The present application relates to a method for preparing a high yield of D-psicose crystals from a D-psicose-containing solution.
background
[2]
D-Pscicose is an epimer of fructose and is a kind of functional saccharide known as a rare saccharide. It is known to be effective in prevention and improvement. Psychos is also known to have excellent solubility, and is one of the materials attracting attention for its use in food. The reaction solution containing D-psicose produced by the epimerization reaction is low-purity containing about 20 to 30% (w/w) of D-psicose solids, and it is 98% or more of D-psicose crystal formulation. Separation and purification techniques such as chromatography and crystallization techniques are required for manufacturing. In general, as a crystallization method of saccharides, the principle of inducing crystal growth within a metastable zone (range between the saturation concentration and the lowest supersaturation concentration in which spontaneous crystals are precipitated) in a supersaturated state is used. Since D-psicose shows little change in crystal formation rate and crystal growth rate even in the supersaturated concentration range, it can be classified as a saccharide with difficult crystallization conditions for grain size growth.
[3]
At the concentration of the metastable region, crystallization such as crystal nucleation does not occur, but when a new crystal is added from the outside, crystal growth occurs and the crystal size increases. That is, when a seed crystal is added to a solution having a saturated concentration or higher in order to generate a crystal, the seed crystal grows in a metastable zone and the crystal is grown.
[4]
As for D-psicose, the crystal growth rate is significantly lower than that of sucrose, so it is common to apply a cooling crystallization method, and for industrialization, high economical technology development is required.
[5]
Although a method using a large amount of ethanol in the process of crystallizing D-psicose has been reported (Kei T, et, al., J. Biosci. Bioeng., 90(4), 453-455, 2000), the above literature According to the method of , crystal nucleation occurs but crystal growth does not occur. Specifically, during the crystallization process, a blocking phenomenon due to amorphous masses appears, which causes the generation of fine particle crystals. If a block occurs during the process, it is difficult to transfer to the outside of the crystallizer, and grinding cannot be performed, resulting in a low production yield. And in general, the size of the grain size is known as an important factor in the sugar crystallization industry, and when the crystals produced in the mass production system are fine particles, they easily escape with the crystal mother liquor from the crystal centrifuge facility and remain in the separation. When the particles are dried, they cause agglomeration between crystals and are excluded from the sieving process, resulting in reduced packaging volume of the final product or poor marketability. Therefore, such fine particle crystals are not suitable for mass production. In addition, in the crystallization of saccharides using ethanol, if the final residual ethanol concentration is high, an off-flavor is generated, thereby lowering the commercial value of the saccharide crystal product.
[6]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[7]
The present inventors have confirmed that it is possible to increase the recovery rate of crystals and grow the crystal grain size by using the D-psicose concentrated solution and organic solvent in the allowed range of operation, and the flowability of the crystallization manufacturing process and the commercialization as a product are improved A method for producing high-purity D-psicose with no off-flavor over 98% (w/w) purity with a grain size of MA200 or higher was developed.
means of solving the problem
[8]
One object of the present application is as a D-psicose crystal, based on the total crystal 100 (w/w), 98% (w/w) or more of D-psicose and 0.001 to 0.05% (w/w) It is to provide a crystal of D-psicose, containing the ethanol of.
[9]
Another object of the present application is a first step of mixing a solution containing D- and an organic solvent; And it is to provide a method for producing a crystal between D-, including a second step of obtaining a mask containing the crystal between D- by adding a seed crystal (seed) to the mixed solution according to the first step and then cooling .
[10]
Effects of the Invention
[11]
In the manufacturing method of the crystal between D- of the present application, through the crystallization method of slowly cooling by adding seed crystals after mixing the organic solvent with the solution containing D- between course, the yield of the crystal between D- between the course D- Not only can it be increased, but also it is possible to prepare D-psicose crystals of sufficient size and appropriate shape to be used in mass production without having an off-flavor.
[12]
Brief description of the drawing
[13]
1 is a micrograph of the D-psicose crystal prepared in Example 1.
[14]
2 is a particle size analysis result of the D- between the crystals prepared in Example 1.
[15]
3 is a particle size analysis result of the crystal composition between D- prepared in Example 2.
[16]
4 is a view showing the shape of the crystal cake between D- generated in Example 6.
[17]
5 (a) is a scanning electron microscope (SEM) photograph of the surface of the crystal surface between D- of Example 1. Figure 5 (b) is a scanning electron microscope (SEM) photograph of the crystal surface between D- of Comparative Example.
[18]
6 is a diagram illustrating a method for measuring an angle of repose.
[19]
Best mode for carrying out the invention
[20]
Hereinafter, the present application will be described in more detail.
[21]
On the other hand, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of this application. In addition, it cannot be said that the scope of the present application is limited by the specific description to be described below.
[22]
In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.
[23]
[24]
As one aspect of the present application for solving the above problems, the present application is a D-psicose crystal, based on 100% (w/w) of the total crystal, 98% (w/w) or more of D-between and D-psicose crystals comprising 0.001 to 0.05% (w/w) of ethanol.
[25]
[26]
In the present application, "D-psicose" is a low-calorie monosaccharide, and has a molecular formula of C 6 H 12 O 6 and the following structural formula. In addition to the chain-like structure, alpha-type and beta-type hexagonal ring-shaped structures are also included in the scope of D-psicose.
[27]

[28]
As used herein, "D-psicose crystal" is a solid in which D-psicose molecules are arranged in a regular repeating structure, and is different from an amorphous solid mass that does not have a repeating structure. D- of the present application of course crystals may be 98% (w/w) or more, specifically 98.5% (w/w) or more, and more specifically, having a purity of 99% (w/w) or more.
