Preparation method for crystalline psicose

Abstract

The present invention relates to the technical field of bioengineering, and provides a preparation method for crystalline psicose, comprising: preparing an immobilized enzyme containing a D-psicose-3-epimerase preparation, and a chromatographic separation system containing the immobilized enzyme and a gel-type polystyrene calcium-type chromatographic resin, so as to prepare a psicose material, and then sequentially performing decoloration, ion exchange desalination, vacuum concentration, evaporation feeding crystallization, and cooling crystallization on an obtained psicose material solution to obtain crystalline psicose. By means of the preparation method, continuous efficient conversion of psicose is achieved, the purity of discharged psicose reaches 98-99%, and the treatment capacity with a unit of resin per unit time reaches 0.038-0.045 t / m3·h. In this way, the purity of the psicose is much higher than that obtained in a general saccharification process, continuous production can be achieved, and processes such as transfer and temporary storage are reduced, thereby improving the production efficiency.

Description

A preparation method of crystalline psicose

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311258473.7, filed on September 27, 2023, entitled “A method for preparing crystalline allulose,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of bioengineering technology, and in particular to a method for preparing crystalline psicose. Background Art

[0004] D-allulose, also known as allulose, is a low-calorie natural sweetener with 70% the sweetness of sucrose but only 10% of its calories. Allulose was approved as a food additive by the Food and Drug Administration (FDA) in 2012 and is generally recognized as safe (GRAS). However, the high cost of allulose limits its use as a sweetener.

[0005] Due to its extremely low abundance, allulose is difficult to extract from nature and difficult to purify via chemical synthesis. Enzymatic conversion avoids the complex feedback regulation of the microbial total synthesis pathway during fermentation, achieving remarkably high product concentrations and simplifying the product extraction process, undoubtedly offering considerable development potential. However, pure D-psicose-3-epimerase lacks stability and is easily affected by pH, temperature, and organic matter. This leads to a high loss of enzymatic activity in industrial applications and renders it difficult to reuse repeatedly. Modern scientific developments have spurred the development of immobilized enzyme processing techniques by chemically or physically immobilizing enzymes in supports. This not only maintains the enzyme's original catalytic properties but also mitigates the inherent limitations of the free enzyme, facilitating automated and continuous reactions and minimizing costs. Therefore, the development of immobilized D-psicose-3-epimerase technology is of vital importance to the industrial development of allulose.

[0006] As psicose has attracted significant attention as a sweetener, the need to develop efficient methods for its production has grown within the food industry. In 2006, CJ CheilJedang Co., Ltd. in South Korea produced D-psicose through enzymatic epimerization using fructose as a substrate. They immobilized psicose-3-epimerase from Agrobacterium tumefaciens and converted fructose to D-psicose using a fructose solution supplemented with manganese dichloride. However, fructose has similar physical properties to psicose, making separation of psicose from fructose difficult. Therefore, a method for producing psicose that can efficiently produce and easily separate psicose is urgently needed.

[0007] Summary of the Invention

[0008] In order to solve the problems existing in the prior art, the present invention provides a method for preparing crystalline psicose.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a method for preparing crystalline psicose, comprising the following steps:

[0011] 1) Co 2+ The type resin was used as an immobilized carrier to adsorb the psicose epimerase, and chitosan was added for cross-linking after adsorption to obtain the immobilized enzyme.

[0012] 2) respectively loading the obtained immobilized enzyme and gel-type polystyrene calcium type chromatographic resin into chromatographic columns of a chromatographic separation system to obtain an immobilized enzyme chromatographic column and a gel-type polystyrene calcium type chromatographic column;

[0013] 3) passing the fructose solution through a chromatographic column of immobilized enzyme for enzymatic conversion to obtain an enzyme conversion solution;

[0014] 4) chromatographically separating the obtained enzyme conversion solution through a gel-type polystyrene calcium chromatography column to obtain a fructose solution and residual enzyme conversion solution; combining the obtained fructose solution with the fructose solution in step 3);

[0015] 5) The remaining enzyme conversion solution is further passed through a gel-type polystyrene calcium chromatography column to obtain a psicose solution;

[0016] 6) sequentially decolorizing, ion-exchange desalting, and vacuum concentrating the obtained psicose liquid to obtain a psicose solution;

[0017] 7) The obtained psicose solution is sequentially subjected to evaporation-fed crystallization and cooling crystallization to obtain crystalline psicose.

[0018] Preferably, in step 1) Co 2+ The preparation method of the type resin is:

[0019] The resin was sequentially soaked in distilled water, sodium hydroxide solution, hydrochloric acid and CoCl2 solution to obtain the final Co 2+ Type resin.

[0020] Preferably, the distilled water immersion, sodium hydroxide solution immersion, and hydrochloric acid solution immersion time are 4 to 5 hours respectively, and the CoCl2 solution immersion time is 8 to 12 hours.

[0021] Preferably, the dosage of the psicose epimerase in step 1) is 100-120 U / g resin, the mass concentration of the chitosan solution is 1-1.5%, and the chitosan solution is mixed with Co 2+ The volume ratio of the molding resin is 0.2 to 0.4:1.

[0022] Preferably, the specific composition of the chromatographic separation system in step 2) is:

[0023] The chromatographic separation system includes 8 chromatographic columns numbered in sequence, wherein chromatographic column No. 1, chromatographic column No. 2, chromatographic column No. 3, chromatographic column No. 5, chromatographic column No. 6, chromatographic column No. 7 and chromatographic column No. 8 are connected in sequence, and chromatographic column No. 4 is reserved; the chromatographic column No. 1, chromatographic column No. 2, chromatographic column No. 3 and chromatographic column No. 4 are loaded with immobilized enzymes, and the chromatographic column No. 5, chromatographic column No. 6, chromatographic column No. 7 and chromatographic column No. 8 are loaded with gel-type polystyrene calcium type chromatographic resins.

[0024] Preferably, the operation process of the chromatographic separation system in step 2) is specifically as follows:

[0025] A fructose solution with a mass concentration of 55-60% enters column 1, undergoes enzyme conversion reaction in columns 1, 2 and 3, enters column 5, enters purified water through column 6, and the fructose solution at the outlet of column 6 that has not been converted by enzymes returns to column 1 along the pipeline. At the same time, the mass concentration of the fructose solution at the feed port is increased to 70-75%, and is mixed with the fructose solution returned from column 6 to obtain a fructose solution with a mass concentration of 55-60%. The enzyme-converted allulose solution is obtained at the outlet of column 8. When switched to the next step, During one operating cycle, the fructose solution with a mass concentration of 55-60% obtained above enters column 1, undergoes enzyme conversion reactions in columns 1, 2, and 3, enters column 6, and enters purified water through column 7. The fructose solution exits column 7 and returns to column 1 along the pipeline. At the same time, the mass concentration of the fructose solution at the feed port is increased to 70-75%, and is mixed with the fructose solution returned from column 7. The enzyme-converted allulose solution is exported from column 5. The subsequent operating cycles are similar, forming continuous production.

[0026] Preferably, the conversion rate of fructose by the immobilized enzyme in step 1) is 30-33%, and the half-life is 25-28 days.

