RU2795523C1 - Method for separating 5'-inosinate disodium - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: method for separating 5'-inosinate disodium from the microbial culture medium containing it. A method for separating 5'-inosinate disodium, including: cultivation of a microorganism producing 5'-inosic acid; adding NaOH to the culture broth of the microorganism obtained by cultivation; adjusting the pH of the culture broth to which NaOH has been added to a value of 7.4 to 8.0; concentrating the pH-adjusted culture broth to form disodium 5'-inosinate crystals; separating the disodium 5'-inosinate crystals from the culture broth containing the disodium 5'-inosinate crystals, and washing the crystals by contacting the disodium 5'-inosinate crystals with a hydrophilic organic solvent.

EFFECT: efficient separation of crystals 5'-inosinate disodium with high separation yield and high purity.

16 cl, 5 tbl, 5 ex

Description

Technical field

The present invention relates to a method for separating disodium 5'-inosinate from a microbial culture.

State of the art

Purine-derived nucleotides such as disodium inosinate (5'-inosinate disodium, IMP2Na) are used as raw materials for the preventive treatment of metabolic disorders, growth promoters and anti-cancer drugs in the pharmaceutical sector, and they are also important substances used as food additives, such as spices in the food industry. Disodium inosinate is obtained by extracting inosinic acid (IMP), a substance naturally present in meat, fish, etc., from fish meat, RNA enzymatic degradation method, microbial fermentation method, combination of fermentation method and synthesis method, combination of chemical degradation and RNA synthesis etc.

Much research has been carried out on the method of obtaining inosinic acid, which is the starting material for the production of disodium inosinate. To obtain disodium 5'-inosinate having qualities suitable for use as a food additive, inosinic acid must be purified from impurities.

As for the method for cultivating microorganisms and obtaining disodium 5'-inosinate from their culture, an organic solvent can be used in the crystallization process. However, many organic solvents must be used, and explosion-proof equipment and worker protection are required accordingly. In addition, the removal of the organic solvent may require a distillation process and appropriate equipment. Accordingly, there is a need for an alternative to such a method.

Description of embodiments

Technical task

The present invention provides a method for separating disodium 5'-inosinate, comprising: cultivating microorganisms producing 5'-inosic acid; bringing the pH of the culture broth of microorganisms to 7.4-8.0; concentrating the pH-adjusted culture broth to form disodium 5'-inosinate crystals; separating the disodium 5'-inosinate crystals from the culture broth containing the disodium 5'-inosinate crystals, and washing the crystals by contacting the separated disodium 5'-inosinate crystals with a hydrophilic organic solvent.

The solution of the problem

The term "5'-inosinic acid" is a nucleoside monophosphate, also called inosine monophosphate (IMP). The terms "5'-inosinic acid" and "its salt" are used interchangeably. The salt includes "5'-inosinate" or 5'-inosinate disodium.

As used in this document, the term "culture" refers to a product obtained by culturing a microorganism. The culture may contain microbial cells or their metabolites. Metabolites can be both intracellular and extracellular. Metabolites may be nucleic acid metabolites. Nucleic acid metabolites may be 5'-inosic acid or a salt thereof.

As used herein, the term "culture broth" refers to the liquid portion obtained by removing microbial cells from culture. The culture broth may contain products isolated from or derived from microorganisms. The culture broth may contain soluble products. The culture broth may contain 5'-inosinic acid or a salt thereof.

In one aspect of the present invention, there is provided a method for separating disodium 5'-inosinate, comprising: cultivating 5'-inosic acid-producing microorganisms; bringing the pH of the culture broth of microorganisms to 7.4-8.0; concentrating the pH-adjusted culture broth to form disodium 5'-inosinate crystals; separating the disodium 5'-inosinate crystals from the culture broth containing the disodium 5'-inosinate crystals, and washing the crystals by contacting the separated disodium 5'-inosinate crystals with a hydrophilic organic solvent.

The method may include obtaining a culture broth by removing the cell body from a microbial culture containing 5'-inosinic acid.

In preparing the culture broth, the microorganism may be a 5'-inosic acid-producing microorganism. The microorganism may be a genetically engineered microorganism to increase the ability to produce 5'-inosic acid. The microorganisms may be bacteria. The microorganism may be a Gram-positive or Gram-negative bacterium. The bacterium may be a bacterium of the genus Corynebacterium or the genus Escherichia. The bacterium may be C. glutamicum, C. ammoniagenes, or £. coli. The microorganism may be designed to release 5'-inosinic acid outside the cell.

