KR102055695B1 - Manufacturing method of D-allose crystal - Google Patents

Abstract

The method for preparing D-allose crystals according to the present invention includes the supersaturation of the unstable region after the addition of D-allose seed to the D-allose-containing mother liquor and the final temperature is 5-20 ° C. lower than the initial temperature. Advancing the crystallization reaction at a temperature gradient condition. The method for producing D-allose crystals according to the present invention is suitable for industrialization because it can produce D-allose crystals of high purity and of appropriate level of particle size in high yield. In addition, the D-allose crystal particles produced by the method for producing D-allose crystals according to the present invention have low hygroscopicity and excellent fluidity and are easy to handle.

Description

Manufacturing method of D-allose crystals

The present invention relates to a method for producing D-allose crystals, and more particularly to a method for producing high purity D-allose crystals.

D-allose is the third carbon epimer of glucose (D-glucose) and is known as a rare monosaccharide, an isomer of allulose, also called psocose. Such allose is used as an anticancer substance because it has a property of inhibiting the proliferation of cancer cells, and may also be used as a long-term preservative because it has a function of inhibiting organ damage due to ischemic action. In addition, allose has been reported to exhibit the function of inhibiting the formation of segmented neutrophil leukocytes, as well as the reduction of platelets. Thus, alloose is one of the rare saccharides of current medical interest. (Hossain, MA, Izuishi, K., and H., Maeta. 2003, J. Hepatobiliary Pancreat Surg., 10: 218225 .; Hossain, MA, Izuishi, K., Tokuda, M., Izumori, K., and H. Maeta. 2004, J. Hepatobiliary Pancreat Surg., 11: 181189 .; Hossain, MA, Wakabayashi, H., Izuishi, K., Okano, K., Yachida, S., Tokuda, M., Izumori, K., and H., Maeta. 2006, J. Biosci. Bioeng., 101: 369371.).

Although alloose is highly valuable in the pharmaceutical industry, there is only a very small amount in the natural world, so it is necessary to secure a sufficient amount of alloose in order to be effectively supplied to medical applications. In addition, until now, allose has been produced mainly by chemical synthesis. However, the chemical synthesis method of allose has a number of serious problems in the production process. Indeed, there are risks in the working environment that require high temperatures and pressures, the formation of additional sugars after chemical reactions, the resulting separation and purification of complex alloses, and environmental pollution from the chemical wastes generated.

In order to solve such a problem, a production method for obtaining a high yield of biologically allotropically using an enzyme produced by a microorganism as a method for producing a large amount of a specific sugar with an environmentally friendly and high specificity has been recently attempted. come. Specifically, the Izumori group of Kagawa University, Japan, studied the biological production of allose. Here we report the first production of allose from allulose using Lhamnose isomerase derived from Pseudomonas stutzeri strain. In addition, the Republic of Korea Patent Publication No. 10-2017-0128720 relates to a method for producing alloose biologically using an enzyme is derived from Clostridium papyrosolvens , allulose allulose A novel ribose-5-phosphate isomerase capable of converting to allose and a method for preparing allose from allulose using the same are disclosed, and Korean Patent Publication No. 10 -0896968 discloses recombinant ribose 5-phosphate isomerases derived from Clostridium thermocellum and methods for making alloses from allulose using the same.

On the other hand, since the reaction solution containing D-allose produced by the enzymatic reaction is a low-purity product containing about 20-30% of D-allose, it is required to separate D-allose with high purity. In fact, industrially produced materials are applied in various ways to separate them with high purity, and in the case of saccharides, high purity liquids are mainly made by chromatography, and then crystallized to produce products. In the case of D-allulose (also known as D-psicose), a rare sugar similar to D-allose, Korean Patent Publication No. 10-11896410 passes a D-psychose solution through a column filled with decolorant. Bleaching; Desalting the decolorized D-psicose solution by ion exchange resin chromatography; Passing the desalted D-psicose solution through a column filled with an ion exchange resin attached with a calcium activator to obtain a purified D-psicose solution; Concentrating the purified D-psicose solution; And a metastable region by adding D-psicose seed to the concentrated D-psicose solution at 0.01-1% (g / g) of the total amount of D-psicose in the concentrated D-psicose solution. A method for preparing purified D-psicose crystals is disclosed that includes crystallizing D-psicose in a supersaturated state of the (metastable zone). In addition, Korean Patent Publication No. 10-1749527 includes the steps of removing impurities from the D- psychose solution to obtain a purified D- psychose solution; Concentrating the purified D-psicose solution from 80 Brix (%) to 85 Brix (%); Cooling the concentrated D-psicose solution through a heat exchanger at 30 ° C. to 40 ° C. at 5 ° C. to 20 ° C. per hour; Seeding the D-psicose solution at 30 ° C. to 40 ° C. within a range of 30 ° C. to 40 ° C. to obtain a mask; And main crystallization at 30 ° C. to 40 ° C. using the seed crystallized mask, and a method for preparing high purity D-psicose crystals having a purity of 98% (w / w) or more and a particle size MA 200 or more is disclosed. It is.

