KR20080007985A - A method for purifying 5'-inosinic acid fermentation broth via crystallization process - Google Patents

Abstract

A method for purifying disodium 5'-inosinate from a microorganism-fermented solution is provided to replace an ion exchange resin column process with a crystallization process by improving a conventional purification process, thereby capable of significantly decreasing the generating amount of the wastewater/waste solution and the amount of chemicals such as acid and alkali. A method for purifying disodium 5'-inosinate from a microorganism-fermented solution comprises the steps of: (a) culturing a 5'-inosinic acid producing microorganism and removing microorganisms therefrom through filtration, centrifugation or heat-treatment to prepare a fermented broth; (b) adding a hydrophilic organic solvent such as methanol, ethanol, propanol, isopropanol and a mixture thereof to the fermented broth to crystallize the disodium 5'-inosinate; (c) re-dissolving the crystals and decolorizing it using activated carbon; and (d) crystallizing the filtrate obtained by removing the activated carbon.

Description

Purification method of 5'-inosinic acid fermentation broth using crystallization process {A method for purifying 5'-Inosinic acid fermentation broth via crystallization process}

The present invention relates to a method for purifying disodium 5'-inosinate (IMP2Na). More specifically, the present invention relates to a method for purifying 5'-inosinate disodium salt from 5'-inosinic acid (IMP) fermentation broth using a crystallization process.

Purine derivative nucleotides such as IMP are used as raw materials for the prophylaxis, growth promoter, and anticancer agent of the large sea in the medical field, and are important substances used as food additives such as seasonings in the food field. In particular, IMP is a net ingredient of food, which is naturally present in meat, fish, etc., and is produced through fish extracting method, RNA enzymatic digestion method, microbial fermentation method, fermentation and synthesis method combination, RNA chemical decomposition and synthesis combination, etc. have. However, no matter which method is produced, the resultant contains pigments, sugars, amino acids, organic acids, inorganic salts, and the like, so that they can be purified to obtain disodium 5'-inosinate of quality that can be used as a food additive. Impurities must be removed.

Purification methods known to date include activated carbon treatment, ion exchange membrane treatment, electrodialysis, precipitation, strong basic anion exchange resin, strong acid cation exchange resin, strong acid cation exchange resin and weakly basic anion exchange resin, etc. .

A conventional method for purifying IMP in a microbial fermentation broth is through a fermentation broth or a membrane filtration process and a filtrate from which the cells in the fermentation broth are removed through an ion exchange resin tower to remove ionic impurities, and The final product is produced through the decolorization process and crystallization process. However, this method has the disadvantage of having a large amount of wastewater / waste solution and complicated management and operation. Improvements are needed in terms of economics and productivity required for industrial use.

Therefore, the present inventors have repeatedly experimented to improve the IMP purification process by the ion exchange resin tower process in order to solve the above disadvantages, and the fermentation is completed with a crystallization process that is easy to manage industrially and generates less waste water / waste solution. It was found that IMP2Na purification in solution was possible and came to complete the present invention.

An object of the present invention is to replace the conventional ion exchange resin process used to purify IMP contained in the fermentation broth of IMP producing microorganisms with a crystallization process, the amount of waste water / waste generated in the purification process and the amount of chemicals such as acid and base It is to provide a method for purifying disodium 5'-inosinate which can greatly reduce the amount of.

In order to achieve the above object, the present invention provides a method for purifying disodium 5'-inosinate from a microbial fermentation broth, the method comprising:

Culturing the 5'-inosinic acid producing strain, and removing the cells to obtain a fermentation broth containing 5'-inosinic acid;

Crystallizing disodium 5'-inosinate by adding a hydrophilic organic solvent to the fermentation broth;

Redissolution of the crystals and decolorizing with activated carbon; And

And removing the activated carbon and crystallizing the filtrate obtained.

In the present invention, any strain can be used as long as the strain used for fermentation is a strain producing IMP. Preferably, Corynebacterium (Corynebacterium) in strain that is used frequently, the IMP fermentation. The fermentation conditions of the microorganisms are not particularly limited, and preferably, media and fermentation conditions of the following composition can be used:

It contains 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 g of biotin, and 5 mg of thiamine-HCl. Incubate for about 140 hours at a culture temperature of 30-32 ° C. in 18 L of 6-8 medium.

In one embodiment of the present invention by culturing the strain Corynebacterium ( Coynebacterium ) is used to obtain a fermentation broth containing about 5% to 10% IMP.