[29]
As the step of adding ethanol is involved in preparing the D- between the crystals of the present application, the D- between the crystals may contain a very small amount of ethanol. The content of the ethanol may be 0.05% (w/w) or less, specifically 0.04% (w/w) or less, more specifically 0.03% (w/w) or less. The ethanol content is, for example, 0.001 to 0.05% (w / w), 0.001 to 0.04% (w / w), 0.001 to 0.03% (w / w), 0.005 to 0.05% (w / w), 0.005 to 0.04% (w/w), 0.005 to 0.03 % (w/w), 0.01 to 0.05 % (w/w), 0.01 to 0.04 % (w/w), 0.01 to 0.03 % (w/w), 0.02 to 0.05 % (w/w), 0.02 to 0.04 % (w/w), or 0.02 to 0.03 % (w/w). According to one embodiment, which is not limited herein, when ethanol is included in the same amount as described above, it can be used equally with D-psicose crystals without ethanol added because there is no already / off-flavor (Test Example 2) ), due to the inclusion of a trace amount of ethanol, the surface of the D-psicose crystal may be smooth, and may be glossy or shiny compared to the D-psicose crystal without the addition of ethanol (Test Example 3). In addition, by containing a very small amount of ethanol, the fluidity of the D-psicose crystals increases and thus the seed crystals are crystallized in a dispersed state, and thus a high yield and crystallinity can be obtained (Test Example 4). The fluidity of the crystal can be confirmed by measuring the fluidity and the angle of repose, The angle of repose refers to the maximum angle of inclination that can be deposited without flowing down when the sediment that has not yet been consolidated is deposited on the slope. The angle of repose can be measured with a protractor after passing the D-psicose crystal composition through a funnel installed on a horizontal plate surface at a constant speed (FIG. 6).
[30]
The % (w/w) may be used interchangeably with weight %. This may mean a crystal weight relative to 100 parts by weight of the entire crystal or a crystal weight relative to 100 parts by weight of a solution including crystals. Specifically, it may refer to the crystal weight of D- between the crystals compared to 100 parts by weight of the total crystals, or the crystal weight of D- between the crystals compared to 100 parts by weight of the solution including the crystals. The crystals may include dry solids (DS).
[31]
In addition, the term “crystal average particle size” herein is a measure indicating the average size of crystals. The method for measuring the grain size of D- between courses is not limited and a method commonly used in the art may be used. Non-limiting examples of measuring the grain size include a comparative method (FGC), a cleavage method (FGI), a flat method (FGP), and the like.
[32]
The D-psicose crystal of the present application has an average particle size (MA: area average) of crystals of 60 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 230 μm or more, 250 μm or more, 300 μm or more, or 320 μm. may be more than For example, it may be 100 μm to 500 μm, 150 μm to 450 μm, 200 μm to 400 μm, 230 μm to 400 μm, 300 μm to 400 μm, 320 μm to 400 μm, or 320 μm to 390 μm.
[33]
d10 of the present D-psicose crystal is a value corresponding to the lower 10% of the crystal distribution, and may be 100 μm or more, 110 μm or more, 130 μm or more, or 150 μm or more. 100 μm to 200 μm, 100 μm to 180 μm, 110 μm to 180 μm, 110 μm to 160 μm, 130 μm to 180 μm, 130 μm to 160 μm, 150 μm to 180 μm, or 150 μm to 160 μm can be
[34]
The d50 of the D-psicose crystal of the present application is a value corresponding to the lower 50% of the crystal distribution and may be used in combination with the median, and 100 μm or more, 150 μm or more, 200 μm or more, 230 μm or more, 250 μm or more , 300 μm or more, or 310 μm or more. For example, it may be 100 μm to 500 μm, 150 μm to 450 μm, 200 μm to 400 μm, 230 μm to 400 μm, 300 μm to 400 μm, 320 μm to 400 μm, or 310 μm to 390 μm.
[35]
The d90 of the present D-psicose crystal is a value corresponding to the lower 90% of the crystal distribution, and may be 200 μm or more, 250 μm or more, 300 μm or more, 330 μm or more, 350 μm or more, 400 μm or more, or 500 μm or more. have. For example, it may be 200 μm to 800 μm, 250 μm to 700 μm, 300 μm to 650 μm, 330 μm to 600 μm, 350 μm to 600 μm, 400 μm to 600 μm, or 500 μm to 590 μm.
[36]
The particle size distribution of the crystals between the present D- course can be confirmed as a relative standard deviation or a relative particle size distribution.
[37]
The relative standard deviation is a percentage value obtained by dividing the standard deviation by the average particle size, and may be 30% to 60%, 35% to 55%, 37% to 50%, 38% to 48%, or 40% to 46%.
[38]
The relative particle size distribution is a value obtained by dividing the difference between d90 and d10 by d50, and may be 0.8 to 1.5, 0.9 to 1.4, or 1.0 to 1.3.
[39]
Accordingly, it is possible to reduce the amount of D- between the crystals lost together with the mother liquor in the crystal centrifugation device facility, and the agglomeration between the crystals can be reduced. Therefore, the D-psicose crystal of the present application having a particle size as described above is suitable for mass production.
[40]
[41]
As another aspect of the present application for solving the above problems, the present application is a first step of mixing the D- between the course-containing solution and the organic solvent; And it provides a method for producing a D- between course crystals, including a second step of obtaining a mask containing the D- between course crystals by adding a seed crystal (seed) to the mixed solution according to the first step.
