CN109022521B

CN109022521B - Method for preparing D-psicose from starch

Info

Publication number: CN109022521B
Application number: CN201811087533.2A
Authority: CN
Inventors: 任世阔; 牛志国; 韩子明; 赵红兵; 吴会广
Original assignee: Livingzone Shanghai Bio Chem Technology Co ltd
Current assignee: Livingzone Shanghai Bio Chem Technology Co ltd
Priority date: 2018-09-18
Filing date: 2018-09-18
Publication date: 2023-05-05
Anticipated expiration: 2038-09-18
Also published as: WO2020057561A1; CN109022521A

Abstract

The invention provides a method for preparing D-psicose from starch, which specifically uses starch as a raw material, and can obtain the D-psicose with the purity of 98-99% through saccharification, isomerization, concentration and chromatographic separation. The method can prepare the D-psicose efficiently and at low cost.

Description

Method for preparing D-psicose from starch

Technical Field

The invention relates to the field of functional foods, in particular to a method for preparing D-psicose from starch.

Background

D-psicose is a rare sugar existing in nature, and research reports that D-psicose is a novel functional monosaccharide, and compared with sucrose, the D-psicose has low heat, pure taste, can not be metabolized by human body, and can well control blood sugar. In 2011, the U.S. FDA has approved D-psicose as a food raw material and is considered as a safe food; therefore, D-psicose is expected to be a substitute of sucrose and is widely used in the fields of diet, health care, medicine and the like as food or food additive.

Currently, in order to obtain D-psicose with higher purity, one method is to directly use fructose as a raw material to prepare D-psicose, however, there is a disadvantage in that: high purity fructose increases production costs, while lower purity fructose introduces other impurities (including other mono-or disaccharides).

Another approach is to separate D-psicose from the existing syrup containing D-psicose (if glucose syrup), however, the D-psicose purity is still low (about 10% or less based on the total amount of all sugar) and still contains many other monosaccharides (especially fructose) although the concentration of D-psicose in the separated product is improved.

Thus, the production costs of D-psicose are also relatively high at present. D-psicose is used as a novel sweetener, so that the novel sweetener is widely popularized and used, and the production cost is further reduced while the high-purity D-psicose is prepared.

Thus, there is a strong need in the art to develop a low cost, efficient process suitable for the industrial preparation of high purity psicose products.

Disclosure of Invention

The invention aims to provide a low-cost, high-efficiency and suitable for industrialized preparation of high-purity psicose products.

In a first aspect the present invention provides a method for producing D-psicose, comprising the steps of:

(a) Saccharifying a starch feedstock to form a first mixture comprising a saccharified product comprising: glucose, maltose, isomaltose, maltotriose and maltotetraose;

(b) Subjecting the first mixture to an isomerization treatment to isomerize glucose to fructose and further to D-psicose, thereby forming a second mixture comprising D-psicose; and

(c) Separating D-psicose from the second mixture, thereby obtaining an isolated product containing D-psicose.

In another preferred embodiment, steps (a) and (b) do not include a step of separating sugar.

In another preferred embodiment, the starch is selected from the group consisting of: corn starch, potato starch, or a combination thereof.

In another preferred embodiment, the starch feedstock is a liquefied starch feedstock.

In another preferred embodiment, step (a 1) is further included before step (a): liquefying starch to form starch raw material liquid.

In another preferred embodiment, the starch is subjected to liquefaction in the presence of an amylase.

In another preferred embodiment, the raw starch is mixed with water to form a starch water mixture prior to liquefaction.

In another preferred embodiment, the mass ratio of starch to the starch water mixture is 25-35:100.

In another preferred embodiment, the liquefaction process has one or more of the following characteristics:

the addition amount of amylase is 10-50u/g starch;

The pH is 5.5-6.5;

the temperature of the liquefaction treatment is 100-130 ℃; and/or

The liquefying time is 30-60min.

In another preferred embodiment, the amylase is added in an amount of 10-25u/g starch.

In another preferred embodiment, in step (a), the starch feedstock is saccharified in the presence of a saccharifying enzyme to form a first mixture.

In another preferred embodiment, in step (a), the saccharification process has one or more of the following features:

the addition amount of the saccharifying enzyme is 10-200u/g starch;

the pH of saccharification treatment is 4.0-5.5;

saccharification time is 20-40h; and/or

The saccharification treatment temperature is 35-70 ℃.

In another preferred embodiment, in the step (a), the pH of the saccharification treatment is 4.3-4.8.

In another preferred embodiment, in step (a), the saccharifying enzyme is added in an amount of 50 to 100u/g starch.

In another preferred example, in the step (a), the saccharification treatment is carried out at a treatment temperature of 45-65 ℃; more preferably 55 to 60 ℃.

In another preferred embodiment, step (a) further comprises the steps of: inactivating the saccharifying enzyme and the liquefying enzyme in the first mixture at the temperature of between 90 and 110 ℃ to obtain an enzyme inactivated first mixture.

In another preferred embodiment, step (a) further comprises the steps of: and adding activated carbon into the first mixture, and performing decolorization treatment to obtain a decolorized first mixture.

In another preferred embodiment, step (a) may or may not include a step of filtering to remove the inactivated saccharifying enzyme and liquefying enzyme.

