KR20170005502A - A method of manufacturing a d-psicose crystal - Google Patents

Abstract

The present invention relates to a method for producing high purity D-psicose crystal of which purity is greater than or equal to 98% (w/w) and the particle diameter is greater than or equal to MA 200. To this end, the method for producing high purity D-psicose crystal comprises the following steps: acquiring a purified D-psicose solution by removing impurities from a D-psicose solution; concentrating the purified D-psicose solution; cooling the concentrated D-psicose solution down to 30-40C using a heat exchanger; obtaining masket after seed-crystalizing the D-psicose solution at 30-40C; and crystalizing using the seed-crystalized masket. The use of the production method of the present invention enables the production of D-psicose crystal which is pure and suitable for industrial applications through an economic crystallization process using the D-psicose solution without organic solvents.

Description

A METHOD OF MANUFACTURING A D-PSICOSE CRYSTAL [0002]

The present invention relates to a process for preparing high-purity D-Cycose crystals using supersaturation from a D-Sicose solution.

D- psicose is not being metabolized in the human body, unlike fructose or sugar has virtually no calories and, in inhibiting fat formation reported in less impact on weight gain sweeteners (Matuo, T. et. Al .. Asia Pac . J. Clin . Untr ., 10, 233-237, 2001; Matsuo, T. and K. Izumori, Asia Pac . J. Clin . Nutr ., 13, S127, 2004).

Recently, the present inventors have reported a method of economically producing D-psicose by reacting glucose with isobutylated fructose and then reacting it with immobilized cells producing D-psicose epimerase (Korean Patent Application No. 10-2009 -0118465). Since the reaction solution containing D-cyclos produced by the enzyme reaction is a low-purity product containing about 20 to 30% (w / w) of D-psicose solid content, In order to prepare D-Cycose crystal grains, it is required to produce crystallization by high purity separation using chromatography. However, up to now, in case of D-Sikos, a high purity crystallization product of 98% (w / There is no.

In order to prepare D-psicose as a crystalline form, a method of fermenting unreacted fructose in the reaction solution of D-psicose by yeast and using a large amount of ethanol has been reported (Kei T, et al., J Biosci. Bioeng., 90 (4), 453-455, 2000). However, since this method completely removes unreacted fructose from the process stream by yeast fermentation, it can not be expected to reduce the manufacturing cost such as by-product deductions using unreacted fructose. The addition of a large amount of ethanol used in the crystallization production leads to an increase in the raw material cost In addition, a distillation type ethanol recovery facility and explosion proof design are required to recover the added ethanol, so that the cost of D-SiCoC manufacturing increases. Therefore, industrialization technology for mass production is limited due to high manufacturing cost. Also, the use of a large amount of ethanol in the D-Sycocoronian crystallization method has a limitation of the technology in the material suitability because the ethanol product remains in the final product and the flavor has a low preference in the market.

Generally, the operation methods in the crystallization industry of saccharides are classified into two types. One is the concentrated crystallization method and the second is the cooled crystallization method. Both methods use the principle of inducing the growth of crystals in the supersaturated and metastable zones by the crystallization method of saccharides. A concentrated crystallization method is used for the crystallization of a saccharide having a high crystal growth rate, such as sucrose, and a cooling crystallization method is generally used for crystallization of a saccharide whose growth rate is significantly lower than that of sucrose.

In addition, conventionally, the method of crystallization of saccharides is performed in a metastable zone, which means that the concentration of the solution ranges from the equilibrium concentration, that is, the range from the saturation concentration to the lowest supersaturation where the crystals are spontaneously precipitated from the saturated concentration. Crystallization such as crystal nucleation does not occur in the concentration of this region, but when a new crystal is added from the outside, crystal growth occurs and crystal size increases. That is, when a seed is added to a solution having a saturation concentration or higher to produce crystals, seed crystals grow in the metastable zone to grow crystals. If the solution for crystallization is excessively concentrated or abruptly cooled, it becomes a supersaturated state exceeding the metastable zone, so that new crystal nuclei are generated instead of crystal growth, which is an inhibiting factor of crystal growth due to the increase of the population Temperature conditions and initial entrainment saturation concentrations are important for crystal growth.

