KR20160062349A - A method of manufacturing high purity d-psicose - Google Patents

Abstract

The present invention relates to a method for producing D-psicose having high purity of 99% (w/w) or higher. The method comprises the steps of: preparing a fructose solution for an enzyme reaction; performing an enzyme reaction on the fructose solution, performing heat exchange cooling on a D-psicose solution, and performing ion refinement on the D-psicose solution; concentrating the refined D-psicose solution having impurity removed therefrom, performing heat exchange cooling on the refined D-psicose solution, and performing continuous chromatography separation on the refined D-psicose solution; performing heat exchange cooling on a continuous chromatography separation base solution, and re-circulating the continuous chromatography separation base solution within a procedure; concentrating the D-psicose solution obtained after the continuous chromatography separation, performing heat exchange cooling on the D-psicose solution obtained after the continuous chromatography separation, and crystallizing the D-psicose solution obtained after the continuous chromatography separation; and performing heat exchange cooling on a crystallization separation base solution, and re-circulating the crystallization separation base solution within a procedure. According to the method for producing D-psicose of the present invention, a temperature is controlled by using a heat exchanger during a procedure, so D-psicose is prevented from being thermally deformed and D-psicose may be produced at a high yield through a stabilized scheme. Moreover, a continuous chromatography separation fructose base solution and a crystallization separation base solution are collected and fed during an enzyme reaction procedure, so D-psicose may be stably separated through continuous chromatography despite long-term recirculation thereof. Accordingly, a high production yield of D-psicose may be ensured.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing high purity D-psicose,

The present invention relates to a process for preparing D-psicose, and more particularly to a process for producing high-purity D-psicose at a high yield without heat denaturation during the production process.

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-cicles, it is required to prepare crystallization through high-purity separation using continuous chromatography.

In the case of a single process (see FIG. 2) in which the mother liquor recovered from the production of D-psicose is not reused, the conversion rate of the enzyme reaction of D-psicose produced from fructose is 20 to 30% (w / The amount of mother liquor generated relative to the amount of product produced in the course, that is, the amount of fructose mother liquor and crystallization liquor mother liquor separated by continuous chromatography, is high. As a result, the manufacturing cost is increased due to the increase of the unit cost due to the decrease of the D-Sicose production, which is an uneconomical problem in the industrial production.

In order to reduce the rise of the unit level due to the low water content as described above, it is necessary to produce a high yield of D-Sicose. To produce such a high yield of D-Sicose, continuous chromatographic separation (See FIG. 1) of the mother liquor for recovering and reusing the mother liquor as an enzyme reaction process. However, when D-Sicose is continuously exposed at a certain temperature, D-Sicose is destroyed by thermal deformation, resulting in a decrease in D-Sicose purity in the manufacturing process flow, (See Figs. 3 to 7).

The decrease in purity due to the thermal deformation of D-cicosso eventually means an increase in the amount of impurities generated, and the impurity accumulation amount in the mother liquor circulation process is increased due to the increase of impurities in this manufacturing process flow, Purity and separation yield are problematic, which causes troubles in the entire process balance. Therefore, it is difficult to produce D-cyoses with high yield.

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

Therefore, in order to produce D-Sicos in high yield, the present invention includes a circulation process of a mother liquor recovering continuous mother liquor and crystallization mother liquor for the production of D-psicose and reusing it as an enzyme reaction process, It is another object of the present invention to provide a method for producing a high-purity D-cucosa at a high yield, including a D-Sikos process without thermal denaturation even in a long-term mother liquor circulating process flow.

In one example of the present invention, there is provided a method for preparing a fructose solution comprising: preparing a fructose solution for an enzyme reaction; Performing an enzymatic reaction on the fructose solution and then heat-exchanging and cooling the D-cycozu solution to purify the ion; Concentrating the purified D-cycozu solution from which the impurities have been removed, subjecting the D-Cycoç solution to heat exchange cooling to separate it by continuous chromatography; Subjecting the continuous chromatographic separation mother liquor to heat exchange cooling to recycle it in the process; Concentrating the D-cicosol solution separated by the continuous chromatography to effect heat exchange cooling to crystallize; And recycling the crystallization-separated mother liquor to heat-exchange cooling and recycling the mother liquor to a high-purity D-psicose having a purity of 99% (w / w) or more.

In another example of the present invention, a high purity D-Sikos is prepared according to the process for preparing the high purity D-Sikos described herein.

According to the process for producing D-psicose of the present invention, heat-denaturation of D-psicose is prevented by controlling the temperature in the process using a heat exchanger, and D-psicos can be produced with high yield by a stabilized process.

