Method for efficient production of psicose

Abstract

The present invention relates to a method for effectively utilizing fructose raffinate obtained in the process for separating psicose conversion product with a high purity chromatography in the process for preparing psicose, and more specifically, it is utilized for preparation of fructose-containing raw material solution for preparation of psicose by putting fructose raffinate obtained in the process for preparing psicose into the process for preparing fructose.

Claims

1. A method of preparation for psicose comprising: separating psicose-conversion product with a simulated moving bed (SMB) chromatography to obtain a psicose fraction and a fructose raffinate, providing the fructose raffinate into a separation process of fructose preparation to prepare fructose-containing raw material, and conducting psicose conversion reaction by using the fructose-containing raw material, wherein the psicose conversion product are obtained by a biological psicose conversion process on fructose raw material using a biocatalyst having a conversion activity from fructose to psicose, and wherein the separation process of fructose preparation comprises an ion purification step which removes ion from fructose isomerization product, and a separation step using a SMB chromatography, and the fructose raffinate is provided to one or more kinds of steps selected from the group consisting of the ion purification step and the separation step using SMB chromatography.

2. The method of preparation of claim 1, wherein the separation process of fructose preparation comprises a primary ion purification step which removes ion from fructose isomerization product, a separation step using a SMB chromatography, a secondary ion purification step and a concentration step, and the fructose raffinate is provided to one or more kinds of steps selected from the group consisting of the primary ion purification step, the high purity separation step using the SMB chromatography and the secondary ion purification step.

3. The method of preparation of claim 1, wherein the fructose-containing raw material has an electrical conductivity of 0 to 15 μs/cm.

4. The method of preparation of claim 1, wherein the fructose-containing raw material has a calcium ion concentration of 0.05 mM or lower.

5. The method of preparation of claim 1, wherein the fructose raffinate obtained in the separating step with using the SMB chromatography has an electrical conductivity of 20 to 200 μs/cm.

6. The method of preparation of claim 1, wherein 65 to 95% by weight of a fructose solution obtained in the fructose preparation is mixed with 5 to 35% by weight of the fructose raffinate based on the total 100% by weight of the fructose solution and the fructose raffinate, when the fructose solution and the fructose raffinate are adjusted to 50 Brix each.

7. The method of preparation of claim 1, wherein the fructose content of fructose-containing raw material which is put into the psicose conversion reaction is 85% by weight or higher based on 100% by weight of the total saccharides content.

8. The method of preparation of claim 1, wherein the fructose raffinate obtained in the separating step with the SMB chromatography comprises 85 to 99% by weight of fructose and 2.0% by weight or lower of psicose based on 100% by weight of the total saccharides.

9. The method of preparation of claim 1, wherein the separating step with SMB chromatography process is carried out with a column chromatograph packed with cation exchange resin in which a calcium active group is attached.

10. The method of preparation of claim 1, wherein the psicose conversion reaction uses a biological catalyst having a psicose coversion ratio of 15% to 70%.

11. The method of preparation of claim 1, further comprising a step of cooling the fructose raffinate obtained in the separating step with the SMB chromatography by a heat exchanger.

12. The method of preparation of claim 1, further comprising a step of concentrating the psicose fraction and crystallizing psicose from the concentrates to obtain psicose crystal and crystallization mother liquor.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a diagram showing one example of general SMB process.

(2) FIG. 2 is schematized one example of a series of process for preparing psicose which is preparing fructose-containing raw material solution by putting fructose raffinate obtained in the step of high purity separation among the process for preparing psicose into the fructose isomerization process and is conducting the psicose conversion reaction by using thereof, according to one example of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(3) Hereinafter, the present invention will be described in more detail with the following examples. However, these examples are only for illustrative purpose, and the scope of the present invention is not limited by these examples.

Preparative Example 1. Preparation of Psicose Syrup

(4) A psicose syrup was prepared from a fructose substrate by the biological method substantially same with the preparation method disclosed in the Korean laid-open patent publication No. 2014-0054997.

(5) Specifically, the encoding gene of psicose epimerase derived from Clostridium scindens (Clostridium scindens ATCC 35704) (DPE gene; Gene bank: EDS06411.1) was introduced into a recombinant vector (pCES_sodCDPE), and Corynebacterium glutaricum was transformed by using the prepared recombinant vector (pCES_sodCDPE) plasmid with electroporation. A bead including the transformed Corynebacterium glutaricum cell was prepared and packed into an immobilization reaction column, and a psicose syrup was prepared from 40 brix of 88% by weight of fructose or 95% by weight of fructose. That is, the psicose syrup of 21˜23 (w/w) % of which solid mixture weight ratio of glucose:fructose:psicose:oligosaccharide is 41:39:15:5 from 88% by weight of fructose-containing substrate (psicose syrup A), and the psicose syrup of 24˜26 (w/w) % of which glucose:fructose:psicose:oligosaccharide=6:67:25:2 from the raw material comprising 95% by weight of fructose content (psicose syrup B).