A microbial culture can be obtained by culturing the microorganism in a medium under conditions that produce 5'-inosinic acid. The microbial culture may contain 5'-inosinic acid. The medium may be a known medium used for the fermentation of 5'-inosinic acid. The environment may contain a carbon source, a nitrogen source, and trace elements. The medium may contain molasses or a corn immersion solution. The environment may be an LB environment. The medium may contain per liter of distilled water 46 g glucose, 30 g fructose, 10 g yeast extract, 18 g KH ₂ PO ₄ , 42 g K ₂ HPO ₄ , 6 g urea, 10 g MgSO ₄ *7H ₂ O, 30 µg biotin and 5 mg thiamine hydrochloride.

The microbial culture may contain 5'-inosinic acid in an amount of 0.5 wt. % up to 20 wt. %, from 1.0 wt. % up to 20 wt. %, from 2.5 wt. % up to 20 wt. %, from 5 wt. % up to 20 wt. % or from 5 wt. % up to 10 wt. %, based on the total weight of the culture. 5'-inosinic acid may be present in the extracellular environment. The microbial culture may have a pH of 6 to 9. The microbial culture may not be subjected to ion exchange chromatography or crystallization using an organic solvent such as methanol.

Cell body removal is the removal of cells or cell debris from liquid components in culture. The removal of the cell body can be carried out in a known manner. Removal of the cell body can be accomplished by filtration, centrifugation, or a combination thereof. The method may include adjusting the pH of the culture broth from 7.4 to 8.0.

The pH adjustment can be done by adding an acid or a base to the culture broth. The acid may be hydrochloric acid. The base may be NaOH. The pH value may be 7.4 to 8.0, 7.6 to 8.0, 7.8 to 8.0, 7.4 to 7.8, or 7.4 to 7.6.

The method may include concentrating the pH adjusted culture broth to form disodium 5'-inosinate crystals. Concentration may be a crystallization process in which the culture broth is concentrated to convert 5'-inosinic acid to disodium 5'-inosinate crystals. This crystallization process is carried out by simply increasing the concentration of 5'-inosinic acid in the culture broth by concentrating the culture broth, and no organic solvent is used. The organic solvent not used here may be a hydrophilic organic solvent. The hydrophilic organic solvent not used here can be a C1-C5 alcohol or a mixture of water and a C1-C5 alcohol. The alcohol may be at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, and mixtures thereof. The hydrophilic organic solvent not used here may be ethanol. The concentration of ethanol can be from 50% to 70% by volume in water.

The concentration of the culture broth with adjusted pH can be directed to reduce the amount of moisture in the culture broth. Concentration may be the evaporation of moisture by heating the culture broth. Concentration can be carried out in relation to the culture broth at a temperature of 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, 50°C to 90°C, from 50 °C to 80°C, 50°C to 70°C, 50°C to 60°C, 60°C to 90°C, 60°C to 80°C, 60°C to 70°C C or from 60°C to 65°C. The concentration can be carried out as concentration under reduced pressure. Concentration can be performed to form disodium 5'-inosinate crystals by increasing the concentration of 5'-inosic acid in the culture broth. Concentration can be carried out until the concentration of 5'-inosinic acid in the culture broth becomes from 380 g/l to 600 g/l, from 400 g/l to 550 g/l, from 400 g/l to 500 g/l l, from 400 g/l to 475 g/l or from 425 g/l to 475 g/l. Concentration can be carried out until crystals of disodium 5'-inosinate are formed and the culture broth is in the form of a suspension containing crystals. Hereinafter, a suspension solution containing crystals is also referred to as a "crystal suspension". As used herein, the term "suspension" refers to a mixture of solids, eg, crystals, suspended in a liquid, typically water, with a specific gravity greater than 1. The crystals may have a particle size of 20 microns to 400 microns.

