METHOD FOR PRODUCING PSICOSE FROM FRUCTOSE-CONTAINING SUBSTRATE
20230183764 · 2023-06-15
Inventors
- Soun Gyu KWON (Cheongju-si, KR)
- Hye Jung KIM (Daejeon, KR)
- Bu-Soo PARK (Daejeon, KR)
- Chong Jin PARK (Daejeon, KR)
- Kang Pyo Lee (Seoul, KR)
- Hee Soon CHEON (Seoul, KR)
Cpc classification
C12Y501/03
CHEMISTRY; METALLURGY
C12N11/04
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a method of obtaining a psicose-containing product from a fructose-containing substrate with high productivity in a short time on an industrial scale by an immobilization reaction using a biocatalyst for producing a psicose, and a method of preparing a liquid type or powder type of psicose by isolating the psicose-containing product obtained by the method and preparing a psicose continuously by inputting a byproduct of isolation process into a process of production of psicose-containing product.
Claims
1. A method for preparing a psicose-containing product from a fructose-containing substrate by an immobilization reaction using a biocatalyst for producing a psicose, wherein the biocatalyst has 4% to 29% of the conversion rate of psicose from the fructose-containing substrate at a reaction temperature of 50 to 60° C. within one (1) hour, wherein the immobilization reaction is performed at the reaction flow rate of 8.5 to 20, based on 1 of the reaction flow rate at which the psicose contained in the psicose-containing product is produced at an amount of 25% by weight or higher with respect to 100% by weight solid content of the total saccharides in the psicose-containing product, and wherein the solid content of psicose and glucose is lower than 20% by weight, with respect to 100% by weight of solid content of psicose, glucose and fructose contained in the psicose-containing product.
2. A method performed to achieve that the solid content of psicose and glucose is 9% by weight or higher to lower than 20% by weight, based on 100% by weight total solid content of saccharide and psicose.
3. The method of claim 1, wherein the immobilization reaction is performed at the reaction flow rate of 10 to 18, based on 1 of the reaction flow rate at which the psicose contained in the psicose-containing product is produced at an amount of 25% by weight or higher with respect to 100% by weight solid content of the total saccharides in the psicose-containing product.
4. The method of claim 1, wherein the immobilization reaction is performed to achieve that the solid content of psicose and glucose is 9% by weight to lower than 20% by weight, and the solid content of fructose is 80 to 91% by weight, based on 100% by weight of total content of fructose, glucose and psicose in the psicose-containing product.
5. The method of claim 1, wherein the fructose-containing substrate comprises 75% by weight to 95% by weight of fructose in 100% by weight of solid content of total saccharides in the substrate.
6. The method of claim 1, wherein the immobilization reaction is performed by using a biocatalyst for producing a psicose having 20 to 29% of the psicose conversion rate from a fructose-containing substrate, and a substrate comprising 80% by weight to 95% by weight of fructose in 100% by weight of solid content of total saccharides in the fructose-containing substrate.
7. The method of claim 1, wherein the fructose-containing substrate is an isomerized saccharide syrup.
8. The method of claim 1, wherein the biocatalyst is an enzyme for producing a psicose or a microbial cell producing a psicose.
9. The method of claim 1, wherein the immobilization reaction is performed by using an immobilization column filled with a bead containing an enzyme for producing a psicose or a microbial cell producing a psicose.
10. The method of claim 9, wherein the immobilization reaction is performed by using a bead containing the microbial cell or enzyme which is treated with divalent metal ion and coated with one or more swelling inhibitors selected from the group consisting of chitosan, chitin, polyethylene glycol (PEG), polyethylene amine (PEI), chito-oligosaccharide and polylysine.
11. The method of claim 9, wherein the immobilization reaction is performed by using a bead containing the microbial cell or enzyme which is treated by freeze-drying and compressed.
12. The method of claim 8, wherein the microbial cell producing a psicose is a Corynebacterium strain transformed with a gene of psicose epimerase.
13. The method of claim 12, wherein the Corynebacterium strain is one or more kinds of Corynebacterium sp. strains selected from the group consisting of Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola and Corynebacterium efficiens.