However, in the case of D-Allose, there have been no reports of methods for preparing crystals suitable for the level of industrialization.

As derived from the conventional technical background of the present invention, it is an object of the present invention to provide a method for producing high purity D-allose crystals in high yield.

The inventors of the present invention apply a metastable zone, which is a condition adopted in the prior art to crystallize D-allulose in a D-allulose-containing solution, to a D-allulose-containing solution. Although crystals were obtained, the crystal yield was remarkably low, and thus it was difficult to apply this crystallization method to a commercialization level commercialization process, thus completing the present invention.

As used herein, the term "supersaturated state" is an unstable state in which the solute is dissolved beyond the solubility of the solvent, and means a state in which the solute may precipitate as a solid. Therefore, in order to separate the solute in the solution by crystallization, a supersaturated state is necessary. In general, the supersaturation state of the solution may be affected by external conditions, impurities, temperature, concentration, pH, and the like.

As used herein, the term "saturable zone" supersaturated state means a state in which the concentration of the solution is in the range from the saturation concentration to the lowest supersaturation concentration that spontaneously precipitates crystals. At this concentration, crystallization such as crystal nucleation does not occur, but since it is a supersaturated concentration, crystal growth spontaneously occurs when crystals are introduced from the outside, thereby increasing the crystal size. That is, when seed crystals are added to a solution having a saturation concentration or higher to generate crystals, seed crystals grow in the metastable region to form large crystals.

As used herein, the term "unstable zone" refers to a state in which the concentration of the solution is in a range above the lowest supersaturation concentration. At this concentration, spontaneous nucleation occurs.

As used herein, the term "stable zone" means a state in which the concentration of the solution does not exceed the saturation concentration. Crystallization does not occur at the concentration in this region, and remains the same at all times unless there is a change in temperature.

In the present specification, the weight% of the solid concentration unit of the D-allose-containing mother liquor may be used interchangeably with Brix.

The term "crystallinity" as used herein is a value defined in Example 3 described later.

In order to achieve the above object, the present invention adds a D-allose seed to the D-allose-containing mother liquor, and then crystallization reaction under a temperature gradient that reduces the temperature of the allose-containing mother liquor from the initial temperature T _i to the final temperature T _f . It provides a method for producing a D-allose crystals comprising the step of proceeding.

In the present invention, the initial solid concentration of the D-allose-containing mother liquor is preferably 65 to 75% by weight based on the total weight of the mother liquor in consideration of the degree of crystallization to D-allose and the size of the D-allose crystal particles. It is more preferable that it is 66-72 weight%. In addition, the D-allose-containing mother liquor preferably contains 92 to 97% by weight of allose based on the total weight of sugars in the mother liquor, given the degree of crystallization to D-allose and the purity of the D-allose crystal grains. More preferably 93 to 96% by weight of allose. For example, the sugar composition in the mother liquor containing D-allose may be composed of 92 to 97 wt% of allose, 0.5 to 3 wt% of allulose, and 1 to 5 wt% of fructose, based on the total weight of sugars in the mother liquor. It may be composed of 93 to 96% by weight, 1 to 2.5% by weight of allulose and 2 to 4.5% by weight fructose. The D-allose-containing mother liquor used as starting material in the present invention may be prepared by various known methods. For example, the D-allose-containing mother liquor is reacted with fructose syrup with D-psicose 3-epimerase to prepare an allulose-containing solution, and again an allulose-containing solution. Was prepared by reacting with ribose-5-phosphate isomerase or L-rhamnose isomerase to prepare an allose-containing solution, followed by desalting, decolorizing, chromatographic fractionation and concentration. Can be.