[0027] Preferably, the cross-linking temperature in step 1) is 25-35° C., and the cross-linking time is 1-2 hours.

[0028] Preferably, the mass concentration of the fructose solution in step 3) is 55-60%.

[0029] Preferably, the temperature of the enzymatic conversion in step 3) is 55-65°C.

[0030] Preferably, the specific steps of decolorization in step 6) are:

[0031] The allulose liquid is decolorized by passing it through a granular activated carbon column at a temperature of 55 to 65°C, and then filtered through an Ama filter at a temperature of 50 to 60°C and a working pressure of 0.2 to 0.4 MPa.

[0032] Preferably, the specific steps of ion exchange desalination in step 6) are:

[0033] The decolorized allulose liquid is sequentially passed through a cation exchange column, an anion exchange column, a cation exchange column, an anion exchange column, and a cation exchange column, with the liquid temperature being 35-45° C. and the flow rate being 2-4 BV / h.

[0034] Preferably, the resin used in the cation exchange column is D001 type; the resin used in the anion exchange column is 301P type.

[0035] Preferably, the specific steps of vacuum concentration in step 6) are:

[0036] The allulose solution after ion exchange desalination was concentrated using a four-effect evaporator at a concentration temperature of 65-75° C. and a pressure of -0.06-0.1 MPa to obtain a allulose solution with a soluble solid mass concentration of 71-73%.

[0037] Preferably, the evaporation fed-batch crystallization in step 7) comprises the following steps:

[0038] 1) mixing the psicose solution with seed crystals and then growing the crystals to obtain a grown solution;

[0039] 2) continuously evaporating and feeding the feed solution after crystallization to obtain a feed solution after evaporation and feeding crystallization.

[0040] Preferably, the evaporation fed-batch crystallization in step 7) specifically comprises the following steps:

[0041] 1) mixing a psicose solution with seed crystals and then growing the crystals to obtain a grown solution; wherein the psicose solution temperature is 43-45° C., the seed crystal amount is 0.8-1% of the dry mass concentration, the seed crystal diameter is 48-58 μm, the growing time is 2-4 hours, and the stirring speed during the growing process is 3-5 rpm.

[0042] 2) continuously evaporating and feeding the feed solution after crystallization to obtain a feed solution after evaporation and feeding crystallization; wherein the feeding solution is a psicose solution with a soluble solid mass concentration of 71-73%, and the soluble solid mass concentration of the material in the sugar boiling tank is adjusted to 79-81% by adjusting the feeding rate and the amount of water evaporation. The rotation speed during the feeding process is 5-10 rpm and the temperature is 44-46° C. When the feeding tank is full, 45-55% of the feed solution in the tank is transferred to a horizontal crystallizer for cooling and crystallization. The sugar boiling tank continues to be fed, and when it is full, 45-55% of the feed solution in the tank is transferred to the horizontal crystallizer, and so on.

[0043] Preferably, the cooling crystallization in step 7) comprises the following steps:

[0044] Cooling the resulting liquid after evaporation and crystallization to a first temperature, and then continuing to cool it to a second temperature;

[0045] The cooling rate is 0.4-0.55°C / h, and the first temperature is 38-41°C;

[0046] The rate of continuing to cool is 0.8-1.1°C / h, and the second temperature is 28-31°C.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1. The present invention sets the mass concentration of the fructose solution fed into the immobilized enzyme-chromatographic separation system to 55-60%. This concentration range can prevent bacterial contamination and improve the efficiency of chromatographic separation. After the fructose solution is returned to the chromatographic separation, since the mass concentration of the returned fructose solution is relatively low, the feed mass concentration of the fructose solution is increased to 70-75% to ensure that the mass concentration of the fructose solution entering the immobilized enzyme-chromatographic separation system is maintained at 55-60%. This system achieves continuous and efficient conversion of allulose, with the purity of the discharged allulose reaching 98-99% and the processing capacity per unit resin per unit time reaching 0.038-0.045 t / m 3 ·h, which is much higher than the purity obtained in the general saccharification process. Compared with the general saccharification, decolorization, separation, concentration, chromatography and other processes, the production is continuous, reducing the transfer and temporary storage processes, and improving production efficiency.

[0049] 2. The evaporation-feed crystallization process described in the present invention is a continuous process, while the cooling crystallization process is a batch process. The present invention sets the feed volume of the sugar boiling tank to 40-50% of the tank volume. After the evaporation-feed is full, 45-55% of the tank volume is transferred to the horizontal crystallizer for cooling crystallization. The sugar boiling tank continues to evaporate and feed. When the tank is full again, another 45-55% of the tank volume is transferred to the next horizontal crystallizer. This achieves semi-continuous crystallization of psicose. Compared to batch crystallization of psicose, semi-continuous crystallization produces a more stable product quality, eliminates differences in crystal quality between batches, is easier to operate, and has higher production efficiency.

[0050] 3. Crystallization Process: To a psicose solution with a soluble solids concentration of 78-79% and a temperature of 43-45°C, seed crystals with a dry weight concentration of 0.8-1% and a diameter of 48-58 μm are added. The crystals are grown for 2-4 hours before evaporation-feed crystallization begins. This seed size and quantity ensures a sufficient number of seed crystals to meet the seed quantity requirements for continuous evaporation-feed crystallization. Furthermore, newly formed nuclei during the continuous evaporation-feed crystallization process can also be used as seeds to replenish seed crystals lost during each feed transfer. During the evaporation-feed crystallization process, the temperature is controlled at 44-46°C, the soluble solids concentration of the feed solution is controlled at 71-73%, and the soluble solids concentration of the sugar-boiling material is controlled at 79-81%. At this temperature and concentration, the material viscosity is 500-600 mPa·s, making it suitable for crystal growth. Furthermore, at this temperature, evaporation efficiency is higher than at 30-40°C, shortening the crystallization cycle and reducing production costs. During the evaporation feed crystallization process, the rotation speed is controlled at 5-10 rpm. This speed promotes mass transfer and heat conduction, while allowing large crystals to settle naturally. Small crystals and newly formed nuclei from the evaporation feed crystallization process remain in the upper portion of the sugar boiling tank. Each time the tank is full, the material is discharged from the bottom of the sugar boiling tank into the horizontal crystallizer. Large crystals can enter the horizontal crystallizer, while small crystals and newly formed nuclei remain in the sugar boiling tank, serving as seed crystals for continued growth.