The method may further include concentrating the culture broth before adjusting the pH of the culture broth. With this concentration, disodium 5'-inosinate may not be formed in the culture broth or may not be substantially formed. Concentration may be aimed at reducing the amount of moisture in the culture broth. Concentration can be directed to evaporation of moisture by heating the culture broth. Concentration can be carried out until the concentration of 5'-inosinic acid in the culture broth is between 150 g/L and 360 g/L. When concentrated, the concentration of 5'-inosinic acid can be from 150 g/l to 360 g/l, from 200 g/l to 300 g/l, from 220 g/l to 280 g/l, or from 240 g/l to 280 g/l. Concentration can be carried out on culture broth at a temperature of 50°C and above, 60°C and above, 70°C and above, °C and above, 90°C and above, from 50°C to 90°C, from 50°C C to 80°C, 50°C to 70°C, 50°C to 60°C, 60°C to 90°C, 60°C to 80°C, 60°C to 70°C or from 60°C to 65°C.

The concentration may be carried out under reduced pressure. The method may further include cooling the culture broth comprising the crystals formed after the concentration. Cooling can be carried out at a temperature of 25°C to 45°C, 25°C to 40°C, 25°C to 35°C, or 25°C to 30°C. speed. Cooling can be carried out at a cooling temperature for 1.0 to 3.0 hours, such as 1.5 to 2.5 hours or 2 hours. °C/h, e.g. 10.0°C/h to 15.0°C/h, 1.0°C/h to 14.0°C/h, 2.0°C/h to 13.0°C/h, or about 12.5°C/h. Cooling can be carried out while the resulting crystal suspension is introduced into a jacketed glass tube and mixed. When cooling is carried out at a lower temperature, the solubility decreases and more crystals can be recovered.

In addition, when cooling is performed at a uniform rate, a harder and better crystal can be obtained. The method may further include aging the crystals by holding the slurry at a cooling temperature after cooling is completed. The exposure can be carried out for 1 hour to 5 hours, 1 hour to 4 hours, 1 hour to 3 hours, 1.5 hours to 2.5 hours, or about 2 hours. crystals are introduced into a glass tube with a shirt and mixed.

The method may further include separating the disodium 5'-inosinate crystals from the culture broth containing the disodium 5'-inosinate crystals formed.

The separation of the crystal can be carried out in a known manner for the separation of crystals within the scope of the objectives of the present invention. The separation of the crystals can be carried out, for example, by centrifugation, filtration, or a combination thereof. Centrifugation can be carried out at a rotation speed of 100 g to 1000 g, 100 g to 800 g, 100 g to 500 g, 300 g to 1000 g, or 500 g to 1000 g. Centrifugation can be carried out for a rotation time of 10 to 30 minutes.

The method may include washing the crystals by contacting the separated disodium 5'-inosinate crystals with a hydrophilic organic solvent.

During the washing process, contacting may include mixing the crystals with a hydrophilic organic solvent. Washing can be aimed at reducing the amount of impurities by removing impurities from the crystal. The hydrophilic organic solvent may be a C1-C5 alcohol or a mixture of water and a C1-C5 alcohol. The alcohol may be methanol, ethanol, propanol, isopropanol, butanol, pentanol, or a mixture thereof. The hydrophilic organic solvent may have a concentration of 50% to 70%, 60% to 70%, 55.0% to 65.0%, or 60% by volume in water. The hydrophilic organic solvent may be ethanol or a mixture of water and ethanol. Ethanol may have the advantage of being inexpensive and of low toxicity. The hydrophilic organic solvent may be 10% to 50%, such as 20% to 50%, 15% to 40%, 20% to 40%, 25% to 40%, 25% to 35%, or from 25% to 50% of the volume of the resulting sample containing crystals.

In the method, crystal separation and crystal washing can be performed simultaneously. In addition, the separation of the crystal and the washing of the crystal can be performed separately.

The method of the present invention may further include removing the coloring material by contacting the washed crystals with activated carbon in an aqueous solvent. Bringing into contact may be mixing the washed crystals with activated carbon in an aqueous solvent and keeping with it. The aqueous solvent may be water.

Removal of the coloring material may include dissolving the washed crystals in water to a concentration of 250 g/l to 500 g/l and adding 0.1% to 10% activated charcoal to the aqueous solution containing the crystals, based on the weight of 5'-inosinic acid. . Bringing into contact can be carried out at a temperature of from 40°C to 80°C.

