到含D-阿洛酮糖的分离产品。
在另一优选例中，所述色谱分离法为模拟移动床色谱分离法。
在另一优选例中，所述色谱分离法包括步骤
(1)提供第二混合物作为进料F；以及
(2)色谱分离：通过基于移动床色谱的色谱分离设备对所述含糖混合液进行分离，得到所述含D-阿洛酮糖的分离产品；
所述色谱分离包括：
(2.1)进料步骤：将所述的含糖混合液通入的色谱柱中；
(2.2)洗脱步骤：将洗脱液D通入色谱柱中进行洗脱，且所述洗脱液D为水；
(2.3)出料步骤：收集出料液，其中，所述出料液包括D-阿洛酮糖的分离产品；
其中，所述色谱分离设备包括2-20个所述色谱柱和/或色谱柱段，且所述色谱柱和/或色谱柱段的填料为阳离子树脂，所述的各个色谱柱和/或色谱柱段串联连接。
在另一优选例中，步骤(2.1)及步骤(2.2)间歇进行；和/或步骤(2.3)连续进行。
在另一优选例中，料水比为1:(0.5-3.0)；其中，所述料水比为进料F:洗脱液D的质量比；优选地，料水比为1:(0.8-2.5)；优选地，为1:(1.0-2.0)。
在另一优选例中，D-阿洛酮糖的分离产品与进料F总量的质量比为(0.9～1.5):1；优选地，(1.0～1.3):1。
在另一优选例中，步骤(2.1)中，所述方法的进料的流量为0.002-0.150BV(床体积)/h；优选地，所述方法的进料的流量为0.005-0.10BV/h；更优选地，为0.01-0.05BV/h。
在另一优选例中，步骤(2.2)中，洗脱液的流量为0.005-0.375BV(床体积)/h，；优选地，所述方法的进料的流量为0.0125-0.10BV/h；更优选地，为0.025-0.125BV/h。
在另一优选例中，步骤(2.3)中，所述出料液的流量为0.002-0.150BV(床体积)/h。
在另一优选例中，所述方法的色谱柱和/或色谱柱段的柱温为20-80℃；优选地，为 30-70℃；更优选地，为50-65℃。
在另一优选例中，所述阳离子树脂的粒径为：50-500um。
在另一优选例中，单个所述色谱柱和/或色谱柱段填充的所述阳离子树脂的密度为0.85～0.95g/cm 3。
所述色谱柱的单柱和/或色谱柱段的单段长度为50-200cm。
在另一优选例中，所述色谱柱的单柱和/或色谱柱段的单段径高比1/20-1/0.4；优选地，为1/15-1/1。
在另一优选例中，所述的移动床色谱包括4-12个(优选地5-10个)色谱柱和/或色谱柱段。
在另一优选例中，所述方法的色谱柱和/或色谱柱段的切换时间为3～15min；优选地，为5-8min。
在另一优选例中，所述的阳离子树脂选自：钙型阳离子树脂、钠型阳离子树脂、钾型阳离子树脂、镁型阳离子树脂、锂型阳离子树脂，或其组合；优选地，为钙型阳离子树脂、镁型阳离子树脂，或其组合。
在另一优选例中，步骤(c)中，在所述色谱分离前还包括：对所述第二混合物进行脱盐处理，从而获得经脱盐的第二混合物。
在另一优选例中，所述脱盐处理包括离子交换柱脱盐。
在另一优选例中，经脱盐的第二混合物的电导率≤20μs。
在另一优选例中，所述的含D-阿洛酮糖的分离产品中，D-阿洛酮糖的纯度≥95％，以干物质总质量为基准；优选地，纯度为96-99％；更优选地，纯度为98-99％。
在另一优选例中，在步骤(c)中，还包括步骤：从所述第二混合物分离回收果糖和葡萄糖。
在另一优选例中，在所述分离回收中，以含“果糖+葡萄糖”的溶液形式回收果糖和葡萄糖。
在另一优选例中，在所述“果糖+葡萄糖”溶液中，葡萄糖和果糖的总浓度为15-40wt％，优选地，为20-35wt％，以总溶液质量为基准。
在另一优选例中，所述“果糖+葡萄糖”溶液包括：葡萄糖40-70wt％的葡萄糖及30-55wt％的果糖，优选地，50-60wt％的葡萄糖及40-50wt％的果糖，以干物质总质量为基准。