In another preferred embodiment, the glucose content in the first mixture is greater than or equal to 70%, preferably greater than or equal to 80%, more preferably greater than or equal to 90%, most preferably greater than or equal to 95%, based on the total mass of dry matter in the first mixture.

In another preferred embodiment, the first mixture comprises from 90 to 99wt% glucose, preferably from 95 to 99wt% glucose, based on the total mass of dry matter in the first mixture.

In another preferred embodiment, in the first mixture, the Y value is not less than 80%

Y＝C1/(C1+C2+C3+C4+C5)

In the method, in the process of the invention,

c1 is the concentration of glucose;

c2 is the concentration of maltose;

c3 is the concentration of isomaltose;

c4 is the concentration of maltotriose;

c5 is the concentration of maltotetraose.

In another preferred embodiment, step (a) further comprises the steps of: the first mixture is subjected to a concentration process, thereby forming a concentrated first mixture.

In another preferred embodiment, the concentration of dry matter in the first mixture, which is not concentrated, is 10-70wt%, preferably 20-65wt%, more preferably 30-60wt%, based on the total mass of the first mixture.

In another preferred embodiment, the dry matter concentration in the concentrated first mixture is 45-55 wt.%, based on the total mass of the first mixture.

In another preferred embodiment, the concentration is performed using an MVR evaporator.

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

(b1) Subjecting the first mixture to a first enzymatic reaction with glucose isomerase, thereby obtaining a fructose-containing mixture; and

(b2) Subjecting said fructose-containing mixture to a second enzymatic reaction with a C-3 isomerase, thereby obtaining said second mixture.

In another preferred embodiment, before step (b 1), the method further comprises the steps of: adding metal ions to the first mixture; preferably, the metal ion is selected from: mn (Mn) ²⁺ 、Co ²⁺ Or a combination thereof.

In another preferred embodiment, the final concentration of the metal ions is each independently 1-10mM.

In another preferred embodiment, steps (b 1) and (b 2) are carried out at 40-80 ℃; preferably, it is carried out at 50-70 ℃.

In another preferred embodiment, the steps (b 1) and (b 2) are performed simultaneously or sequentially.

In another preferred embodiment, said step (b) or said steps (b 1) and (b 2) are performed in the same reactor.

In another preferred embodiment, in the step (b), glucose isomerase and C-3 isomerase are added to the first mixture, and the second mixture is obtained by reaction.

In another preferred embodiment, in the step (b 1), the first mixture is reacted through a reaction column to which glucose isomerase is immobilized to obtain a mixture containing fructose; and/or

In step (b 2), the fructose-containing mixture is passed through a reaction column in which a C-3 isomerase is immobilized to obtain the second mixture.

In another preferred example, in the step (b), the first mixture is passed through a reaction column to which glucose isomerase and a reaction column to which C-3 isomerase are immobilized, thereby obtaining the second mixture.

In another preferred embodiment, the total concentration of saccharide of the second mixture is 30-68wt% (preferably 50-65 wt%) based on the total mass of the second mixture.

In another preferred embodiment, the psicose purity in the second mixture is greater than 5wt%, preferably greater than 11wt%; more preferably greater than 14wt% based on the total mass of dry matter in the second mixture.

In another preferred embodiment, the second mixture comprises: psicose, glucose, fructose, and oligosaccharides (including disaccharides).

In another preferred embodiment, the oligosaccharide is selected from the group consisting of: maltose, isomaltose, maltotriose, maltotetraose, or a combination thereof.

In another preferred embodiment, the content of the oligosaccharide (including disaccharide) in the second mixture is 2-10% by weight based on the total mass of dry matter in the second mixture.

In another preferred embodiment, the glucose is present in the second mixture in an amount of 5 to 55wt%, preferably 10 to 40wt%, more preferably 20 to 30wt%, based on the total mass of dry matter in the second mixture.

In another preferred embodiment, the fructose is present in the second mixture in an amount of 1 to 50wt%, preferably 5 to 30wt%, more preferably 10 to 25wt%, based on the total mass of dry matter in the second mixture.

In another preferred embodiment, in step (c), an isolated product comprising D-psicose is isolated from the second mixture by chromatographic separation.

In another preferred embodiment, the chromatographic separation is simulated moving bed chromatographic separation.

In another preferred embodiment, the chromatographic separation method comprises the steps of

(1) Providing a second mixture as feed F; and

(2) Chromatographic separation: separating the sugar-containing mixed solution by a chromatographic separation device based on moving bed chromatography to obtain a separation product containing D-psicose;

The chromatographic separation comprises:

(2.1) feeding step: introducing the sugar-containing mixed solution into a chromatographic column;

(2.2) elution step: introducing the eluent D into a chromatographic column for eluting, wherein the eluent D is water;

(2.3) discharging: collecting a discharge liquid, wherein the discharge liquid comprises a separated product of D-psicose;

the chromatographic separation device comprises 2-20 chromatographic columns and/or chromatographic column sections, wherein the packing of the chromatographic columns and/or the chromatographic column sections is cationic resin, and the chromatographic columns and/or the chromatographic column sections are connected in series.

In another preferred embodiment, steps (2.1) and (2.2) are performed intermittently; and/or step (2.3) is carried out continuously.