Benefits of the Invention

According to the separation method of disodium 5'-inosinate according to the present invention, crystals of disodium 5'-inosinate can be separated efficiently. In particular, according to the method, since the organic solvent is only used in the washing process, the amount of the organic solvent is small. According to the method, the separated disodium 5'-inosinate crystals have a high separation yield and high purity.

In particular, by adjusting the pH of the culture broth, the separation yield of disodium 5'-inosinate crystals can be significantly increased. In addition, by bringing the separated disodium 5'-inosinate crystals into contact with a hydrophilic organic solvent to wash the crystals, the decrease in yield compared to the case of washing with water can be prevented, the color of the resulting crystals is improved, and the amount of harmful organic solvents in the crystal is greatly reduced. Accordingly, economic feasibility can be ensured.

Implementation of the invention

Hereinafter, the present invention is described in more detail by way of examples. However, these examples are for illustrative purposes only, and the protection scope of the present invention is not limited to these examples.

Example 1 Fermentation of IMP and Isolation of IMP from Culture

1. Preparation of IMP and cultivation of microorganisms

Corynebacterium glutamicum was inoculated in a 30 L fermentation incubator containing 18 L of medium and cultured at 31° C. for 140 hours to form 5'-inosinic acid (IMP) in culture. 5'-inosinic acid was secreted outside the cell and was present in the culture broth. The medium had an initial pH of 7.2 and contained per liter of distilled water 46 g glucose, 30 g fructose, 10 g yeast extract, 18 g KH ₂ PO ₄ , 42 g K ₂ HPO ₄ , 6 g urea, 10 g MgSO ₄ *7H ₂ O, 30 µg biotin and 5 mg thiamine hydrochloride. The pH of the final culture was 6.8.

After adjusting the pH to 10.5 by adding 50 wt. % NaOH to the resulting cell culture was removed by filtration through a microfilter and a supernatant was obtained. The concentration of 5'-IMP in the supernatant was measured by HPLC. The supernatant contained 140 g/l 5'-IMP.

2. Crystal formation of disodium 5'-inosinate

2 L of the filtrate was placed in a rotary evaporator (N-1110V: EYELA, Japan), the moisture was evaporated from the filtrate, and the filtrate was concentrated to reach a concentration of 5'-IMP of 260 g/L. Evaporation was carried out using an NVC-2200 vacuum regulator at an internal pressure of the evaporator of 16 kPa (120 mm Hg), a vessel temperature of 65° C. and an evaporation rate of 1 l/h. At this time, the temperature of the filtrate inside the vessel was 55°C. This concentration process is hereinafter referred to as "primary concentration".

The resulting concentrated filtrate had a pH of 9.0 and a temperature of 55°C. The change in the pH of the initial filtrate from 10.5 to 9.0 is apparently associated with the removal of ammonia during evaporation.

Then, to 1077 ml of the resulting concentrated filtrate was added 4.5 ml of 35 vol. % hydrochloric acid per 1 l of the resulting concentrated filtrate to bring the pH to 7.4 and maintain the temperature at 55°C. Hereinafter, this process is referred to as the “pH adjustment process”.

1082 ml of the concentrated filtrate, the pH of which was adjusted, was placed in a rotary evaporator, and under the same conditions as described above, the concentrated filtrate was concentrated to a supersaturated solution in which 460 g/l of 5'-IMP crystals and the liquid in the filtrate were mixed, then have received a state of suspension. Here, the concentration "460 g/l"5'-IMP means the amount of dissolved crystals of both 5'-IMP and 5'-IMP2Na. As a result, 5'-IMP and 2Na ⁺ ions in 608 ml of a supersaturated solution precipitated as disodium 5'-inosinate crystals (hereinafter also "5'-IMP2Na"), and thus the concentrated filtrate became a viscous suspension in which solid crystals were mixed with the liquid filtrate (hereinafter also referred to as "suspension"). Immediately after the completion of crystallization, the temperature of the resulting suspension was 55° C. and its pH was 8.0. This process is hereinafter referred to as the "secondary concentration and crystallization process". That is, since 5'-IMP2Na crystals were formed by the secondary concentration and crystallization process, it indicates that 5'-1MP can be converted into 5'-IMP2Na crystals by concentration without using an organic solvent. This is a significant effect, which is unexpected for a person skilled in the art.