[0051] 4. The cooling crystallization process is a batch crystallization process. In the range of 45-40℃, the temperature is lowered by 0.5℃ per hour. In this temperature range, the corresponding saturated material has a large viscosity (400-600mPa·S). Setting a temperature reduction of 0.5℃ per hour can ensure sufficient material transfer and heat conduction. In the range of 40-30℃, the temperature is lowered by 1℃ per hour. In this temperature range, the corresponding saturated material has a low viscosity (300-400mPa·S). Setting a temperature reduction of 1℃ per hour can ensure sufficient mass transfer and heat conduction, and improve production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0053] Figure 1 is a schematic diagram of the initial cycle operation of the immobilized enzyme-chromatographic separation system; wherein columns 1, 2, 3, and 4 are columns loaded with immobilized enzymes, and columns 5, 6, 7, and 8 are columns loaded with gel-type polystyrene calcium chromatographic resins;

[0054] Figure 2 is a schematic diagram of the secondary circulation operation of the immobilized enzyme-chromatographic separation system; wherein columns 1, 2, 3, and 4 are columns loaded with immobilized enzymes, and columns 5, 6, 7, and 8 are columns loaded with gel-type polystyrene calcium chromatographic resins;

[0055] FIG3 is the microscopic morphology of crystallized psicose in Example 1;

[0056] FIG4 is a microscopic morphology of crystallized psicose in Example 2;

[0057] FIG5 shows the microscopic morphology of crystallized psicose in Comparative Example 11;

[0058] FIG6 shows the microscopic morphology of crystallized psicose in Comparative Example 12;

[0059] FIG7 shows the microscopic morphology of crystallized psicose in Comparative Example 13;

[0060] FIG8 shows the microscopic morphology of crystallized psicose in Comparative Example 14;

[0061] FIG9 shows the microscopic morphology of crystallized psicose in Comparative Example 15. DETAILED DESCRIPTION

[0062] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention are only used to describe the technical solutions of the present invention and are not intended to limit the present invention.

[0063] The reagents used in the examples of the present invention are all commercially available products. The resin used in the cation exchange column used in the following examples is D001 type; the resin used in the anion exchange column is 301P type.

[0064] Example 1 A method for preparing crystalline psicose

[0065] 1. Preparation of D-psicose-3-epimerase enzyme preparation

[0066] D-psicose 3-epimerase was used to convert D-fructose to D-psicose. The D-psicose 3-epimerase was derived from Bacillus subtilis BLCY-010 and the donor was Ruminococcus sp. CAG55. Bacillus subtilis BLCY-010 was deposited at the China General Microbiology Center (CGMCC) on July 17, 2023, with the accession number CGMCC No. 27923. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0067] 1. Primary seed culture: Inoculate a glycerol tube of Bacillus subtilis BLCY-010 into liquid LB medium at an inoculum size of 0.5% and culture at 33°C with shaking at 220 rpm for 10 h to prepare a primary seed solution.

[0068] 2. Secondary seed culture: The primary seed solution was inoculated into a seed tank with an inoculation amount of 5%, cultured at 33°C for 6 hours, and the ventilation ratio was controlled at 1:1 (V / Vm), thereby preparing the secondary seed solution.

[0069] The secondary seed culture medium comprises the following components (all in weight to volume ratio): 0.5% yeast extract powder; 1% peptone; 0.8% sodium chloride; 0.5% glucose; and the balance water.

[0070] 3. Fermentation culture: The secondary seed liquid was inoculated into a fermenter at an inoculum size of 5%. The culture was carried out at 33°C for 30 h to obtain the fermentation liquid. The pH of the culture process was controlled at 6.8, the flow rate was controlled at 1:1 (V / Vm), and the dissolved oxygen was controlled at 20%. After inoculation, the culture was cultured until a dissolved oxygen trough appeared. Then, the feed medium was added at a rate of 5 mL / L / h.

[0071] The fermentation medium comprises the following components (all in weight to volume ratio): 1.8% corn steep liquor, 2.7% peptone, 0.1% diammonium hydrogen phosphate, 0.2% sodium sulfite, 1.4% dipotassium hydrogen phosphate, 0.4% sodium dihydrogen phosphate, 0.1% magnesium sulfate, 1.5% glycerol, and the balance water.

[0072] The components of the feed medium are (all weight to volume ratios): 35% glycerol, 0.8% magnesium sulfate, 6% diammonium hydrogen phosphate, and the balance water.

[0073] 4. Post-fermentation extraction: The fermentation broth is centrifuged in a disc centrifuge at 2000 rpm and controlled at 25°C. After centrifugation, the cells are collected and washed with tap water twice their weight. The cells are then centrifuged a second time to obtain the enzyme preparation.

[0074] 2. Preparation of immobilized enzyme

[0075] 1. Pretreatment of D101 resin

[0076] Soak the resin in distilled water 4 times the volume of D101 resin for 4 hours. After filtering out the distilled water, soak the resin in 5% mass concentration NaOH solution for 5 hours. After filtering out the NaOH solution, wash the D101 resin with distilled water to pH 7.0. Then soak the resin in 5% mass concentration HCl solution for 5 hours. After filtering out the HCl solution, wash the resin with distilled water to pH 7.0. Then soak the resin in 30mM CoCl2·6H2O for 12 hours. Filter out the excess liquid to complete the resin pretreatment.

[0077] 2. Preparation of immobilized enzyme

[0078] The enzyme preparation was added to the resin at an enzyme dosage of 120 U / g resin. After adsorption at 30°C for 5 hours, a chitosan solution with a mass concentration of 1% and a volume 0.3 times that of the resin was added for cross-linking. The cross-linking temperature was controlled at 35°C and the cross-linking time was 2 hours to complete the preparation of the immobilized enzyme.

[0079] 3. Determination of activity of immobilized enzyme:

[0080] 1) Conversion rate refers to the percentage of allulose produced in the dry matter of the solution when the reaction of fructose conversion by immobilized enzyme reaches equilibrium.

[0081] Using 55% fructose solution as substrate and adjusting the pH to 6.0, immobilized enzyme particles with a dry mass concentration of 20% were added to the fructose solution. After reacting at 60°C for 4 hours, the conversion rate was 32.84% as determined by HPLC.

[0082] 2) Half-life detection

[0083] The enzyme activity units of the immobilized enzyme were tested every 24 hours, and the time taken for the enzyme activity units to drop to half was the half-life.

[0084] Enzyme activity units were defined by adding 200 mg of immobilized enzyme particles to 300 μL of a 55% fructose solution at 60°C for 10 minutes. The psicose content was analyzed by HPLC, and one unit of enzyme activity was defined as 1 μmol of psicose produced per minute. The changes in the activity units of the immobilized enzyme are shown in Table 1 below. On day 27 (648 hours), the enzyme activity units were 48.96 U / g, approximately half the value, indicating a half-life of the immobilized enzyme of 27 days.

[0085] Table 1 Changes in activity units of immobilized enzymes

[0086] 3. Preparation and operation of immobilized enzyme-chromatographic separation system

[0087] 1. Preparation of immobilized enzyme-chromatographic separation system

[0088] An eight-column immobilized enzyme-chromatographic separation system was prepared, with columns 1, 2, 3, and 4 being filled with immobilized enzymes, and columns 5, 6, 7, and 8 being filled with gel-type polystyrene calcium chromatographic resins.

[0089] 2. Operation of the immobilized enzyme-chromatographic separation system

[0090] As shown in Figure 1, the fructose solution enters column 1, undergoes enzymatic conversion reactions in columns 1, 2, and 3, then enters column 5, enters purified water via column 6, and the material at the outlet of column 6 returns to column 1, with the psicose liquid material exiting column 8. During the next cycle, as shown in Figure 2, the fructose solution enters column 1, undergoes enzymatic conversion reactions in columns 1, 2, and 3, then enters column 6, enters purified water via column 7, and the material at the outlet of column 7 returns to column 1, with the psicose liquid material exiting column 5.