在另一优选例中，所述含D-阿洛酮糖的分离产品包括液态产品和固态产品。
在另一优选例中，所述的方法还包括步骤:
(d)对所述的含D-阿洛酮糖的分离产品进行浓缩和结晶(优选地，降温结晶)，从而得到固体D-阿洛酮糖。
在另一优选例中，所述对所述的含D-阿洛酮糖的分离产品进行浓缩通过MVR蒸发器 进行。
在另一优选例中，步骤(d)中，所述进行浓缩为进行减压(优选地，真空度为-0.07～-0.1MPa)浓缩。
在另一优选例中，步骤(d)中，在74～76℃下,进行浓缩的。
在另一优选例中，所述方法中，阿洛酮糖的单程转化率为4.5-14.5g阿洛酮糖/100g淀粉。
在另一优选例中，在步骤(a)中，所述的淀粉原料液的量为1～5吨；优选地，为1～3吨；更优选地，为2吨。
在另一优选例中，所述的方法的D-阿洛酮糖的生量为100～1250kg/天；优选地，为100～750kg/天；更优选地，为200～500kg/天。
在另一优选例中，在步骤(b2)中，所述C-3异构化酶为D-阿洛酮糖-3-差向异构酶。
在另一优选例中，所述葡萄糖异构酶和/或C-3差向异构酶选自下组：酶液、酶干粉、固定化酶、或其组合。
在另一优选例中，所述D-阿洛酮糖-3-差向异构酶为类芽孢杆菌(Paenibacillus senegalensis)的D-阿洛酮糖-3-差向异构酶。
在另一优选例中，所述的D-阿洛酮糖-3-差向异构酶选自下组：
(i)氨基酸序列如SEQ ID NO.:1所示的多肽；
(ii)将SEQ ID NO.:1所示氨基酸序列的多肽经过一个或多个氨基酸残基(较佳地，1-50个，更佳地，1-30个，更佳地，1-10个，最佳地，1-6个)的取代、缺失或添加而形成的、或是添加信号肽序列后形成的、并具有催化产生阿洛酮糖活性的衍生多肽；
(iii)序列中含有(i)或(ii)中所述按氨基酸序列的衍生多肽；
(iv)氨基酸序列与SEQ ID NO.:1所示氨基酸序列的同源性≥70％(较佳地≥80％，更佳地≥90％)，并具有催化产生阿洛酮糖活性的衍生多肽。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为本发明的连续色谱分离流程示意图。
具体实施方式
经过广泛而深入的研究，通过大量工艺路线的筛选，本发明人首次意外地开发了一种基于淀粉原料生产D-阿洛酮糖的方法。在本发明方法中，通过连续的和特别优化的糖化处理、异构化处理和分离处理，不仅可以连续工业化生产D-阿洛酮糖，而且可以获得高纯度(可高达98-99％)的D-阿洛酮糖，并且可显著降低D-阿洛酮糖的生产成本。在 此基础上，本发明人完成了本发明。
术语
如本文所用，术语“干物质”是指除去水以外的所有物质的总和。
如本文所用，术语“浓度”是指特定物质重量占溶液总重量的百分比，例如，阿洛酮糖的浓度为阿洛酮糖重量/溶液总重量*100％。
如本文所用，术语“纯度”是指特定物质重量占除水以外物质总重量的百分比，例如，阿洛酮糖的纯度为阿洛酮糖重量/溶液中干物质的重量*100％。
如本文所用，术语“糖类物质”由C、H、O三种元素组成的分子化合物，在本申请中所述糖类物质为阿洛酮糖、果糖、葡萄糖等。
如本文所用，术语“重量比”在一定时间段内或者分离一定量的物料时各物质之间的平均重量比。
如本文所用，术语“酶活”是指每分钟转化为1umol相对应产品的酶量，为1u。
如本文所用，术语“干物质”是指混合物中除水以外的组分，在本发明中主要为糖类物质。
如本文所用，术语“单程转化率”是指，不回收利用分离阿洛酮糖时得到“果糖+葡萄糖”的溶液时，单位质量的淀粉制备得到的阿洛酮糖的量。
C-3异构化酶
