WO2020057560A1 - Procédé de production de d-psicose - Google Patents

Procédé de production de d-psicose Download PDF

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Publication number
WO2020057560A1
WO2020057560A1 PCT/CN2019/106497 CN2019106497W WO2020057560A1 WO 2020057560 A1 WO2020057560 A1 WO 2020057560A1 CN 2019106497 W CN2019106497 W CN 2019106497W WO 2020057560 A1 WO2020057560 A1 WO 2020057560A1
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Prior art keywords
mixture
glucose
metal ion
fructose
psicose
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Chinese (zh)
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任世阔
牛志国
韩子明
赵红兵
吴会广
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Livingzone Shanghai Bio Chem Technology Co Ltd
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Livingzone Shanghai Bio Chem Technology Co Ltd
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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
    • C12P19/14—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 belongs to the field of food raw material processing, and particularly relates to a production process of alloxulose.
  • D-psicose is an epimer of fructose. It is a kind of monosaccharide that exists in nature but has very little content. Its sweet taste is similar to sucrose, but its calorie is much lower than that of sucrose. It is an ideal substitute for sucrose; compared with D-glucose and D-fructose, it also has a stronger ability to scavenge active oxygen.
  • D-psicose is scarce in nature, it is not suitable for large-scale industrial production, and chemical synthesis will produce more by-products, high costs, large pollution, and the development of biosynthetic technology is relatively mature; the products produced are relatively single.
  • the current production technology generally has many unfavorable factors such as the production of many pollutants, high environmental protection pressure, low substrate conversion rate, and high production costs. If the waste water generated by ion exchange can be reduced, the expensive metal ions can be recycled, and the re-reaction can be realized, it will greatly reduce the production cost and environmental pollution.
  • the purpose of the present invention is to provide a method for producing psicose, which can effectively utilize metal ions and recycle them, reduce waste water generated by ion exchange, generate low cost, and cause little environmental pollution.
  • a method for producing psicose from starch comprising the following steps:
  • step (3) Isomerizing the first mixture containing the enzymatic metal ion in step (2), isomerizing glucose to fructose and then to D-psicose, thereby forming D-containing A second mixture of ptoseose;
  • step (4) The enzymatic metal ion recovered in step (4) is recycled as the enzymatic metal ion in step (2).
  • the saccharified product further includes maltose, isomaltose, maltotriose and / or maltotetraose.
  • the method further includes step (6) concentrating the isolated product containing D-psicose in step (5) to obtain a concentrated isolated product containing D-psicose.
  • the step (6) uses the MVR method for evaporation and concentration.
  • the evaporation and concentration is evaporation and concentration under reduced pressure (preferably, the degree of vacuum is -0.07 to -0.1 MPa).
  • the temperature of the evaporation and concentration is 74-76 ° C.
  • the method further includes step (7) allowing the D-psicose in the D-psicose-containing isolated product concentrated in step (6) to be crystallized (preferably, crystallized by cooling). ) Precipitated, and D-psicose crystals were isolated.
  • the chromatographic separation is a simulated moving bed chromatographic separation.
  • step (5) includes steps:
  • the chromatographic separation includes:
  • feeding step passing the third mixture into the chromatographic column;
  • 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 includes the D-psicose-containing isolated product;
  • the chromatographic separation device includes 2 to 20 (preferably, 4 to 12; more preferably, 5 to 10) the chromatographic column and / or the column segment, and the chromatographic column and / or The packing of the chromatographic column segment is a cationic resin, and each of the chromatographic columns and / or the chromatographic column segments are connected in series.
  • steps (5.2.1), (5.2.2), and (5.2.3) are performed sequentially or independently.
  • steps (5.2.1) and (5.2.2) are performed sequentially or independently.
  • step (5.2.1) and step (5.2.2) are performed intermittently; and / or step (5.2.3) is performed continuously.