After culturing the 5'-inosinic acid producing strain, the cells are removed to obtain a fermentation broth containing 5'-inosine acid. Removing the cells from the microbial fermentation broth may be performed by a method selected from the group consisting of filtration, centrifugation, and heat treatment.

In one embodiment of the invention, the fermentation broth is filtered by a membrane filtration process. By this filtration process, the fermentation broth is separated into filtrate and cell sludge. Any membrane filter to be used may be used as long as it can separate the cells from the fermentation broth. The operating conditions of the membrane filter can also be easily set by those skilled in the art in order to separate the cells from the fermentation broth. For example, the fermentation broth may be heated to about 40-50 ° C. in advance, and the fermentation broth may be performed at a temperature of about 40-50 ° C. and a pressure (TMP) between the membranes of 1.2 to 1.5 atm. The size of the pores of the membrane used can also be readily selected by those skilled in the art.

In one embodiment of the present invention, the filtrate obtained by membrane filtration is dried so that the content of IMP is 15-30%. This drying process reduces the water content in the filtrate to reduce the total volume of the filtrate, while simultaneously increasing the content of IMP. Drying procedures are well known in the art. Examples of drying processes include reduced pressure drying.

When the concentration is completed, the hydrophilic organic solvent is added to the fermentation broth from which the cells are removed to adjust the pH of the concentrated fermentation broth before crystallization. Crystal precipitation occurs in the region where 5'-inosinic acid disodium salt is produced, that is, pH 6-10, so that the pH of the fermentation broth is adjusted to this range. Preferably, the pH of the fermentation broth from which the cells are removed is adjusted to 7.5 to 9.5.

As the hydrophilic organic solvent to be used, methanol, ethanol, propanol, isopropanol, or mixtures thereof can be used. Preferably, methanol is used in which the shape of the crystal obtained is most suitable for solid-liquid separation. In addition, although the organic solvent used may be diluted with water and used, since the addition amount of a solvent increases as a dilution magnification increases, industrially, the concentration range of a solvent is preferable from 80 to 100 volume%.

The temperature during organic solvent injection addition can be set to 50 to 80 ° C, preferably 65 ± 10 ° C. In addition, the addition of the organic solvent injection may be accompanied by a temperature change such as a cooling operation.

After completion of the organic solvent injection addition, it is also possible to immediately carry out solid-liquid separation, but may further comprise the step of cooling to improve the yield.

The proportion (concentration) of the organic solvent in the liquid phase of the crystal precipitation system may be in the range of 40 to 80% by volume, but industrially, 50% by volume or more is preferable to secure a yield.

Crystals obtained through this process can be obtained in the same way as the conventional method for purifying disodium inosine acid, and further through decolorization and crystallization using activated carbon one or two times to obtain a final disodium inosinate product. The crystallization process at this time may use a hydrophilic organic solvent or a concentrated / cooled crystal method.

The decolorization process using activated carbon is performed by adding activated carbon to a solution in which the crystals obtained through the crystallization process are re-dissolved and stirring for a predetermined time so that the pigment component is adsorbed onto the activated carbon and then separating the activated carbon.

The temperature during adsorbing the pigment component by adding activated carbon can be set to 30 to 80 ° C., but it is preferably carried out at 50 to 70 ° C.

The process of removing activated carbon and crystallizing the obtained filtrate may use a hydrophilic organic solvent or a concentrated crystallization method in the same manner as in the above crystallization process.

The final crystal of IMP2Na obtained according to the method of the present invention has a characteristic of IMP2Na content of 97.0 to 102.0%, moisture of 28.5% or less and pH (5% solution) of 7.0 to 8.5 with respect to dry weight.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are intended to illustrate the present invention by way of example, but the scope of the present invention is not limited to these embodiments.

Example  1: application of crystallization (primary to tertiary) by hydrophilic organic solvent

In this embodiment, 6,000 ml of the fermentation broth containing IMP is heated to 40 ° C., and then a membrane filter (pore size: 0.1 μm, area of 1.0 m) under conditions of 0.5 to 3.0 atm pressure (TMP) between the membranes. Filtration was carried out by ² ) to separate the cell sludge and the filtrate. The filtrate was dried under reduced pressure at a vacuum degree of 680 mmHg and a container temperature of 55 ° C. to obtain 1,950 ml of a concentrate having an IMP content of 25%. 3,120 mL of an aqueous solution of 95% by volume methanol was added for 6 hours. The crystal precipitation tube temperature was 70 ° C. at the start of methanol injection and 40 ° C. when the methanol injection addition was completed. After completion of the methanol injection addition the solution was cooled again to 30 ° C. and crystals were separated (primary).