In the present invention, the initial temperature T _i for the crystallization reaction is preferably selected from 35 to 55 ℃, preferably from 38 to 52 ℃. When the initial temperature T _i is less than 35 ° C, there is a risk of freezing in the course of the crystallization reaction, low crystallinity may be difficult to apply to industrialization, and the size of D-allose crystal particles is small, such as washing, dehydration and drying. Losses can be significant at post-processing stages. In addition, it may be difficult for the D-allose-containing mother liquor to exist in the supersaturated state of the labile region during the crystallization reaction if the initial temperature T _i exceeds 55 ° C. In the present invention, the final temperature T _f for the crystallization reaction is preferably 5-20 ° C. lower than the initial temperature T _i , and more preferably 8-15 ° C. lower. For example, the final temperature T _f may be selected from 25 ~ 45 ℃ and preferably selected from 28 ~ 42 ℃.

The temperature gradient in the present invention preferably maintains the first stage and D- allo temperature of agarose containing the mother liquor to reduce the temperature of the mother liquor containing D- allo agarose to a final temperature T _f at the initial temperature T _i to a final temperature T _f It may be configured as a second step. In addition, the first step is preferably performed over 24 to 72 hr, more preferably over 40 to 86 hr. In addition, the second step is preferably performed over 10 to 22 hr, more preferably over 12 to 20 hr. In the present invention, the formation of D-allose microcrystals and the growth of D-allose crystals may be performed in harmony through the first and second steps.

In the present invention, the D-allose-containing mother liquor is 1) present in the supersaturated state of the unstable region, or 2) transition from the supersaturated state of the unstable region to the supersaturated state of the metastable region, or 3) the metastable region. It may have three modes, such as transition from the supersaturated state of to the supersaturated state of the unstable region. In the present invention, the mode of the D-allose-containing mother liquor during the crystallization reaction is such as the initial solid concentration of the D-allose-containing mother liquor, the initial temperature T _i for the crystallization reaction and the cooling rate at the temperature gradient for the crystallization reaction, and the like. Can be determined by parameters. When the D-allose-containing mother liquor is in the supersaturated state of the unstable region during the crystallization reaction, large amounts of D-allose microcrystals are formed, and the D-allose-containing mother liquor is in the supersaturated state of the metastable region. -Allose crystals are grown, but not necessarily limited thereto, and the formation of D-allose microcrystals and the growth of D-allose crystals are balanced to satisfy the high crystallinity and the appropriate level of crystal grain size required for industrialization. You can.

The method for producing D-allose crystals according to the present invention may preferably further comprise the step of washing, dehydrating and drying the mother liquor having undergone the crystallization reaction to obtain D-allose crystal particles. The process of washing and dehydrating the mother liquor may be implemented by various known methods such as centrifugation, reduced pressure filtration using filter paper or filter cloth. In addition, the drying process may be implemented by various known methods such as hot air drying, spray dry, fluidized bed drying.

In the case of producing D-allose crystals by the method of the present invention, the crystallinity has a value of 25 to 40%, preferably 27 to 38%. In addition, the size of the D-allose crystal particles produced by the method of the present invention has a value of 100 to 250 µm, preferably 120 to 220 µm. In addition, the D-allose crystal particles produced by the method of the present invention has a purity of 98% by weight or more (for example, 98 to 99.9% by weight) of D-allose, and preferably 98.5% or more (for example, 98.5 to 99.5 weight percent). In addition, the D-allose crystal particles produced by the method of the present invention have low hygroscopicity and do not solidify even after long-term storage and have excellent fluidity.

The method for producing D-allose crystals according to the present invention is suitable for industrialization because it can produce D-allose crystals of high purity and of appropriate level of particle size in high yield. In addition, the D-allose crystal particles produced by the method for producing D-allose crystals according to the present invention have low hygroscopicity and excellent fluidity and are easy to handle.

FIG. 1 shows a stable zone, a metastable zone and an unstable zone according to a saturation curve and a supersaturation curve of a high purity D-allose crystal.
Figure 2 is a graph showing the change in moisture content of storage time of the allose crystal particles obtained in Preparation Example 11 and Preparation Example 12 of the present invention.

Hereinafter, the present invention will be described in detail through the following examples. However, the following examples are only intended to clearly illustrate the technical features of the present invention, and do not limit the protection scope of the present invention.