如本文所用，术语“C-3异构化酶”、“C-3果糖差相异构酶”与“C-3差相异构酶”可互换使用，指可高效催化产生阿洛酮糖的酶。
在本发明中，一种典型的C-3异构化酶为D-阿洛酮糖-3-差向异构酶。在本发明中，典型的所述D-阿洛酮糖-3-差向异构酶为SEQ ID NO.:1所示的蛋白(即野生型D-阿洛酮糖-3-差向异构酶)或其衍生蛋白(例如，SEQ ID NO.:2所示的突变型D-阿洛酮糖-3-差向异构酶)，所述D-阿洛酮糖-3-差向异构酶来自类芽孢杆菌(Paenibacillus senegalensis)。
本文所用的术语“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。因此，本文所用的术语“分离的D-阿洛酮糖-3-差向异构酶”是指所述蛋白基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化本发明的D-阿洛酮糖-3-差向异构酶。基本上纯的蛋白在非还原聚丙烯酰胺凝胶上能产生单一的条带。然而，鉴于本发明的教导以及现有技术，本领域技术人员还应明白“D-阿洛酮糖-3-差向异构酶”还应包括所述蛋白的变异形式，所述变异形式具有与“本发明的D-阿洛酮糖-3-差向异构酶”相同或相似的功能，但其氨基酸序列与野生型D-阿洛酮糖-3-差向异构酶所示氨基酸序列有少量差异。这些变异形式包括(但 不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个，还更佳如1-8个、1-6个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或多个(通常为20个以内，较佳地为10个以内，更佳地为6个以内)氨基酸。例如，本领域技术人员熟知，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括D-阿洛酮糖-3-差向异构酶蛋白的活性片段和活性衍生物。
多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严格性条件下能与“本发明的黄D-阿洛酮糖-3-差向异构酶”的编码DNA杂交的DNA所编码的蛋白。本发明还包括其他多肽，如包含“本发明的D-阿洛酮糖-3-差向异构酶”或其片段的融合蛋白。除了几乎全长的多肽外，本发明还应包括“本发明的D-阿洛酮糖-3-差向异构酶”的活性片段。通常，该片段具有“本发明的D-阿洛酮糖-3-差向异构酶”的氨基酸序列的至少约10个连续氨基酸，通常至少约30个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
本发明还提供“D-阿洛酮糖-3-差向异构酶”的类似物。这些类似物与天然“本发明的D-阿洛酮糖-3-差向异构酶”的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性蛋白。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的蛋白。
在本发明中，“D-阿洛酮糖-3-差向异构酶”的保守性变异多肽指与野生型D-阿洛酮糖-3-差向异构酶所示氨基酸序列相比，有至多20个，较佳地至多10个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽，但所述保守性变异多肽依然具有与氨基酸序列如SEQ ID NO:1所示蛋白相同或相似的活性，即，催化产生阿洛酮糖的活性。
因此，鉴于本发明的教导和现有技术，本领域技术人员可根据，例如下表所示进行氨基酸替换而产生保守性变异的突变体。