In another preferred example, the material-water ratio is 1 (0.5-3.0); wherein the feed water ratio is the mass ratio of the feed F to the eluent D; preferably, the ratio of the feed to the water is 1 (0.8-2.5); preferably 1 (1.0-2.0).

In another preferred embodiment, 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 to 1.3): 1.

In another preferred embodiment, in step (2.1), the flow rate of the feed to the process is from 0.002 to 0.150BV (bed volume)/h; preferably, the flow rate of the feed to the process is from 0.005 to 0.10BV/h; more preferably 0.01-0.05BV/h.

In another preferred embodiment, in step (2.2), the flow rate of the eluent is 0.005-0.375BV (bed volume)/h; preferably, the flow rate of the feed to the process is from 0.0125 to 0.10BV/h; more preferably, 0.025-0.125BV/h.

In another preferred embodiment, in the step (2.3), the flow rate of the discharging liquid is 0.002-0.150BV (bed volume)/h.

In another preferred embodiment, the column temperature of the chromatographic column and/or chromatographic column section of the method is 20-80 ℃; preferably, 30-70deg.C; more preferably 50-65 ℃.

In another preferred example, the cationic resin has a particle size of: 50-500um.

In another preferred example, the cationic resin packed in the individual column and/or column section has a density of 0.85 to 0.95g/cm ³ 。

The single column and/or single section length of the chromatographic column is 50-200cm.

In another preferred embodiment, the single column and/or single section aspect ratio of the column sections is 1/20-1/0.4; preferably 1/15-1/1.

In another preferred embodiment, the moving bed chromatography comprises 4-12 (preferably 5-10) chromatography columns and/or column sections.

In another preferred example, the switching time of the chromatographic column and/or chromatographic column section of the method is 3-15 min; preferably 5-8min.

In another preferred embodiment, the cationic resin is selected from the group consisting of: a calcium-type cationic resin, a sodium-type cationic resin, a potassium-type cationic resin, a magnesium-type cationic resin, a lithium-type cationic resin, or a combination thereof; preferably, a calcium type cationic resin, a magnesium type cationic resin, or a combination thereof.

In another preferred embodiment, in step (c), before the chromatographic separation, the method further comprises: desalting the second mixture to obtain a desalted second mixture.

In another preferred embodiment, the desalting treatment comprises ion exchange column desalting.

In another preferred embodiment, the conductivity of the desalted second mixture is less than or equal to 20. Mu.s.

In another preferred embodiment, the purity of the D-psicose in the separated product containing D-psicose is more than or equal to 95 percent based on the total mass of dry matters; preferably, the purity is 96-99%; more preferably, the purity is 98-99%.

In another preferred embodiment, in step (c), the method further comprises the steps of: fructose and glucose are separated and recovered from the second mixture.

In another preferred embodiment, in the separation recovery, fructose and glucose are recovered as a solution containing "fructose+glucose".

In another preferred embodiment, the total concentration of glucose and fructose in the "fructose+glucose" solution is 15-40wt%, preferably 20-35wt%, based on the total solution mass.

In another preferred embodiment, the "fructose+glucose" solution comprises: glucose 40-70wt% glucose and fructose 30-55wt%, preferably glucose 50-60wt% and fructose 40-50wt% based on the total mass of dry matter.

In another preferred embodiment, the D-psicose-containing isolated product comprises a liquid product and a solid product.

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

(d) Concentrating and crystallizing (preferably, cooling crystallization) the separated product containing D-psicose, thereby obtaining solid D-psicose.

In another preferred embodiment, said concentrating said D-psicose containing isolated product is performed by a MVR evaporator.

In another preferred embodiment, in the step (d), the concentration is performed under reduced pressure (preferably, the vacuum degree is-0.07 to-0.1 MPa).

In another preferred embodiment, in step (d), the concentration is carried out at 74-76 ℃.

In another preferred embodiment, the process has a single pass conversion of psicose of 4.5-14.5g psicose per 100g starch.

In another preferred embodiment, in the step (a), the amount of the starch raw material liquid is 1 to 5 tons; preferably 1 to 3 tons; more preferably 2 tons.

In another preferred embodiment, the method has a D-psicose yield of 100 to 1250 kg/day; preferably, 100 to 750 kg/day; more preferably 200 to 500 kg/day.

In another preferred embodiment, in step (b 2), the C-3 isomerase is D-psicose-3-epimerase.

In another preferred embodiment, the glucose isomerase and/or C-3 epimerase is selected from the group consisting of: enzyme solution, enzyme dry powder, immobilized enzyme, or a combination thereof.

In another preferred embodiment, the D-psicose-3-epimerase is a D-psicose-3-epimerase of Paenibacillus sp (Paenibacillus sp.).

In another preferred embodiment, the D-psicose-3-epimerase is selected from the group consisting of:

(i) A polypeptide with an amino acid sequence shown as SEQ ID NO. 1;

(ii) A polypeptide of the amino acid sequence set forth in SEQ ID No. 1 by substitution, deletion or addition of one or more amino acid residues (preferably, 1 to 50, more preferably, 1 to 30, more preferably, 1 to 10, most preferably, 1 to 6) or by addition of a signal peptide sequence and having catalytic activity to produce psicose;

(iii) The sequence contains the derivative polypeptide according to the amino acid sequence in (i) or (ii);

(iv) The amino acid sequence has more than or equal to 70 percent (preferably more than or equal to 80 percent, more preferably more than or equal to 90 percent) homology with the amino acid sequence shown in SEQ ID No. 1 and has the activity of catalyzing the generation of allose.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a schematic diagram of a continuous chromatographic separation scheme in accordance with the present invention.