Example  1: high purity D- Allos  Containing solution, high purity D- Allos  Preparation of Concentrated Concentrates and D-Allose Seed Crystals

(1) Preparation of High Purity D-Allulose-Containing Solutions

High-fructose corn syrup (HFCS; sugar composition: about 56.0% by weight fructose, about 39.4% by weight glucose, about 3.3% by weight maltose, about 1.3% by weight glucose oligomer of polymerization degree 3 or higher) After dilution to 30 Brix, add D-psicose 3-epimerase to the level of about 0.6% by weight relative to the total solids of the high fructose corn syrup dilution and 60 The fructose contained in the diluent of high fructose corn syrup was converted to allulose by reacting for about 12 hr at ℃. D-psicose 3-epimerase used in the embodiment of the present invention is disclosed in Korean Patent No. 10-1473918 owned by the applicant of the present invention. Specifically, the D-psicose 3-epimerase is Flavonifractor plautii ), the optimum activity temperature considering the thermal stability is about 65 ℃, the optimum active pH is about 7.0 (see FIGS. 5 and 6 of the Republic of Korea Patent No. 10-1473918). The present invention refers to the contents disclosed in Korean Patent No. 10-1473918 with respect to the preparation of a high purity allulose-containing solution using D-psicose 3-epimerase.

Thereafter, the D-psicose 3-epimerase reaction solution was filtered through diatomaceous earth (Celite 512J) to obtain a filtrate. Thereafter, activated carbon was added to the filtrate in an amount of about 3% by weight based on the solid content, and the mixture was decolorized by reacting at a pH of 4.5 and a temperature of 70 ° C for about 1 hr. The decolorized filtrate is passed through a cation exchange resin (SCR-B, Samyang) substituted with a hydrogen group and an anion exchange resin (S4268, Lanxess) substituted with a hydroxyl group in order to perform ion purification and a low purity allulose-containing solution ( Sugar composition: about 40% by weight fructose, about 44% by weight glucose, about 15% by weight allulose, about 1% by weight glucose oligomer of 2 or more). Thereafter, the low purity allulose containing solution was concentrated to be about 50 Brix to obtain a low purity allulose containing concentrate.

Thereafter, chromatography was performed by passing the low purity allulose-containing concentrate through a calcium group-substituted ion exchange resin (MUK 555), and a high purity allulose-containing solution (sugar composition: about 97% by weight of allulose and about 3 fructose). % By weight) was obtained.

(2) Preparation of high purity D-allose containing solution

Thereafter, the high purity allulose-containing solution is concentrated to about 30 brix, and ribose-5-phosphate isomerase is added to the total solids of the high purity allulose-containing concentrate. Allulose contained in the high purity allulose containing concentrate was converted to allose by addition at a level of 3% by weight and reaction at 53 ° C. for about 12 hr. Ribose-5-phosphate isomerase used in the embodiments of the present invention is R132E mutant ribose- disclosed in Korean Patent No. 10-1217670 owned by the applicant of the present invention. 5-phosphate isomerase. Specifically, the R132E mutant ribose-5-phosphate isomerase is arginine (Arg), which is the 132th residue amino acid of the wild type ribose-5-phosphate isomerase isolated from the Clostridium thermocellum strain. Is converted to glutamic acid (Glu). The optimal activity temperature considering the thermal stability of the R132E mutant ribose-5-phosphate isomerase is about 65 ° C., and the optimum activity pH is about 7.5 (see FIGS. 1 to 4 of Korean Patent No. 10-1217670). The present invention refers to the contents disclosed in Korean Patent No. 10-1217670 with respect to the preparation of a high purity allose containing solution using the R132E mutant ribose-5-phosphate isomerase.

Thereafter, the ribose-5-phosphate isomerase reaction solution was filtered through diatomaceous earth (Celite 512J) to obtain a filtrate. Thereafter, activated carbon was added to the filtrate in an amount of about 3% by weight based on the solid content, and the mixture was decolorized by reacting at a pH of 4.5 and a temperature of 70 ° C for about 1 hr. The decolorized filtrate is passed through a hydrogen-substituted cation exchange resin (SCR-B, Samyang Corporation) and a hydroxyl-substituted anion exchange resin (S4268, Lanxess) in order to perform ion purification and a low-purity allose-containing solution (saccharides). Composition: about 69% by weight allulose, about 28% by weight allose, about 3% by weight fructose). Thereafter, the low-purity allose-containing solution was concentrated to about 50 Brix to obtain a low-purity allose-containing concentrate.

Thereafter, chromatography was carried out by passing the low purity allose-containing concentrate through a calcium group-substituted ion exchange resin (MUK 555), and a high purity allose-containing solution (sugar composition: about 95.2 wt% of allose and about 1.6 wt% of allulose). %, About 2.6% by weight fructose). The high purity allose containing solution was concentrated to about 60 to 70 Brix to obtain a high purity allose containing concentrate, and the high purity allose containing concentrate was used as a mother liquor for allose crystallization.