初始残基 代表性的取代残基 优选的取代残基
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
因此，本文所用的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
本发明的蛋白可以是重组蛋白、天然蛋白、合成蛋白，优选重组蛋白。本发明的蛋白可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的蛋白可以是糖基化的，或可以是非糖基化的。本发明的蛋白还可包括或不包括起始的甲硫氨酸残基。
本领域技术人员明白，本发明的“D-阿洛酮糖-3-差向异构酶”还包括“D-阿洛酮糖-3-差向异构酶”的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的“D-阿洛酮糖-3-差向异构酶”相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本文的定义这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
鉴于本领域现有技术和本发明的教导，本领域技术人员不难获得本发明D-阿洛酮糖-3-差向异构酶的活性片段。因此，任何一种“D-阿洛酮糖-3-差向异构酶”的生物活性片段都可以应用于本发明。在本文中，“D-阿洛酮糖-3-差向异构酶”的生物活性片段是指“D- 阿洛酮糖-3-差向异构酶”的片段，但其仍然能保持全长“D-阿洛酮糖-3-差向异构酶”的全部或部分功能。通常情况下，所述的生物活性片段至少保持全长“D-阿洛酮糖-3-差向异构酶”的50％的活性。在更优选的条件下，所述活性片段能够保持全长“D-阿洛酮糖-3-差向异构酶”的60％、70％、80％、90％、95％、99％、或100％的活性。
基于本发明的教导和现有技术，本领域技术人员还可以明白，可以将D-阿洛酮糖-3-差向异构酶制成固定化酶等其它利用形式。
如本文所用“淀粉酶”是指能水解淀粉、糖原和有关多糖中的O-葡萄糖键的酶(例如，α-淀粉)，其作用于淀粉时从淀粉分子内部以随机的方式切断α-1，4糖苷键。
如本文所用术语“糖化酶”，又称葡萄糖淀粉酶[Glucoamylase，EC.3.2.1.3.]，这种酶能把淀粉从非还原性未端水解a-1.4葡萄糖苷键产生葡萄糖，也能缓慢水解a-1.6葡萄糖苷键，转化为葡萄糖的酶。同时也能水解糊精，糖原的非还原末端释放β-D-葡萄糖。
如本文所用，术语“快组分”是指保留时间较短，先出(较快离开色谱分离)的组分。类似地，术语“慢组分”是指保留时间较长，后出(较慢离开色谱分离)的组分。
制备D-阿洛酮糖的方法
在本发明中，还提供了一种制备D-阿洛酮糖的方法，包括步骤：
(a)对淀粉进行糖化处理，得到第一混合物；
(b)对所述第一混合物进行异构化处理，得到第二混合物；和
(c)对所述第二混合物进行后处理，得到D-阿洛酮糖。
在一优选实施方式中，制备D-阿洛酮糖的方法如下所示：
Figure PCTCN2019106498-appb-000001
步骤(a)中，淀粉经过淀粉酶液化和糖化酶糖化，形成淀粉糖化液(所述淀粉糖化液即步骤(a)中的第一混合物)。
在本发明中，在所述淀粉糖化液中，含有一定浓度(如≥70％，≥80％，≥90％或≥95％)的葡萄糖以及余量的其它糖类物质(包括麦芽糖、异麦芽糖、麦芽三糖等)(例如，淀粉糖化液成分为：葡萄糖约95.4％、麦芽糖1.9％、异麦芽糖0.9％、麦芽三糖0.5％、麦芽四糖以上多糖1.4％，以干物质总质量为基准)。
所述淀粉糖化液经过葡萄糖异构酶和C-3差相异构酶异构化。