D-Sicose exhibits almost no change in crystal formation rate and crystal growth rate even in the supersaturation concentration range, so that it can be classified as a sugar having difficulty in grain growth crystallization conditions. In general, the size of crystal grains in the sugar crystallization industry is known to be an important factor. When crystals produced in a mass production system are in the form of atomized crystals, separation of crystals and mother liquor in a crystal centrifuge apparatus is facilitated due to the viscosity of the supersaturated concentration section The purity of the final product is lowered due to the residual mother liquor, and the residual mother liquor is aggregated between the crystals when dried, resulting in a reduced amount of the final product or lowering the merchantability. Therefore, such fine particle crystals are not suitable as a mass production method.

Korean Patent Application Publication No. 2011-0035805 (Published April 4, 2011)

Accordingly, it is an object of the present invention to provide a method for obtaining a high-purity D-Cyclosporin separation liquid which can be reused without removing unreacted fructose from the process stream by using continuous chromatographic separation, without using yeast fermentation. In addition, the high-purity D-SiCoCo separation liquid obtained can be used to grow crystal grains without using an organic solvent such as ethanol, thereby facilitating separation of crystals and mother liquor, thereby minimizing separation, And a method for producing the same.

In addition, the present invention provides a D-Sycocoridation crystallization method which reduces the manufacturing cost by reducing the variable and fixed costs, and provides a 98% (w / w) solution having a crystal grain size of MA200 or more improved in flowability of the crystallization- ) ≪ / RTI > of high-purity D-psicose.

In one example of the present invention, the step of removing impurities from the D-Sicose solution to obtain a purified D-Sicose solution; Concentrating the purified D-cytochrome solution; Cooling the concentrated D-cycozu solution to 30 to 40 占 폚; Terminating the D-Psycose solution at 30 to 40 占 폚 to obtain a mask; Purity D-psicose crystals having a purity of 98% (w / w) or more and a particle size of MA 200 or more, comprising the step of crystallizing the crystals using the final purified mascot.

By using the production method of the present invention, it is possible to produce D-Sycose crystals which are pure and suitable for industrial application through an economical crystallization process without using an organic solvent from D-Sicose solution.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the change in thermal denaturation purity with temperature in an 80 Brix (%) solution of D-Sicose.
Fig. 2 is a graph showing crystal grain size of D-cyucose prepared by seed crystal.
FIG. 3 is a graph showing crystal grain size of D-Sicose prepared by the present crystal.

Hereinafter, the present invention will be described in more detail. Those skilled in the art will appreciate that various modifications, additions, and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

One example of the present invention comprises the steps of: removing impurities from a D-cycozu solution to obtain a purified D-cycozu solution; Concentrating the purified D-cytochrome solution; Cooling the concentrated D-cycozu solution to 30 to 40 캜, preferably 35 to 40 캜, through a heat exchanger; Terminating the D-Psycose solution at 30 to 40 占 폚 to obtain a mask; To a process for producing high purity D-Sikos crystals having a purity of 98% (w / w) or more and a particle size of MA 200 or more, comprising the step of D-cyclosporin crystallization using the above-mentioned purified pelletized masks.

The D-psicose solution used in the present invention may be one produced by a strain of Corynebacterium sp. Expressing D-psicose epimerase from fructose or a psicose epimerase isolated therefrom. The term " psicose epimerase " means a psicose-3-epimerase having an activity of converting fructose into psicose.

As a non-limiting example, the D-cytochrome solution may be a bacterial cell obtained by culturing Corynebacterium glutamicum KCTC 13032 producing D-cicoslo epimerase, as described in Korean Patent Application No. 2009-0118465 Immobilized on an immobilized carrier such as sodium alginate, and then supplying fructose to the substrate.