(3) Preparation of D-Allose Seeds

D-allose seed was prepared using the high purity allose containing concentrate of about 70 brix concentration obtained previously as mother liquor. Specifically, the mother liquor is placed in a crystallizer in which stirring and temperature control systems are empty, and the temperature of the mother liquor is adjusted to 50 ° C., and the reagent grade allose particles having a purity of 95% or more is about 0.1% by weight of the solid of the mother liquor. Was added in an amount of and stirred at a speed of about 100 rpm to evenly disperse the reagent grade allose particle product. Thereafter, the agitation rate was reset to about 1.5 rpm, and the crystallization reaction was performed at a temperature gradient condition in which the temperature of the mother liquor was slowly cooled from 50 ° C. to 40 ° C. over about 48 hr and maintained at 40 ° C. for about 16 hr. Thereafter, the solution subjected to crystallization was washed with water by centrifugation, dehydrated, and dried at about 50 ° C. for 12 hr to obtain a D-allose seed. The resulting D-allose seed had a crystal grain size of about 100-200 μm and a sugar composition of 99.2 wt% allose and 0.8 wt% fructose.

Example  2: D- Allos  Determination of Saturation and Supersaturation

Example 1 gave high purity D-allose crystals (saccharide composition: 99.2 wt% allose and 0.8 wt% fructose). Thereafter, a small amount of high-purity D-allose crystal was added to 5 g of distilled water at specific temperature conditions, and the concentration which was no longer dissolved was defined as the saturation concentration at that temperature. In addition, by using a high-purity D- allose crystals to prepare a high-purity D- allose solution of 55 to 70% by weight solid content concentration, while slowly cooling the temperature at the time when the crystal particles start to form is measured, The concentration was defined as the supersaturation concentration at that temperature. Table 1 shows saturation concentrations and supersaturation concentrations of the high-purity D-allose crystals (sugar composition: 99.2 wt% allose and 0.8 wt% fructose) by temperature.

Saturation Curve of High Purity D-Allose Crystals Supersaturation Curve of High Purity D-Allose Crystals Temperature (℃) Concentration (wt% solids) Temperature (℃) Concentration (wt% solids) 25.0 49.0 25.3 55.0 30.0 50.0 29.0 57.0 35.0 53.5 33.0 58.0 40.0 55.6 39.2 62.0 45.0 58.3 44.0 65.0 50.0 59.6 49.0 67.0 55.0 60.2 55.1 70.0

From the results of Table 1, as shown in FIG. 1, a saturation curve and a supersaturation curve of high-purity D-allose crystals are prepared, and a stable zone, a metastable zone, and an unstable zone are divided. It was.

Example  3: high purity Allos  Crystallization Experiment by Solid Concentration of Concentrated Concentrate

D-allose seeds were prepared using high purity allose containing concentrates of various solid concentrations as mother liquors. Specifically, the mother liquor is placed in a crystallizer in which a stirring and temperature control system is empty, and the temperature of the mother liquor is adjusted to a specific temperature, and then the D-allose seed obtained in Example 1 is about 0.1% by weight of the solid of the mother liquor. Was added in an amount of% and stirred at a rate of about 100 rpm to evenly disperse the D-allose seed. Thereafter, the stirring speed was reset to about 1.5 rpm and the mother liquor was maintained at a specific temperature for about 48 hr without a temperature gradient to proceed with the crystallization reaction. Thereafter, the solution subjected to crystallization was washed with water by centrifugation, dehydrated, and dried at about 50 ° C. for 12 hr to obtain D-allose crystals. Tables 2 to 4 show the results according to the crystallization conditions.

In the following table, the degree of crystallinity (%) is a parameter in proportion to the crystallization yield, and was measured using the following method. The crystallization yield may be changed in part by additional processes such as water washing, dehydration and drying after the crystallization reaction is completed.

As the crystallization reaction proceeds, the concentration of the crystal mother liquor decreases.

-The crystal mother liquor undergoing crystallization reaction was centrifuged at 5,000 rpm to obtain a supernatant, and the solid concentration of the supernatant was measured.