在一个优选的实施例中，可不分离该糖化液，淀粉糖化液经过适当浓缩，直接经过葡萄糖异构酶和C-3差相异构酶异构化，形成混合液
在另一优选例中，在步骤(a)中，所述淀粉糖化液经过加热处理(温度80-100℃)使其中的淀粉酶和糖化酶失活。
在另一优选例中，过滤除去所述淀粉糖化液中经加热处理后失活的淀粉酶液化和糖化酶糖化。
所述淀粉糖化液经葡萄糖异构和C-3差向异构后混合液(混合液即步骤(b)的第二混 合液)的成分为：
1)浓度30-60％(例如，50％),以混合液的总质量为基准；
2)组分：阿洛酮糖5.5～7.5％、果糖18.6～20.5％、葡萄糖20.5～25.9％、麦芽糖1.9％、异麦芽糖0.9％、麦芽三糖0.5％、麦芽四糖以上多糖1.4％，以混合液的总质量为基准；
3)经过脱盐电导率≤20μs(中性)。
在另一优选例中，所述的异构化为通过向淀粉糖化液中直接添加葡萄糖异构化酶和C-3异构化酶，或者使淀粉糖化液流经固定有葡萄糖异构化酶和C-3异构化酶反应柱。
在另一优选例中，所述的异构化为通过向淀粉糖化液中直接添加葡萄糖异构化酶和C-3异构化酶可以不分离步骤(a)中的使用的经失活处理酶。
进入色谱分离，分离后得到两部分溶液，其中阿洛酮糖溶液成分如下：
1)含阿洛酮糖部分溶液：阿洛酮糖的纯度达到97％～99％，分离收率95％以上，浓度4～7.2％。(所述经过浓缩、结晶等工艺加工成阿洛酮糖产品。)
2)含“果糖+葡萄糖”的溶液：干物浓度25～35％，“果糖+葡萄糖”的溶液中各组分的纯度为果糖30～55％、葡萄糖40～70％、麦芽糖0.6～1.0％、异麦芽糖0.3～0.5％、麦芽三糖0.13～0.3％、麦芽四糖以上多糖0.4～0.7％、阿洛酮糖0.05～0.3％。
在另一优选例中，所示方法包括如下步骤：
1)淀粉液化与糖化制备淀粉糖化液：
混合(淀粉与水，浓度25-35％、加入淀粉酶10-50u/g淀粉、调pH)、液化(100-130℃液化并维持30-60分钟)、中和(调pH)、糖化(加入糖化酶10-200u/g淀粉、58-62℃糖化)得到所述淀粉糖化液(所述淀粉糖化液成分为葡萄糖约95.4％、麦芽糖1.9％、异麦芽糖0.9％、麦芽三糖0.5％、麦芽四糖以上多糖1.4％，以干物质总质量为基准)；优选地，加热所述淀粉糖化液使酶失活，加入活性炭脱色并过滤。
在另一优选例中，淀粉用纯化水调成25-35wt％(例如30wt％)的乳液，调节pH至6.2-6.4(如用碳酸钠调解)，加入淀粉重量0.05％的淀粉酶(20-50u/g淀粉)，调配均匀，在110±5℃喷射液化，液化DE值为15-20％。将该液化液冷却至55-60℃，调节pH为4.3-4.8，加入淀粉重量0.05％的糖化酶(50-100u/g淀粉)，在55-60℃开始糖化，至DE>98％,升温至100±10℃灭活2-3min。降温至50℃，加入淀粉重量0.5～1.5％(如1.0％)的活性炭，搅拌15～45min(如30min)脱色，过虑除去不溶性杂质和酶蛋白，得到稀糖化液，经过减压浓缩脱水，得到高浓度的淀粉糖化液。
在另一优选例中，1kg淀粉制备得到含50±2wt％的葡萄糖(以淀粉糖化液总重量为基准)的1.5～2.5kg淀粉糖化液。
2)将上述淀粉糖化液(例如，采用MVR或其它蒸发器)浓缩到干物浓度30-60wt％(优选地，45-55wt％)，以总质量计；并加入金属离子。按照一定流速通过装有“葡萄糖异构酶”和“C-3果糖差相异构酶”的酶固定化柱子。过柱得到的所述混合液(混合液即步骤(b)的第二混合物)成分为：阿洛酮糖5.5～7.5％、果糖18.6～20.5％、葡萄糖20.5～ 29％、麦芽糖1.9％、异麦芽糖0.9％、麦芽三糖0.5％、麦芽四糖以上多糖1.4％，以混合液的总质量为基准。