To obtain D-cicosol crystals from the D-psicose solution, purification of D-psicose and other materials that may affect crystallization must be removed to form the necessary state for efficient crystallization. Thus, a method for preparing D-psicose crystals according to the present invention can include the step of removing impurities from a D-psicose solution to obtain a purified D-psicose solution.

Specifically, the step of obtaining the purified D-psicose solution comprises: passing the D-psicose solution through a column filled with a bleaching agent to decolorize the solution; Desalting the decolorized D-cytochrome solution by ion exchange resin chromatography; And passing the desalted D-cycozu solution through continuous chromatography packed with an ion exchange resin with a calcium activator to obtain a purified D-Cycozu solution.

The step of passing the continuous chromatography may be a simulated moving bed (SMB) process.

More specifically, the step of desalting the D-Sicose solution may be carried out by chromatography through a column packed with a cation exchange resin, and a column packed with an anion exchange resin. In the case of using a strong basic anion resin in the ion exchange resin purification method for preparing the D-Cyclo purified liquid, the purity is lowered due to denaturation of D-cyoses. Therefore, in order to prepare a high purity D- . Modification of D-Cycose is also confirmed in a mixed resin of a strongly acidic cationic resin and a strongly alkaline anionic resin used in a conventional saccharide purification. Therefore, in the step of desalting the D-Sicose solution, 100% weakly alkaline anionic resin should be utilized to effect effective desalting purification without denaturing of D-Sicose.

Separation by continuous chromatography can also be carried out in order to purify high-purity D-Cycose. The content of D-cyucose in the solution of D-psicose to obtain D-psicose crystals should be 90 to 95% or more, more preferably 95% or more. Direct crystallization can not be performed because the purity of D-cyucose in the D-cytochrome solution prepared by D-psicose epimerase is as low as 24% (w / w). In order to obtain high-purity D-Cycose crystals, the impurities are removed by decolorization and desalting before the crystallization step and purified by continuous chromatography, that is, chromatography on a column packed with an ion exchange resin to which a calcium activator is attached, Can be isolated and purified.

The method of producing D-psicose crystals according to the present invention is characterized in that 80 to 85 Brix (%) of D-psicose crystals are produced, unlike the method of gradually cooling from a high temperature to a constant temperature section within a supersaturation concentration range, The D-Sicose solution concentrated to a supersaturation concentration range (D-Sicose solution × 100 / total solution) was rapidly cooled through a heat exchanger at a temperature of 30 to 40 ° C at 5 to 20 ° C per hour, use. When cooled to a temperature of 30 to 40 占 폚, thermal denaturation of the D-Sicose solution concentrated to a supersaturation concentration range of 80 to 85 Brix (%) (D-Sicose solution × 100 / whole solution) The purity of the D-psicose is preserved, so that the crystal yield and particle size can be improved during the crystallization operation. In order to transfer the D-SiCoCo solution concentrated to a supersaturation concentration range of 80 to 85 Brix (%) in a concentration apparatus (D-SiCoCo solution × 100 / whole solution) to the crystallization apparatus, 20 to 100% v) is concentrated in the intermediate storage tank, and the temperature is 60 to 75 ° C, which corresponds to the heat denaturation temperature of D-Sicose.

Specifically, the crystallization time of the D-psicose should be 80 to 120 hours or more, and the concentrated D-psicose solution is rapidly heated at a temperature of 30 to 40 ° C at 5 to 20 ° C per hour through a heat exchanger within a supersaturation concentration range , And the mixture is put into a crystallization apparatus. The temperature is raised and cooled 5 to 10 times in the crystallization temperature range of 30 to 40 占 폚 to crystallize the D-psicose. That is, a D-Sicose solution of 30 to 40 캜, which is concentrated to a supersaturation concentration range of 80 to 85 Brix (%) at 5 to 20% (v / v) of the crystallization apparatus working amount, The temperature was allowed to circulate within the range of 30 to 35 ° C and the D-Sycorid seeds were added at a rate of 10 to 100 ppm (v / v) based on the initial charge, Lt; / RTI > In the present invention, the term "mask" means a slurry state in which a crystal and a solution are mixed with each other when the D-cyclosporin starts the crystallization reaction.

