KR100926253B1 - A preparation method of disodium 5'-inosinate by using crystallization process - Google Patents

Abstract

The present invention relates to a method for producing 5'-inosinate (IMP2Na) from 5'-inosinic acid (IMP) fermentation broth produced by microbial fermentation. According to the present method, the amount of waste water generated is lower than that of the ion exchange resin method, and it is possible to produce high purity disodium inosinate only by controlling temperature and using a small amount of organic solvent.

Inosinic acid (IMP), disodium inosinate (IMP2Na), organic solvent, cooling crystallization, supersaturation

Description

A preparation method of disodium 5'-inosinate by using crystallization process

The present invention relates to a production method of disodium 5'-inosinate (IMP2Na) using a crystallization method, and more particularly, in the purification of disodium inosine acid from inosine acid (IMP) -producing strain fermentation broth, a conventional method of using an ion exchange resin It relates to a method of producing high purity disodium inosinate by the crystallization method in place of.

In other words, by improving the crystallization method using a conventional organic solvent, by controlling the solubility and the crystallization time and growth point of the specific properties of the physical properties to drastically reduce the organic solvent consumption, the same crystal yield and the purity of the disodium inosinate crystals It is about how to raise.

Purine derivative nucleotides, such as disosium 5'-inosinate (IMP2Na), are used in the pharmaceutical sector as raw materials for the prophylaxis, growth promoter, and anticancer agent of the Great Sea. .

Disodium inosinate is used as a seasoning agent by incorporating Inosonic acid (IMP), a substance naturally present in meat and fish, through fish meat extraction, RNA enzyme digestion, microbial fermentation, combination of fermentation and synthesis, RNA chemical decomposition and synthesis It is to prepare a disodium inosine acid component.

The present inventors have conducted a lot of research focusing on the production of disodium inosinate by the microbial fermentation method, and has received a number of patent technologies related thereto.

However, the resultant produced by the above method contains pigments, sugars, amino acids, organic acids, inorganic salts, etc., so as to obtain a disodium 5'-inosinate that can be used as food additives, the impurities are purified by impurities. Should be removed.

In the conventional method for purifying disodium inosine using ion exchange resin, a method of obtaining disodium inosinate crystals by separating organics and inorganics using a cation exchange resin and an anion exchange resin in multiple stages, obtaining a high purity inosinic acid solution, and concentrating crystallization (Japanese Patent Laid-Open No. 40-39715). However, the purification method using the ion exchange resin has a disadvantage of generating a large amount of waste water.

As a method for overcoming the disadvantages of wastewater generation, as a prior art, microbial fermentation broth is separated without using an ion exchange resin, the supernatant is concentrated, and high purity disodium inosinate is obtained by repeating crystallization several times using a hydrophilic organic solvent. There is a way. Looking at the known patented technology using this method, the purification method of the mixture of sodium 5'-inosinate and sodium 5'-guanylate (Korean Patent No. 1987-0007197), 5'- disodium guanylate and disodium 5'-inosinate Method for preparing mixed crystals (Korean Patent Publication No. 1988-0010694), crystallization method of sodium 5'-guanylate (Korean Patent Publication No. 1993-0009984), 5'- disodium guanylate and 5'-inosinoate mixed crystal Manufacturing method (Korean Patent Publication No. 2004-0018435) and the like. However, this also has a problem that a large amount of hydrophilic organic solvent used when the disodium inosinate is obtained using the concentrate obtained after the cell separation of the microbial fermentation broth and impurities are simultaneously precipitated due to the addition of the hydrophilic organic solvent. In addition, it is necessary to optimize the amount of the organic solvent to be added to the cell separation solution because lower inosinic acid content due to the simultaneous precipitation of impurities may affect the subsequent crystallization process.

Therefore, the present inventors focused on the hydrophilic organic solvent crystallization method which is part of the inosinic acid purification method from the microbial fermentation broth. In particular, in order to solve the problems described above, experiments have been conducted to improve the yield and the purity of crystals while minimizing the amount of organic solvents used. It has come to complete the industrially applicable invention which can improve the yield while improving.

SUMMARY OF THE INVENTION An object of the present invention is to provide a purification method in which a conventional method for producing disodium inosinate using a microbial fermentation method is to improve the conventional problems, that is, significantly reduce the amount of hydrophilic organic solvent used and improve the purity of crystals.

The above object of the present invention was achieved in the crystallization method by establishing the optimum crystallization condition using the solubility according to the temperature and the addition of the organic solvent and thereby measuring the purity of purified disodium inosinoate.