[0091] The operating conditions of the immobilized enzyme-chromatographic separation system are:

[0092] The initial feed fructose solution mass concentration is 55%. After the second cycle starts, the feed fructose solution mass concentration is 70%. The system operating temperature is 55°C, and the material-water ratio is 1:2.5. When the enzyme activity unit of column No. 1 drops to half, columns No. 2, 3, and 4 are replaced to flow, and the immobilized enzyme in column No. 1 is replaced, and so on, to form continuous production.

[0093] The specific chromatographic cycle process of the immobilized enzyme-chromatographic separation system is as follows:

[0094] In the first chromatographic cycle, the material was taken from the outlet of column 6, and the purity of fructose was 95.31% after HPLC analysis. The material was taken from the outlet of column 8, and the purity of psicose was 98.23% after HPLC analysis.

[0095] In the second chromatographic cycle, the material was taken from the outlet of column 7, and the purity of fructose was 96.17% after HPLC analysis. The material was taken from the outlet of column 5, and the purity of psicose was 98.31% after HPLC analysis.

[0096] In the third chromatographic cycle, the material was taken from the outlet of column 8, and the purity of fructose was 97.08% after HPLC analysis. The material was taken from the outlet of column 6, and the purity of psicose was 98.56% after HPLC analysis.

[0097] The fructose conversion rate in the immobilized enzyme-chromatographic separation system is defined as:

[0098] Over a period of time, the dry basis of allulose leaving the system / the dry basis of fructose entering the system × 100%. When the system was running for 60 days, the fructose conversion rate during this period of operation was 96.72%. The processing capacity per unit resin per unit time was 0.041t / m 3 ·h.

[0099] 4. Preparation of Crystallized Allulose

[0100] 1. The psicose solution discharged from the immobilized enzyme-chromatographic separation system was subjected to decolorization, cross-linking, and four-effect evaporation concentration to obtain a psicose solution with a soluble solids concentration of 73%.

[0101] The decolorization process involves passing the allulose material through a granular activated carbon column at a temperature of 65°C, followed by filtration through an Ama filter at a temperature of 60°C and an operating pressure of 0.4 MPa.

[0102] The ion exchange process is to pass the decolorized material through a cation exchange column, anion exchange column, cation exchange column, anion exchange column, and cation exchange column in sequence, with a material temperature of 45° C. and a flow rate of 4 BV / h.

[0103] The vacuum concentration process is to use a four-effect evaporator to concentrate the material after the separation, the concentration temperature is 75°C, the pressure is -0.06Mpa, and a psicose solution with a soluble solid mass concentration of 73% is obtained;

[0104] The mass concentration of soluble solids in the present invention × the purity of psicose = the mass concentration of psicose.

[0105] 2. Continuous evaporation crystallization

[0106] A 73% soluble solids solution was pumped into a sugar boiling tank, filling the tank at a volume of 50% of the tank's volume. Vacuum evaporate the water in the tank to raise the soluble solids concentration to 78.25%. The temperature was adjusted to 43.5°C, and seed crystals with a dry weight concentration of 1% and a diameter of 48 μm were added. The crystals were grown for 3 hours at a stirring speed of 3 rpm. After the crystals were grown, evaporation and feeding crystallization began. The temperature was controlled at 45°C, and the feeding liquid was a 73% soluble solids solution. The feeding rate was adjusted to maintain a soluble solids concentration of 80.5% in the sugar boiling tank. The stirring speed during the evaporation and feeding process was maintained at 5 rpm. When the tank was full, 50% of the solution was transferred to a horizontal crystallizer. Evaporation and feeding continued until the tank was full again, and another 50% of the solution was transferred to the next horizontal crystallizer, and so on.

[0107] 3. Batch cooling crystallization

[0108] After transferring to a horizontal crystallizer, stabilize at 45°C for 3 hours with agitation at 3 rpm. Cool by 0.5°C per hour within the 45-40°C range and by 1°C per hour within the 40-30°C range. Once the temperature reaches 30°C, stabilize for 2 hours. This concludes the cooling crystallization process.

[0109] 4. Centrifugation and drying of crystallized allulose

[0110] Crystallized allulose was centrifuged and dried. During centrifugation, the speed was maintained at 600 rpm for 600 seconds, then increased to 900 rpm and maintained for 1200 seconds. The centrifugation was followed by a 10-second water wash at 900 rpm, at the 300-second mark, and again at the 900-second mark. During drying, the hot air temperature was controlled at 60°C, and the drying time was 60 minutes.

[0111] The microscopic morphology (×100) and mesh size distribution of the crystalline psicose are shown in FIG3 and Table 2:

[0112] Table 2 Mesh size distribution of crystalline psicose

[0113] Example 2 A method for preparing crystalline psicose

[0114] 1. Preparation of D-psicose-3-epimerase enzyme preparation

[0115] D-psicose 3-epimerase was used to convert D-fructose to D-psicose. The D-psicose 3-epimerase was derived from Bacillus subtilis BLCY-010 and the donor was Ruminococcus sp. CAG55. Bacillus subtilis BLCY-010 was deposited at the China General Microbiology Center (CGMCC) on July 17, 2023, with the accession number CGMCC No. 27923. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0116] 1. Primary seed culture: Inoculate a glycerol tube of Bacillus subtilis BLCY-010 into liquid LB medium at an inoculum size of 1%. Culture at 37°C with shaking at 220 rpm for 15 h to prepare a primary seed solution.

[0117] 2. Secondary seed culture: The primary seed solution was inoculated into a seed tank with an inoculation amount of 10%, and cultured at 37°C for 9 hours with a ventilation ratio of 1:1.5 (V / Vm) to obtain the secondary seed solution.

[0118] The secondary seed culture medium comprises the following components (all in weight to volume ratio): 0.5% yeast extract powder; 1% peptone; 0.8% sodium chloride; 0.5% glucose; and the balance water.

[0119] Fermentation culture: The secondary seed liquid was inoculated into the fermenter at an inoculation amount of 10%. The fermentation liquid was cultured at 37°C for 36 h to obtain the fermentation liquid. The pH was controlled at 7.2 during the culture process, the flow ratio was controlled at 1:1.5 (V / Vm), and the dissolved oxygen was controlled at 40%. After inoculation, the culture was cultured until a dissolved oxygen trough appeared, and then the feed medium was started at a feeding rate of 10 mL / L / h.

[0120] The fermentation medium comprises the following components (all in weight to volume ratio): 1.8% corn steep liquor, 2.7% peptone, 0.1% diammonium hydrogen phosphate, 0.2% sodium sulfite, 1.4% dipotassium hydrogen phosphate, 0.4% sodium dihydrogen phosphate, 0.1% magnesium sulfate, 1.5% glycerol, and the balance water.

[0121] The components of the feed medium are (all weight to volume ratios): 35% glycerol, 0.8% magnesium sulfate, 6% diammonium hydrogen phosphate, and the balance water.

[0122] Post-fermentation extraction: The fermentation broth is centrifuged in a disc-type centrifuge at 2000 rpm and controlled at 35°C. After centrifugation, the cells are collected and washed with tap water three times their weight. The enzyme is then centrifuged a second time and the cells are collected.