Further, by the method for producing D-psicose according to the present invention, the continuous-chromatographed fructose mother liquor and the crystallization separation mother liquor can be recovered and added to the enzyme reaction step, so that even if recirculated for a long period of time, Separation can be performed, and a high production yield of D-Sicose can be realized.

Fig. 1 is a schematic view showing a process of a D-psicose production process according to the present invention.
Fig. 2 is a schematic view showing a single process without re-use of the mother liquor recovered in the D-psicose production process.
3 to 7 are graphs showing the change in the% (w / w) purity of D-psicose according to the temperature and the concentration of D-psicose.

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: preparing a fructose solution for an enzyme reaction; Performing an enzymatic reaction on the fructose solution and then heat-exchanging and cooling the D-cycozu solution to purify the ion; Concentrating the purified D-cycozu solution from which the impurities have been removed, subjecting the D-Cycoç solution to heat exchange cooling to separate it by continuous chromatography; Subjecting the continuous chromatographic separation mother liquor to heat exchange cooling to recycle it in the process; Concentrating the D-cicosol solution separated by the continuous chromatography to effect heat exchange cooling to crystallize; Dissolving the mother liquor and heat-exchanging and cooling the crystallization-separated mother liquor, thereby producing a high-purity D-psicose having a purity of 99% (w / w) or more.

The step of preparing the fructose solution for the enzyme reaction may include dissolving fructose at 30 to 50 brix (%) at a temperature of 30 to 40 캜. More specifically, the step is a step of mixing the recovered crystallization mother liquid with the recovered crystallization mother liquor at 30 DEG C and the recovered crystallized fructose of at least 99% (w / w) To 50 brix (%) in an enzyme reaction.

In the step of performing ion-purification by heat-exchanging and cooling the D-Sucose solution after the enzymatic reaction of the fructose solution, the enzyme reaction specifically includes a strain of the genus Corynebacterium expressing D- The enzyme may be one which is capable of converting a fructose into a cyclosporin. The term 'cyclosporidase' may refer to a cyclosporin-3-epimerase having an activity of converting fructose into a cyclosporine.

The heat-exchange cooling of the enzyme-treated D-cytoplasmic solution is carried out by cooling the solution at 40 ° C at a rate of 5 to 10 ° C per hour through a heat exchanger to obtain SV (Space Velocity: flow rate (L) / hour (L)] 0.5 to 3.

The enzyme-reacted D-Sicose solution can be ion-purified by chromatography through a column packed with strongly acidic cation exchange resin and a column packed with weakly basic anion exchange resin, and strongly basic anion resin The use of 100% weakly alkaline anionic resin can be utilized at a low temperature of 30 to 40 DEG C in order to produce a high yield of D-Sicos because D-Sicose is denatured at a low temperature of 30 to 40 DEG C when used .

The step of performing the enzyme reaction and the heat exchange cooling to purify the ions may be specifically as follows: D-Sicosol solution is prepared by dissolving D-psicose epimerase An enzyme isolated from the cells or cells obtained by culturing Corynebacterium glutamicum KCTC 13032 was immobilized on a immobilized carrier such as sodium alginate and then immobilized on a immobilized carrier such as sodium alginate, The solution was heated to a temperature of 50 ° C at a rate of 5 to 20 ° C per hour through a heat exchanger to the substrate to obtain a 100% weakly alkaline anion at a SV (Space Velocity: flow rate (L) / hour (Hr) / resin amount Can be obtained by using a resin.

In the step of concentrating the purified D-cytochrome solution from which the impurities have been removed and then conducting a heat exchange cooling of the D-cytochrome solution to perform continuous chromatography separation, specifically, the step of concentrating the purified D- The D-psicose solution was put into a low-temperature concentrator and concentrated in a short time at a temperature of 65 to 75 ° C for 10 to 15 minutes to adjust the concentration to 60 brix (%) (D-Sicose solution × 100 / total solution) .

The concentrated D-cicosol solution is heat-exchanged and cooled and separated by continuous chromatography. The column is cooled at a temperature of 5 to 25 ° C per hour through a heat exchanger, and a column packed with a strong acidic cation- To a purity of 90 to 95% (w / w) or more by D-psicose.

The step of heat-exchange cooling and recycling the continuous chromatographic separation mother liquor may include recycling the separated mother liquor to the enzyme reaction process at 30 < 0 > C through a heat exchanger at 20 to 30 [deg.] C / hour.