608 ml of the suspension was poured into a jacketed glass tube, a stirrer (eyela, zz-2121) was placed on top and the suspension was cooled at a cooling rate of 12.5°C/h to 30°C for 2 h while stirring at a speed of 200 rpm . A jacketed glass tube was used to cool the suspension at a constant rate. After cooling was completed, exposure was carried out at 30°C for 2 hours while stirring the suspension contained in a glass tube with a shirt. This process corresponds to the aging process, allowing the growth of crystals to continue. Hereinafter, this process is referred to as the “cooling and aging process”.

3. Extraction of disodium 5'-inosinate crystals

5'-IMP2Na crystals were isolated from the resulting aged suspension. Specifically, 608 ml of the resulting aged suspension was placed in an H-110F basket separator (KOKUSAN Co. Ltd., Japan) and centrifuged at 340xg for 20 minutes. The H-110F centrifuge has a perforated basket that is connected to an external rotation source. The perforated basket is made of polyamide multifilament filter cloth, and the air permeability of the filter is 250 l/m ² /s at 0.2 kPa (2 mbar). As a result of centrifugation, 300 g of a cake containing IMP2Na crystals was obtained, which was prepared by removing the liquid from the suspension.

Then 150 ml of 60 vol. % aqueous ethanol solution to wash the IMP2Na crystals (hereinafter also referred to as "ethanol wash"). As a result of washing, 272 g of 5'-IMP2Na crystals were recovered and then they were dried at room temperature for 24 hours to obtain 265 g of dried 5'-IMP2Na columnar crystals. This 5'-IMP2Na crystal is also referred to as the 5'-IMP2Na hydrate crystal 7.5.

The purity and transmittance of the resulting dried 5'-IMP2Na crystal were measured. As a result, the dried 5'-IMP2Na crystal had a yield of 94.6%, a purity of 95.7%, and a transmittance of 86.10%. Yield and purity were calculated using the following formula.

Equation 1

Yield = mass of obtained 5'-IMP2Na crystal/Macca of 5'-IMP2Na crystal in 2 L filtrate × 100

To determine the purity, each of 1.0 g of dried 5'-IMP2Na crystal and 1.0 g of standard 5'-IMP2Na crystal (Sigma, ≥9.0% (HPLC)) was dissolved in 1 L of triple distilled water to obtain a concentration of 1. 0 g/l test group and 1.0 g/l concentration of standard product solution, respectively. 5 µl of test solution and 5 µl of standard solutions were loaded onto a column on an Agilent 1260 Infinity Quaternary LC system (Agilent Technology Inc.). The column was a Shiseido CAPCELLPAK C18 ACR column (150 mm x 4.6 mm, 3 µm). Then, at 254 nm, the absorption coefficient of the eluate flowing out when applying 2 vol. % acetonitrile / 98 vol. % phosphate buffer (pH 2.4) into the column at a flow rate of 1 ml/min. The phosphate buffer contained 2 g/l ammonium phosphate, 0.2 g/l tetrabutylammonium phosphate and 0.82 g/l phosphoric acid. At this time the temperature was 35°C. These HPLC conditions were also used to measure the concentration of 5-IMP in the filtrate. As a result, the purity was calculated by the following formula.

Equation 2

Purity = mass 5'-IMP/mass solids × 100

In addition, a solution obtained by dissolving the test group in water at a concentration of 5 (w/v)% was placed in a rectangular cell of the CARY 100 UV-VIS instrument (Agilent Technology Inc.) to measure the transmittance, and its transmittance was measured at 420 nm.

As control 1 test group, 5'-IMP2Na crystals were recovered in the same manner as described above, except that 60 vol. % aqueous ethanol solution during washing (hereinafter also referred to as "no washing"). As a result, the resulting purified 5'-IMP2Na crystal had a dry weight of 266 g, a yield of 95%, a purity of 86.40%, and a transmittance of 40.48%.

As control 2 of the test group, 5'-IMP2Na crystals were recovered in the same manner as described above, except that in the "discovery of disodium 5'-inosinate crystals" deionized water was used in an amount of 25% relative to the volume of the 5' crystal suspension -IMP2Na instead of 60 rpm % ethanol aqueous solution (hereinafter also referred to as "washing with water"). As a result, the resulting purified 5'-IMP2Na crystal had a dry weight of 240 g, a yield of 85.7%, a purity of 94.64%, and a transmittance of 72.72%.