14. The method of claim 8, wherein the microbial cell producing a psicose is a strain producing a psicose epimerase.
15. The method of claim 1, wherein the immobilization reaction is performed at 40 to 80° C. for 0.5 to 48 hours.
16. A method for preparing a psicose by obtaining a psicose-containing liquid product comprising, obtaining a psicose-containing product according to claim 1, decoloring or desalting the produced psicose-containing product, and concentrating the decolored or desalted product to 75 Brix (%) or higher of solid content of psicose.
17. A method of preparing a psicose comprising preparing a psicose-containing powder comprising, obtaining a psicose-containing product according to claim 1, decoloring or desalting the produced psicose-containing product, separating the decolored or desalted product to separate a psicose fraction of 90% by weight or higher of psicose content and a raffinate fraction, concentrating the separated psicose fraction, preparing a concentrate in a psicose supersaturated state to crystallize a psicose, and separating the crystallized psicose from a crystal mother liquor and drying.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0052]
[0053]
[0054]
[0055]
DETAILED DESCRIPTION OF THE EMBODIMENTS
Example 1: Establishment of Psicose Production System
[0056] 1-1: Preparation of a Strain Producing Psicose
[0057] The gene encoding a psicose epimerase derived from Clostridiuim scindens (Clostridiuim scindens ATCC 35704) (DPE gene; Gene bank: EDS06411.1) was synthesized as a modified polynucleotide form optimized for E. coli and called CDPE. The polynucleotide optimized for E. coli and sod promoter and T7 terminator obtained from pET21a vector were obtained as each template through PCR, and these were linked as one template by overlap PCR method and cloned into pGEM T-easy vector through T-vector cloning, to confirm the sequence including sod promoter (SEQ ID NO: 1), optimized CDPE sequence of SEQ ID NO: 8 and T7-terminator.
[0058] The whole polynucleotide was inserted into the same restriction enzyme site of an expression vector pCES208 (J. Microbiol. Biotechnol., 18:639-647, 2008) with restriction enzymes NotI and XbaI(NEB), to produce a recombinant vector pCES208/psicose epimerase (pCES_sodCDPE). The cleavage map of the produced recombinant vector (pCES_sodCDPE) was shown in
[0059] Corynebacterium glutamicum was transformed with the prepared recombinant vector (pCES_sodCDPE) using electroporation. Colonies were picked and inoculated in 4 ml of LB medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L) supplemented with Kanamycin at final concentration of 15 ug/ml, and then cultured for approximately 16 hours under the culture conditions of 30° C. and 250 rpm. Then, 1 ml of the culture solution was collected and inoculated in 100 ml LB medium including 15 ug/ml of Kanamycin, and the culture was continued for over 16 hours. After lysis of cultured cells with beadbeater, only supernatant was obtained and mixed with a sample buffer at a mixing ratio of 1:1, and then heated at 100° C. for 5 minutes. The prepared samples were subjected to electrophoresis on a 12% SDS-PAGE gel (composition: running gel—3.3 ml H2O, 4.0 ml 30% acrylamide, 2.5 ml 1.5M Tris buffer (pH 8.8), 100 μl 10% SDS, 100 μl, 10% APS, 4 μl TEMED/stacking gel—1.4 ml H2O, 0.33 ml 30% acrylamide, 0.25 ml 1.0M Tris buffer (pH 6.8), 20 μl 10% SDS, 20 μl 10% APS, 2 μl TEMED) at 180V for approximately 50 minutes, to confirm the protein expression. After the expression of CDPE was confirmed on the SDS-PAGE gel, His-tag purification was proceeded using Ni-NTA resin and the expression rate was calculated using a calculation formula (expression rate (%)=(Purified protein (mg)/Total soluble protein (mg))×100), for accurate measurement of expression level. The prepared Corynebacterium glutamicum transformant produced 16.62 mg of the total water-soluble proteins and 1.74 mg of purified enzyme protein.
[0060] 1-2: Preparation of Immobilization Bead
[0061] In order to prepare psicose from fructose using the recombinant strain producing psicose epimerase in Example 1-1, the cells were collected by centrifugation in the cell culture.