Specifically, the separation mother liquor may be one in which fractions of fructose separated by the continuous chromatography are recovered and used.

In the present invention, there is an advantage that the production yield of D-Sicose can be increased by including the step of re-introducing the separated mother liquor into the enzyme reaction process.

In the step of concentrating the D-Psychosis solution separated by the continuous chromatography and performing heat exchange cooling to crystallize, the concentration may include concentration at a temperature of 65 to 75 ° C for 10 to 15 minutes.

Further, the step of cooling and crystallizing by heat exchange may be performed by rapidly cooling the mixture at 30 to 40 ° C at a temperature of 5 to 20 ° C per hour through a heat exchanger, and then heating and cooling are repeatedly performed 5 to 10 times within a temperature range of 30 to 40 ° C. To induce crystal growth for 80 to 120 hours.

The step of heat-exchange cooling the crystallization separation mother liquor and recirculating the recovered mother liquor to the enzyme reaction process at 30 ° C by cooling the recovered mother liquor through a heat exchanger at 20 to 30 ° C per hour.

In the present invention, not only the continuous chromatographic separation mother liquor but also the crystallization separation mother liquor can be re-introduced into the enzyme reaction process, thereby maximizing the production yield of D-psicose and reducing the cost of production of D-psicose.

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  One

D-Sicose was prepared according to the method shown in Fig.

(B) 50% by weight (bp) of the crystalline fructose with a purity of 99% (w / w) or more and the recovered 30 ° C continuous chromatography column and the recovered 30 ° C crystallization mother liquor and 30 ° C water. To prepare an enzyme reaction substrate solution.

The enzyme reaction is carried out by fermentation of Corynebacterium glutamicum KCTC 13032 and the above-mentioned microorganism or D-Sucoschis epimerase isolated therefrom on a sodium alginate carrier as disclosed in Korean Patent Application No. 10-2009-0118465 The enzyme reaction substrate solution prepared above was heated to 50 ° C. at a rate of 5 to 20 ° C. per hour through a heat exchanger to obtain SV (Space Velocity: flow rate (L)), / Hour (Hr) / resin amount (L)] of 0.5, and the purity of D-Cyoses was about 24% (w / w).

The D-Sicose solution having a purity of 24% (w / w) was cooled through a heat exchanger at a temperature of 30 to 40 ° C at a rate of 5 to 10 ° C per hour, and then a strongly acidic cation exchange resin (Bayer S1668) (L) / hour (Hr) / resin (L)] 3 in a column packed with a weakly basic anion exchange resin (Bayer Bayer S4528) The removal of the ion components was confirmed to be less than 10 micro-millimeters per cm 2 by measuring the electrical conductivity. The purity of D-Sicose of the purified enzyme reaction solution was maintained at 24% (w / w) .

The purified D-cycozu solution was put into a low-temperature concentrator (Forced Thin Film Evaporator, Welkornheim) and concentrated in a short time at a temperature of 65 to 75 ° C for 10 to 15 minutes to adjust the concentration to 60 brix (D-Psychosolution solution × 100 / total solution), cooled at 5 to 25 ° C. per hour through a heat exchanger, and then subjected to continuous treatment in a column packed with a strong acidic cation-exchange resin having a calcium active group at 60 ° C. The fractions having a purity of 95% (w / w) or more and a purity of fructose of 95% (w / w) or more were separated by means of a chromatography.

The separated mother liquor, that is, the fructose fraction having a purity of 95% (w / w) or more in the continuous chromatography was recovered, cooled at 20 to 30 ° C per hour and recycled to the enzyme reaction process at 30 ° C.

The D-Sicosol solution having a purity of 95% (w / w) or more separated in the continuous chromatography was concentrated in a short time for 10 to 15 minutes at a temperature of 65 to 75 캜 to adjust the concentration to 80 brix (%), The D-Psychosolution solution having a concentration of 95% (w / w) or more of purity is rapidly cooled through a heat exchanger at 5 to 20 ° C at a temperature of 40 ° C, and then heated and cooled at a temperature of 30 to 40 ° C for 5 to 10 And crystallization was carried out for 80 to 120 hours so as to obtain D-Sicose. Further, the mother liquor separated from the crystallization, that is, the D-psicose fraction having a purity of 90% or more was recovered and recycled to the enzyme reaction process at 30 ° C.

Through the above process, a high-purity D-Sikosu of 99% or more, which is superior in production yield by more than 75%, can be obtained by controlling the temperature in the process at a low temperature by using a heat exchanger while concentrating in a short time in a low temperature thickener .