[0123] 2. Preparation of immobilized enzyme

[0124] 1. Pretreatment of D101 resin

[0125] Soak the resin in distilled water 3 times the volume of D101 resin for 5 hours. After filtering out the distilled water, soak the resin in 4% mass concentration NaOH solution for 4 hours. After filtering out the NaOH solution, wash the resin with distilled water to pH 6.5. Then soak the resin in 4% mass concentration HCl solution for 4 hours. After filtering out the HCl solution, wash the resin with distilled water to pH 6.5. Then soak the resin in 25mM CoCl2·6H2O with a molar concentration of 8 hours. Filter out the excess liquid to complete the resin pretreatment.

[0126] 2. Preparation of immobilized enzyme

[0127] The enzyme preparation was added to the resin at an enzyme dosage of 100 U / g resin. After adsorption at 35°C for 5 hours, a chitosan solution with a mass concentration of 1.5% and a volume 0.4 times that of the resin was added for cross-linking. The cross-linking temperature was controlled at 35°C and the cross-linking time was 2 hours to complete the preparation of the immobilized enzyme.

[0128] 3. Determination of activity of immobilized enzyme:

[0129] 1) Conversion rate refers to the percentage of allulose produced in the dry matter of the solution when the reaction of fructose conversion by immobilized enzyme reaches equilibrium.

[0130] Using 55% fructose solution as substrate and adjusting the pH to 6.0, immobilized enzyme particles with a dry mass concentration of 20% were added to the fructose solution. After reacting at 60°C for 4 hours, the conversion rate was 31.68% as determined by HPLC.

[0131] 2) Half-life detection

[0132] The enzyme activity units of the immobilized enzyme were tested every 24 hours, and the time taken for the enzyme activity units to drop to half was the half-life.

[0133] Enzyme activity units were defined by adding 200 mg of immobilized enzyme particles to 300 μL of a 55% fructose solution at 60°C for 10 minutes. The psicose content was analyzed by HPLC, and one unit of enzyme activity was defined as 1 μmol of psicose produced per minute. The changes in the activity units of the immobilized enzyme are shown in Table 3 below. On day 26 (624 hours), the activity units were 44.87 U / g, approximately half the value, indicating a half-life of the immobilized enzyme of 26 days.

[0134] Table 3 Changes in activity units of immobilized enzymes

[0135] 3. Preparation and operation of immobilized enzyme-chromatographic separation system

[0136] 1. Preparation of immobilized enzyme-chromatographic separation system

[0137] An eight-column immobilized enzyme-chromatographic separation system was prepared, with columns 1, 2, 3, and 4 being filled with immobilized enzymes, and columns 5, 6, 7, and 8 being filled with gel-type polystyrene calcium chromatographic resins.

[0138] 2. Operation of the immobilized enzyme-chromatographic separation system

[0139] As shown in Figure 1, the fructose solution enters column 1, undergoes enzymatic conversion reactions in columns 1, 2, and 3, then enters column 5, enters purified water via column 6, and the material at the outlet of column 6 returns to column 1, with the psicose liquid material exiting column 8. During the next cycle, as shown in Figure 2, the fructose solution enters column 1, undergoes enzymatic conversion reactions in columns 1, 2, and 3, then enters column 6, enters purified water via column 7, and the material at the outlet of column 7 returns to column 1, with the psicose liquid material exiting column 5.

[0140] The operating conditions of the immobilized enzyme-chromatographic separation system are:

[0141] The initial feed fructose solution mass concentration is 60%. After the second cycle starts, the feed fructose solution mass concentration is 75%. The system operating temperature is 55°C, and the material-water ratio is 1:2.0. When the enzyme activity unit of column 1 drops to half, columns 2, 3, and 4 are replaced, and the immobilized enzyme in column 1 is replaced, and so on, to form continuous production.

[0142] The specific chromatographic cycle process of the immobilized enzyme-chromatographic separation system is as follows:

[0143] In the first chromatographic cycle, the material was taken from the outlet of column 6, and the purity of fructose was 95.16% after HPLC analysis. The material was taken from the outlet of column 8, and the purity of psicose was 98.19% after HPLC analysis.

[0144] In the second chromatographic cycle, the material was taken from the outlet of column 7, and the purity of fructose was 95.86% after HPLC analysis. The material was taken from the outlet of column 5, and the purity of psicose was 98.46% after HPLC analysis.

[0145] In the third chromatographic cycle, the material was taken from the outlet of column 8, and the purity of fructose was 96.63% after HPLC analysis. The material was taken from the outlet of column 6, and the purity of psicose was 98.52% after HPLC analysis.

[0146] In the fourth chromatographic cycle, material was taken from the outlet of column 5, and the purity of fructose was 96.41% after HPLC analysis. Material was taken from the outlet of column 7, and the purity of psicose was 98.60% after HPLC analysis.

[0147] The fructose conversion rate in the immobilized enzyme-chromatographic separation system is defined as:

[0148] Over a period of time, the dry basis of allulose leaving the system / the dry basis of fructose entering the system × 100%. When the system was running for 60 days, the fructose conversion rate during this period of operation was 97.08%. The processing capacity per unit resin per unit time was 0.043t / m 3 ·h.

[0149] 4. Preparation of Allulose

[0150] 1. The psicose solution discharged from the immobilized enzyme-chromatographic separation system was decolorized, cross-linked, and concentrated by four-effect evaporation to obtain a psicose solution with a soluble solids concentration of 71%.

[0151] The decolorization process is to pass the allulose material through a granular activated carbon column for decolorization, the material temperature is 55°C, and then filter it through an Ama filter, the material temperature is 50°C, and the working pressure of the Ama filter is 0.2Mpa.

[0152] The ion exchange process is to pass the decolorized material through a cation exchange column, anion exchange column, cation exchange column, anion exchange column, and cation exchange column in sequence, with a material temperature of 35° C. and a flow rate of 2 BV / h.

[0153] The vacuum concentration process is to use a four-effect evaporator to concentrate the material after the centrifugation, the concentration temperature is 65 ° C, the pressure is -0.1 MPa, and a psicose solution with a soluble solid mass concentration of 71% is obtained;

[0154] The mass concentration of soluble solids in the present invention × the purity of psicose = the mass concentration of psicose.

[0155] 2. Continuous evaporation crystallization

[0156] A 71% soluble solids solution was pumped into a sugar cooking tank, filling the tank at a volume of 40% of the tank's volume. Vacuum evaporate the water in the tank to raise the soluble solids concentration to 79%. The temperature was adjusted to 45°C, and seed crystals with a dry weight concentration of 0.8% and a diameter of 58 μm were added. The crystals were grown for 3 hours at a stirring speed of 3 rpm. After the crystals were grown, evaporation and feeding crystallization began. The temperature was controlled at 44°C, and the feeding liquid was a 71% soluble solids solution. The feeding rate was adjusted to maintain a soluble solids concentration of 79% in the sugar cooking tank. The stirring speed during the evaporation and feeding process was maintained at 10 rpm. When the tank was full, 45% of the solution was transferred to a horizontal crystallizer. Evaporation and feeding continued until the tank was full again, and another 45% of the solution was transferred to the next horizontal crystallizer, and so on.