  • the material-to-water ratio is 1: (0.5 to 3.0); wherein the material-to-water ratio is the mass ratio of feed F: eluent D; preferably, the material-to-water ratio is 1: (0.8 ⁇ 2.5); preferably 1: (1.0 ⁇ 2.0).
  • the mass ratio of the D-psicose-containing isolated product 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 to 0.150 BV (bed volume) / h; preferably, the feed flow rate of the method is 0.005 to 0.10 BV / h; more preferably, 0.01 to 0.05 BV / h.
  • the flow rate of the eluent is 0.005 to 0.375 BV (bed volume) / h; preferably, the feed flow rate of the method is 0.0125 to 0.10 BV / h; more preferably, 0.025 to 0.125 BV / h.
  • step (5.2.3) the flow rate of the discharge liquid is 0.002 to 0.150 BV (bed volume) / h.
  • the column temperature of the chromatographic column and / or column segment is 20-80 ° C; preferably, 30-70 ° C; more preferably, 50-65 ° C .
  • 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 (5) the particle size of the cationic resin is: 50-500um.
  • step (5) the density of the cationic resin filled in a single column and / or column segment is 0.85 to 0.95 g / cm 3 .
  • step (5) the single column of the chromatographic column and / or the single segment length of the chromatographic column segment is 50-200 cm;
  • a single column of the chromatographic column and / or a single segment diameter-to-height ratio of the column segment is 1/20 to 1 / 0.4; preferably, 1/15 to 1 / 1.
  • the switching time of the chromatographic column and / or the chromatographic column segment of the method is 3 to 15 minutes; preferably, it is 5 to 8 minutes.
  • the purity of D-psicose is ⁇ 95%, based on the total dry matter mass; preferably, the purity is 96-99 %; More preferably, the purity is 98-99%.
  • the enzymatic metal ion is selected from: Mn 2+ , Co 2+ , Mg 2+ , Fe 2+ , Ca 2+ or a combination thereof; preferably, the enzymatic metal ion is selected from : Mn 2+ , Co 2+ , Mg 2+ , Fe 2+ or a combination thereof; more preferably, the enzymatic metal ion is Mn 2+ , Co 2+ or a combination thereof.
  • step (4) the enzymatic metal ion is recovered by the following steps:
  • the recovered enzymatic metal ion obtained in step (ii) is a recovered enzymatic metal ion in the form of an aqueous solution.
  • the recovered enzymatic metal ion obtained in step (ii) is directly used in step (2) as an aqueous solution.
  • Ca 2+ concentration of the solution containing Ca 2+ was 0.1 ⁇ 1.0M.
  • the solute in the Ca 2+ -containing solution is CaCl 2 .
  • step (ii) after the ion exchange resin in step (ii) is eluted, it can be used as the ion exchange resin in step (i).
  • the ion exchange resin is packed in an ion exchange column, and the packing density of the ion exchange column is 0.7 to 1.2 g / mL.
  • the ion exchange resin is a calcium-type cationic resin.
  • the second mixture flows through the ion exchange column at 10-30 Kg / min.
  • step (1) includes the following steps:
  • the liquefied starch is saccharified in the presence of a saccharifying enzyme to obtain a first mixture containing glucose.
  • step (1.1) comprises emulsifying the starch by adding water to a mixture containing 25% to 30% by weight of starch.
  • amylase is a-amylase.
  • step (1) has one or more of the following characteristics:
  • step (1.2) the pH of the liquefaction treatment is 5.5 to 6.5 (preferably, the pH is 5.8 to 6.2);
  • step (1.2) the temperature of the liquefaction treatment is 100 to 120 ° C (preferably, 105 to 115 ° C);
  • step (1.2) the DE value after the liquefaction treatment is 15 to 25% (preferably 15 to 20%);
  • step (1.3) the pH of the saccharification treatment is 4.1 to 4.9;
  • step (1.3) the temperature of the saccharification treatment is 50 to 70 ° C; and / or
  • step (1.3) the DE value after saccharification is 97 to 100%.