[42]
[43]
The "D-psicose-containing solution" is not limited as long as "D-psicose is a dissolved or dispersed solution. The D-psicose-containing solution contains 90% (w/w) or more of D-psicose, Specifically, high purity of 91% (w/w) or more, 92% (w/w, DS) or more, 93% (w/w) or more, 94% (w/w) or more, or 95% (w/w) or more It may be a solution containing D-psicose, but is not limited thereto.
[44]
The D-psicose-containing solution may be a solution obtained by treating an enzyme for the D-psicose production substrate to an epimerization reaction, or may be separated or purified. Examples, but not limited to the substrate and enzyme, include fructose and D-psicose epimerase, fructose-6-phosphate and D-psicose-6-phosphate epimerase and D-psicose-6-phosphate dephosphorylation. There are enzymes. Specifically, the D-psicose-containing solution may be a purified solution obtained by subjecting fructose to D-psicose epimerization reaction. Specifically, fructose used as a substrate in the epimerization reaction may be dissolved in water at a concentration of 30 to 50 brix (%) at a temperature of 30 to 40° C. and used. Herein, brix (%) means a percentage of the weight of D-psicose, or fructose, based on the weight of the total solution. Here, fructose may be mixed with a fructose-containing solution that is then separated by chromatography, and may be used at a temperature of 30 to 40° C. at a concentration of 30 to 50 brix (%). Here, as the fructose-containing solution separated by the chromatography, a fructose-containing fraction having a purity of 70% (w/w) or more, specifically 75% (w/w) or more, may be used.
[45]
The D-psicose epimerization reaction may be to generate D-psicose by epimerizing fructose in the presence of a psychoepimerization enzyme, a mutant thereof, a strain producing the enzyme, or a culture thereof. The D-psicose epimerase that can be used herein is an enzyme derived from various donor microorganisms, such as Agrobacterium tumefaciens, Flavonifractor plauti, Clostridium hylemonae. or a variant. The transformation strain includes Escherichia coli, Corynebacterium, Bacillus, and Aspergillus, but is not limited to this strain. As the strain transformed into the E. coli, for example, BL21(DE3)/pET24-ATPE [Korean Patent Publication No. 10-211-0035805], BL21(DE3)/pET24-ATPE-2 [Korea Patent No. 10 -1203856] and the like, and the Corynebacterium sp. strain is Corynebacterium glutamicum ATCC13032/pCJ-1-ATPE [Korea Patent Publication No. 10-2011-0035805 Accession No. KCCM11046], Corynebacterium glutamicum ATCC13032/pFIS-1-ATPE -2 [Accession No. KCCM11204P of Korean Patent No. 10-1203856], Corynebacterium glutamicum CJ KY [Accession No. KCCM11403P of Korean Patent No. 10-1455759],
[46]
In one example, the epimerization reaction is a psychoepimerization enzyme, a variant thereof, a strain producing the enzyme or a culture thereof, and a carrier, for example, sodium alginate immobilized on a carrier, for example, sodium alginate, the immobilized enzyme, isomerization After filling the reaction equipment, for example, in a column, it may be to supply a solution containing fructose to the packed column. The temperature in the facility may be maintained at a temperature of 40 to 70° C., for example, 40 to 55° C. for the epimerization reaction. At this time, the temperature of the solution containing the supplied fructose is, for example, 5 to 20 ° C per hour, to a temperature of 40 to 60 ° C, for example, to a temperature of 50 ° C. L)/hour (Hr)/resin amount (L)] 0.5 to 3. The purity of D-psicose produced by the epimerization reaction may be about 15 to about 35% (w/w), for example, about 20 to about 30% (w/w).
[47]
The solution containing the epimerized D-psicose may be cooled. The cooling is cooling the temperature or ambient temperature of the solution to a range of 25 to 45°C, specifically 30 to 40°C. Specifically, it may be cooled slowly at 1 to 10° C. per hour, and a heat exchanger may be used for the cooling.
[48]
[49]
The D-psicose-containing solution may be obtained by separating and/or purifying a stock solution containing D-psicose. That is, the manufacturing method of the crystal between the D- of the present application may include the step of separating and/or purifying the stock solution containing the D- between the course before the first step.
[50]
The D-psicose-containing solution may be obtained by purifying a stock solution containing D-psicose. Specifically, the purification may be performed by any one or more selected from the group consisting of decolorization according to passage of a column filled with a decolorizing agent, desalting by ion exchange resin chromatography, and continuous chromatography.
[51]
Specifically, after cooling the solution containing the epimerized D-psicose, it is passed through a column filled with a decolorizing agent to decolorize and purified through a column filled with a strongly acidic cation exchange resin and a weakly basic anion exchange resin. . When using a strongly basic anion resin, even at a low temperature of 25 to 45 ° C., since a decrease in purity may occur by denaturing D-psicose, a weakly basic anion exchange resin can be used for manufacturing a high yield of D-psicose, and more Specifically, 100% weakly basic anion exchange resin may be used. For effective removal of ion components, a cation exchange resin and an anion exchange resin can be used at the same time. In this case, the ratio of the cation exchange resin to the anion exchange resin may be 1:0.5 to 1:3. During the ion purification, a temperature of 25 to 45° C., specifically, a temperature of 30 to 40° C. is maintained to prevent denaturation of D-psicose. Thereby, it is possible to remove the ionic component included as an impurity in the D-psicose-containing solution. After the ion purification, the content of the ionic component may be 20 microsiemans or less, specifically, 10 microsiemans or less per unit cm as measured by an electrical conductivity meter. The purity of D-psicose in the ion purified solution is about 10 to about 35% (w/w). Here, in the cooling and ion purification, the mother liquid generated in the subsequent D-psicose crystallization step can be reused by removing ethanol through distillation. The purified D-psicose-containing solution may be concentrated and cooled.