Preparative Example 2. Preparation of Fructose Raffinate

(6) Two kinds of psicose syrups obtained from Preparative Example 1 were flew through the column at a room temperature which was packed with resin in which cation exchange resin, anion exchange resin and cation and anion exchange resin mixed resin at a rate of twice the volume of ion exchange resin per hour and desalted in order to remove impurities such as colored and ion components, etc.

(7) Then, after separating high purity of psicose fraction by using a chromatography packed with calcium (Ca.sup.2+) type of ion exchange resin, the remainder was collected as raffinate. The raffinate collected from the psicose syrup obtained from the raw material of 77% by weight of fructose content (psicose syrup A) included 85 to 98% by weight of fructose, 1 to 10% by weight of glucose, and 1 to 5% by weight of reducing sugar.

(8) The raffinate collected from the psicose syrup obtained from the raw material of 95% by weight of fructose content (psicose syrup B) included 88 to 98% by weight of fructose, 1 to 8% by weight of glucose, and 1 to 4% by weight of reducing sugar.

Example 1. Production of Psicose Using Fructose Raffinate

(9) To produce 10 tons of solids of 95% by weight of psicose content by using the fructose-containing raw material solution of 88% by weight of fructose content obtained in Preparative Example 2, the psicose conversion process and separation process were carried out at flow rate 3.8 m.sup.3/hr. The psicose content of product collected through the psicose conversion process was 20 to 23% by weight, and they passed through the separation process at a concentration of 45 to 50% by weight after ion purification. The raffinate which was generated when separated by using Ca+ type separation (SMB) resin was generated by 3 m.sup.3 per hour.

(10) Specifically, in the preparation process of fructose, after mixing corn starch with water so as to be 30 to 35% by weight, enzymatic hydrolysis was progressed to collect saccharification liquid of 88% by weight or higher of glucose content. Then, the saccharification liquid was under vacuum drum filtration and insoluble materials were removed, thereby obtaining fructose isomerization product (fructose content 42% by weight syrup).

(11) The fructose raffinate of psicose preparation process obtained in Preparative Example 2 was concentrated to 88% by weight of fructose content and 50% by weight of the total solids (50 Brix) and mixed with the product which passed through the fructose isomerization process (fructose content 42% by weight syrup), and put into the ion purification process consisting of strongly acidic resin, weakly basic resin and strongly acidic and weakly basic mixed resin. That is, by adjusting the total solid content of fructose isomerization product and fructose raffinate put into the primary ion purification process to 50% by weight (50 Brix), the mixture ratio of the fructose isomerization product and fructose raffinate of Preparative Example 2 was 8:2 to 9:1 of weight ratio. After the mixed fructose syrup massed through the primary ion purification and was concentrated to the total solid 50% by weight, it passed through the SMB chromatography process. The glucose raffinate and fructose fraction were obtained by performing the SMB process and the fructose-containing solution of 88% by weight of fructose content was obtained by performing the secondary ion purification and concentration process. To the fructose-containing solution, the psicose conversion reaction and separation process were carried out with the method substantially same as Preparative Example 1.

(12) The process was carried out, repeated 10 times, and the mixture substrate and the composition of saccharides of raffinate by each recycle were analyzed and shown in the following Table 1. The mixture solution shown in the following Table 1 means the fructose-containing raw material obtained after putting the fructose raffinate obtained from the separation process of psicose preparation into the separation process of fructose preparation.

(13) TABLE-US-00001 TABLE 1 Saccharides including disaccharides or higher degree of Reducing Psicose Classification polymerization Glucose Fructose Psicose sugar yield F95 raw material 1.1% 5.0% 88.1% 0.0% 5.8% 22.9% raffinate 1.5% 8.3% 85.6% 0.3% 4.3% 1st mixture 1.1% 5.0% 88.2% 0.0% 5.7% 22.9% solution 1st raffinate 1.5% 8.3% 85.5% 0.4% 4.3% 5th mixture 1.3% 4.9% 88.2% 0.0% 5.6% 22.9% solution 5th raffinate 1.5% 8.3% 85.4% 0.4% 4.3% 10th mixture 1.3% 4.8% 88.3% 0.0% 5.8% 22.9% solution 10th raffinate 1.5% 8.3% 85.1% 0.4% 4.3%

(14) As can be seen in the Table 1, it was demonstrated that the fructose purity of raw material put into the psicose conversion process maintained constantly even if the number of recycle times increased, by putting the fructose raffinate obtained in the psicose preparation process into the fructose preparation process.