Further, in order to induce the growth of the D-psicose crystal, a supersaturation concentration range of 80 to 85 Brix (%) or more in a range of 5 to 20% (v / v) And the D-Sicose solution at 30 to 40 캜, which is concentrated at a temperature of 30 to 40 캜, is repeated twice or more, specifically 5 to 10 times. Times, specifically 5 to 10 times, so as to induce D-SiCoCo crystal growth for 80 to 120 hours.

The D-psicose crystals obtained by the production method of the present invention may specifically have a crystal size of MA 200 or more, more specifically, a crystal size of MA 300 or more.

The grain size is a measure of the average size of the crystals. In JIS and ASTM, the number of crystals contained in a square area of 25 mm on one side is measured with a microscope photo magnified 100 times, and the number is referred to as a grain size number and indicates the grain size. In recent years, it has also been used to mean the average size of crystals. In the present invention, the mean size of crystals was used.

The method of measuring the crystal grain size of D-psicose is not limited and a method commonly used in the art can be used. Non-limiting examples of measuring grain size include the comparison method (FGC), the cutting method (FGI), and the flat method (FGP).

In order to obtain D-SiCoOy crystal having a crystal grain size of MA 200 or more, it is necessary to proceed to the second stage of the final crystallization and the main crystallization under the same conditions as above, and the final purification is carried out at 5 to 20% (v / v) . The grain size of the final purification produced in the present invention is in the range of MA 100 to MA 150, and the thus-produced seed crystal mass is transferred to the present crystallization apparatus, and the crystallization is proceeded by the above- (W / w) or more having a crystal grain size of MA 300 or more can be produced.

In the crystallization apparatus of the present invention, conventionally used saccharide crystallization apparatus can be used as it is or modified appropriately, and a device for separating mother liquid from high purity D-psicose crystals from a final crystal mask, Or may be suitably modified and utilized.

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to preferred embodiments of the present invention. It is to be understood, however, that the same is by way of illustration and example only and is not to be construed in a limiting sense.

The contents not described here are sufficiently technically inferior to those skilled in the art, and a description thereof will be omitted.

Example

Manufacturing example

As disclosed in Korean Patent Application No. 10-2009-0118465, the fermentation of Corynebacterium glutamicum KCTC 13032 and the fermentation of D-fucose by a carrier immobilized on the microorganism or D-cicoslo epimerase separated therefrom -Cyclo-D-glucitol, < / RTI > The purity of D-Sicose prepared by this method was about 24% (w / w) and was low enough to be directly crystallized. The resulting D-cycozu solution was concentrated to 50 Brix (%), and passed through a decolorizing column filled with granulated active carbon to remove the colored material in the concentrate.

Example 1

The purity of the resultant solution obtained in the above Preparation Example is 24% (w / w) and the purity of the crystallized D-Sycose should be increased to 90 to 95% (w / w) or more for crystallization. For efficient separation of D-cyucose by continuous chromatography, the ions in the D-Cypro solution should be removed. If the ion component is present in the solution to be separated, the ionic component and the active agent of the separation resin are substituted, and the separation efficiency is lowered. Therefore, it is impossible to carry out continuous separation of the D-psicose with a purity of 90 to 95% (w / w) or more using the separation resin.

Thus, a strongly acidic cation exchange resin (Bayer S1668) substituted with a hydrogen group and a weakly basic anion exchange resin (Bayer Bayer S4528) substituted with a hydroxyl group were prepared by dissolving a D-psicose solution of 24% (w / The packed column was passed through to remove ionic components in the solution. Confirmation of ion component removal was measured with an electric conductivity meter and adjusted to be less than 10 micro-millimeters per unit cm, and the purity of D-Sicose was maintained at 24% (w / w). In the case of weakly basic anion exchange resins, there is a strong basicity characteristic, so 100% weakly basic anion resin which does not have strong basic properties should be used in order to proceed desalination without loss due to decrease in purity of D-Sicose .