[0157] 3. Batch cooling crystallization

[0158] After transferring to a horizontal crystallizer, stabilize at 45°C for 5 hours with a stirring speed of 5 rpm. Within the 45-40°C range, cool by 0.5°C per hour, and within the 40-30°C range, cool by 1°C per hour. Once the temperature drops to 30°C, stabilize for 2 hours. This concludes the cooling crystallization process.

[0159] 4. Centrifugation and drying of crystallized allulose

[0160] Crystallized allulose was centrifuged and dried. During centrifugation, the speed was maintained at 600 rpm for 600 seconds, then increased to 900 rpm and maintained for 1200 seconds. The mixture was washed with water for 10 seconds at 900 rpm for 300 seconds, and again at 900 rpm for 10 seconds at 900 seconds. During drying, the hot air temperature was controlled at 55°C, and the drying time was 60 minutes.

[0161] The microscopic morphology (×100) and mesh size distribution of the crystalline psicose are shown in FIG4 and Table 4:

[0162] Table 4 Mesh size distribution of allulose crystals

[0163] Comparative Example 1

[0164] The molar concentration of CoCl2·6H2O in Example 1 was replaced with 5 mM, and the remaining steps were the same.

[0165] The activity of the immobilized enzyme was determined as follows:

[0166] 1. Determination of conversion rate

[0167] The conversion rate refers to the percentage of allulose produced in the dry matter of the solution when the reaction of fructose conversion by the immobilized enzyme reaches equilibrium. Using a 55% fructose solution as the substrate, adjusted to a pH of 6.0, and adding 20% ​​of the dry weight of immobilized enzyme particles to the fructose solution, the reaction was carried out at 60°C for 4 hours. The conversion rate, as determined by HPLC, was 27.31%.

[0168] 2. Detection of half-life

[0169] The enzyme activity units of the immobilized enzyme were measured every 24 hours, and the time it took for the enzyme activity units to drop to half was defined as the half-life. The enzyme activity unit was defined as adding 200 mg of immobilized enzyme particles to 300 μL of a 55% fructose solution at 60°C for 10 minutes. The psicose content was analyzed by HPLC, and one enzyme activity unit was defined as 1 μmol of psicose produced per minute. The changes in the enzyme activity units of the immobilized enzyme were shown in Table 5. On the 20th day (480 hours), the enzyme activity units were 38.62 U / g, approximately half the value, and the half-life of the immobilized enzyme was 20 days.

[0170] Table 5 Changes in activity units of immobilized enzymes

[0171] Comparative Example 2

[0172] The mass concentration of the chitosan solution in Example 1 was replaced with 0.5%, and the remaining steps were the same.

[0173] The activity of the immobilized enzyme was determined as follows:

[0174] 1. Determination of conversion rate

[0175] The conversion rate refers to the percentage of allulose produced in the dry matter of the solution when the reaction of fructose conversion by the immobilized enzyme reaches equilibrium. Using a 55% fructose solution as the substrate, adjusted to a pH of 6.0, and adding 20% ​​of the dry weight of immobilized enzyme particles to the fructose solution, the reaction was carried out at 60°C for 4 hours. The conversion rate, as determined by HPLC, was 31.82%.

[0176] 2. Half-life detection

[0177] The enzyme activity units of the immobilized enzyme were measured every 24 hours, and the time it takes for the enzyme activity units to drop to half is the half-life. The enzyme activity unit was defined as adding 200 mg of immobilized enzyme particles to 300 μL of a 55% fructose solution, reacting at 60°C for 10 minutes. The psicose content was analyzed by HPLC, and one enzyme activity unit was defined as 1 μmol of psicose produced per minute. The changes in the immobilized enzyme activity units are shown in Table 6 below. On the 15th day (360 hours), the enzyme activity units were 45.81 U / g, and the half-life of the immobilized enzyme, which is approximately 15 days, is 50%.

[0178] Table 6 Changes in activity units of immobilized enzymes

[0179] Comparative Example 3

[0180] The mass concentration of the chitosan solution in Example 1 was replaced with 2%, and the remaining steps were the same.

[0181] The activity of the immobilized enzyme was determined as follows:

[0182] 1. Determination of conversion rate

[0183] The conversion rate refers to the percentage of allulose produced in the dry matter of the solution when the reaction of fructose conversion by the immobilized enzyme reaches equilibrium. Using a 55% fructose solution as the substrate, adjusted to a pH of 6.0, and adding 20% ​​of the dry weight of immobilized enzyme particles to the fructose solution, the reaction was carried out at 60°C for 4 hours. The conversion rate, as determined by HPLC, was 24.58%.

[0184] 2. Half-life detection

[0185] The enzyme activity units of the immobilized enzyme were measured every 24 hours, and the time it took for the enzyme activity units to drop to half was defined as the half-life. The enzyme activity unit was defined as follows: 200 mg of immobilized enzyme particles were added to 300 μL of a 55% fructose solution, reacted at 60°C for 10 minutes, and analyzed by HPLC for psicose content. One enzyme activity unit was defined as 1 μmol of psicose produced per minute. The changes in the immobilized enzyme activity units were shown in Table 7. On the 23rd day (504 hours), the enzyme activity units were 41.13 U / g, and the half-life of the immobilized enzyme, which is approximately 23 days, was halved.

[0186] Table 7 Changes in immobilized enzyme activity units

[0187] Comparative Example 4

[0188] The amount of chitosan solution in Example 1 was changed to 0.1 times the volume of the resin, and the remaining steps were the same.

[0189] The activity of the immobilized enzyme was determined as follows:

[0190] 1. Determination of conversion rate

[0191] The conversion rate refers to the percentage of allulose produced in the dry matter of the solution when the reaction of fructose conversion by the immobilized enzyme reaches equilibrium. Using a 55% fructose solution as the substrate, adjusted to a pH of 6.0, and adding 20% ​​of the dry weight of immobilized enzyme particles to the fructose solution, the reaction was carried out at 60°C for 4 hours. The conversion rate, as determined by HPLC, was 30.26%.

[0192] 2. Half-life detection

[0193] The enzyme activity units of the immobilized enzyme were measured every 24 hours, and the time it takes for the enzyme activity units to drop to half is the half-life. The enzyme activity unit was defined as adding 200 mg of immobilized enzyme particles to 300 μL of a 55% fructose solution at 60°C for 10 minutes. The psicose content was analyzed by HPLC, and one enzyme activity unit was defined as 1 μmol of psicose produced per minute. The changes in the immobilized enzyme activity units are shown in Table 8 below. On the 10th day (240 hours), the enzyme activity units were 45.58 U / g, which is approximately 10 days. The half-life of the immobilized enzyme is 10 days.

[0194] Table 8 Changes in immobilized enzyme activity units

[0195] Comparative Example 5

[0196] The amount of chitosan solution in Example 1 was changed to 0.5 times the volume of the resin, and the remaining steps were the same.