Comparative Example  One

The procedure of Example 1 was repeated except that the process of cooling with a heat exchanger was omitted in Example 1 to produce a high-purity D-psicose having a purity of 99% or more. It was confirmed that the yield was 60% there was.

This is because the D-psicose has a characteristic that the purity decreases as a certain concentration and a certain period of time elapse (see Figs. 3 to 7), so that the continuous chroat separation without the cooling heat exchange step in the process When the number of times is continuously accumulated, the purity of D-Sycorose added to the continuous chromatography is decreased in proportion to the number of repetitions at the initial 24% (w / w) and 95% (w / (W / w) or less. The low recovery rate of the high purity D-psicose of more than 95% (w / w) is a cause of the decrease in the yield of high purity D-psicose production with a purity of 99% or more . In addition, since the denatured impurities derived from D-psicose are separated into fructose fractions in the continuous chromatographic separation step, as the number of repetition cycles in the process accumulates, problems arise in continuous chromatographic separation and control of accumulated impurities About 5 to 10% (v / v) of the separated fructose fraction is discarded, resulting in a reduction in the yield of high-purity D-psicose production of 99% or more in purity.

Continuous Chromatography Separation When the d-Sikosu purity of the stock solution of continuous chromatography is continuously decreased by the accumulation of impurities when about 5 to 10% (v / v) of fructose mother liquor is not discarded, 95% (w / w) separating program conditions for continuous chromatographic separation for high-purity D-Cycose separation of more than 95% (w / w) in continuous chromatography, The amount of deionized water used for the separation of D-psicose also increases, resulting in a load of concentration process, which causes a bottleneck in the process flow, resulting in a decrease in production amount and an increase in manufacturing cost.

Comparative Example  2

The production process was performed in the same manner as in Example 1, except that the step of recovering and reusing the mother liquor was omitted according to the method shown in FIG. 2 to produce a high-purity D-psicose having a purity of 99% or more. The yield was found to be 20%.

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 (10)

Preparing a fructose solution for an enzyme reaction; Performing an enzymatic reaction on the fructose solution and then heat-exchanging and cooling the D-cycozu solution to purify the ion; Concentrating the purified D-cycozu solution from which the impurities have been removed, subjecting the D-Cycoç solution to heat exchange cooling to separate it by continuous chromatography; Subjecting the continuous chromatographic separation mother liquor to heat exchange cooling to recycle it in the process; Concentrating the D-cicosol solution separated by the continuous chromatography to effect heat exchange cooling to crystallize; And recrystallizing the crystallization separation mother liquor by heat exchange cooling to recycle in the process. The method according to claim 1, wherein the step of preparing the fructose solution for the enzyme reaction comprises the step of separating the supernatant fraction having a purity of 90% (w / w) or more and the crystallization separation mother liquid having a purity of 90% ) Of D-Cycose fraction is prepared by mixing with fructose at a concentration of 30 to 50 brix (%) at a temperature of 30 to 40 占 폚. The method of claim 1, wherein the step of subjecting the fructose solution to an enzyme reaction followed by heat-exchange cooling the D-cycozu solution to perform ion purification is performed at a temperature of 30 to 40 ° C. The process according to claim 1, wherein the step of concentrating the purified D-cicosol solution followed by heat-exchange cooling the D-cicosol solution to perform a continuous chromatographic separation is carried out at a temperature of 55 to 65 ° C. ≪ / RTI > 2. The process according to claim 1, wherein the step of heat-exchanging and cooling the separation mother liquor of the continuous chromatography is heat-exchange-cooling a fructose fraction having a purity of at least 90% (w / w) ≪ RTI ID = 0.0 > D-cyacose < / RTI > The method according to claim 1, wherein the step of concentrating the D-Psychosolution separated by the continuous chromatography to perform heat exchange cooling and crystallization comprises concentrating the D-Psychosis solution having a purity of at least 95% (w / w) And crystallizing the resulting mixture at a temperature of 30 to 40 占 폚. The process according to claim 1, wherein the step of heat-exchanging and cooling the crystallization separation mother liquor to recycle in the process comprises heat-exchanging and cooling a D-Sycose fraction having a purity of at least 90% (w / w) ≪ RTI ID = 0.0 > recycling < / RTI > 8. The process according to any one of claims 1 to 7, wherein the process is a process for preparing D-cicles at a purity of at least 99% (w / w). 8. A process for preparing D-cyoses, which is prepared according to the process of any one of claims 1 to 7. 10. The D-psicose according to claim 9, wherein the D-psicose has a purity of at least 99% (w / w).

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