Example 2

In Example 1, a 24% (w / w) purity D-CyCos solution was obtained by removing impurities such as coloring matter and ion components by decolorizing and desalting, and concentrated to 60 Brix (% Was passed through an ion-exchange resin (Amberlite CR1310Ca) substituted with a group to obtain a purified D-cycozu solution. The amount of the ion-exchange resin filled in the ion exchange resin column was 10 L, and the amount of the purified D-Sikos solution prepared in Example 1 was 2 L / cycle and the operation temperature was 60 ° C. After the sample was injected and eluted with 5 L / cycle of deionized water, the purity of the obtained D-cyoses was 95% (w / w).

Example 3

A solution of 95% (w / w) purity D-psicose purified by Example 2 was obtained and concentrated to 80.0 Brix (%). The solution was rapidly cooled at a rate of 10 DEG C per hour through a heat exchanger, cooled to 40 DEG C and then charged with 20% (v / v) of the crystallization apparatus operation amount of the D- C, and the prepared D-psicose seed is introduced at the initial dose of 100 ppm (v / v) to start the D-psicose final purification reaction. In order to induce the growth of D-psicose crystals, a D-Sikos solution at 40 ° C was added at a concentration of 80.0 Brix (%) at 20% (v / v) , So that the temperature inside the crystal muscle is repeatedly raised and cooled four times within the range of 35 to 40 ° C. Finally, the D-psicose seed crystal reaction is performed for 60 hours from the start of the crystallization. The purity of the final D-psicose crystals was 99.4% (w / w) after separating, drying and fractionating the D-psicose cleansing mace by using a high-purity D-psicose crystal and a crystal centrifugal separator through the mother liquid separation. w), the particle size was MA 135.5, and the yield was 35.2%.

Example 4

D-Cyclosporin crystallization is carried out with the D-SiCOSO final purification mask prepared in Example 3 above. After the D-Sikos final cleansing mask was introduced at 20% (v / v) of the crystallization apparatus operation amount, the cooling water temperature of the crystallization apparatus was circulated at 35 ° C, and the purified purity of 95% The solution was concentrated to 80.0 Brix (%) through a heat exchanger and rapidly cooled to 40 DEG C at a rate of 10 DEG C per hour. The solution was simultaneously supplied at 20% (v / v) The total amount of the present crystallization is disclosed as 40% (v / v) of the crystallization apparatus operation amount. In order to induce the growth of the D-psicose crystal, an additional 40 ° C D-Sicose solution concentrated at 80.0 Brix (%) at 20% (v / v) Is repeated three times so that the temperature inside the crystal muscle is repeatedly raised and cooled three times within the range of 35 to 40 ° C. Finally, the D-cicosane-based crystallization reaction is performed for 80 hours from the start of the crystallization. The purity of the final D-psicose crystal was 99.8% (w / w) after separating, drying and fractionating the high-purity D-psicose crystal and the mother liquid using a crystal centrifugal separator through separation of the mother liquid. w), the particle size was MA 373.9, and the yield was 52.8%.

Comparative Example 1

(Bayer S1668) substituted with a hydrogen group and a weakly basic anion exchange resin (Bayer S4268) substituted with a hydroxyl group were charged in a 24% (w / w) purity solution of the decolorized D- After passing through the column, the ion component in the solution was removed through an ion exchange column (Bayer NM60) in which a strongly acidic cation exchange resin and a strong basic anion exchange resin were mixed and filled in the final stage. Confirmation of removal of the ion component was measured with an electric conductivity meter, and the purity of D-Sicose was reduced to 21.2% (w / w).