[0197] The activity of the immobilized enzyme was determined as follows:

[0198] 1. Determination of conversion rate

[0199] The conversion rate refers to the percentage of allulose produced in the dry matter of the solution when the reaction of fructose conversion by the immobilized enzyme reaches equilibrium. Using a 55% fructose solution as the substrate, adjusted to a pH of 6.0, and adding 20% ​​of the dry weight of immobilized enzyme particles to the fructose solution, the reaction was carried out at 60°C for 4 hours. The conversion rate, as determined by HPLC, was 21.92%.

[0200] 2. Half-life detection

[0201] The enzyme activity units of the immobilized enzyme were measured every 24 hours, and the time it took for the enzyme activity units to drop to half was defined as the half-life. The enzyme activity unit was defined as adding 200 mg of immobilized enzyme particles to 300 μL of a 55% fructose solution, reacting at 60°C for 10 minutes. The psicose content was analyzed by HPLC, and one enzyme activity unit was defined as 1 μmol of psicose produced per minute. The changes in the immobilized enzyme activity units were shown in Table 9. On the 24th day (576 hours), the enzyme activity units were 40.36 U / g, approximately half the time. The half-life of the immobilized enzyme is 24 days.

[0202] Table 9 Changes in activity units of immobilized enzymes

[0203] Comparative Example 6

[0204] The enzyme dosage in Example 1 was replaced with 80 U / g, and the remaining steps were the same.

[0205] The activity of the immobilized enzyme was determined as follows:

[0206] 1. Determination of conversion rate

[0207] The conversion rate refers to the percentage of allulose produced in the dry matter of the solution when the reaction of fructose conversion by the immobilized enzyme reaches equilibrium. Using a 55% fructose solution as the substrate, adjusted to a pH of 6.0, and adding 20% ​​of the dry weight of immobilized enzyme particles to the fructose solution, the reaction was carried out at 60°C for 4 hours. The conversion rate, as determined by HPLC, was 22.29%.

[0208] 2. Half-life detection

[0209] The enzyme activity units of the immobilized enzyme were measured every 24 hours, and the time it took for the enzyme activity units to drop to half was defined as the half-life. The enzyme activity unit was defined as adding 200 mg of immobilized enzyme particles to 300 μL of a 55% fructose solution, reacting at 60°C for 10 minutes. The psicose content was analyzed by HPLC, and one enzyme activity unit was defined as 1 μmol of psicose produced per minute. The changes in the immobilized enzyme activity units were shown in Table 10. On day 16 (384 hours), the enzyme activity units were 30.46 U / g, representing a decrease of approximately half. The half-life of the immobilized enzyme is 16 days.

[0210] Table 10 Changes in immobilized enzyme activity units

[0211] Comparative Example 7

[0212] The initial feed fructose solution mass concentration in Example 1 was replaced with 40%, and after the circulation operation, the feed fructose solution mass concentration was replaced with 60%, and the remaining steps were the same.

[0213] In the first chromatographic cycle, the material was taken from the outlet of column 6, and the purity of fructose was 95.23% after HPLC analysis. The material was taken from the outlet of column 8, and the purity of psicose was 98.31% after HPLC analysis.

[0214] In the second chromatographic cycle, the material was taken from the outlet of column 7, and the purity of fructose was 96.08% after HPLC analysis. The material was taken from the outlet of column 5, and the purity of psicose was 98.42% after HPLC analysis.

[0215] In the third chromatographic cycle, the material was taken from the outlet of column 8, and the purity of fructose was 96.89% after HPLC analysis. The material was taken from the outlet of column 6, and the purity of psicose was 99.61% after HPLC analysis.

[0216] After testing, the system was operated for 60 days and the conversion rate of fructose during this period of operation was 96.75%. The processing capacity per unit resin per unit time was 0.027t / m 3 ·h.

[0217] Comparative Example 8

[0218] The system operating temperature in the operating conditions of the immobilized enzyme-chromatographic separation system described in Example 1 was replaced with 40° C., and the remaining steps were the same.

[0219] In the first chromatographic cycle, the material was taken from the outlet of column 6, and the purity of fructose was 90.16% after HPLC analysis. The material was taken from the outlet of column 8, and the purity of psicose was 92.87% after HPLC analysis.

[0220] In the second chromatographic cycle, the material was taken from the outlet of column 7, and the purity of fructose was 90.74% after HPLC analysis. The material was taken from the outlet of column 5, and the purity of psicose was 93.10% after HPLC analysis.

[0221] In the third chromatographic cycle, the material was taken from the outlet of column 8, and the purity of fructose was 91.12% after HPLC analysis. The material was taken from the outlet of column 6, and the purity of psicose was 93.25% after HPLC analysis.

[0222] After testing, the system was operated for 60 days and the conversion rate of fructose was 91.86%. The processing capacity per unit resin per unit time was 0.039t / m 3 ·h.

[0223] Comparative Example 9

[0224] The material-water ratio in the operating conditions of the immobilized enzyme-chromatographic separation system described in Example 1 was replaced with 1:1.5, and the remaining steps were the same.

[0225] In the first chromatographic cycle, the material was taken from the outlet of column 6, and the purity of fructose was 84.13% after HPLC analysis. The material was taken from the outlet of column 8, and the purity of psicose was 87.62% after HPLC analysis.

[0226] In the second chromatographic cycle, the material was taken from the outlet of column 7, and the purity of fructose was 84.56% after HPLC analysis. The material was taken from the outlet of column 5, and the purity of psicose was 87.85% after HPLC analysis.

[0227] In the third chromatographic cycle, the material was taken from the outlet of column 8, and the purity of fructose was 84.87% after HPLC analysis. The material was taken from the outlet of column 6, and the purity of psicose was 87.92% after HPLC analysis.

[0228] After testing, the system was operated for 60 days and the conversion rate of fructose was 85.63%. The processing capacity per unit resin per unit time was 0.05t / m 3 ·h.

[0229] Comparative Example 10

[0230] The operating mode of the immobilized enzyme-chromatographic separation system in Example 1 was modified so that the fructose solution entered column 1, underwent enzymatic conversion reactions in columns 1 and 2, then entered column 5, then entered purified water via column 6. The material at the outlet of column 6 was returned to column 1, and a psicose liquid material was discharged from column 8. In the next cycle, the fructose solution entered column 1, underwent enzymatic conversion reactions in columns 1 and 2, then entered column 6. The material at the outlet of column 7 was returned to purified water, and the psicose liquid material was discharged from column 5. When the enzyme activity units in columns 1 and 2 dropped to half, the material was switched to columns 3 and 4, and the immobilized enzymes in columns 1 and 2 were replaced. The remaining steps remained unchanged.

[0231] In the first chromatographic cycle, the material was taken from the outlet of column 6, and the purity of fructose was 95.12% after HPLC analysis. The material was taken from the outlet of column 8, and the purity of psicose was 86.24% after HPLC analysis.

[0232] In the second chromatographic cycle, the material was taken from the outlet of column 7, and the purity of fructose was 95.65% after HPLC analysis. The material was taken from the outlet of column 5, and the purity of psicose was 87.23% after HPLC analysis.

[0233] In the third chromatographic cycle, the material was taken from the outlet of column 8, and the purity of fructose was 95.59% after HPLC analysis. The material was taken from the outlet of column 6, and the purity of psicose was 87.27% after HPLC analysis.