Table 1 shows the dry weight, yield, purity and transmittance of 5'-IMP2Na crystals obtained for test group, control 1 and control 2. As shown in table 1, when 60 vol. % aqueous ethanol solution in the process of separating the crystals, the purity and transmittance of the crystals improved simultaneously compared with the case where washing was not performed or the case when washing with water. In particular, in the case of the test group, the purity increased by 10.8% and 1.1%, and the transmittance increased by 112.7% and 18.4% compared to control 1 and control 2, respectively. This increase in purity and transmittance is a significant effect that is unexpected in light of the prior art. In particular, the fact that the transmittance increased by 112.7% and 18.4% indicates that the resulting 5'-IMP2Na crystal can easily meet food standards. In more detail, in order to meet food standards, the transmittance must be at 98%, and to achieve this level of transmittance for control 1 and control 2, it is necessary to recrystallize two or more times, while the test group can reach the food standard already during one additional recrystallization. In addition, to bring control 1 and control 2 to the level of the test group, an increase in the number of washes is required, and the recovery may decrease.

4. Influence of pH on the crystallization process

In this section, the influence of solution pH on crystallization of 5'-1MP during crystallization was confirmed.

In particular, the pH was adjusted to 7.2 by adding 35 vol. % hydrochloric acid to 2000 ml of concentrated filtrate (pH 9.0) obtained after “primary concentration” in section “2. The formation of crystals of 5'-inosinate disodium", and the temperature was maintained at 55°C.

2000 ml of the concentrated filtrate, the pH of which was adjusted, was placed in a rotary evaporator, and under the same conditions as described above, the concentrated filtrate was concentrated to a supersaturated solution in which 400 g/l of 5'-IMP crystals and the liquid in the filtrate were mixed, that is, a state of suspension was obtained. Here, the concentration "400 g/l"5'-IMP means the number of crystals of both 5'-IMP and 5'-IMP2Na. As a result, 5'-IMP and 2Na ⁺ ions in 608 ml of supersaturated solution precipitated as 5'-IMP2Na crystals, and the concentrated filtrate became a viscous suspension in which solid crystals and liquid filtrate (hereinafter also referred to as "suspension") were mixed. . Immediately after the completion of crystallization, the temperature of the resulting suspension was 55° C. and its pH was 8.0.

608 ml of the suspension was poured into a jacketed glass tube, a stirrer (eyela, zz-2121) was placed on top and the suspension was cooled at a cooling rate of 12.5°C/h to 30°C for 2 h while stirring at a speed of 200 rpm . A jacketed glass tube was used to cool the suspension at a constant rate. After cooling was completed, exposure was carried out at 30°C for 2 h while stirring the suspension contained in a jacketed glass tube. This process corresponds to the aging process, allowing the growth of crystals to continue. Hereinafter, this process is referred to as the “cooling and aging process”.

The pH of 608 ml of the cooled filtrate was adjusted to 7.4-9.0 with 35 vol. % hydrochloric acid and 50% NaOH solution. The filtrate supernatant at each pH was loaded onto the HPLC instrument as described above and eluted to measure the 5'-1MP concentration in the filtrate. Table 2 shows the concentration of 5'-1MP in the filtrate as a function of pH.

As shown in Table 2, the concentration of 5'-1MP was lowest at pH 7.4 to 8.0. In Table 2, low solubility at pH 7.4 to 8.0 means that 5'-IMP2Na crystals form well in this pH range.

5. Influence of organic solvent, concentration and amount used in

separation of disodium 5'-inosinate crystals In this section, in relation to section “3. Extraction of 5'-inosinate disodium", the influence of other organic solvents instead of 60 vol. was confirmed. % aqueous ethanol solution, concentration and quantity on solubility of 5'-IMP, color of 5'-IMP2Na crystals and purity of 5'-IMP2Na crystals, amount of moisture and absorption coefficient.

(1) Solubility of 5'-IMP depending on organic solvents and absorption coefficient of the solution in which the crystals are dissolved

608 ml of the aged suspension obtained in section “2. Disodium 5'-inosinate crystal formation" was placed in 1000 ml of the organic solvent shown in Table 3 and stirred at 25° C. for 1 hour to allow the 5HMP to dissolve in the organic solvent suspension. This solution was centrifuged at 1500 rpm for 10 minutes to obtain a supernatant.