Detailed Description

Through extensive and intensive studies, the present inventors have unexpectedly developed a method for producing D-psicose based on a starch raw material for the first time through screening of a large number of process routes. In the method of the present invention, by continuous and particularly optimized saccharification, isomerization and separation treatments, not only D-psicose can be continuously industrially produced, but also D-psicose of high purity (up to 98-99%) can be obtained, and the production cost of D-psicose can be significantly reduced. On this basis, the present inventors have completed the present invention.

Terminology

As used herein, the term "dry matter" refers to the sum of all but water.

As used herein, the term "concentration" refers to the weight percent of a particular substance based on the total weight of the solution, e.g., the concentration of psicose is 100% by weight psicose per total weight of the solution.

As used herein, the term "purity" refers to the percentage by weight of a particular substance relative to the total weight of the substance other than water, e.g., the purity of psicose is 100% by weight of psicose per weight of dry matter in solution.

As used herein, the term "saccharide" is a molecular compound consisting of three elements C, H, O, which in this application is psicose, fructose, glucose, etc.

As used herein, the term "weight ratio" is the average weight ratio between the substances over a period of time or when a certain amount of material is separated.

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

As used herein, the term "dry matter" refers to components of the mixture other than water, primarily saccharide materials in the present invention.

As used herein, the term "single pass conversion" refers to the amount of psicose produced per unit mass of starch when a solution of "fructose + glucose" is obtained without recycling the separated psicose.

C-3 isomerase

As used herein, the terms "C-3 isomerase", "C-3 fructose epimerase" and "C-3 epimerase" are used interchangeably to refer to enzymes that can catalyze the production of psicose with high efficiency.

In the present invention, a typical C-3 isomerase is D-psicose-3-epimerase. In the present invention, the typical D-psicose-3-epimerase is a protein shown in SEQ ID NO. 1 (i.e., wild-type D-psicose-3-epimerase) or a protein derived therefrom (e.g., mutant D-psicose-3-epimerase shown in SEQ ID NO. 2), which is derived from Paenibacillus (Paenibacillus senegalensis).

The term "isolated" as used herein refers to a substance that is separated from its original environment (i.e., the natural environment if it is a natural substance). If the naturally occurring polynucleotide and polypeptide are not isolated or purified in vivo, the same polynucleotide or polypeptide is isolated or purified from other naturally occurring substances. Thus, the term "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. The person skilled in the art is able to purify the D-psicose-3-epimerase of the present invention using standard protein purification techniques. Substantially pure proteins can produce a single band on a non-reducing polyacrylamide gel. However, it will be apparent to those skilled in the art in view of the teachings of the present invention and the prior art that "D-psicose-3-epimerase" shall also include variants of the protein which have the same or similar function as "D-psicose-3-epimerase of the present invention" but differ in their amino acid sequence by a small amount from the amino acid sequence shown for the wild-type D-psicose-3-epimerase. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 6) amino acids, and addition of one or more (usually 20 or less, preferably 10 or less, more preferably 6 or less) amino acids at the C-terminal and/or N-terminal. For example, it is well known to those skilled in the art that substitution with amino acids having similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of D-psicose-3-epimerase protein.

Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the DNA encoding "yellow D-psicose-3-epimerase of the invention". The invention also includes other polypeptides, such as fusion proteins comprising the "D-psicose-3-epimerase of the invention" or fragments thereof. In addition to the almost full-length polypeptide, the invention shall also include an active fragment of the "D-psicose-3-epimerase of the invention". Typically, the fragment has at least about 10 contiguous amino acids, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the amino acid sequence of the "D-psicose-3-epimerase of the present invention".

The invention also provides analogs of "D-psicose-3-epimerase". The difference between these analogs and the natural "D-psicose-3-epimerase of the present invention" may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, by site-directed mutagenesis or other known techniques of molecular biology. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

Modified (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their proteolytic resistance or to optimize their solubility properties are also included.

In the present invention, a conservatively variant polypeptide of "D-psicose-3-epimerase" refers to a polypeptide having up to 20, preferably up to 10, more preferably up to 5, most preferably up to 3 amino acids replaced by amino acids having similar or similar properties, as compared to the amino acid sequence shown in wild-type D-psicose-3-epimerase, but still having the same or similar activity as the protein having the amino acid sequence shown in SEQ ID NO:1, i.e.an activity of catalyzing the production of psicose.

Thus, given the teachings of the present invention and the prior art, one skilled in the art can make conservative variant mutants based on, for example, amino acid substitutions as shown in the following table.

Thus, as used herein, "containing," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are under the notion of "containing", "having" or "including".

The protein of the present invention may be a recombinant protein, a natural protein, a synthetic protein, preferably a recombinant protein. The proteins of the invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, higher plant, insect, 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 or may not also include an initial methionine residue.