3)脱盐：采用钙型离子交换柱脱盐，然后进入色谱分离设备。
4)色谱分离：采用4-8个装有专用色谱分离树脂并用氯化钙再生好的柱子，流程如图1所示，以4柱为例。
如图1所示，出料连续进行，进行间歇性进料(进料间隔时间比如30-60min，与树脂的量和填充密度等有关)和洗脱(洗脱间隔时间比如30-60min，与树脂的量和填充密度等有关)，分别收集慢组分(主要成分是阿洛酮糖，即D-阿洛酮糖的分离产品)和快组分(主要成分是葡萄糖和果糖)。
具体地，4个色谱柱串联连接，流动相依次流过(流速为0.002-0.150BV/h，其中，4个柱子中流速是相同或不同的)这4个色谱柱，同时色谱柱可向流动相流动方向移动。
首先进料(进料流量0.002-0.150BV/h)，进料0.01-0.02BV后进水洗脱，且通过移动色谱柱来使进水点(加入洗脱剂的位置)位于快组分出料位置和慢组分出料位置之间，色谱柱的切换时间t为5-8min。其中，所述切换时间是指，色谱柱在某一位置，在t时间时，色谱柱开始沿流动相流动方向的反方向移动并移动到流动相流动方向的反方向上的下一个位置；例如，如图1所示，某一色谱柱移动到色谱柱1位置后，再经过t时间后，该色谱柱由色谱柱1位置快速移动到了图中色谱柱4的位置。
在另一优选例中，所述色谱分离方法的料水比(物料质量：洗脱水的质量)为1：(1.5～3)、快组分/慢组分(阿洛酮糖溶液)为(2～3)：1。
例如，色谱条件可以为：
进料F＝0.4Kg/h(与树脂量有关)、洗脱剂D＝0.6Kg/h、出料BD(慢组分)＝0.33Kg/h、AD(快组分)＝0.66Kg/h、处理量0.03Kg料液/Kg树脂、料水比1:1.5、AD/BD＝2；
瞬时操作条件：F→A＝33ml/min(进料时快组分出料)、D→B(洗脱时慢组分出料)＝33ml/min、循环量(与切换时间成反比)R＝33ml/min、D→A(洗脱时快组分出料)＝33ml/min。
结果：
(1)得到阿洛酮糖溶液纯度98-99％，(以干物质总质量为基准)；阿洛酮糖收率95％以上；
(2)得到“果糖+葡萄糖”混合液，其干物质浓度25-35wt％。
在干物质中果糖的含量为30-55％、葡萄糖含量为40-70％。
(3)得到“麦芽糖+异麦芽糖+麦芽三糖+多糖+阿洛酮糖”混合液，其干物浓度5-20％。
其中：麦芽糖3-10wt％、异麦芽糖3-10wt％、麦芽三糖1-5wt％、多糖0.5-5wt％、阿洛酮糖1-6wt％(以“麦芽糖+异麦芽糖+麦芽三糖+多糖+阿洛酮糖”混合液的质量为基准)。
本发明的主要优点包括：
(1)本发明首次以淀粉为原料，经过糖化、异构化、浓缩、色谱分离等处理过程， 得到纯度高达98-99％的D-阿洛酮糖。
(2)本发明首次采用特殊的C-3异构化酶,它为来自于类芽孢杆菌(Paenibacillus senegalensis)的D-阿洛酮糖-3-差向异构酶，可高效催化产生D-阿洛酮糖。
(3)用本发明的方法生产D-阿洛酮糖，可显著降低D-阿洛酮糖的生产成本(成本可下降30％)。
(4)本发明的方法还可循环使用原料(葡萄糖、果糖和金属离子)，极大降低了成本。
(5)本发明方法适用于工业生产。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例中所用的葡萄糖异构酶、淀粉酶和糖化酶购自诺维信。
实施例中所用的C-3差向异构酶(即C-3异构化酶)，其序列如SEQ ID No.:2所示。
如果没有特别说明，实施例中所用的材料和试剂均为市售产品。
实施例1
淀粉糖化液制备