[0062] Then, the cell suspension was treated with 0.05% (v/v) of en emulsifier (Ryoto(tkSugar Ester, M-1695) in a final volume and treated at 35° C. (±5° C.) for 60 minutes. When the reaction was completed, the microbial cells were collected after removing the supernatant containing the emulsifier again by centrifugation.
[0063] To prepare immobilized beads, the collected microbial cells were mixed with distilled water to a final microbial cell concentration of 5% (v/v), and 5% (v/v) of the collected microbial cells were mixed with 4% (v/v) of alginic acid dissolved in water at a mixing ratio of 1:1, and refrigerated at 4° C. to remove bubbles generated during mixing. The refrigerated mixture solution was extruded through Needle (inner diameter 0.20˜0.30 mm) and formed into a droplet shape due to the weight, and the dropped mixture solution was cured by being dropped into the prepared 100 mM calcium chloride (CaCl.sub.2) solution, to form spherical or elliptical beads (diameter 2.0˜2.2 mm). The formed beads were soaked in a 100 mM calcium chloride solution and mixed evenly by a stirrer so as to be further cured.
[0064] After all mixture solution was extruded, the beads were further cured by keeping refrigerator for 4˜6 hours, and then cured for approximately 6 hours in a refrigerated state with replacing with a new 100 mM calcium chloride solution. After the beads were completely cured, the beads were skimmed and the moisture was removed completely. The beads were stirred for 10 minutes after addition of water at 3 times as much as the volume of the beads. The process was repeated three times to remove the calcium chloride solution. After completely removing moisture of washed beads, a fructose-containing substrate (50 brix of fructose and 1 mM MnCl.sub.2.Math.4H.sub.2O) was inputted at 3 times as much as the volume of the beads, and then it was stirred for 10 minutes. Such treatment was carried out at 2 times or more to replace with a fructose-containing substrate used as a reaction substrate. The reaction substrate was adjusted to pH 6.8˜7.2 with 3N NaOH. The liquid fructose or crystalline fructose can be used as the substrate depending on the product kinds.
[0065] The beads replaced with the reaction substrate were filled into an immobilization reaction column, and then used for producing a psicose syrup.
[0066] 1-3: Preparation of Psicose Syrup
[0067] After filling the prepared beads in Example 1-2 into an immobilization reaction column, a psicose syrup was produced under the following reaction conditions. A raw material which was 50% of solid content, and included 88.8% by weight of fructose and 4.8% by weight of glucose, among 100% by weight of solid content of total saccharides of fructose-containing substrate (pH 6.8˜7.1) with 50% (w/w) or more of solid content, was provided to the following immobilization reaction column, thereby preparing the mixed saccharide of the psicose syrup.
[0068] <Immobilization Column Reaction Conditions>
[0069] (1) Reaction temperature: 50 r of internal temperature of the column jacket
[0070] (2) Reaction substrate: substrate containing 88.8% by weight of fructose and 4.8% by weight of glucose in the total solid content of fructose-containing substrate with 50% (w/w) or higher of solid content, and including less than 6.4% by weight of other saccharides of 1 or higher DP (Degree of Polymerization) except for the fructose and glucose.
[0071] (3) Production standard: production of syrup including 24% by weight of psicose content in 100% by weight of solid content of total saccharides in the product.
[0072] As the result of the reaction, 24% by weight of psicose syrup with the weight ratio of glucose:fructose:psicose:oligosaccharide of 5:65:24:6 was collected from the reaction solution.
Example 2: Psicose Production Depending on the Increased Reaction Flow Rate
[0073] After filling beads prepared in Example 1-2 into an immobilization reaction column, the amounts of produced psicose syrup depending on the column flow rate under the following reaction conditions were compared by feeding the reaction substrates at 5 times of column volume for 1 hour according to the ratio of increased flow rate, at the point when the psicose content (%) in the column was stabilized. The column was fed by a raw material of reaction substrate solution with 50% (w/w) of solid content including 88.8% by weight or more of the fructose content and 4.8% by weight of glucose content, based on 100% by weight of the solid content of total saccharides.