The present invention is a first step of separating and removing the cells from the fermentation broth of the inosinic acid producing strain, the second step of concentrating the fermentation broth, the third step of adjusting the pH of the fermentation broth to 6 to 10, the concentrated solution is adjusted pH A fourth step of completely dissolving the pre-crystallized disodium inosinate, a fifth step of cooling the concentrate in which the disodium inosinate crystal is dissolved, and a step of crystallizing disodium inosinate by adding a hydrophilic organic solvent to the cooled concentrate. It is characterized by providing a method for producing disodium inosine which consists of six steps.

In the present invention, the microbial fermentation broth used was inoculated with the inoculation of the inosinic acid-producing strain, and the strain used for the culture may be any strain as long as it produces the inosinic acid, preferably Corynebacterium ( Corynebacterium ) The fungal strain can be used.

The strain Corynebacterium ( Coynebacterium ) is preferably cultured to produce about 5% to 10% of inosinic acid in the fermentation broth.

The first step of the present invention may be carried out by any one method selected from the group consisting of filtration, centrifugation and heat treatment, preferably by filtration by a membrane filtration process. The membrane filtration process can be easily set by the person skilled in the art to separate the cells from the fermentation broth. For example, the fermentation broth may be heated to about 40 to 50 ° C. in advance, and may be carried out under conditions of a pressure between membranes (TMP) of 1.2 to 1.5 atm.

In the second step, the concentration of inosinic acid is preferably 200 to 300 g / L, and most preferably, 230 to 260 g / L.

The third step may be preferably performed by adjusting the pH of the concentrate to 7.5 ~ 9.5.

The fourth step may be preferably performed by heating the temperature to 55 ~ 75 ℃.

The fifth step is cooled at a constant rate in order to induce the natural precipitation of the crystal after heating, at this time it is preferable to cool to a temperature of 10 ~ 30 ℃ for 3 to 6 hours, based on this preferred cooling rate It is preferable that they are 5 degrees C / hr-10 degrees C / hr.

The sixth step is preferably performed by adding an organic solvent at a temperature of 20 ℃ of the culture concentrate, the hydrophilic organic solvent can be carried out by adding so as to be in the range of 30 ~ 60 (v / v)% of the total liquid phase. have.

The hydrophilic organic solvent may also be used with lower alcohols, preferably methanol, ethanol, propanol, isopropanol or mixtures thereof, and most preferably, the resulting crystals may be carried out using methanol which is most suitable for solid-liquid separation. Can be.

The organic solvent may be diluted with water and used, but since the addition amount of the solvent increases as the dilution ratio increases, industrially, the concentration range of the solvent is preferably 70 to 95 (v / v)%.

The present invention relates to a method for producing 5'-inosinoate (IMP2Na) using a crystallization method, the amount of waste water generated by the method compared to the ion exchange resin method, and only by controlling the temperature and using a small amount of organic solvent It is possible to produce high purity disodium inosine, which is an excellent invention in the food industry.

Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these examples.

Example  One : Of disodium inosine  Establish crystallization conditions

In order to establish the optimum crystallization condition of disodium inosinate, the solubility of disodium inosinate was measured according to temperature and organic solvent. The method measured the solubility of disodium inosinoate by using HPLC by adjusting the concentration (v / v) of the organic solvent in the total liquid at 20, 30, 40, 50, and 60 ° C to 0 to 60%. (See Figure 1)

The supersaturation curve of the concentrate measures the solubility of the disodium inosinate solution in the fermentation broth and the nucleus suddenly increases when the temperature increases and the temperature at which the nucleation increases abruptly with temperature at each organic solvent concentration (v / v). Missing spots were recorded using an on-line particle size analyzer, and obtained by careful analysis of particle number, average size, and concentration of particles. In addition, the solubility of the fermentation broth by the temperature of disodium inosinate and the addition of the organic solvent was obtained through experiments to determine the proper cooling temperature by calculating the crystal formation rate according to the cooling temperature, and compared with the addition of the hydrophilic organic solvent from the beginning. .

Experimental results are shown in FIGS. 1 and 2. As a result of the experiment, the precipitation of crystals is based on the inosinic acid supersaturation of the culture concentrate.In comparison with the supersaturation curve, the inosinic acid concentration curve according to the actual crystallization time and condition of the inoculic acid solubility curve and the supersaturation was compared with the forced introduction of the organic solvent. It was found to exist stably in the metastable region between the curves, thereby improving the grain size of the crystals by suppressing amorphous generation.

Example  2: application of crystallization method of culture concentrate by crystallization of organic solvent

Based on the results of Example 1, disodium inosinoate was produced from the fermentation broth in the following manner.