Comparative Example 2

A strong acidic cation exchange resin (Bayer S1668) substituted with a hydrogen group and a weakly basic anion exchange resin (Bayer S4268) substituted with a hydroxyl group in a 24% (w / w) purity solution of D- The packed column was passed through to remove ionic components in the solution. Confirmation of removal of ion components was measured with an electric conductivity meter, and the purity of D-Sicose was reduced to 22.8% (w / w) at a rate of 10 micro-millimeters or less per unit cm.

Comparative Example 3

A 95% (w / w) purity 95% (w / w) solution of D-psicose purified by Example 2 was obtained and concentrated to 85 Brix (% Crystallization was carried out by cooling the D-Sikos solution temperature from 50 ° C to 35 ° C at 0.31 ° C for 48 hours. Crystallization was carried out using a crystal centrifugal separator through separation of high purity D- As a result, the high-purity D-Sikos crystals and the mother liquor were not separated in the final determination mask.

Comparative Example 4

(W / w) purity 95% (w / w) in the crystallization apparatus after concentrating it to 82.5 Brix (%) after obtaining a D- The crystallization proceeded by cooling the D-Sikosu solution temperature from 50 ° C to 35 ° C at 0.30 ° C for 50 hours. The crystallization proceeded from the final crystal mask, and a high-purity D-Sikos crystal was separated from the mother liquor using a crystal centrifuge , The purity of the final D-psicose crystals obtained was 98.5% (w / w), the particle size was MA 82, and the yield was 20.9%.

Comparative Example 5

(W / w) purity 95% (w / w) in the crystallization apparatus after concentrating it to 82.5 Brix (%) after obtaining a D- Crystallization was carried out by cooling the D-Sikosu solution temperature from 50 ° C to 35 ° C at 0.15 ° C per hour for 100 hours. Crystallization was carried out using a crystal centrifugal separator by separating the mother liquor from high purity D- , The purity of the final D-psicose crystals obtained was 98.9% (w / w), the particle size was MA 95, and the yield was 24.5%.

Comparative Example 6

(W / w) purity 95% (w / w) in the crystallization apparatus after concentrating it to 82.5 Brix (%) after obtaining a D- Crystallization was carried out by cooling the D-Sikosu solution temperature from 50 ° C to 35 ° C at 0.08 ° C for 200 hours, and the crystal was centrifuged from the final crystal mask through high-purity D- , The purity of the final D-psicose crystals obtained was 97.9% (w / w), the particle size was MA 75, and the yield was 17.2%.

In the above Examples and Comparative Examples, Comparative Example 1 using a strong basic ion exchange resin and Comparative Example 2 using a weakly basic weakly basic ion exchange resin that is not a weakly basic 100% had a purity of D- Low. Also, in Comparative Examples 3 to 5 using a method of gradually cooling from a high temperature to a constant temperature range within a supersaturation concentration range of a conventional saccharide crystallization system, separation of the D-psicose crystals and the mother liquor was not performed, Purity, particle size and yield were all low. From these results, it was confirmed that the D-psicose crystal manufacturing method of the present invention can produce D-psicose which is superior in commerciality to the conventional cooling crystallization method.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

D-Sikos crystals having a purity of 98% (w / w) or more and a particle size of MA 300 or more. The D-psicose crystal according to claim 1, wherein the D-psicose crystal contains no ethanol. The D-psicose crystal according to claim 1, wherein the coefficient of variation (C.V.) of the D-psicose crystal is less than 67.3%. 4. The D-psicose crystal according to claim 3, wherein the coefficient of variation (C.V.) of the D-psicose crystal is about 57.5%. The D-psicose crystal according to claim 1, wherein the D-psicose crystal is formed by forming seed crystals having a size of MA 100 to 150, and crystallizing the seed crystals to a size of MA 300 or more. 6. The D-Sycose crystal according to claim 5, characterized in that, by virtue of said crystallization, the coefficient of variation (C.V.) of the D-SiCoCo crystal is smaller than the coefficient of variation of the termination definition (C.V.). 7. The D-psicose crystal according to claim 6, wherein a variation coefficient (CV) of the D-psicose crystal is less than 67.3% by the main crystallization.

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