X: initial solid content concentration of crystal mother liquor

Y: solid content of supernatant after centrifugation

Z: concentration of crystallized solid content

Item division Preparation Example 1 Preparation Example 2 Crystallization reaction conditions Mother liquor initial solids concentration
(weight%) 60 65 Crystallization reaction temperature and reaction time Held at 25 ° C. for 48 hr Held at 25 ° C. for 48 hr Change of reaction temperature, solid concentration of crystal mother liquor and crystallization region by crystallization reaction time (0 hr, 25 ° C, 60% by weight, unstable area) → (48 hr, 25 ° C, 55.6% by weight, unstable area) (0 hr, 25 ° C, 65% by weight, unstable area) → (48 hr, 25 ° C, 56.8% by weight, unstable area) Seed addition amount (% by weight of mother liquor initial solids) 0.1 0.1 result Crystallinity (%) 16.5 29.2 Grain size (μm) 50-100 50-100 Crystal picture

Item division Preparation Example 3 Preparation Example 4 Crystallization reaction conditions Mother liquor initial solids concentration
(weight%) 67 70 Crystallization reaction temperature and reaction time Held at 25 ° C. for 48 hr Held at 25 ° C. for 48 hr Change of reaction temperature, solid concentration of crystal mother liquor and crystallization region by crystallization reaction time Solidification during the crystallization reaction, making measurement impossible Solidification during the crystallization reaction, making measurement impossible Seed addition amount (% by weight of mother liquor initial solids) 0.1 0.1 result Crystallinity (%) Solidification during the crystallization reaction, making measurement impossible Solidification during the crystallization reaction, making measurement impossible Grain size (μm) Solidification during the crystallization reaction, making measurement impossible Solidification during the crystallization reaction, making measurement impossible Crystal picture Solidification during the crystallization reaction, making measurement impossible Solidification during the crystallization reaction, making measurement impossible

Item division Preparation Example 5 Preparation Example 6 Crystallization reaction conditions Mother liquor initial solids concentration
(weight%) 67 70 Crystallization reaction temperature and reaction time Held at 30 ° C. for 48 hr Held at 30 ° C. for 48 hr Change of reaction temperature, solid concentration of crystal mother liquor and crystallization region by crystallization reaction time (0 hr, 30 ° C, 67% by weight, unstable area) → (48 hr, 30 ° C, 60% by weight, unstable area) Solidification during the crystallization reaction, making measurement impossible Seed addition amount (% by weight of mother liquor initial solids) 0.1 0.1 result Crystallinity (%) 26.1 Solidification during the crystallization reaction, making measurement impossible Grain size (μm) 60-120 Solidification during the crystallization reaction, making measurement impossible Crystal picture

Solidification during the crystallization reaction, making measurement impossible

From the experimental results of Preparation Examples 1 to 6, the lower the solid content concentration of the crystal mother liquor, the lower the crystallinity and fine crystals are formed, and the higher the solid content concentration of the crystal mother liquor, the higher the crystallization reaction temperature, the higher the crystallization degree. It can be seen that the increase and the crystal grain size improve.

Example  4: high purity Allos  Containing concentrate Metastable  Temperature in the zone On gradient  According to different concentrations

D-allose seeds were prepared using high purity allose containing concentrates of various solid concentrations as mother liquors. Specifically, the mother liquor is placed in a crystallizer in which a stirring and temperature control system is empty, and the temperature of the mother liquor is adjusted to a specific temperature, and then the D-allose seed obtained in Example 1 is about 0.1% by weight of the solid of the mother liquor. Was added in an amount of% and stirred at a rate of about 100 rpm to evenly disperse the D-allose seed. Thereafter, the stirring speed was reset to about 1.5 rpm and the mother liquor was slowly cooled over a predetermined time or maintained at a cooled temperature for a predetermined time to proceed with the crystallization reaction at a temperature gradient condition corresponding to the metastable region. Thereafter, the solution subjected to crystallization was washed with water by centrifugation, dehydrated, and dried at about 50 ° C. for 12 hr to obtain D-allose crystals. Table 5 shows the results according to the crystallization conditions.