Example 2. Production of Fructose Using Fructose Raffinate

(15) The present experiment was carried out to confirm the difference of saccharide composition and yield according to presence/absence of recycling of fructose raffinate.

(16) Specifically, in the fructose preparation process, after mixing corn starch with water to be 30 to 35% by weight, enzymatic hydrolysis was progressed to collect saccharification liquid of 88% by weight or higher of glucose content. Then, the saccharification liquid was under vacuum drum filtration and insoluble materials were removed, thereby obtaining fructose isomerization product (fructose content 42% by weight syrup).

(17) The fructose raffinate of psicose preparation process obtained in Preparative Example 2 was concentrated to 88% by weight of fructose content and 50% by weight of the total solids (50 Brix) and mixed with the product which passed through the fructose isomerization process (fructose content 42% by weight syrup), and put into the ion purification process consisting of strongly acidic resin, weakly basic resin and strongly acidic and weakly basic mixed resin. That is, by adjusting the total solid content of fructose isomerization product and fructose raffinate put into the primary ion purification process to 50% by weight (50 Brix), the mixture ratio of the fructose isomerization product and fructose raffinate of Preparative Example 2 was 8:2 to 9:1 of weight ratio. After the mixed fructose syrup massed through the primary ion purification and was concentrated to the total solid 50% by weight, it passed through the SMB chromatography process. The glucose raffinate and fructose fraction were obtained by performing the SMB process. The saccharide composition of mixture and raffinate by each process was analyzed and shown in the following Table 2.

(18) TABLE-US-00002 TABLE 2 Saccharides Solid over Reducing separation Classification disccharide Glucose Fructose Psicose sugar yield No Fructose 2.6% 54.5% 42.3% 0.0% 0.6% — cycle isomerization reactant Fructose fraction 0.3% 10.4% 88.0% 0.0% 1.2% 48.1% after SMB separation Glucose raffinate 4.7% 95.3% 0.0% 0.0% 0.0% 51.9% after SMB separation With Mixture of 2.6% 54.5% 48.8% 0.0% 0.6% — cycle fructose isomerization reactant and fructose raffinate Fructose fraction 0.4% 10.0% 88.5% 0.0% 1.1% 55.4% after high purity separation Glucose raffinate 4.7% 95.3% 0.0% 0.0% 0.0% 44.6% after high purity separation

(19) In the Table 2, based on that the total solid content of fructose isomerization reactant, fructose fraction after SMB separation, glucose raffinate after SMB separation, and the mixture of fructose isomerization reactant and fructose raffinate was 100% by weight, the solid content of each composition component was shown as % by weight. The separation yield was shown as the total solid content of fructose fraction after SMB separation (% by weight) and the total solid content of glucose raffinate (% by weight), based on the total solid 100% by weight of feed materials provided in the SMB separation process.

(20) As can be seen in the Table 2, when the fructose separation process was carried out by mixing the raffinate fraction of psicose conversion process with the fructose isomerization reactant of fructose preparation process, based on the total solid 100% by weight of feed materials provided in the SMB separation process, the total solid content of fructose fraction after SMB separation of 48.1% by weight and the glucose raffinate of 51.9% by weight were separated. When mixing the fructose raffinate according to the present invention, the total solid of fructose fraction after SMB separation of 55.4% by weight and the glucose raffinate of 44.6% by weight were separated.

(21) Thus, it was demonstrated that when putting the fructose raffinate of psicose preparation into the separation process of fructose preparation, the fructose content in fructose fraction obtained in the SMB process was increased, and in addition, the solid separation yield was increased by 115%. Therefore, equal fructose purity and yield increasing effect was demonstrated even if mixing the fructose raffinate fraction obtained in the high purity chromatography of psicose preparation process with products which passed through the fructose isomerization process and separating it.

Example 3. Production of Psicose Using Raffinate

(22) The psicose isomerization process and separation process were carried out with the same method as Example 1, but at flow rate of 3.8 m.sup.3/hr, in order to produce 10 tons of solids of 95% by weight of psicose content using syrup of 95% by weight of fructose content. The psicose content of products which were passed through the psicose conversion process and collected was 21 to 25% by weight, and they passed through the separation process at a concentration of 45 to 50% by weight after ion purification. The raffinate which was generated when separated by using Ca+ type separation resin was generated by 3 m.sup.3 per hour.

(23) The process was carried out, repeated 10 times, and the mixture substrate and the composition of saccharides of raffinate by each process were analyzed and shown in the following Table 3.