[52]
The concentration is a process of concentrating the ion-purified D-psicose-containing solution so that the D-psicose concentration is in the range of 50 to 70 brix (%), for example, in the range of 55 to 65 brix (%). Specifically, it may be concentrated at a temperature of 50 to 80 °C, for example, at a temperature of 55 to 70 °C. At this time, a low-temperature concentrator may be used for concentration to prevent D-psicose denaturation. After the concentration, cooling may be performed. In the case of cooling, the solution or the ambient temperature may be cooled so as to be at least 10°C lower than the temperature at the time of concentration. For example, the cooling temperature may range from 40 to 60°C. The cooling rate may be gradually cooled by 5 to 25° C. per hour, and a heat exchanger may be used for the cooling. Here, for concentration and cooling, the mother liquor generated in the subsequent D-psicose crystallization process can be reused by removing ethanol through distillation.
[53]
[54]
The D-psicose-containing solution may be a solution obtained by separating fructose from a solution containing D-psicose. Specifically, the fructose-containing solution may be separated through chromatography.
[55]
Chromatography is to separate D- between courses by using the difference in the weak binding force between the D- and metal ions attached to the ion resin, for example, continuous chromatography can be used. The ion resin used for chromatography may be a strongly acidic cation exchange resin to which K, Na, Ca, and Mg residues are attached. Specifically, it may be an ionic resin capable of separating D-psicose and fructose, and may be, for example, K, Ca, or Na. A solution containing fructose and a purified solution containing D-psicose can be obtained by the above chromatography. The purified D-psicose-containing solution may be a solution containing a purity of 90% (w/w) or more, for example, a purity of 95% (w/w) or more of D-psicose. Specifically, the purity of D-psicose may be 90 to 99% (w/w) or more. The fructose-containing solution may be a solution containing fructose with a purity of 70% (w/w) or more. In the chromatography, the separated fructose-containing solution may be reused for the D-psicose epimerization reaction. As fructose is separated by chromatography without denaturing and separating, the overall yield can be increased by reuse in the D-psicose epimerization reaction. Before reuse, it may further comprise cooling to a range of 25 to 45 °C, 30 to 40 °C.
[56]
[57]
The D-psicose-containing solution of the present application may be concentrated. For example, by concentrating the purified D-psicose solution of 95% (w/w) or more of the obtained purity, the concentration of D-psicose is 75 brix (%) or more, for example, 80 brix (%) or more can make it happen The concentration may be specifically concentrated under a temperature of 50 to 80 ℃, for example, under a temperature of 55 to 70 ℃. At this time, a low-temperature concentrator may be used for concentration to prevent D-psicose denaturation.
[58]
[59]
The manufacturing method of the crystal between D- of the present application includes a first step of mixing the solution containing D- and the organic solvent between the course. Accordingly, it is possible to partially precipitate D-psicose. In addition, it may contribute to separation of crystals in particularly high yield through addition and cooling of seed crystals, which will be described later.
[60]
The mixing of the first step may be carried out under 20 to 60 ℃. Specifically, it may be carried out under 35 to 60 ℃, more specifically, it may be carried out at 40 to 60 ℃. When the temperature is lower than the above temperature, it becomes a supersaturated state exceeding the metastable region, which causes the formation of new crystal nuclei rather than the growth of crystals, which may cause a problem that crystal growth is inhibited. is volatilized, resulting in a decrease in recovery rate and generation of oil vapor, which may cause safety problems.
[61]
The organic solvent may be alcohol, and specifically, may include any one or more selected from ethanol, methanol, and isopropyl alcohol.
[62]
The organic solvent may have moisture: organic solvent of 1:0.5 or more, 1:0.7 or more, or 1:1 or more, for example, 1:0.5 to 1:10, 1:0.7 to 1:10, 1:1 to 1 :10, 1:1 to 1:9, or a mixture of 1:1 to 1:8.
[63]
[64]
The manufacturing method of the D- between course crystals of the present application includes a second step of obtaining a mask (masscuite) containing the D- between course crystals by cooling after adding the D- between course seed crystals (seed) to the mixed solution do. Here, the mask refers to a slurry state in which the crystal and the solution are mixed when the D-psicose seed crystal starts the crystallization reaction.
[65]
The most difficult thing to achieve in the manufacturing method of the D-psicose crystal is the control of the size or shape of the crystal. In the present application, after mixing the organic solvent in the solution containing D- between course, based on the crystallization method of slowly cooling by adding seed crystals, this can be achieved.
[66]
Herein, the D-psicose seed crystal refers to a fine crystal mainly composed of D-psicose, and is added while maintaining the supersaturation of D-psicose in the organic solvent mixture in a metastable zone constantly. The organic solvent mixture has a low viscosity, and the added D-psicose seed crystals can be easily dispersed. Under such conditions of low viscosity and high dispersing force, crystal growth may be excellent. The D-psicose seed crystal may be a seed crystal of 100 μm or less, and specifically, a seed crystal having a size of 40 to 100 μm. The D-psicose seed crystal may be added in an amount of 0.01 to 1% (w/w) by weight based on the total weight of the mixed solution.