Item division Preparation Example 7 Preparation Example 8 Preparation Example 9 Crystallization reaction
Condition Mother liquor initial solids concentration
(weight%) 65 65 67 Crystallization reaction temperature and reaction time Held at 45 ° C. for 48 hr Slowly cool from 50 ° C to 45 ° C over 48hr 1st: Slow cooling from 50 ° C to 40 ° C over 48h
Secondary: hold at 40 ° C. for 16 hr Change of reaction temperature, solid concentration of crystal mother liquor and crystallization region by crystallization reaction time (0 hr, 45 ° C, 65% by weight, metastable area) → (48 hr, 45 ° C, 63% by weight, metastable area) (0 hr, 50 ° C, 65% by weight, metastable area) → (48 hr, 45 ° C, 63% by weight, metastable area) (0 hr, 50 ° C, 67% by weight, metastable area) → (24 hr, 45 ° C, 62% by weight, metastable area) → (48 hr, 40 ° C, 61.6% by weight, metastable area) → (64 hr, 40 ° C., 61.1 wt%, metastable area) Seed addition amount (% by weight of mother liquor initial solids) 0.1 0.1 0.1 result Crystallinity (%) 8.6 8.3 22.6 Grain size (μm) 400-1,100 400-1,000 1st: 100 ~ 120
2nd: 100 ~ 250 Crystal picture

1st decision

2nd decision

From the experimental results of Preparation Examples 7 to 9 it can be seen that when the crystallization reaction is carried out in the metastable region conditions, the allose particles grow in a thin and long needle shape and the crystal grain size increases. However, when the crystallization reaction is performed under metastable region conditions, it is difficult to industrialize because of low crystallinity of allose.

Example  5: high purity Allos  Temperature in unstable region of containing concentrate On gradient  According to different concentrations

D-allose seeds were prepared using high purity allose containing concentrates of various solid concentrations as mother liquors. Specifically, the mother liquor is placed in a crystallizer in which a stirring and temperature control system is empty, and the temperature of the mother liquor is adjusted to a specific temperature, and then the D-allose seed obtained in Example 1 is about 0.1% by weight of the solid of the mother liquor. Was added in an amount of% and stirred at a rate of about 100 rpm to evenly disperse the D-allose seed. Thereafter, the stirring speed was reset to about 1.5 rpm and the mother liquor was slowly cooled over a predetermined time or maintained at a cooled temperature for a predetermined time to proceed with the crystallization reaction at a temperature gradient condition including an unstable region. Thereafter, the solution subjected to crystallization was washed with water by centrifugation, dehydrated, and dried at about 50 ° C. for 12 hr to obtain D-allose crystals. Table 6 and Table 7 show the results according to the crystallization conditions.

Item division Preparation Example 10 Preparation Example 11 Crystallization reaction
Condition Mother liquor initial solids concentration
(weight%) 67 67 Crystallization reaction temperature and reaction time 1st: Slow cooling from 40 ° C to 30 ° C over 48h
Secondary: hold at 30 ° C. for 16 hr 1st: Slowly cool from 47 ℃ to 37 ℃ over 48h
Secondary: hold at 37 ° C. for 16 hr Change of reaction temperature, solid concentration of crystal mother liquor and crystallization region by crystallization reaction time (0 hr, 40 ° C, 67% by weight, unstable area) → (24 hr, 35 ° C, 59.4% by weight, unstable area) → (48 hr, 30 ° C, 58% by weight, unstable area) → (64 hr, 30 ℃, 57.6% by weight, unstable region) (0 hr, 47 ° C, 67% by weight, unstable area) → (24 hr, 42 ° C, 61% by weight, metastable area) → (48 hr, 37 ° C, 59% by weight, unstable area) → (64 hr, 37 ° C, 59% by weight, metastable area) Seed addition amount (% by weight of mother liquor initial solids) 0.1 0.1 result Crystallinity (%) 33.1 29.1 Grain size (μm) 1st: 100 ~ 120
2nd: 100 ~ 150 1st: 100 ~ 150
2nd: 100 ~ 150 Crystal picture

2차 결정

1st decision

2nd decision

1st decision

2nd decision

Item division Preparation Example 12 Preparation Example 13 Crystallization reaction
Condition Mother liquor initial solids concentration
(weight%) 70 67 Crystallization reaction temperature and reaction time 1st: Slow cooling from 50 ° C to 40 ° C over 48h
Secondary: hold at 40 ° C. for 16 hr 1st: Slow cooling from 50 ° C to 30 ° C over 48h
Secondary: hold at 30 ° C. for 16 hr Change of reaction temperature, solid concentration of crystal mother liquor and crystallization region by crystallization reaction time (0 hr, 50 ° C, 70% by weight, unstable area) → (24 hr, 45 ° C, 65.4% by weight, unstable area) → (48 hr, 40 ° C, 61.2% by weight, metastable area) → (64 hr, 40 ° C., 60.2% by weight, metastable area) (0 hr, 50 ° C, 67% by weight, metastable area) → (24 hr, 40 ° C, 62% by weight, metastable area) → (48 hr, 30 ° C, 60% by weight, unstable area) → (64 hr , 30 ° C, 59.5% by weight, metastable area) Seed addition amount (% by weight of mother liquor initial solids) 0.1 0.1 result Crystallinity (%) 35.2 28.5 Grain size (μm) 1st: 100 ~ 150
2nd: 150 ~ 200 Crystal picture