  • step (1) further includes step (1.4) of performing a concentration treatment on the first mixture to obtain a concentrated first mixture.
  • the concentration of the dry matter in the first mixture without concentration is 10 to 70% by weight, preferably 20 to 65% by weight, more preferably 30 to 60% by weight, and the concentration is 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 45-60% by weight (preferably, 50-60% by weight), based on the total mass of the first mixture.
  • the concentration treatment is performed using an MVR evaporator.
  • the method before concentrating the first mixture, further includes the steps of inactivation by heating, decolorization of activated carbon, filtration, removal of ionic proteins, and / or removal of amino acids.
  • the isomerization treatment means that the first mixture containing the enzymatic metal ion is formed in the presence of glucose isomerase and fructose isomerase. A second mixture containing D-psicose.
  • the fructose isomerase is a C-3 epimerase.
  • the fructose isomerase is D-psicose-3-epimerase of Paenibacillus senegalensis.
  • the C-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.
  • glucose isomerase and fructose isomerase are fixed on a reaction column in advance.
  • the isomerization section includes the step: the first mixture containing the enzymatic metal ion flows through a reaction column immobilized with glucose isomerase and fructose immobilized in sequence. Isomerase reaction column.
  • step (3) the temperature catalyzed by the enzyme is 55-65 ° C.
  • the flow rate of the first mixture containing the enzymatic metal ion flowing through the reaction column with the glucose isomerase immobilized is 10-30 Kg / min; and / or
  • the flow rate through the reaction column to which the fructose isomerase is immobilized is 10 to 30 Kg / min.
  • the flow rates are the same or different when flowing through different reaction columns.
  • the first mixture flows through the reaction column immobilized with glucose isomerase and the reaction column immobilized with fructose isomerase at one time, and then directly flows through the ion exchange column to obtain a third mixed solution.
  • the second mixture and / or the third mixture contain 10 to 20% by weight of alloxulose, 30 to 50% by weight of fructose, and 30 to 50% by weight of glucose, based on the total dry matter mass. As a benchmark.
  • step (5) an isolated product containing fructose and glucose can be isolated; the obtained isolated product containing fructose and glucose is recovered and used as the first mixture.
  • the output further includes an isolated product containing fructose and glucose.
  • the isolated product containing fructose and glucose recovered in step (5) is used as a first mixture after being subjected to a concentration treatment (preferably, an MVR concentration treatment).
  • the total concentration of glucose and fructose is 20 to 40% by weight, preferably 25 to 35% by weight, based on the mass of the isolated product containing fructose and glucose. As a benchmark.
  • the isolated product containing fructose and glucose includes: 40 to 70 wt% glucose and 30 to 55 wt% fructose, preferably, 50 to 60 wt% glucose and 40 to 50 wt% fructose, and The total mass of dry matter in the isolated product containing fructose and glucose is used as a reference.
  • the method further includes the step of: performing a concentration treatment on the fructose and glucose-containing isolated product.
  • the concentration treatment is a concentration treatment by MVR.
  • the total concentration of fructose and glucose in the concentrated fructose and glucose-containing isolated product is 30-60% by weight, based on the total mass of the fructose and glucose-containing isolated product.
  • 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 to 99 wt% glucose, preferably 95 to 99 wt% glucose, based on the total dry matter mass in the first mixture.
  • 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 allosexose, glucose, fructose, and oligosaccharides (including disaccharides), based on the total dry matter mass of the second mixture.
  • the conversion rate of allosexose is 60-85 g of allosexose / 100 g of starch (including grape candy reuse).
  • step (a) the amount of the raw starch is 0.1 to 2.0 tons.
  • the production amount of D-psicose in the method is 100-1500 kg / day; preferably, 100-750 kg / day; more preferably, 200-500 kg / day.