[67]
If the D-psicose seed crystal is not added in the manufacturing method of the present application, an amorphous D-psicose mass is produced and D-psicose crystals of the desired size or shape cannot be produced. The D-psicose seed crystal is preferably added to the mixed solution in a dispersed form. After the input of the seed crystal, the crystal is grown by controlling the cooling conditions.
[68]
Regarding the cooling conditions, the temperature of the first step of mixing before cooling is as described above, and the final temperature cooled according to the second step is 8 to 30 ℃, 8 to 25 ℃, 8 to 20 ℃, 10 to 30 °C, 10 to 25 °C, or 10 to 20 °C. According to such temperature control, it is possible to suppress the formation of new crystals to increase the size of the crystals and to increase the yield. When the final temperature cooled by the second step is higher than the above range, the recovery rate of D-psicose crystals is lowered, and when it is low, nucleation is caused to generate a large amount of fine particles of less than 100 μm, which is agglomerated between crystals during drying. As a result, the packaging volume of the final product is small or the marketability is deteriorated.
[69]
Here, by controlling the rate of cooling, the growth rate of the D-psicose crystals can be controlled. Specifically, the cooling may be performed at a rate of 0.05 to 1.4°C/hour, 0.6 to 1.4°C/hour, 0.7 to 1.3°C/hour, 0.8 to 1.2°C/hour, or 0.9 to 1.1°C/hour. In another example, the cooling rate may be adjusted to cool in the range of the metastable region. When the cooling rate is faster than the above range, the mixed solution rapidly enters the supersaturation region out of the metastable region, causing a large amount of particulates to be generated. Also, too low cooling rate reduces productivity per unit time, resulting in inefficiency in mass production.
[70]
The cooling and/or crystallization in the second step may be performed for 20 to 70 hours, or 30 to 70 hours.
[71]
According to one embodiment, which is not limited herein, when the D-psicose crystals are prepared through the use of an organic solvent and control of the cooling rate, a shaped intercourse crystal block may be generated.
[72]
The manufacturing method of the present application may further include a third step of separating and drying the D- between the crystals from the mask. Specifically, from the mask obtained by the first and second steps, it is possible to separate D-psicose crystals containing an excess of organic solvent.
[73]
The step of separating the D- between the crystals from the mask can be used without limitation as long as it is a method capable of separating the crystals, but for example, a centrifugal dehydrator can be used. The separated crystal may have an organic solvent of 0.07% (w/w) or more, 0.1% (w/w), or 0.13% (w/w) or more, based on 100% (w/w) of the total crystal. 0.07 % (w/w) to 0.5 % (w/w), 0.1 % (w/w) to 0.3 % (w/w), or 0.13 % (w/w) to 0.2 % (w/w) days can
[74]
By drying the D-psicose crystals containing an excess of the organic solvent, 98% (w/w) or more of D-psicose and 0.05% (w/w) or less based on 100% (w/w) of the total crystals D-psicose crystals containing an organic solvent can be obtained. The description of the obtained D-psicose crystal is as described above.
[75]
The manufacturing method of the present application may further include a fourth step of reusing the organic solvent as the organic solvent of the first step after recovering the organic solvent from the crystal mother liquid from which the D-psicose crystals are separated according to the third step.
[76]
In addition, it may further include a fifth step of reusing the crystal mother liquid from which the organic solvent has been removed according to the fourth step in the preparation of the D-psicose solution of the first step.
[77]
[78]
The yield according to the manufacturing method of the present application, that is, the weight percentage of the final obtained D- between the course crystals relative to the weight of the D- between the course present in the solution containing D- of the first step is 65% (w / w) or more , 70% (w/w) or more, 75% (w/w) or more, or 80% (w/w) or more.
[79]
That is, the yield herein may be expressed by the following equation.
[80]
[Equation 1]
[81]
Yield (%) = (weight of dehydrated and dried D-psicose crystals / D-psicose weight of stock solution before crystallization)Х 100
[82]
When calculating the yield, the weight of D-psicose in the stock solution before crystallization is measured by measuring the g/L of D-psicose in the stock solution through HPLC analysis, and then the measured D- according to the previously measured amount of crystallization stock solution (L) By substituting g/L of psicose, the weight (g) of D-psicose included in the specific stock amount (L) can be calculated.
[83]
In addition, the mother liquid separated in the crystallization, that is, the supernatant dehydrated from the mask can be reused in the first step of mixing the organic solvent with the D-psicose solution by recovering the organic solvent through distillation, and the organic solvent is removed after distillation D- The solution containing psicose is cooled to 30 ° C., and recirculated to a column filled with a strong acid cation exchange resin substituted with a hydrogen group and a weakly basic anion exchange resin substituted with a hydroxyl group, recycled by continuous chromatography, or the first step can be recycled. The mother liquor generated during crystallization of D-psicose may be a D-psicose-containing fraction having a purity of 75% (w/w) or more, a purity of 85% (w/w) or more, and a purity of 95% (w/w) or more.
[84]
Modes for carrying out the invention
[85]
Hereinafter, the present application will be described in more detail by way of Examples. However, the following examples are only for illustrating the present application, and the scope of the present application is not limited thereto.