(24) TABLE-US-00003 TABLE 3 Saccharides including disaccharides or higher degree of Reducing Psicose Classification polymerization Glucose Fructose Psicose sugar yield F95 raw material 0.1% 3.5% 95.4% 0.0% 1.0% 27.1% Raffinate of psicose 0.1% 3.9% 94.2% 1.2% 0.6% conversion reaction 5th mixture solution 0.1% 3.5% 95.4% 0.0% 1.0% 27.1% 5th raffinate of 0.1% 3.9% 94.0% 1.2% 0.6% psicose conversion reaction 10th mixture solution 0.1% 3.5% 95.4% 0.0% 1.0% 27.1% 10th raffinate of 0.1% 3.9% 94.2% 1.2% 0.6% psicose conversion reaction

(25) As can be seen in the Table 3, it was demonstrated that the fructose content of raw material put into the psicose conversion process maintained constantly even if the number of recycle times increased, by putting the raffinate fraction of the psicose preparation process into the fructose preparation process.

Example 4. Production of Fructose Using Raffinate

(26) To confirm the difference of saccharide composition and yield according to presence/absence of recycling of fructose raffinate, an experiment was carried out with the substantially same method as Example 2, but by using the raw material of 95% by weight of fructose content, instead of the raw material of 88% by weight of fructose content in Example 2, the psicose conversion reaction was carried out.

(27) The glucose raffinate and fructose fraction were obtained respectively, by performing the SMB process. The saccharide composition of mixture and raffinate by each process was analyzed and shown in the following Table 4.

(28) TABLE-US-00004 TABLE 4 Saccharides including disaccharides or higher Solid degree of Reducing separation Classification polymerization Glucose Fructose Psicose sugar yield No Fructose syrup 2.6% 54.5% 42.3% 0.0% 0.6% — cycle after fructose isomerization process Fructose fraction 0.0% 3.2% 95.4% 0.0% 1.4% 44.3% after SMB separation Glucose fraction 4.7% 95.3% 0.0% 0.0% 0.0% 55.7% after SMB separation With Mixture of 1.9% 45.3% 52.1% 0.0% 0.7% — cycle fructose isomerization reactant and fructose raffinate Fructose fraction 0.1% 3.2% 95.5% 0.0% 1.3% 54.6% after SMB separation Glucose fraction 4.2% 95.8% 0.0% 0.0% 0.0% 45.4% after SMB separation

(29) As can be seen in the Table 4, when performing the fructose separation process by mixing the raffinate fraction of psicose conversion process with the fructose isomerization reactant of fructose preparation process, based on that total solid of feed materials provided in the SMB separation process was 100% by weight, The total solid of fructose fraction after SMB separation of 44.3% by weight and the glucose raffinate of 55.7% by weight were separated. When mixing the fructose raffinate according to the present invention, the total solid of fructose fraction after SMB separation of 54.6% by weight and the glucose raffinate of 45.4% by weight were separated. Thus, it was demonstrated that when putting the fructose raffinate of psicose preparation into the separation process of fructose preparation, the fructose content in fructose fraction obtained in the SMB process was increased, and in addition, the solid separation yield was increased by 123%. Therefore, equal fructose purity and yield increasing effect was demonstrated even if mixing the fructose raffinate fraction obtained in the high purity chromatography of psicose preparation process with products which passed through the fructose isomerization process and separating it.

Example 5. Comparative Experiment of Psicose Conversion Ratio According to Mixed Metal Ion

(30) The psicose conversion ratio in the composition similar to the fructose raffinate obtained in the psicose preparation process was evaluated. That is, the psicose conversion process was confirmed by diluting the syrup of fructose purity 95% by weight syrup to 50% by weight and adding Ca.sup.2+ ion by 0.005˜0.01 mM, and then additionally adding 1.0 mM of Mn, and shown in Table 5.

(31) TABLE-US-00005 TABLE 5 Ca ion Mn ion Relative activity compared to non- concentration concentration additive control group (%) non-additive non-additive 100.00 non-additive 1.0 mM 152.0 0.005 mM 1.0 mM 141.2 0.008 mM 1.0 mM 136.5 0.010 mM 1.0 mM 127.1

(32) It was demonstrated that the relative activity of 152% of under the treatment of manganese increasing the activity of psicose conversion reaction, was reduced by about 16% at the maximum when the addition of calcium. The relative activity tended to be reduced gradually as the concentration of calcium ion was increased.

(33) This result shows why it is necessary to purify Ca ion of 0.01 mM or lower precipitated in the separation chromatography process, in reusing the fructose raffinate obtained in the high purity separation chromatography process. In case of no ion purification of fructose raffinate, the activity is lower than the case of using only manganese, thereby negatively affecting the yield of psicose.