  • FIG. 1 is a reaction flowchart of the method of the present invention.
  • FIG. 2 is a process flow chart of the method of the present invention.
  • FIG. 3 is a liquid chromatogram of the obtained mixed liquid in the method of the present invention.
  • FIG. 4 is a schematic diagram of a chromatographic separation process of the present invention.
  • the content (g / 100ml saccharified solution) refers to the mass of reducing sugar in 100ml saccharified solution, and the dry matter content (g / 100g) refers to the mass of dry matter contained in 100g saccharified solution.
  • 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.
  • fructose isomerase is a C-3 phase isomerase.
  • C-3 heterophase isomerase and "D-psicose-3-epimase” are used interchangeably and refer to an enzyme that can efficiently catalyze the production of psulose.
  • the fructose isomerase used in the present invention is the protein shown in SEQ ID No .: 1 (that is, wild-type D-psicose-3-epimerase) or a derivative protein thereof (for example, SEQ ID-NO.:2 mutant D-psicose-3-epimerase), and the fructose isomerase is derived from Paenibacillus senegalensis.
  • C-3 phase isomerase should also include the variant form of the protein, the variant form having the same or similar to the “C-3 phase isomerase of the present invention” Function, but its amino acid sequence is slightly different from that shown by wild-type C-3 phase isomerase.
  • 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.
  • polypeptide variants include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, which can interact with "D-psicose of the present invention- "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).
  • 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. Therefore, given the teachings of the present invention and the prior art, those skilled in the art can make conservative variant mutants based on, for example, amino acid substitutions as shown in the table below.
  • substitution residues 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
  • 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. Depending on the host used in the recombinant production protocol, 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. Those skilled in the art will appreciate that the "D-psicose-3-epimase" of the present invention also includes fragments, derivatives and similar of "D-psicose-3-epimase" Thing.
  • 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
  • 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. Based on the teachings of the present invention and the prior art, those skilled in the art can also understand that D-psicose-3-epimerase can be made into other utilization forms such as immobilized enzyme.
  • dry matter refers to the components of the mixture other than water, and in the present invention are primarily sugars.
  • 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.
  • MVR refers to a mechanical vapor recompression evaporator.
  • 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).
  • the present invention provides a complete set of efficient and environmentally friendly production processes.
  • the invention provides a high-efficiency and environmentally-friendly biocatalytic conversion process for alloxulose (as shown in Fig. 1). Utilizing cheap high-quality starch to convert glucose (e.g., starch liquefaction enzyme, saccharifying enzyme, and then starch to glucose in turn), and catalyzing the conversion to glucose by isomerase and fructose isomerase (preferably, Alloxulose is removed by ion exchange to remove excess metal ions, and then purified by chromatography to obtain pure alloxulose).
  • This process uses recycling technology to reduce pollution, reduce costs, and increase production capacity, thereby achieving environmental protection and efficient production of alloxulose.
  • the present invention uses traditional starch sugar-making technology to produce glucose, which is further converted into psicose by bioenzyme catalysis.
  • This process uses metal ions (ie, enzymatic metal ions) Mn 2+ , Co 2+ , Mg 2+ , Fe
  • metal ions ie, enzymatic metal ions
  • Mn 2+ , Co 2+ , Mg 2+ , Fe One or more of 2+ and Ca 2+ promote the catalytic efficiency of the enzyme, and improve the stability of the enzyme, and prolong the life of the enzyme;
  • these metals need to be separated or recovered Ions, and intermediates and raw materials are easy to recycle and reuse, thereby avoiding the environmental pollution caused by the abandonment of these metal ions, intermediates and raw materials.
  • a cheap ion exchange resin such as a calcium-type cationic resin
  • the metal ions used in the reaction are eluted with an aqueous solution of calcium chloride, and the recovered metal ions are repeatedly used in the biocatalysis process.