Thereafter, the concentration of 5'-IMP in the supernatant was measured by HPLC. The crystals were separated as described in section "3. Extraction of disodium 5'-inosinate crystals, and dissolved in water to give a 5 (m/v) aqueous solution, and their absorbance at 420 nm was measured. Since the main impurities in the crystal were yellow-brown in color, absorption at 420 nm was chosen to measure the amount of these coloring substances. Absorbance was measured using a CARY 100 UV-VIS instrument (Agilent Technology Inc.).

Table 3 shows the 5'-1MP solubility according to the type and concentration of the organic solvent. Tables 3 and 4 show % by volume.

Table 4 shows the absorption coefficient of a solution obtained by dissolving 5'-IMP2Na crystals using various types and concentrations of organic solvents and in water.

As shown in Tables 3 and 4, when the concentration of the hydrophilic organic solvent was 50% and 70%, it was confirmed that the concentration of 5'-IMP in the mother liquor was low and the absorption coefficient of the washed crystals was low.

(2) Characteristics of crystals according to the amount of organic solvent

The 5'-IMP2Na crystals were separated and the purity of the crystals, the moisture content of the crystals, and the absorption coefficient were measured in the same manner as described above, except that the amount of 60 vol. % aqueous ethanol solution used in section “3. Extraction of crystals of 5'-inosinate disodium", changed from 0 vol. % up to 40 vol. % relative to the volume of the suspension. The moisture content of the crystals was measured by drying in a thermostat, in which the mass was measured before and after drying at 125°C for 3 h in a thermostat. Crystal purity and absorbance were determined as described above.

As shown in table 5, in the case of 60 about. % aqueous ethanol solution, which is a washing solution, when its amount is 25% or more, for example, 25% to 35% of the volume of the crystal suspension, the purity of the crystals, the moisture content of the crystals, and the absorbance improved simultaneously.

Claims (21)

1. A method for separating 5'-inosinate disodium, including: cultivation of a microorganism producing 5'-inosinic acid; adding NaOH to the culture broth of the microorganism obtained by cultivation; adjusting the pH of the culture broth to which NaOH is added to a value of 7.4 to 8.0; concentrating the pH-adjusted culture broth to form disodium 5'-inosinate crystals; separating the disodium 5'-inosinate crystals from the culture broth containing the disodium 5'-inosinate crystals, and washing the crystals by contacting the disodium 5'-inosinate crystals with a hydrophilic organic solvent. 2. The method according to p. 1, in which the culture broth of the microorganism contains from 5 to 20 wt. % 5'-inosinic acid, based on the total mass of the culture broth. 3. The method according to claim 1, wherein the microorganism is a bacterium of the genus Corynebacterium or the genus Escherichia. 4. The method according to p. 1, in which the concentration is carried out under reduced pressure. 5. The method according to claim 1, wherein the concentration is carried out until the concentration of 5'-inosinic acid in the culture broth is between 400 and 550 g/L. 6. The method of claim 1 further comprising concentrating the culture broth to which NaOH has been added prior to adjusting the pH. 7. The method according to claim 6, wherein the concentration of the culture broth before adjusting the pH is carried out until the concentration of 5'-inosinic acid in the culture broth is between 150 and 360 g/L. 8. The method according to claim 6, wherein the concentration of the culture broth before adjusting the pH consists in performing the concentration under reduced pressure. 9. The method according to p. 1, including cooling the formed crystals after concentration. 10. The method according to p. 9, in which the cooling is carried out at a temperature of from 25 to 30°C. 11. The method of claim 1 wherein the hydrophilic organic solvent is a C1-C5 alcohol. 12. The method of claim 11 wherein the alcohol is at least one selected from the group consisting of methanol, ethanol, propanol and isopropanol. 13. The method of claim 1 wherein the hydrophilic organic solvent is ethanol. 14. The method of claim. 1, in which the concentration of the hydrophilic organic solvent is from 50 to 70 vol.%. 15. The method of claim. 1, in which the volume of the hydrophilic organic solvent is from 20 to 50% of the total volume of the sample, including the crystals formed. 16. The method of claim 1 further comprising contacting the washed crystals with activated charcoal in an aqueous solvent to remove the coloring material.