[0074] The analysis of saccharide content was detected with RI by injecting 10 μL of samples suitably diluted with water solvent at the flow rate of 0.6 ml/min by using Aminex HPX-87C column (80° C.) of Biorad company, and within 30 minutes of analysis time, the fructose, psicose and other DP 1 or more saccharides were integrated, thereby analyzing each area. In addition, to analyze the content by each saccharide composition, values corresponding to area by each saccharide composition were analyzed as the content of each saccharide, when the value of sum of total areas of fructose, psicose and other DP1 or more saccharides shown within 30 minutes of analysis time.
[0075] <Immobilization Column Reaction Conditions>
[0076] (1) Reaction temperature: 50° C. of internal temperature of the column jacket,
[0077] (2) Reaction substrate: substrate including 88.8% by weight of fructose and 4.8% by weight of glucose among 100% by weight of solid of total saccharides of fructose-containing substrate with the solid content of 50% (w/w) (pH 6.8˜7.2) and containing less than 6.4% of other saccharides of DP 1 or more except for fructose and glucose, which is a raw material including 94.9% by weight of fructose content and 5.1% by weight of glucose in the raw material substrate, when they were converted on the basis of 100% by weight of total content of fructose, glucose and psicose,
[0078] (3) Column flow rate: the psicose content in the product is evaluated depending on the ratio of increased flow rate, as the reaction is carried out by increasing the reaction flow rate by 1 to 20 times, based on 1 of the reaction flow rate at which the psicose contained in the psicose-containing product is produced at an amount of 25% by weight or higher with respect to 100% by weight solid content of the total saccharides in the psicose-containing product,
[0079] (4) Production standard: total content of glucose and psicose ranges from 9% by weight or more to less than 20% by weight, in 100% by weight of total content of fructose, glucose and psicose in the product.
[0080] The experimental result was shown in the following Table 2 and
TABLE-US-00002 TABLE 2 Fructose Psicose Glucose Total content of Increase ratio of content content content psicose and flow rate (wt %) (wt %) (wt %) glucose (wt %) Substrate before 94.9 0 5.1 5.1 reaction 1 67.8 27.1 5.1 32.2 2 69.7 25.2 5.1 30.3 4 73.5 21.4 5.1 26.5 6 76.7 18.2 5.1 23.3 8 79.6 15.3 5.1 20.4 10 81.8 13.1 5.1 18.2 12 83.8 11.1 5.1 16.2 16 87.5 7.4 5.1 12.5 20 90.3 4.6 5.1 9.7
[0081] As shown in Table 2, when the total solid content of fructose, glucose and psicose included in the reaction substrate was 100% by weight, a raw material including 94.9% of fructose content and 5.1% of glucose was used. Since the fructose was converted into psicose, as the conversion reaction was progressed, 94.9% of fructose content as the substrate was gradually reduced, and the psicose content was increased, with maintaining the same content of 5.1% of glucose.
[0082] According to the increase of column flow rate, the psicose conversion rate was gradually reduced. The psicose content was 4.6%, when reacted at the flow rate of 20 times higher than the flow rate of producing 27.1% psicose content in Table 2. When reacted at the flow rate increased 10 times higher than the flow rate of producing 25% psicose content of Example 1, a syrup composition having less than 20% by weight of total content of glucose and psicose was produced.
[0083] The change in psicose content by the change in the column flow rate in Table 2 was shown in
[0084] As shown in
[0085] In addition, as the result of analyzing the content of produced materials in which the production was completed in the reaction with 10 times higher of the column flow rate under the immobilization reaction conditions by using the formula of
Example 3: Evaluation of Reaction Stability
[0086] After filling beads prepared in Example 1-2 into an immobilization reaction column, the fructose-containing substrate and the reaction condition were the same as those of Example 2.
[0087] By using the formula of
[0088] <Immobilization Reaction Conditions>
[0089] (1) Reaction temperature: 50° C.,
[0090] (2) Reaction substrate: the same reaction substrate of Example 2,
[0091] (3) Column condition: flow rate increased at 8.5 times higher than the flow rate of producing 25% by weight of psicose content in 10% by weight of solid content of total saccharides in Example 1, and
[0092] (4) Production standard: total content of glucose and psicose ranges from 15% by weight or more to less than 20% by weight, when the total content of fructose, glucose and psicose in the product was 100% by weight.