2차 결정

1st decision

2nd decision

From the experimental results of Preparation Examples 10 to 13 it can be seen that when the crystallization reaction proceeds in a temperature gradient condition including an unstable region, the crystallinity is high and the crystal grain size is also large and suitable for industrialization.

Example  6: Allos  Sugar Composition of Crystal Particles

The sugar composition of the allose crystal particles obtained in Preparation Example 11 and Preparation Example 12 was measured using HPLC, and the results are shown in Table 8 below.

division Preparation Example 11 Preparation Example 12 Allose Crystal Particle Sugar Composition 98.9 wt% allose, 1.1 wt% fructose 99.2 wt% allose, 0.8 wt% fructose

Example  7: Allos  Hygroscopicity of Crystal Particles

After separating the allose crystal particles obtained in Preparation Examples 11 and 12 with a 20 mesh sieve and stored at a temperature of 30 ℃ and 75% relative humidity conditions, the change of moisture content with storage time was measured. .

Figure 2 is a graph showing the change in moisture content of storage time of the allose crystal particles obtained in Preparation Example 11 and Preparation Example 12 of the present invention. In FIG. 2, # 11 represents Preparation Example 11 and # 12 represents Preparation Example 12. FIG. As shown in FIG. 2, the allose crystal particles obtained in Preparation Example 11 and Preparation Example 12 had a moisture content of less than 1% for a long time, and no caking occurred during the storage period. Therefore, the allose crystal particles obtained in Production Examples 11 and 12 of the present invention have low hygroscopicity and excellent fluidity, and are easy to handle.

Although the present invention has been described through the above embodiments as described above, the present invention is not necessarily limited thereto, and various modifications can be made without departing from the scope and spirit of the present invention. Therefore, the protection scope of the present invention should be construed as including all embodiments falling within the scope of the claims appended to the present invention.

Claims (9)

Adding a D-allose seed to the D-allose-containing mother liquor and then proceeding the crystallization reaction under a temperature gradient that reduces the temperature of the allose-containing mother liquor from the initial temperature T _i to the final temperature T _f . ,
The initial solid content concentration of the D-allose-containing mother liquor is 65 to 75% by weight based on the total weight of the mother liquor,
The D-allose-containing mother liquor comprises 92-97 wt% of allose based on the total weight of sugars in the mother liquor,
The initial temperature T _i is selected from 38 ~ 52 ℃,
The final temperature T _f is 8-20 ° C. lower than the initial temperature T _i ,
The temperature gradient comprises a temperature condition corresponding to the supersaturation state of the unstable region.
The method of claim 1, wherein the initial solid concentration of the D-allose-containing mother liquor is 66-72 wt% based on the total weight of the mother liquor.
The method of claim 1, wherein the D-allose-containing mother liquor comprises 93-96 wt% of allose based on the total weight of sugars in the mother liquor.
The method of claim 1, wherein the temperature gradient is maintained the first stage and D- allo temperature of agarose containing the mother liquor to reduce the temperature of the mother liquor containing D- allo agarose to a final temperature T _f at the initial temperature T _i to a final temperature T _f Method for producing a D-allose crystals, characterized in that consisting of a second step.
5. The method of claim 4, wherein the first step is performed over 24-72 hr and the second step is over 10-22 hr. 6.
The method of claim 1, wherein the D-allose-containing mother liquor is present in the supersaturated state of the unstable region during the progress of the crystallization reaction.
The method of claim 1, wherein the D-allose-containing mother liquor is converted from the supersaturation of the unstable region to the supersaturation of the metastable region during the progress of the crystallization reaction.
The method of claim 1, wherein the D-allose-containing mother liquor is converted from the supersaturation of the metastable region to the supersaturation of the unstable region during the progress of the crystallization reaction.
The D-allose crystal according to any one of claims 1 to 8, further comprising washing, dehydrating and drying the mother liquor subjected to the crystallization reaction to obtain D-allose crystal particles. How to prepare.

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