It will be apparent to those skilled in the art that the "D-psicose-3-epimerase" of the present invention also includes fragments, derivatives and analogues of "D-psicose-3-epimerase". As used herein, the terms "fragment," "derivative" and "analog" refer to polypeptides that retain substantially the same biological function or activity of the "D-psicose-3-epimerase" of the present invention. The polypeptide fragments, derivatives or analogues of the invention may be (i) polypeptides having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein. Such fragments, derivatives and analogs are within the purview of one skilled in the art in view of the definitions herein.

In view of the prior art and the teachings of the present invention, it will be readily apparent to those skilled in the art to obtain active fragments of the D-psicose-3-epimerase of the present invention. Thus, any biologically active fragment of "D-psicose-3-epimerase" can be used in the present invention. As used herein, a biologically active fragment of "D-psicose-3-epimerase" refers to a fragment of "D-psicose-3-epimerase" that is still capable of retaining all or part of the function of the full length "D-psicose-3-epimerase". Typically, 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 capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full length "D-psicose-3-epimerase".

It will also be apparent to those skilled in the art from the teachings and teachings of the present invention that D-psicose-3-epimerase can be prepared in other useful forms such as immobilized enzymes.

As used herein, "amylase" refers to an enzyme capable of hydrolyzing O-glucose bonds in starch, glycogen and related polysaccharides (e.g., alpha-starch), which cleaves alpha-1, 4 glycosidic bonds in a random manner from the interior of the starch molecule when acting on starch.

As used herein, the term "saccharifying enzyme," also known as Glucoamylase [ Glucoamylase, EC.3.2.1.3 ], is an enzyme that hydrolyzes starch from the non-reducing end to glucose by hydrolysis of the a-1.4 glucosidic bond, and also slowly hydrolyzes the a-1.6 glucosidic bond to glucose. And simultaneously, the non-reducing end of the glycogen can release the beta-D-glucose.

Method for preparing D-psicose

In the present invention, there is also provided a method for preparing D-psicose, comprising the steps of:

(a) Saccharification is carried out on starch to obtain a first mixture;

(b) Carrying out isomerization treatment on the first mixture to obtain a second mixture; and

(c) And (3) carrying out post-treatment on the second mixture to obtain D-psicose.

In a preferred embodiment, the process for preparing D-psicose is as follows:

in step (a), the starch is liquefied by amylase and saccharified by saccharifying enzyme to form a starch saccharification liquid (the starch saccharification liquid is the first mixture in step (a)).

In the invention, the starch saccharification liquid contains glucose with a certain concentration (such as more than or equal to 70 percent, more than or equal to 80 percent, more than or equal to 90 percent or more than or equal to 95 percent) and other sugar substances (including maltose, isomaltose, maltotriose and the like) in balance (for example, the starch saccharification liquid comprises about 95.4 percent of glucose, 1.9 percent of maltose, 0.9 percent of isomaltose, 0.5 percent of maltotriose and more than 1.4 percent of polysaccharide based on the total mass of dry matters).

The starch saccharification liquid is isomerized by glucose isomerase and C-3 differential phase isomerase.

In a preferred embodiment, the saccharified solution is not isolated, and the starch saccharified solution is suitably concentrated and isomerized directly to a mixed solution by glucose isomerase and C-3 differential isomerase

In another preferred embodiment, in step (a), the saccharification liquid of starch is subjected to a heat treatment (temperature 80-100 ℃) to inactivate amylase and saccharifying enzymes therein.

In another preferred embodiment, the amylase liquefied and saccharified by heat-treated and deactivated amylase in the saccharification liquid is removed by filtration.

The components of the mixed solution (the mixed solution is the second mixed solution in the step (b)) after glucose isomerization and C-3 epimerization of the starch saccharification liquid are as follows:

1) A concentration of 30-60% (e.g., 50%) based on the total mass of the mixture;

2) The components are as follows: 5.5 to 7.5 percent of psicose, 18.6 to 20.5 percent of fructose, 20.5 to 25.9 percent of glucose, 1.9 percent of maltose, 0.9 percent of isomaltose, 0.5 percent of maltotriose and 1.4 percent of polysaccharide above maltotetraose, based on the total mass of the mixed solution;

3) The electric conductivity after desalination is less than or equal to 20 mu s (neutral).

In another preferred embodiment, the isomerization is carried out by adding glucose isomerase and C-3 isomerase directly to the saccharification liquid, or passing the saccharification liquid through a column to which glucose isomerase and C-3 isomerase are immobilized.

In another preferred embodiment, the isomerization is performed without isolation of the inactivated treatment enzyme used in step (a) by adding glucose isomerase and C-3 isomerase directly to the starch saccharification liquid.

Separating by chromatography to obtain two parts of solution, wherein the psicose solution comprises the following components:

1) Psicose-containing partial solution: the purity of the psicose reaches 97% -99%, the separation yield is more than 95%, and the concentration is 4% -7.2%. (said processing to psicose product by concentration, crystallization, etc.)

2) Solutions containing "fructose + glucose": the dry matter concentration is 25-35%, the purity of each component in the solution of fructose and glucose is 30-55%, glucose is 40-70%, maltose is 0.6-1.0%, isomaltose is 0.3-0.5%, maltotriose is 0.13-0.3%, polysaccharide above maltotetraose is 0.4-0.7% and psicose is 0.05-0.3%.