TABLE-US-00003 TABLE 3 Psicose Total content of Reaction time Fructose content psicose and (day) content(wt %) (wt %) glucose (wt %) 0 80.5 14.4 19.5 1 80.1 14.8 19.9 2 80.1 14.8 19.9 4 80.1 14.8 19.9 6 80.1 14.8 19.9 8 80.1 14.8 19.9 9 80.2 14.7 19.8 12 80.3 14.6 19.7 13 80.5 14.4 19.5 14 80.6 14.3 19.4 15 80.7 14.2 19.3
[0093] As shown in Table 3, the psicose content in the product were measured during the reaction for 15 days at the high reaction flow rate, a composition including 10% by weight or more to less than 15% by weight of psicose content and 15% by weight or more to less than 20% by weight of total content of psicose and glucose, on the basis of 100% by weight of the total content of fructose, glucose and psicose, could be stably produced for 15 days.
Example 5: Continuous Recycle Production of Psicose
[0094] A psicose production system was established as a continuous production process by continuously producing a psicose syrup having less than 20% by weight of total content of glucose and psicose, when the total content of fructose, glucose and psicose contained in the product was 100% by weight, and separating a psicose fraction from the product and a fructose raffinate fraction.
[0095] Specifically, the psicose conversion reaction was performed by controlling the flow rate of psicose conversion reaction column under the condition of producing less than 20% by weight of glucose and psicose based on 100% by weight of total content of fructose, glucose and psicose contained in the product, and psicose fraction with 90% or more of psicose purity and fructose raffinate fraction containing excess amount of fructose were separated from the psicose-containing product by using high-purity separation process.
[0096] The schematic diagram of the entire reaction process of psicose production was shown in
[0097] After liquid fructose with 75 Brix (%) of solid content (when the total solid content was 100% by weight, the content of fructose was contained as 88.8% by weight) was added by water to 50 Brix (%) of the solid content, and adjusted to pH 7.0 by adding with 5N NaOH, to prepare a raw material of reaction. The raw material was provided into a reaction column which were filled with microbial cell immobilization beads of Example 1-2 (50° C. constant temperature) and a syrup containing less than 20% by weight of glucose and psicose was produced according to the substantially same method of Example 3, when the fructose in the raw material was converted into a psicose, on the basis of 100% by weight of the total content of fructose, glucose and psicose of reaction raw material.
[0098] As a process for separating the produced psicose syrup, the produced psicose-containing syrup was decolored at 50° C. for 30 minutes by adding 0.05% (w/w) of activated carbon to the solid content in the syrup, and the completely decolored psicose-containing syrup was passed through a micro filter, thereby removing the activated carbon.
[0099] The psicose-containing syrup in which the activated carbon was removed was desalted by flowing through the column filled with cation exchange resin, anion exchange resin and a mixed resin thereof at room temperature in order to remove impurities such as ion component, etc., at the volumetric rate of 2 times (1˜2 times) of ion exchange resin per hour. The psicose was separated at the ratio of 0.15 based 1 of total solid weight in the raw material used for psicose conversion reaction, in the SMB (simulated moving bed) filled with calcium (Ca.sup.2+) type ion exchange resin at the flow rate of 0.06SV, under the condition that the separation ratio of psicose:fructose was 0.7:1, to obtain a high-purity psicose fraction of 96% psicose purity with 4 Brix (%) of solid content. The triple-distilled water was used for the mobile phase of column because of food usage.
[0100] At the same time, raffinate fraction including 5.5% glucose, 83.9% fructose, 3.4% psicose, and 4.2% other saccharides as the content of composition except for the high-purity psicose fraction were collected at the ratio of 0.85, when the total solid weight of raw material used for psicose conversion reaction was 1. After the fractionized raffinate was transferred and set to the solid content 50 Brix (%) as the fructose-containing substrate of psicose conversion, and adjusted to pH 7.0 by using 5N NaOH, to be provided into a reaction column and recycled to perform a psicose conversion reaction.
[0101] The high-purity psicose separated in the SMB was transferred to a storage tank and concentrated to be the solid content 80 Brix (%) or more at 60° C., and was cooled to the supersaturated state according to the cooling crystallization method, to produce a psicose crystal. Then, the psicose powder with 99% psicose purity was finally obtained at 81% yield by centrifugal dehydration and drying.