[0234] After testing, the system was operated for 60 days and the conversion rate of fructose was 85.13%. The processing capacity per unit resin per unit time was 0.038t / m 3 ·h.

[0235] Comparative Example 11

[0236] The preparation method of psicose in step 4 of Example 1 was replaced, and the remaining steps were the same;

[0237] The specific steps are as follows:

[0238] 1. Continuous evaporation crystallization:

[0239] The volume of the injected psicose solution in Example 1 was changed to 70%. When the tank was full, the volume of the liquid fed into the horizontal crystallizer was changed to 30% of the tank volume. The remaining steps were the same.

[0240] 2. Batch cooling crystallization: the steps are the same.

[0241] 3. Centrifugation and drying of crystallized allulose: the steps are the same.

[0242] After testing, the morphology (×100) and mesh size distribution of crystalline allulose are shown in Figure 5 and Table 11 below:

[0243] Table 11 Mesh size distribution of crystalline psicose

[0244] Comparative Example 12

[0245] The preparation method of psicose in step 4 of Example 1 was replaced, and the remaining steps were the same;

[0246] The specific steps are as follows:

[0247] 1. Continuous evaporation crystallization:

[0248] In Example 1, the amount of seed crystals added was changed to seed crystals with a dry mass concentration of 0.5%, and the remaining steps were the same.

[0249] 2. Batch cooling crystallization: the steps are the same.

[0250] 3. Centrifugation and drying of crystallized allulose: the steps are the same.

[0251] After testing, the morphology (×100) and mesh size distribution of crystalline allulose are shown in Figure 6 and Table 12 below:

[0252] Table 12 Mesh size distribution of crystalline psicose

[0253] Comparative Example 13

[0254] The preparation method of psicose in step 4 of Example 2 was replaced, and the remaining steps were the same;

[0255] The specific steps are as follows:

[0256] 1. Continuous evaporation crystallization:

[0257] In Example 1, the diameter of the seed crystal added was changed to 75 μm, and the remaining steps were the same.

[0258] 2. Batch cooling crystallization: the steps are the same.

[0259] 3. Centrifugation and drying of crystallized allulose: the steps are the same.

[0260] After testing, the morphology (×100) and mesh size distribution of crystalline allulose are shown in Figure 7 and Table 13:

[0261] Table 13 Mesh size distribution of crystalline psicose

[0262] Comparative Example 14

[0263] The preparation method of psicose in step 4 of Example 1 was replaced, and the remaining steps were the same;

[0264] The specific steps are as follows:

[0265] 1. Continuous evaporation crystallization:

[0266] In Example 1, the amount of seed crystals added was changed to seed crystals with a dry mass concentration of 2%, and the remaining steps were the same.

[0267] 2. Batch cooling crystallization: the steps are the same.

[0268] 3. Centrifugation and drying of crystallized allulose: the steps are the same.

[0269] After testing, the morphology (×100) and mesh size distribution of crystalline allulose are shown in Figure 8 and Table 14 below:

[0270] Table 14 Mesh size distribution of crystalline psicose

[0271] Comparative Example 15

[0272] The preparation method of psicose in step 4 of Example 1 was replaced, and the remaining steps were the same;

[0273] The specific steps are as follows:

[0274] 1. Continuous evaporation crystallization:

[0275] In Example 1, the stirring speed during crystal growth was changed to 10 rpm, and the stirring speed during evaporation and feeding was adjusted to 30 rpm. The remaining steps were the same.

[0276] 2. Batch cooling crystallization: the steps are the same.

[0277] 3. Centrifugation and drying of crystallized allulose: the steps are the same.

[0278] After testing, the morphology (×100) and mesh size distribution of crystalline allulose are shown in Figure 9 and Table 15 below:

[0279] Table 15 Mesh size distribution of crystalline psicose

[0280] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing crystalline psicose, characterized in that: The steps include: 1) Co 2+ The type resin was used as an immobilized carrier to adsorb the psicose epimerase, and after the adsorption was completed, chitosan solution was added for cross-linking to obtain the immobilized enzyme; 2) respectively loading the obtained immobilized enzyme and gel-type polystyrene calcium type chromatographic resin into chromatographic columns of a chromatographic separation system to obtain an immobilized enzyme chromatographic column and a gel-type polystyrene calcium type chromatographic column; 3) passing the fructose solution through a chromatographic column of immobilized enzyme for enzyme conversion to obtain an enzyme conversion solution; 4) performing chromatographic separation on the obtained enzyme conversion solution through a gel-type polystyrene calcium type chromatographic column to obtain a fructose solution and remaining enzyme conversion solution; combining the obtained fructose solution with the fructose solution in step 3); 5) continuing to pass the remaining enzyme conversion liquid through a gel-type polystyrene calcium chromatography column to obtain a D-psicose liquid; 6) sequentially decolorizing, ion-exchanging and desalting the obtained psicose liquid, and vacuum concentrating the liquid to obtain a psicose solution; 7) The obtained psicose solution is subjected to evaporation feeding crystallization and cooling crystallization in sequence to obtain crystalline psicose.

2. The preparation method according to claim 1, characterized in that: In the step 1), Co 2+ The preparation method of the type resin is: The resin was sequentially soaked in distilled water, sodium hydroxide solution, hydrochloric acid and CoCl2 solution to obtain the final Co 2+ Type resin.

3. The preparation method according to claim 2, characterized in that: The distilled water immersion, sodium hydroxide solution immersion and hydrochloric acid solution immersion time are all 4 to 5 hours, and the CoCl2 solution immersion time is 8 to 12 hours.

4. The preparation method according to claim 1, characterized in that: The dosage of the D-psicose isomerase in step 1) is 100-120 U / g resin, the mass concentration of the chitosan solution is 1-1.5%, and the chitosan solution and Co 2+ The volume ratio of the type resin is 0.2-0.4:

1.

5. The preparation method according to claim 1, characterized in that: In the step 1), the conversion rate of the immobilized enzyme to fructose is 30-33%, and the half-life is 25-28 days.

6. The preparation method according to claim 1, characterized in that: The cross-linking temperature in step 1) is 25-35° C., and the cross-linking time is 1-2 hours.

7. The preparation method according to claim 1, characterized in that: The mass concentration of the fructose solution in step 3) is 55-60%.

8. The preparation method according to claim 1, characterized in that: The temperature of the enzyme conversion in step 3) is 55-65°C.

9. The preparation method according to claim 1, characterized in that: The evaporation feed crystallization in step 7) comprises the following steps: 1) mixing the psicose solution with seed crystals and then growing the crystals to obtain a solution after growing the crystals; 2) The feed liquid after crystal growth is continuously evaporated and fed to obtain a feed liquid after evaporation and feeding crystallization.

10. The preparation method according to claim 1, characterized in that: The cooling crystallization in step 7) comprises the following steps: Cooling the resulting liquid after evaporation and crystallization to a first temperature, and then continuing to cool it to a second temperature; The cooling rate is 0.4-0.55°C / h, and the first temperature is 38-41°C; The rate of continued cooling is 0.8-1.1°C / h, and the second temperature is 28-31°C.

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