  • the process of recovering intermediates and raw materials uses chromatographic separation and concentrated under reduced pressure System, immobilized glucose isomerase and fructose isomerization (for example, both glucose isomerase and fructose isomerase use immobilized enzymes and use them in jacketed reaction columns) Repeated conversion, after multiple (such as 10 and above) cycle conversion, the glucose conversion rate can reach more than 75%, thereby greatly improving production efficiency.
  • the a-amylase, saccharifying enzyme, glucose isomerase and fructose isomerase used in the process may be commercially purchased or produced by themselves.
  • a catalytic amount of one or more of metal ions Mn 2+ , Co 2+ , Mg 2+ , Fe 2+ and Ca 2+ is added during the biological enzyme-catalyzed reaction. In order to improve the conversion efficiency and improve the stability of the immobilized enzyme.
  • a high-quality starch is sequentially biocatalyzed with starch liquefaction enzyme, saccharification enzyme, glucose isomerase, and fructose isomerase to obtain a mixed solution of glucose, fructose, and allose (I.e. the second mixture).
  • the obtained mixed solution (ie, the second mixture) contains one or more of metal ions Mn 2+ , Co 2+ , Mg 2+ , Fe 2+ and Ca 2+ .
  • the obtained mixed solution of glucose, fructose, and alloseose (that is, the second mixture) is subjected to chromatographic separation, and the added metal ions are recovered with a calcium-type cationic resin and recycled.
  • the chromatographic separation filler used is a cationic resin (such as a calcium-type resin), and the isolated axulose aqueous solution is concentrated under reduced pressure to obtain a high-purity axulose with a purity of 98. %the above.
  • the glucose and allosexose solution ie, the fructose and glucose-containing separation products obtained by chromatographic separation are concentrated under reduced pressure and used as raw materials for glucose isomerization and fructose isomerization. .
  • the recovered metal ion is returned to the aforementioned glucose isomerization and fructose isomerization biocatalysis processes.
  • the calcium-type cationic resin used is eluted with an aqueous solution of calcium chloride to recover the added metal ions, and then reused.
  • the isomerization of glucose and fructose and further purification and separation use a series-circulation reactor.
  • Step 1 Prepare a glucose solution (ie, the first mixture)
  • Emulsify high-quality starch at 25-30%, adjust pH 6.0 ⁇ 0.2 with Na 2 CO 3 , add a ⁇ amylase to adjust the slurry uniformly, then control the temperature (110 ⁇ 5 °C) to perform spray liquefaction, and control the liquefaction DE value. 15% to 20%. Then, the liquefied liquid is cooled to about 60 ° C, the pH value is adjusted to about 4.5, and human saccharifying enzyme is added to perform thermal saccharification (for example, saccharification at 50 to 70 ° C), and the reaction reaches the required DE value (for example, 97 to 100%) After heating, the enzyme is deactivated, and activated carbon is added for adsorption filtration.
  • the solution is concentrated by MVR (the concentrated water can be used as the eluent for the third step of chromatographic separation) to obtain a certain concentration of glucose solution.
  • MVR the concentrated water can be used as the eluent for the third step of chromatographic separation
  • the amount of one or more of the metal ions Mn 2+ , Co 2+ , Mg 2+ , and Fe 2+ can be converted into the next step.
  • Second step Isomerization of glucose (that is, the step of obtaining a second mixture or a third mixture)
  • the glucose solution containing metal ions ie, the first mixture
  • the glucose solution obtained in the first step is passed through glucose isomerase, fructose isomerase, and calcium-type cationic resin in sequence at a certain speed (for example, 10-30 Kg / min).
  • the obtained reaction conversion solution contains glucose and fructose And psicose.
  • the reaction conversion solution obtained in the second step (with the metal ions added in the first step removed) is subjected to chromatographic separation using a simulated moving bed.
  • the column packing is made of calcium resin, and a certain ratio of material to water is controlled for purification and separation.