[86]
[87]
Example 1. Purity 98% (w / w) or more, ethanol 0.05% (w / w) or less of high-purity D-psicose crystal composition preparation
[88]
[89]
(1) Production of low-purity D-psicose solution using microorganisms
[90]
A 50 brix (%) fructose solution (enzyme reaction substrate solution) with a purity of 95% (w/w) or more was prepared. The epimerase reaction is D-psicose isolated from a strain of Corynebacterium glutamicum KCCM 11046P, as disclosed in Korean Patent Application Publication No. KR 10-2011-0035805 A (Korean Patent Application No. 10-2009-0118465). After the epimerization enzyme was immobilized on a sodium alginate carrier and filled in an isomerization reaction facility (Isomerization Tower, Hangi Machinery Industry), the prepared enzyme reaction substrate solution was heated to 50° C. at a rate of 5 to 20° C. per hour through a heat exchanger to SV [Space Velocity: flow rate (L)/time (Hr)/resin amount (L)] 0.5 to give an epimerized D-psicose solution. At this time, the purity of D-psicose was about 24% (w/w).
[91]
[92]
(2) Purification of D-psicose solution
[93]
After the first cooling of the epimerized D-psicose solution to a temperature of 30 to 40 ° C. at 5 to 10 ° C. per hour through a heat exchanger, the decolorization was passed through a column filled with a decolorant, and a strong acid cation substituted with a hydrogen group It was desalted by passing it through a column filled with an exchange resin and a weakly basic anion exchange resin substituted with a hydroxyl group with SV 3, and the final ion component was adjusted to be 10 micro Siemance or less per unit cm through electrical conductivity measurement, and the desalted enzyme The purity of D-psicose of the reaction solution was maintained at 24% (w/w).
[94]
[95]
(3) Separation of high-purity D-psicose solution using chromatography
[96]
The ion-refined D-psicose-containing solution was put into a low-temperature concentrator (Forced Thin Film Evaporator, Welcron Hantech) and the concentration was 60 brix (%) in a short time for 10 to 15 minutes at a temperature of 55 to 70 ° C. ), and second cooling at 5 to 25 ° C per hour through a heat exchanger at 50 to 60 ° C. By continuous chromatography in a column filled with a strongly acidic cation exchange resin with a calcium active group attached to the D-psicose It was separated into a purified D-psicose solution having a purity of 95% (w/w) or more and a fructose-containing solution having a purity of 70% (w/w) or more of fructose.
[97]
The fructose-containing solution with a purity of 70% (w/w) or more separated by the continuous chromatography was recovered, cooled at 20 to 30°C per hour, and recycled at 30°C to the fructose epimerization process.
[98]
[99]
(4) Concentration of D-psicose solution, organic solvent treatment, and crystallization through cooling
[100]
The purified D-psicose solution of at least 95% (w/w) of purity separated in the continuous chromatography was concentrated under a temperature of 55 to 70° C. and the concentration was adjusted to 85.0 brix (%), and the concentration was adjusted to 95% of the purity (w/w) or more D-psicose solution is rapidly cooled to a temperature of 40 °C at 5 to 20 °C per hour through a heat exchanger, and then parts by weight relative to moisture excluding solids: ethanol = ethanol = 1: 1.13 was mixed.
[101]
After adding an appropriate amount of seed to the D-psicose solution in which the ethanol cooled to 40 °C is mixed, the seed crystal is cooled to a final temperature of 10 °C at a cooling rate of 1 °C per hour and crystallized for 30 hours to determine D-psicose A mask containing this was obtained.
[102]
The mask containing the D-psicose crystals was placed in a high-speed centrifugal dehydrator and rotated at 4,000 rpm for 10 minutes, the supernatant was drained and D-psicose crystals containing an excess of ethanol were obtained. At this time, the residual supernatant can be washed by spraying deionized water or ethanol, and the concentration of ethanol contained in the obtained D-psicose crystals was about 0.15% (w/w).
[103]
The recovered ethanol was transferred to a D-psicose crystal containing an excess of the fluidized bed dryer or vacuum dryer, dried for 1 to 2 hours to remove the excess ethanol, and 0.03% (w/w) of ethanol containing 98% purity ( w/w) or more of D-psicose crystals were obtained. The crystal amount of D-psicose obtained after drying was 2,252 g, separated in the continuous chromatography, and about 81% was recovered compared to 2,780 g present in the concentrated D-psicose solution and the crystal size was MA 336 (Fig. 1). and 2).
[104]
Additionally, the mother liquid separated in the crystallization, that is, the supernatant dehydrated from the mask can be reused as a step of recovering ethanol through distillation and mixing with the D-psicose solution, and contains D-psicose from which the ethanol is removed after distillation The solution is cooled to 30° C., and recirculated to a column filled with the strong acid cation exchange resin substituted with a hydrogen group and a weakly basic anion exchange resin substituted with a hydroxyl group, or can be recycled through a continuous chromatography step.
[105]
[106]
Example 2. Purity 98% (w/w) or more, ethanol 0.05% (w/w) or less of high-purity D-psicose crystal composition preparation
[107]
In Example 1, the D-psicose-containing solution concentrated to 80.0 brix (%) was cooled to 50° C., and the mixture of ethanol corresponding to water by weight parts moisture: ethanol = 1: 9 excluding solids and cooling A high-purity D-psicose crystal composition with a purity of 98% (w/w) or more and ethanol 0.05% (w/w) or less in the same manner as in Example 1 except for 60 hours at a rate of 0.5°C per hour and a final temperature of 20°C prepared.
[108]
The crystal amount of D-psicose contained in the obtained purity 98% (w/w) or more of ethanol 0.05% (w/w) or less was 2,307 g, and about 83% was recovered compared to 2,780 g of D-psicose initially dissolved. The size was MA 241 (Fig. 3).
[109]
[110]
Example 3. Purity 98% (w / w) or more, ethanol 0.05% (w / w) containing D-psicose crystal composition preparation
[111]
In Example 1, in the same manner as in Example 1, except that the drying time was set to 30 minutes to 1 hour, a high-purity D-psicose crystal composition having a purity of 98% (w/w) or more was prepared.