Step 1: Strain Cultivation and Cell Separation

In the present invention, Corynebacterium glutamicum CJM107 (KCCM-10227) strain, which is a strain producing inosine acid, was used. Strains in fermentation broth containing 46 g of glucose, 30 g of fructose, 10 g of yeast extract, 18 g of KH ₂ PO _4, 18 g of K ₂ HPO _4, 42 g of urea, 6 g of MgSO ₄ · 7H ₂ O, 30 μg of biotin, and 5 mg of thiamin-HCl Was inoculated and cultured in 18 L medium having a pH of 6 to 8 at a culture temperature of 30 to 32 ° C. until the inosinic acid content in the fermentation broth was about 5 hours to 10%. Inosine acid content in the fermentation broth was confirmed through HPLC analysis. Even if it is not a genus of Corynebacterium, the same effect can be obtained using any strain as long as the strain produces inosine acid.

The fermentation broth was separated and removed from the cell sludge using a membrane filter to obtain a filtrate. The filtration process is preferably carried out by heating the fermentation broth to 50 ℃, which is to increase the efficiency of cell separation, the rate of the filtrate comes out at about 50 ℃ rather than low temperature can be reduced the filtration time, the This is because productivity can be expected to increase. However, if the temperature rises above 50 ℃, the decomposition of inosinic acid occurs, so care should be taken. The filtration process can be separated using a centrifugation or heat treatment method in addition to the filtration method from the viewpoint of those skilled in the art.

Step 2: Fermentation Filtrate  concentration

The filtrate was concentrated under a vacuum of 680 mmHg and a vessel temperature of 55 ° C. to reduce inosinic acid concentration to 230 to 260 g / L using a vacuum drying method. The same step can be obtained even if the concentration of the inosinic acid concentration is 200 ~ 300 g / L.

Step 3: pH  adjustment

The pH was adjusted to 7.5 by adding 60 mL of 35% hydrochloric acid solution to 1 L of the fermentation broth to the culture concentrate. In order to crystallize disodium inosine from the concentrate, it is preferable to adjust the pH to neutral to basic because the dissolution of disodium inosinate in the neutral pH range is the lowest and the yield by crystallization is the highest. 6-10, preferably adjusted to pH 7.5-9.5. 250 mL of the pH-adjusted concentrate was added to a crystal precipitation tube.

Step 4: Gaon

In order to prevent the crystals from being formed first from the concentrate, the temperature of the crystal precipitation tube into which the concentrate was added was heated to 60 ° C. to keep the crystals completely dissolved. The same effect will be obtained even if it is heated in the range of 55-75 degreeC from a viewpoint of a person skilled in the art.

Step 5: Add Organic Solvent

350 mL of 95% (v / v) methanol aqueous solution was added to the fermentation broth heated to 60 ° C. at a constant flow rate for 5 hours at which time the temperature was maintained at 60 ° C. Lower alcohols may be used instead of methanol as other hydrophilic organic solvents, preferably methanol, ethanol, propanol, isopropanol or mixtures thereof, most preferably the shape of the crystals obtained is most suitable for solid-liquid separation. Methanol can be used.

Although the above organic solvent may be diluted with water and used, since the addition amount of the solvent increases as the dilution ratio increases, industrially, the concentration range of the solvent is preferably 70 to 100 (v / v)%.

Step 6: Cooling and Separation of Crystals

After the completion of the methanol addition, in order to induce natural precipitation of the crystals, the temperature was cooled to 40 ° C. over 2 hours, and the crystals were separated using a basket separator using centrifugation.

Analysis of Separated Crystals

Purity of the disodium inosinate was measured by HPLC quantification, and the water was dried at 105 degrees in a dry oven for 3 hours to measure moisture, and the crystals were dissolved at a concentration of 5% (w / v) to produce a spectrophotometer. The transmittance (T%) was measured at 430 nm using a spectrophotometer, and the weight of the entire crystal obtained was measured using an electronic balance. As a result of measurement, the purity of the separated disodium inosine was 79%, moisture was 29%, and T% was 72.5, and the total weight was 67.5 g.

Example  3: application of fermentation concentrate crystallization method by cooling crystallization

In Example 3, steps 1, 2 and 3 of Example 2 were performed in the same manner. Since step 4 will be described.

Step 4: Gaon

In the same manner as in Example 1, the temperature of the crystal precipitation tube was warmed to 60 ° C. to keep the crystal completely dissolved.

Step 5: Cool

Cooling was performed in the absence of crystals in the heating step. Cooling was cooled for 5 hours at a constant rate to 20 ℃, the crystal production rate was 70% based on the supernatant concentration.

Step 6: Add Organic Solvents and Separate Crystals

150 mL of an aqueous 95 (v / v)% methanol solution was added for 2 hours to precipitate the remaining disodium inosinate as a crystal in the supernatant. The temperature of the crystal precipitation tube was added at a temperature of 20 ° C. during methanol injection.