  • Two parts solution the first part is an aqueous solution of ptoseose (isolated product containing D-psicose) with a purity of more than 98%; the second part is an aqueous solution containing glucose and fructose (that is, fructose and glucose) Isolated product), the sugar content is 20-30%, (the total concentration of glucose and fructose is about 20-30%).
  • the discharging is performed continuously, and intermittent feeding (such as 30 ⁇ 60min of feeding interval, which is related to the amount of resin and packing density) and elution (such as 30 ⁇ 60min of elution interval, and The amount of resin is related to the filling density, etc., and the slow component (the main component is ptoseose, that is, an isolated product containing D-psicose) and the fast component (the main components are glucose and fructose, that is, containing An isolated product of fructose and glucose).
  • intermittent feeding such as 30 ⁇ 60min of feeding interval, which is related to the amount of resin and packing density
  • elution such as 30 ⁇ 60min of elution interval, and The amount of resin is related to the filling density, etc.
  • the slow component the main component is ptoseose, that is, an isolated product containing D-psicose
  • the fast component the main components are glucose and fructose, that is, containing An isolated
  • chromatographic columns are connected in series, and the mobile phase flows through the four chromatographic columns in sequence (flow rate is 0.002 to 0.150 BV / h). At the same time, the chromatographic column can be moved in the mobile phase flow direction.
  • the switching time t of the column is 5-8 minutes.
  • 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 a position above and below the opposite direction of the mobile phase flow direction; for example, as 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 crystals are crystallized by gradient cooling, and the white crystals of the psicose are obtained by centrifugation and filtration, and the purity is more than 99%. .
  • Step 5 Recycling of metal ions
  • reaction solution transformed with glucose isomerase and fructose isomerase that is, the reaction conversion solution in the second step, that is, the second mixture
  • the added metal ions are exchanged and adsorbed on the cationic resin.
  • the calcium resin has been exchanged and the replacement resin is replaced; the used resin is eluted with calcium chloride to enrich and recover the adsorbed metal ions.
  • the recovered ions are added to the glucose and fructose solution obtained in the third step or the glucose solution obtained in the first step to enter the cycle for re-conversion.
  • Step 6 Reuse of recovered glucose and fructose solutions
  • the recovered glucose and fructose solution is reused 3 to 20 times, preferably 5 to 15 times, and more preferably 8 to 12 times.
  • the present invention has a high yield.
  • the present invention adopts recycling technology to reduce pollution, reduce costs, increase productivity, and environmentally and efficiently produce alloxulose.
  • the glucose isomerase, amylase and saccharifying enzyme used in the examples were purchased from Novozymes.
  • the fructose isomerase used in the examples is a C-3 epimerase, and its sequence is shown in SEQ ID No.:2.
  • the temperature is maintained at 60 degrees, and the glucose isomerase and fructose isomerase are controlled at a temperature of 60 degrees Celsius from the bottom to the top at a rate of 20 Kg / min, and then passed through the calcium-type cationic resin from the top to the bottom to remove the metal ion Co 2 +
  • the ratio of the three is 15.3 / 40.9 / 43.8; the liquid phase spectrum detected by the differential detector is shown in Figure 3, and the three main peaks from left to right In turn, glucose, fructose, and allosexose.
  • Example 1 after the calcium-type cationic resin was saturated with Co 2+ exchange adsorption, it was eluted with a 10 wt% CaCl 2 aqueous solution, and the Co 2+ solution was collected and returned to Example 1 for reuse; the calcium-type resin was recycled. After testing, the recovery rate of Co 2+ ions was 95%.
  • Example 1 the chromatographically separated glucose and fructose mixed aqueous solution (concentration: 27%) was concentrated under reduced pressure until the sugar content reached 55%, mixed with the newly produced glucose solution, and the recovered Co 2+ was added to pre-heat and keep it at At 60 degrees Celsius, a new catalytic cycle reaction can be entered.