In another preferred embodiment, the method comprises the steps of:

1) Starch liquefaction and saccharification to prepare starch saccharification liquid:

mixing (starch with water, concentration 25-35%, adding amylase 10-50u/g starch, regulating pH), liquefying (liquefying at 100-130deg.C and maintaining for 30-60 min), neutralizing (regulating pH), saccharifying (adding saccharifying enzyme 10-200u/g starch, saccharifying at 58-62deg.C) to obtain the starch saccharification liquid (the starch saccharification liquid comprises glucose about 95.4%, maltose 1.9%, isomaltose 0.9%, maltotriose 0.5%, and polysaccharides above maltotetraose 1.4%, based on dry matter total mass); preferably, the saccharification of starch is heated to deactivate the enzymes, decolorized with activated carbon and filtered.

In another preferred embodiment, the starch is prepared as an emulsion of 25-35 wt.% (e.g. 30 wt.%) with purified water, the pH is adjusted to 6.2-6.4 (e.g. with sodium carbonate), amylase (20-50 u/g starch) is added in an amount of 0.05% by weight of the starch, the mixture is homogeneously prepared, and the mixture is spray liquefied at 110.+ -. 5 ℃ to a liquefied DE value of 15-20%. Cooling the liquefied solution to 55-60deg.C, adjusting pH to 4.3-4.8, adding saccharifying enzyme (50-100 u/g starch) accounting for 0.05% of starch weight, saccharifying at 55-60deg.C until DE >98%, and heating to 100+ -10deg.C for inactivating for 2-3min. Cooling to 50 ℃, adding active carbon with the weight of starch being 0.5-1.5% (such as 1.0%), stirring for 15-45 min (such as 30 min) for decoloring, filtering to remove insoluble impurities and enzyme protein, obtaining thin saccharification liquid, and concentrating under reduced pressure for dehydration to obtain high-concentration starch saccharification liquid.

In another preferred embodiment, 1kg of starch is prepared to yield 1.5 to 2.5kg of a saccharification liquid containing 50.+ -. 2 wt.% glucose, based on the total weight of the saccharification liquid.

2) Concentrating the above starch saccharification liquid (e.g., using MVR or other evaporator) to a dry matter concentration of 30-60wt% (preferably 45-55 wt%), based on total mass; and metal ions are added. The mixture was passed through an enzyme immobilization column containing "glucose isomerase" and "C-3 fructose differential isomerase" at a certain flow rate. The composition of the mixed solution (mixed solution, namely the second mixture in the step (b)) obtained by passing through the column is as follows: 5.5 to 7.5 percent of psicose, 18.6 to 20.5 percent of fructose, 20.5 to 29 percent of glucose, 1.9 percent of maltose, 0.9 percent of isomaltose, 0.5 percent of maltotriose and 1.4 percent of polysaccharide above maltotetraose, based on the total mass of the mixed solution.

3) Desalting: desalting with calcium ion exchange column, and loading into chromatographic separation device.

4) Chromatographic separation: the procedure is shown in FIG. 1 using 4-8 columns containing a specific chromatographic separation resin and regenerated with calcium chloride, for example, 4 columns.

As shown in fig. 1, the discharge is continuously carried out, and intermittent feeding (feeding interval time such as 30 to 60min, depending on the amount of resin and packing density, etc.) and elution (elution interval time such as 30 to 60min, depending on the amount of resin and packing density, etc.) are carried out, and the fast component (main component is psicose, i.e., separated product of D-psicose) and slow component (main component is glucose and fructose) are collected, respectively.

Specifically, 4 columns are connected in series, and the flow-through (the flow rate is 0.002-0.150BV/h, wherein whether the flow rates are the same or different in the 4 columns) of the 4 columns is sequentially performed, and the columns can move towards the flow direction of the flow-through flow.

Firstly feeding (feeding flow rate is 0.002-0.150 BV/h), then feeding water for eluting after feeding is 0.01-0.02BV, and moving the chromatographic column to enable the water feeding point (the position where the eluent is added) to be positioned between a fast component discharging position and a slow component discharging position, wherein the switching time t of the chromatographic column is 5-8min. The switching time is that the chromatographic column is at a certain position, and at time t, the chromatographic column starts to move along the flowing direction of the mobile phase and moves to the next position in the flowing direction of the mobile phase; for example, as shown in fig. 1, after a certain column is moved to the column 1 position, the column is quickly moved from the column 1 position to the column 2 position in the figure after a further time t.

In another preferred example, the chromatographic separation method has a feed water ratio (mass of feed: mass of wash water) of 1: (1.5-3), and the fast component/slow component (psicose solution) is (2-3): 1.

for example, the chromatographic conditions may be:

feed f=0.4 Kg/h (related to resin amount), eluent d=0.6 Kg/h, discharge AD (slow component) =0.33 Kg/h, BD (fast component) =0.66 Kg/h, throughput 0.03Kg feed/Kg resin, feed water ratio 1:1.5, BD/ad=2;

transient operating conditions: f→b=33 ml/min (fast component discharge at the time of feeding), d→a (slow component discharge at the time of elution) =33 ml/min, circulation amount (inversely proportional to switching time) r=33 ml/min, d→b (fast component discharge at the time of elution) =33 ml/min.