The purity of the resulting disodium inosinoate was 92.5%, moisture 31%, T% 92.2, and the total weight was 66.3 g. Based on the above results, it was confirmed that adding the organic solvent after cooling was more effective in terms of improving the purity of the crystal than adding and cooling the organic solvent.

Example  4: application of fermentation concentrate crystallization method by cooling crystallization (80% methanol)

In Example 4, the same process was performed up to step 5 of Example 3, but the concentration of the organic solvent added in step 6 was added to compare the results. After that, step 6 will be described in detail.

Step 6: Add Organic Solvents and Separate Crystals

In order to precipitate disodium inosinate remaining in the supernatant as crystals, 150 mL of an 80 (v / v)% methanol aqueous solution was added for 3 hours. The temperature of the crystal precipitation tube was added at a temperature of 20 ° C. during methanol injection.

The purity of the resulting disodium inosinoate was 94.9%, water 30%, T% 92.5, and the total weight was 72.4 g.

According to the results of the present example, it was confirmed that the highest purity inosinic acid or sodium was obtained by the method of Example 4.

There may be a difference in purity of the inosinate crystals obtained according to the concentration range of the organic solvent, and there may be a change in yield. It was found that the addition ratio of the hydrophilic organic solvent in the liquid phase of the crystal precipitation system is advantageous to the crystal yield in a constant range of 30 to 40 (v / v)% with respect to the whole liquid phase, and exceeds the volume ratio. In this case, impurities were precipitated and found to act as a cause of lowering inosinate crystal purity.

The content of disodium inosinate obtained according to Examples 1 to 3 was 85 to 95%, and the water content was 28.5% or less. As a result of this embodiment, it was confirmed that disodium inosinate was improved by 10 to 20% in purity from the method of adding the organic solvent from the initial crystallization (Korean Patent Application No. 10-2006-67305).

In addition, compared to the invention of the Republic of Korea Patent Application No. 10-2006-67305 described above, it is carried out by shortening three times the crystallization operation once, reducing the amount of organic solvent introduced through the cooling crystallization and reducing the inosine disodium content There was an effect of raising to 85 to 95% level. This is summarized as follows.

division Organic solvent input (v / v,%) # 1 primary inosine disodium content (%) Crystallization start temperature (℃) Cooling temperature (℃) Organic solvent input temperature (℃) Conventional method 61 70 60 40 60 How to improve 37 85 60 20 20

The conventional method is to add an organic solvent at 60 degrees, and to cool down to 40 degrees after the completion of the input. The cooling crystallization, which is an improvement method, is first cooled from 60 to 20 degrees and then added by adding a small amount of organic solvent at 20 degrees. By the method of obtaining, inosine disodium content was much higher according to the present invention was found to have a significant effect.

Figure 1 shows the inosinic acid solubility according to the content% of the methanol added to the fermentation broth concentrate.

Figure 2 shows the supersaturation curve of the crystals by adding methanol, a hydrophilic organic solvent.

Claims (9)

A first step of separating and removing the cells from the fermentation broth of the inosinic acid producing strain; A second step of concentrating the fermentation broth from which the cells are separated and removed in the first step; A third step of adjusting the pH of the concentrated solution concentrated in the second step to 6 to 10; A fourth step of completely dissolving the pre-crystallized disodium inosinate by heating the concentrate having pH adjusted in the third step; A fifth step of cooling the concentrated solution in which the disodium inosinate crystal is dissolved in the fourth step; And Disodium inosinoate production method comprising a sixth step of crystallizing disodium inosine by adding a hydrophilic organic solvent to the concentrated solution cooled in the fifth step. The method of claim 1, wherein the second step is concentrated inosinic acid production method, characterized in that the concentration of inosinic acid 200 ~ 300g / L. The method of claim 1, wherein the fourth step warms the temperature to 55-75 ° C. 6. The method of claim 1, wherein the fifth step cools the temperature to 20 to 30 ° C. at a rate of 5 ° C./hr to 10 ° C./hr. The method of claim 1, wherein the sixth step is performed by adding an organic solvent at a temperature of 20 ° C. of the culture concentrate. The method of claim 1, wherein the sixth step adds a hydrophilic organic solvent to a range of 30 to 40 (v / v)% of the total liquid phase. The method of claim 1, wherein the sixth step uses a lower alcohol as a hydrophilic organic solvent. 8. The method for producing disodium inosinoate according to claim 7, wherein the lower alcohol is methanol. 9. The method for producing disodium inosinoate according to claim 8, wherein the concentration of methanol is 70 to 95 (v / v)%.

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