先把100Kg淀粉用纯化水调成30％的乳液，用碳酸钠调节pH至6.36.2-6.4，加入淀粉重量0.05％的淀粉酶(20u/g淀粉)，调配均匀，在110度喷射液化，液化DE值为18％。将该液化液冷却至55度，调节pH为4.5，加入淀粉重量0.05％的糖化酶(100u/g淀粉)，在55度开始糖化，至DE>98％，升温至100度灭活3min，降温至50度，加入淀粉重量1.0％的活性炭，搅拌30min脱色，过虑除去不溶性杂质和酶蛋白，得到稀淀粉糖化液，经过减压浓缩脱水，得到200Kg葡萄糖含量50％的高浓度的淀粉糖化液，淀粉糖化液成分：葡萄糖约95.4％、麦芽糖1.9％、异麦芽糖0.9％、麦芽三糖0.5％、麦芽四糖以上多糖1.4％(以干物质为基准)。
实施例2
D-阿洛酮糖的制备
2.1异构化
在5L的反应釜中，加入3.0Kg淀粉糖化液(实施例1中制备，葡萄糖含量为50％，约由1.5Kg淀粉制得)，1.0mM MnSO 4.7H 2O，葡萄糖异构化酶和C-3差向异构酶，总酶活分别为50000u和20000u，在60℃反应，约3h葡萄糖转化达到平衡。获得反应产 物。
对所述反应产物进行测定，结果表明：约14.6％的葡萄糖转化为D-阿洛酮糖。
2.2酶去除
对所述反应产物，通过膜过滤除去两个酶(葡萄糖异构化酶和C-3差向异构酶)。
2.3分离D-阿洛酮糖
经钙型阳离子树脂处理，从反应产物中除去锰离子，并回收含锰离子的溶液。
接着，用模拟移动床进行色谱分离，从而分离获得主要含阿洛酮糖的水溶液以及主要含果糖和葡萄糖的混合液(“果糖+葡萄糖”溶液)。其中所得“果糖+葡萄糖”溶液被回收循环利用，即对其进行浓缩后，再次用葡萄糖异构化酶和C-3差向异构酶进行转化。
结果
(a)获得了“果糖+葡萄糖”混合液6.0Kg，其干物的含量为31％。在干物中，果糖含量为46％,葡萄糖含量为52％，按干物质总重量计。
(b)获得了阿洛酮糖的水溶液3.0Kg，其中，D-阿洛酮糖的含量为7％，D-阿洛酮糖纯度为98.7％；杂质(包括果糖、葡萄糖和其它糖分)的含量＜1.3％，按干物质总重量计。
总产率：0.140kg阿洛酮糖/kg淀粉。
实施例3
D-阿洛酮糖的制备
3.1异构化
在5L的反应釜中，加入3Kg淀粉糖化液(实施例1制备)，1.0mM MnSO 4.7H 2O，预热至60度，由泵输送流经(过柱的流速为1Kg/h)葡萄糖异构化酶和C-3差向异构酶的固定化反应柱，在60℃由生物酶催化转化，流出液即为反应产物。
对所述流出液进行测定，流出液中有14.3％葡萄糖转化为D-阿洛酮糖。
3.2分离D-阿洛酮糖
经钙型阳离子树脂处理，从反应产物中除去锰离子，并回收含锰离子的溶液。
接着，用模拟移动床进行色谱分离，从而分离获得主要含阿洛酮糖的水溶液以及主要含果糖和葡萄糖的混合液(“果糖+葡萄糖”溶液)。其中所得“果糖+葡萄糖”溶液被回收循环利用，即对其进行浓缩后，再次用葡萄糖异构化酶和C-3差向异构酶进行转化。
结果
(a)得到了“果糖+葡萄糖”混合液6.0Kg，其干物的含量为30％。在干物质中果糖的含量为46％、葡萄糖含量为52％；
(b)得到了阿洛酮糖的水溶液3.0Kg，其中，D-阿洛酮糖的含量(干物质浓度)为6.7％，D-阿洛酮糖纯度为98.1％，杂质(包括果糖、葡萄糖和其它糖分)的含量＜1.9％，按干物质的总重量计。
总产率：0.134kg阿洛酮糖/kg淀粉。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种生产D-阿洛酮糖的方法，其特征在于，包括步骤：
    (a)对淀粉原料液进行糖化处理，从而形成含有糖化产物的第一混合物，所述糖化产物包括：葡萄糖、麦芽糖、异麦芽糖、麦芽三糖及麦芽四糖；
    (b)对所述第一混合物进行异构化处理，将葡萄糖异构化为果糖进而异构化为D-阿洛酮糖，从而形成含D-阿洛酮糖的第二混合物；和