Results:

(1) Obtaining the purity of the psicose solution of 98-99% (based on the total mass of dry matters); the yield of psicose is more than 95%;

(2) The "fructose+glucose" mixed solution is obtained, and the dry matter concentration is 25-35wt%.

The content of fructose in dry matter is 30-55%, and the content of glucose is 40-70%.

(3) The mixture of maltose, isomaltose, maltotriose, polysaccharide and allose is obtained, and the dry matter concentration is 5-20%.

Wherein: 3-10wt% of maltose, 3-10wt% of isomaltose, 1-5wt% of maltotriose, 0.5-5wt% of polysaccharide and 1-6wt% of psicose (based on the mass of the mixture of maltose, isomaltose, maltotriose, polysaccharide and psicose).

The main advantages of the invention include:

(1) The invention takes starch as raw material for the first time, and the D-psicose with purity up to 98-99% is obtained through the treatment processes of saccharification, isomerization, concentration, chromatographic separation and the like.

(2) The invention adopts special C-3 isomerase for the first time, which is D-psicose-3-epimerase from paenibacillus (Paenibacillus senegalensis) and can efficiently catalyze and produce D-psicose.

(3) The method for producing D-psicose can obviously reduce the production cost of the D-psicose (the cost can be reduced by 30%).

(4) The method of the invention can also recycle raw materials (glucose, fructose and metal ions), thereby greatly reducing the cost.

(5) The method is suitable for industrial production.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Glucose isomerase, amylase and glucoamylase used in the examples were purchased from novelin.

The C-3 epimerase (i.e., C-3 isomerase) used in the examples has the sequence shown in SEQ ID No. 2.

Materials and reagents used in the examples were commercially available products unless otherwise specified.

Example 1

Preparation of starch saccharification liquid

Firstly, 100Kg of starch is prepared into 30% emulsion by purified water, the pH value is regulated to 6.36.2-6.4 by sodium carbonate, amylase (20 u/g starch) accounting for 0.05% of the weight of the starch is added, the mixture is uniformly prepared, and the mixture is sprayed and liquefied at 110 ℃ to obtain the liquefied DE value of 18%. Cooling the liquefied solution to 55 ℃, regulating the pH value to 4.5, adding saccharifying enzyme (100 u/g starch) accounting for 0.05% of the weight of starch, starting saccharification at 55 ℃, heating to 100 ℃ for inactivation for 3min, cooling to 50 ℃, adding active carbon accounting for 1.0% of the weight of starch, stirring for 30min for decoloration, filtering to remove insoluble impurities and enzyme protein, obtaining dilute starch saccharifying solution, concentrating under reduced pressure for dehydration, and obtaining high-concentration starch saccharifying solution with the glucose content of 50% of 200Kg, wherein the starch saccharifying solution comprises the following components: glucose about 95.4%, maltose 1.9%, isomaltose 0.9%, maltotriose 0.5%, maltotetraose and more than 1.4% (dry matter basis).

Example 2

Preparation of D-psicose

2.1 isomerization

Into a 5L reactor, 3.0Kg of a starch saccharification solution (prepared in example 1, having a glucose content of 50% and prepared from about 1.5Kg of starch) and 1.0mM MnSO were added ₄ .7H ₂ O, glucose isomerase and C-3 epimerase, the total enzyme activities were 50000u and 20000u, respectively, and the glucose conversion was equilibrated at 60℃for about 3 hours. The reaction product is obtained.

The reaction products were measured and the results showed that: about 14.6% of the glucose is converted to D-psicose.

2.2 enzyme removal

For the reaction product, two enzymes (glucose isomerase and C-3 epimerase) were removed by membrane filtration.

2.3 isolation of D-psicose

The manganese ions are removed from the reaction product by treatment with a calcium type cation resin, and a solution containing manganese ions is recovered.

Next, chromatographic separation was performed with a simulated moving bed, thereby separating an aqueous solution mainly containing psicose and a mixed solution mainly containing fructose and glucose ("fructose+glucose" solution). Wherein the obtained fructose and glucose solution is recycled, namely, after being concentrated, the solution is converted by glucose isomerase and C-3 epimerase again.

Results

(a) 6.0Kg of a "fructose+glucose" mixture was obtained, the dry matter content of which was 31%. In the dry matter, the fructose content was 46% and the glucose content was 52% by weight based on the total dry matter.

(b) 3.0Kg of an aqueous solution of psicose was obtained, wherein the content of D-psicose was 7% and the purity of D-psicose was 98.7%; the content of impurities (including fructose, glucose and other sugars) is < 1.3% by weight based on the total dry matter.

Total yield: 0.140kg psicose/kg starch.

Example 3

Preparation of D-psicose

3.1 isomerization

Into a 5L reactor, 3Kg of starch saccharification solution (prepared in example 1) and 1.0mM MINNSO were added ₄ .7H ₂ O, preheated to 60℃and pumped through (flow rate through the column 1 Kg/h) glucose isomerase and C-3 epimerase immobilizationAnd (3) catalyzing and converting the reaction column by using biological enzyme at the temperature of 60 ℃ to obtain an effluent liquid which is a reaction product.