[112]
The obtained D-psicose crystals contained 0.05% (w/w) of ethanol and had a purity of 98% (w/w) or more.
[113]
[114]
Example 4. Figure 98% (w / w) or more, ethanol 0.06% (w / w) containing D-psicose crystal composition preparation
[115]
In Example 1, in the same manner as in Example 1, except that the drying time was set to 10 to 20 minutes, a high-purity D-psicose crystal composition having a purity of 98% (w/w) or more was prepared.
[116]
The obtained D-psicose crystals contained 0.06% (w/w) of ethanol and had a purity of 98% (w/w) or more.
[117]
[118]
Example 5. Preparation of high-purity D-psicose crystals by changing the type of organic solvent to be mixed
[119]
In Example 1, in the same manner as in Example 1, except for mixing methanol and isopropyl alcohol in the concentrated D-psicose-containing solution, high-purity D-psicose crystals with a purity of 98% (w/w) or more were prepared did.
[120]
When methanol was used, the yield of the obtained D- between the crystals was 33%, and the average particle size was MA 109.
[121]
When isopropyl alcohol was used, the yield of the obtained D- between the crystals was 32%, and the average particle size was MA 61.
[122]
[123]
Example 6. Preparation of a high-purity D-psicose crystal composition using an organic solvent but not controlling the cooling rate
[124]
In Example 2, in the concentrated D-psicose-containing solution, the ratio of water by weight to water excluding the solid content: ethanol = 1: 4: Mixing ethanol corresponding to 4 and 30 minutes to 1 without controlling the cooling rate after seed crystal input In the same manner as in Example 2, except for cooling to a final temperature of 20 ° C within an hour and crystallizing for 30 hours, ethanol with a purity of 98% (w/w) or more and less than 0.05% (w/w) of ethanol containing less than 0.05% (w/w) of high purity D-psicose crystal A composition was prepared.
[125]
The obtained D- between the crystal amount of 1,056 g was recovered about 38% compared to the initial dissolved D- between the course and the resulting D- between course crystal block was confirmed to be 1,084 g, 39%.
[126]
[127]
comparative example. Production of high-purity D-psicose crystals by a cooling crystal method that does not use organic solvents
[128]
In Example 2, the concentrated D-psicose-containing solution was cooled to an initial temperature of 40 ° C. and then cooled to 20 ° C. for 80 hours without mixing with ethanol. w/w) or more of high purity D-psicose crystals were prepared.
[129]
The yield of the obtained D- between the crystals was 53%, the average particle size was MA374.
[130]
[131]
Test Example 1. Confirmation of Residual Ethanol Removal Effect
[132]
Example 1 (including residual ethanol 0.03% (w / w) or less) and Comparative Examples, Examples 4, and high-purity D-psicose crystals of Example 3 in powder form to the subject to ingest a certain amount, and then the difference A three-point test was evaluated for the identification of The two test groups were arranged with three options, and the examiner ingested the three options one after another without knowing the sequence, and had them choose one with a different taste. Twenty testers were tested three times in total to evaluate whether there was a difference through the percentage of correct answers among the total number of tests. The D-psicose crystals and residual ethanol concentration % (w/w) used for evaluation are shown in Table 1 and the evaluation results are shown in Table 2. Evaluation counts the number of correct answers and compares the total number of responses and the number of correct answers with the significance test table to determine whether there is statistical significance. If the test was performed 60 times, it is judged that there is a significant quality difference if the number of correct answers is 27 or more.
[133]
[134]
[Table 1]
　 Example 3 Example 1 Example 4 comparative example
Residual ethanol concentration (%, w/w) 0.05 0.03 0.06 0
[135]
[Table 2]
control group number of inspections number of correct answers correct answer rate Test result
% Difference presence/absence
comparative example Example 4 60 48 80.0 you
comparative example Example 3 60 22 36.7 radish
comparative example Example 1 60 12 20.0 radish
Example 4 Example 3 60 43 71.7 you
Example 4 Example 1 60 46 76.7 you
Example 3 Example 1 60 11 18.3 radish
[136]
D-psicose crystals (Examples 1 and 3) containing 0.05% (w/w) or less of residual ethanol compared to D-psicose crystals (Comparative Example) prepared by a method that does not use an organic solvent (Examples 1 and 3) have a sensory difference confirmed that there is no From this, it can be seen that when the residual ethanol is adjusted below a certain level of concentration, it is to realize the same taste as the original D-psicose crystal.
[137]
[138]
Test Example 2. Confirmation of the effect of reducing taste / odor intensity by removing residual ethanol
[139]
In Test Example 1, the taste/flavor intensity for the same test group (Table 1) used in the test was evaluated. After ingesting D-psicose crystals to 20 examiners, the intensity of taste/flavor was expressed as a numerical value (5 points were the intensity of taste/flavor intensity), and the evaluation results are shown in Table 3. Compared to the comparative example, which is a D-psicose crystal prepared by a method not using an organic solvent, the D-psicose crystal (Examples 1 and 3) containing 0.05% (w/w) or less of residual ethanol has already been / It was confirmed that there was no difference in off-flavor. From this, when the residual ethanol is removed below a certain level of concentration, it can be seen that the taste/flavor is equivalent to the original D-psicose crystal.