    (c)从所述第二混合物分离D-阿洛酮糖，从而得到含D-阿洛酮糖的分离产品。
  2. 如权利要求1所述的方法，其特征在于，在步骤(a)之前还包括步骤(a1)：对淀粉进行液化处理，从而形成淀粉原料液。
  3. 如权利要求2所述的方法，其特征在于，在淀粉酶存在下对淀粉进行液化处理。
  4. 如权利要求3所述的方法，其特征在于，所述的液化处理具有下述一个或多个特征：
    淀粉酶的加入量为10-50u/g淀粉；
    pH为5.5～6.5；
    所述液化处理的温度为100-130℃；和/或
    液化时间为30-60min。
  5. 如权利要求1所述的方法，其特征在于，在步骤(a)中，在糖化酶存在下，对淀粉原料液进行糖化处理，从而形成第一混合物。
  6. 如权利要求5所述的方法，其特征在于，在步骤(a)中，所述糖化处理具有下述一个或多个特征：
    糖化酶的加入量为10-200u/g淀粉；
    糖化处理的pH为4.0～5.5；
    糖化时间为20-40h；和/或
    糖化处理的处理温度为35～70℃。
  7. 如权利要求1所述的方法，其特征在于，所述步骤(b)包括：
    (b1)用葡萄糖异构化酶对所述第一混合物进行第一酶促反应，从而得到含有果糖的混合物；和
    (b2)用C-3异构化酶对所述含有果糖的混合物进行第二酶促反应，从而得到所述第二混合物。
  8. 如权利要求7所述的方法，其特征在于，在步骤(b1)前还包括步骤：向所述第一混合物中加入金属离子；优选地，所述金属离子选自：Mn 2+、Co 2+、Mg 2+、Fe 2+或其组合。
  9. 如权利要求1所述的方法，其特征在于，在步骤(c)中，通过色谱分离法，从所述第二混合物分离得到含D-阿洛酮糖的分离产品。
  10. 如权利要求9所述的方法，其特征在于，所述色谱分离法包括步骤
    (1)提供第二混合物作为进料F；以及
    (2)色谱分离：通过基于移动床色谱的色谱分离设备对所述含糖混合液进行分离，得到所述含D-阿洛酮糖的分离产品；
    所述色谱分离包括：
    (2.1)进料步骤：将所述的含糖混合液通入色谱柱中；
    (2.2)洗脱步骤：将洗脱液D通入色谱柱中进行洗脱，且所述洗脱液D为水；
    (2.3)出料步骤：收集出料液，其中，所述出料液包括D-阿洛酮糖的分离产品；
    其中，所述色谱分离设备包括2-20个所述色谱柱和/或色谱柱段，且所述色谱柱和/或色谱柱段的填料为阳离子树脂，所述的各个色谱柱和/或色谱柱段串联连接。
  11. 如权利要求10所述的方法，其特征在于，
    料水比为1:(0.5-3.0)；其中，所述料水比为进料F:洗脱液D的质量比；优选地，料水比为1:(0.8-2.5)；优选地，为1:(1.0-2.0)；和/或
    D-阿洛酮糖的分离产品与进料F总量的质量比为(0.9～1.5):1；优选地，(1.0～1.3):1。
  12. 如权利要求10所述的方法，其特征在于，所述色谱柱的单柱和/或色谱柱段的单段径高比1/20-1/0.4；优选地，为1/15-1/1。
  13. 如权利要求10所述的方法，其特征在于，所述的移动床色谱包括4-12个(优选地5-10个)色谱柱和/或色谱柱段。
  14. 如权利要求10所述的方法，其特征在于，所述方法的色谱柱和/或色谱柱段的切换时间为3～15min；优选地，为5-8min。
  15. 如权利要求1所述的方法，其特征在于，所述方法中，阿洛酮糖的单程转化率为4.5-14.5g阿洛酮糖/100g淀粉。
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