  • Example 1.1 and Example 1.3 were repeated 10 times. Recycling produces and separates a mixed aqueous solution of glucose and fructose
  • the conversion rate of starch to D-psicose is 85%, and the yield is 80%, that is, 500Kg of starch can produce about 400Kg of psicose.
  • step (2) is basically the same as in embodiment 1.1, in which step (2)
  • the process is basically the same as in Example 1.2, in which the recovery of Co 2+ is replaced by the recovery of Mn 2+ .
  • the process is basically the same as in Example 1.3.
  • Example 2.1 and Example 2.3 were repeated 10 times. Recycling produces and separates a mixed aqueous solution of glucose and fructose.

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Abstract

L'invention concerne un procédé de production de D-psicose à l'aide d'amidon, comprenant les étapes consistant à soumettre l'amidon à une liquéfaction et une saccharification pour obtenir un premier mélange; à ajouter des ions métalliques enzymatiques dans le mélange pour l'isomérisation en vue d'obtenir un second mélange; et à opérer une séparation chromatographique sur le second mélange après l'élimination des ions métalliques enzymatiques, de façon à obtenir du D-psicose.
PCT/CN2019/106497 2018-09-18 2019-09-18 Procédé de production de d-psicose Ceased WO2020057560A1 (fr)

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CN117304239A (zh) * 2023-09-25 2023-12-29 江苏久吾高科技股份有限公司 一种d-阿洛酮糖的提纯方法
EP4431614A1 (fr) 2023-03-15 2024-09-18 Annikki GmbH Procédé de préparation de solutions aqueuses contenant de la d-psicose ou de la l-psicose
WO2024189215A2 (fr) 2023-03-15 2024-09-19 Annikki Gmbh Procédé de production d'une solution aqueuse contenant du d-psicose
EP4446422A2 (fr) 2023-03-15 2024-10-16 Annikki GmbH Procédé de préparation d'une solution aqueuse contenant de la l-psicose
EP4464786A1 (fr) 2023-05-15 2024-11-20 Annikki GmbH Procédé de préparation de solutions aqueuses contenant de la d-psicose ou de la l-psicose
WO2024249194A1 (fr) * 2023-05-26 2024-12-05 Corn Products Development, Inc. Procédés permettant d'augmenter la récupération d'allulose à partir de solutions mélangées d'allulose/fructose

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CN113080357B (zh) * 2021-05-17 2023-09-15 江苏赛威分离科技有限公司 一种低热量复配甜味剂及其生产工艺
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CN116987689B (zh) * 2023-09-27 2023-12-15 山东百龙创园生物科技股份有限公司 一种结晶阿洛酮糖的制备方法

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Publication number Priority date Publication date Assignee Title
EP4431614A1 (fr) 2023-03-15 2024-09-18 Annikki GmbH Procédé de préparation de solutions aqueuses contenant de la d-psicose ou de la l-psicose
WO2024189215A2 (fr) 2023-03-15 2024-09-19 Annikki Gmbh Procédé de production d'une solution aqueuse contenant du d-psicose
EP4446422A2 (fr) 2023-03-15 2024-10-16 Annikki GmbH Procédé de préparation d'une solution aqueuse contenant de la l-psicose
EP4520839A2 (fr) 2023-03-15 2025-03-12 Annikki GmbH Procédé de préparation d'une solution aqueuse contenant de la d-psicose
EP4464786A1 (fr) 2023-05-15 2024-11-20 Annikki GmbH Procédé de préparation de solutions aqueuses contenant de la d-psicose ou de la l-psicose
WO2024249194A1 (fr) * 2023-05-26 2024-12-05 Corn Products Development, Inc. Procédés permettant d'augmenter la récupération d'allulose à partir de solutions mélangées d'allulose/fructose
CN117304239A (zh) * 2023-09-25 2023-12-29 江苏久吾高科技股份有限公司 一种d-阿洛酮糖的提纯方法

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