[140]
[Table 3]
comparative example Example 4 Example 3 Example 1
1.9±0.8 3.0±0.4 2.0±0.9 1.9±0.7
[141]
[142]
Test Example 3. Confirmation of smooth surface effect by containing ethanol
[143]
The high-purity D-psicose crystal surface of Example 1 (including residual ethanol 0.03% (w/w) or less) and Comparative Example was SEM-irradiated. The results are shown in FIG. 5 .
[144]
It was confirmed that the surface of the D-psicose crystals (FIG. 5(a)) to which the ethanol of Example 1 was added was smooth compared to the D-psicose crystals (FIG. 5(b)) to which ethanol was not added of Comparative Examples. From this, it can be seen that the smoothness of the surface of the D- between course crystals is increased by the addition of ethanol, so that gloss or luster can be achieved.
[145]
[146]
Test Example 4. Confirmation of the effect of increasing crystal fluidity due to the inclusion of ethanol
[147]
Test Example 4.1 Measurement of Fluidity
[148]
The fluidity of D-psicose crystals (Example 1) containing 0.03% (w/w) or less of residual ethanol compared to D-psicose crystals (Comparative Example) prepared by a method not using an organic solvent was evaluated. . A fluidity meter was used to evaluate the fluidity.
[149]
[Table 4]
inlet diameter 1.5 cm
Tray diameter 5.0 cm
Inlet-Tray gap 15 cm
input 30 g
[150]
Table 4 is a table showing the measurement conditions of the fluidity meter.
[151]
Fluidity = [(weight of powder input - weight of powder remaining in tray) / weight of powder input × 100)]
[152]
The larger the result obtained according to the above formula, the greater the liquidity of the crystal.
[153]
Test Example 4.2 Measurement of angle of repose
[154]
Additionally, the effect of increasing fluidity due to ethanol inclusion was confirmed by measuring the angle of repose. The angle of repose means the maximum angle of inclination that can be deposited without flowing down when the sediment, which has not yet been consolidated, is deposited on the slope, and when the angle of repose is small, the liquidity of the crystal is large (FIG. 6).
[155]
The angle of repose was measured with a protractor after passing the D-psicose crystal composition through a funnel installed on a horizontal plate at a constant speed.
[156]
[Table 5]
　 liquidity angle of repose
Example 1 59.7 35
comparative example 48 45
[157]
Table 5 is a table showing the fluidity and angle of repose measurement results. From Table 5, it was confirmed that the fluidity increased in the case of D-psicose to which ethanol was added, and through this, the ethanol-containing D-psicose had a low viscosity and was crystallized in a dispersed state to have a high yield and crystallinity. imply that it can
[158]
[159]
From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.
[160]
[161]
[162]
Claims
[Claim 1]
As a D-psicose crystal, based on 100% (w/w) of the total crystal, 98% (w/w) or more of D-psicose and 0.001 to 0.05% (w/w) of ethanol, D-Psychos Determination.
[Claim 2]
According to claim 1, wherein the average particle size (MA) is characterized in that 200 ㎛ or more, D-psicose crystal.
[Claim 3]
A first step of mixing the D-psicose-containing solution and the organic solvent; and a second step of adding a seed crystal to the mixed solution according to the first step and then cooling to obtain a mask containing the D- between course crystals, D- method for producing crystals between courses.
[Claim 4]
According to claim 3, wherein the mixing of the first step is carried out under 40 to 60 ℃, the final temperature cooled according to the second step is 10 to 20 ℃, D- method for producing a crystal between courses.
[Claim 5]
The method of claim 3, wherein the second step is to adjust the cooling rate to allow the seed crystal to grow within the metastable region section, the method for producing a D-psicose crystal.
[Claim 6]
The method of claim 5, wherein the cooling rate in the second step is carried out in a range of 0.05 to 1.4 °C/hour, D- method for producing crystals between courses.
[Claim 7]
The method of claim 3, wherein the cooling and crystallization in the second step is performed for 20 to 70 hours, D- method for producing crystals between courses.
[Claim 8]
The method of claim 3, wherein the solution containing D- between courses contains 95% (w/w) or more of D- between courses, D- between the crystals.
[Claim 9]
The method according to claim 3, wherein the weight percentage of the final obtained D-psicose crystals relative to the D-psicose content present in the D-psicose-containing solution of the first step is 65% (w/w) or more, D -Method for producing psycho-crystals.
[Claim 10]
The method of claim 3, wherein the organic solvent is alcohol.
[Claim 11]
The method according to claim 3, wherein the organic solvent includes any one or more selected from ethanol, methanol, and propyl alcohol.
[Claim 12]
The method of claim 3, wherein the organic solvent is a mixture of water: organic solvent 1:0.5 or more in the D-psicose-containing solution.
[Claim 13]
The method according to claim 3, wherein the D-psicose-containing solution has a concentration of 80 to 85 brix between D-psicose, D-psicose crystal manufacturing method.
[Claim 14]
The method of claim 3, further comprising a third step of separating and drying the D- between the crystals from the mask, D- between the crystals.
[Claim 15]
The method according to claim 14, further comprising a fourth step of reusing the organic solvent as the organic solvent of the first step after recovering the organic solvent from the crystal mother liquid from which D-psicose crystals are separated according to the third step, Method for producing D-psicose crystals.
[Claim 16]
15. The method of claim 14, further comprising a fifth step of reusing the crystal mother liquid in the preparation of a D-psicose-containing solution after the organic solvent is removed from the crystal mother liquid from which the D-psicose crystals are separated according to the third step. The method for producing a crystal between D-psicose.
[Claim 17]
The method of claim 14, wherein the organic solvent concentration of the crystals between D- by the drying is adjusted to 0.05% (w/w) or less, the method for producing the crystals between D-.