The effluent was assayed for the conversion of 14.3% of the glucose to D-psicose.

3.2 isolation of D-psicose

Results

(a) 6.0Kg of a "fructose+glucose" mixed solution was obtained, the dry matter content of which was 30%. The fructose content was 46% and the glucose content was 52% in the dry matter;

(b) 3.0Kg of an aqueous solution of psicose was obtained, wherein the content of D-psicose (dry matter concentration) was 6.7%, the purity of D-psicose was 98.1%, and the content of impurities (including fructose, glucose and other sugar) was < 1.9% by total weight of dry matter.

Total yield: 0.134kg psicose/kg starch.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

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Claims

1. A method for producing D-psicose, comprising the steps of:

(b) Subjecting the first mixture to an isomerization treatment to isomerize glucose to fructose and further to D-psicose, thereby forming a second mixture comprising D-psicose;

said step (b) comprises:

(b2) Subjecting said fructose-containing mixture to a second enzymatic reaction with a C-3 isomerase,

thereby obtaining said second mixture; and is also provided with

The method further comprises the following steps before the step (b 1): adding metal ions to the first mixture; the metal ion is selected from: mn (Mn) ² ⁺ 、Co ²⁺ Or a combination thereof; and the final concentration of metal ions is 1-10mM;

wherein in step (b 2) the C-3 isomerase is a D-psicose-3-epimerase; and the D-psicose-3-epimerase is a D-psicose-3-epimerase of paenibacillus (Paenibacillus senegalensis); step (b 1) and step (b 2) are carried out at 40-80 ℃; preferably, at 50-70 ℃;

And

(c) Separating D-psicose from the second mixture by chromatographic separation, thereby obtaining a D-psicose-containing isolated product;

the chromatographic separation method comprises the steps of

(1) Providing a second mixture as feed F; and

the chromatographic separation comprises:

(2.1) feeding step: introducing the sugar-containing mixed solution into a chromatographic column; wherein the flow rate of the feed is 0.005-0.10BV/h;

wherein the chromatographic separation device comprises 4-12 chromatographic columns and/or chromatographic column sections, the packing of the chromatographic columns and/or the chromatographic column sections is cation resin, and the chromatographic columns and/or the chromatographic column sections are connected in series; wherein the cationic resin is calcium type cationic resin;

and, step (2.1) and step (2.2) are intermittently performed; and step (2.3) are carried out continuously;

The material-water ratio is 1 (1.0-2.0); wherein the feed water ratio is the mass ratio of the feed F to the eluent D;

the mass ratio of the separated product of D-psicose to the total amount of the feed F is (0.9-1.5): 1;

the switching time of the chromatographic column and/or chromatographic column section of the method is 5-8 min;

the column temperature of the chromatographic column and/or chromatographic column section of the method is 50-65 ℃;

the density of the cationic resin filled in the chromatographic column and/or chromatographic column section is 0.85-0.95 g/cm ³ ；

The particle size of the cationic resin is as follows: 50-500um;

the single column and/or single section length of the chromatographic column is 50-200cm;

the single-column and/or single-section diameter-height ratio of the chromatographic column is 1/15-1/1;

in the process, the per pass conversion of psicose is 4.5-14.5g psicose per 100g starch.

2. The method of claim 1, further comprising, prior to step (a), step (a 1): liquefying starch to form starch raw material liquid.

3. The method of claim 2, wherein the starch is subjected to liquefaction in the presence of an amylase.

4. A method according to claim 3, wherein the liquefaction process has one or more of the following characteristics:

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

the pH is 5.5-6.5;

the temperature of the liquefaction treatment is 100-130 ℃; and/or

The liquefying time is 30-60min.

5. The method of claim 1, wherein in step (a), the starch feedstock is saccharified in the presence of a saccharifying enzyme to form a first mixture.

6. The method of claim 5, wherein in step (a), the saccharification process has one or more of the following features:

the addition amount of the saccharifying enzyme is 10-200u/g starch;

the pH of saccharification treatment is 4.0-5.5;

saccharification time is 20-40h; and/or

The saccharification treatment temperature is 35-70 ℃.

7. The method of claim 1, wherein the metal ion is Mn ²⁺ 。

8. The process according to claim 1, wherein steps (b 1), (b 2) are performed simultaneously or sequentially and/or steps (b 1) and (b 2) are performed in the same reactor.

9. The method of claim 1, wherein the D-psicose-3-epimerase is selected from the group consisting of:

(i) A polypeptide with an amino acid sequence shown as SEQ ID NO. 1;

(ii) A mutant D-psicose-3-epimerase shown in SEQ ID No. 2.

10. The method of claim 1, wherein the final concentration of metal ions is 1mM.

11. The method of claim 1, wherein,

in the step (2.2), the flow rate of the eluent is 0.005-0.375BV/h; and

in the step (2.3), the flow rate of the discharged liquid is 0.002-0.150BV/h.

12. The method of claim 1, wherein,

in the step (2.2), the flow rate of the eluent is 0.025-0.125BV/h; and

in the step (2.3), the flow rate of the discharging liquid is 0.002-0.150BV/h.

13. The process as claimed in claim 1, wherein the mass ratio of the separated product of D-psicose to the total amount of feed F is from 1.0 to 1.3:1.