The separated crystals were redissolved in deionized water (DIW) at an IMP concentration of 30%, and activated carbon was added thereto and maintained at a temperature of 60 ° C. for 1 hour while stirring to separate activated carbon.

2,190 ml of the filtrate from which activated carbon was removed was added to a crystal precipitation tube, and warmed to 55 ° C., and 1,752 ml of 95% by volume aqueous methanol solution was added for 3 hours. After completion of the methanol injection addition, the resulting solution was cooled to 25 ° C. and crystals were separated (secondary).

The separated crystals were redissolved in deionized water at an IMP concentration of 30%, activated carbon was added thereto, maintained at a temperature of 60 ° C. under stirring for 1 hour, and then activated carbon was separated.

866 ml of the filtrate from which activated carbon was removed was added to a crystal precipitation tube, and then heated to 60 ° C., and 866 ml of 95% by volume aqueous methanol solution was added for 3 hours. After completion of the methanol injection addition, the mixture was cooled to 30 ° C, crystals were separated, and dried (tertiary).

The final IMP2Na crystals obtained in this example had an IMP content of 99.5%, moisture 22.7%, pH 7.6 (5% aqueous solution), and a total weight of 369.5 g.

Example  2: crystallization by hydrophilic organic solvent (1st ~ 2nd) + concentration / crystallization (3rd) method applied

In this embodiment, 3,900 ml of the fermentation broth containing IMP is heated to 50 ° C., and then a membrane filter (pore size 0.1 μm, area 1.0 m) under conditions of 0.5 to 3.0 atm pressure (TMP) between the membranes. Filtration was carried out by ² ) to separate the cell sludge and the filtrate. The filtrate was dried under reduced pressure at a vacuum degree of 680 mmHg and a vessel temperature of 55 ° C. to obtain 2,430 ml of a concentrate having an IMP content of 20%. The concentrate was adjusted to 8.0, and then charged into a crystal precipitation tube. The crystal precipitation tube temperature was 65 ° C. at the start of methanol injection, and the crystal precipitation tube temperature was 30 ° C. when the methanol injection addition was completed. After completion of the methanol injection addition the solution was cooled to 20 ° C. and crystals were separated (primary).

The separated crystals were redissolved in deionized water at an IMP concentration of 30%, and then activated carbon was added thereto and maintained at a temperature of 60 ° C. for 1 hour under stirring to separate activated carbon.

2190 mL of the filtrate from which activated carbon was removed was added to a crystal precipitation tube, and then heated to 45 ° C., and 1,970 mL of a 95% by volume aqueous methanol solution was added for 3 hours. After completion of the methanol injection addition, the resulting solution was cooled to 30 ° C. and crystals were separated (secondary).

The separated crystals were redissolved in deionized water at an IMP concentration of 30%, activated carbon was added, and the activated carbon was separated after being maintained at a temperature of 60 ° C. for 1 hour while stirring.

 866 ml of the filtrate from which activated charcoal was removed was dried under reduced pressure at a vacuum degree of 680 mmHg and a container temperature of 55 ° C., concentrated to 68% IMP concentration, added to a crystal precipitation tube, cooled to 15 ° C. for 5 hours, and crystals were separated. It was then dried (tertiary).

The final IMP2Na crystals obtained in this example were IMP content 101.0%, moisture 20.4%, pH 7.9 (5% aqueous solution), and the total weight was 255.8 g.

Example  3: Crystallization by hydrophilic organic solvent (primary) + concentration / crystallization (second to third) method applied

In this embodiment, 4,600 ml of the fermentation broth containing IMP is heated to 50 ° C., and then a membrane filter (pore size: 0.1 μm, area of 1.0 m) under conditions of 0.5 to 3.0 atm pressure (TMP) between the membranes. Filtration was carried out by ² ) to separate the cell sludge and the filtrate. The filtrate was dried under reduced pressure at a vacuum degree of 680 mmHg and a container temperature of 55 ° C. to obtain 1,010 ml of a concentrate having an IMP content of 30%. The pH of the concentrate was adjusted to 8.5, and then poured into a crystal precipitation tube. 1,515 ml of 95% by volume aqueous methanol solution was added for 4 hours. The crystal precipitation tube temperature was 75 ° C. at the start of methanol injection, and the crystal precipitation tube temperature was 35 ° C. when the methanol injection addition was completed. After completion of the methanol injection addition the solution was cooled to 25 ° C. and crystals were separated (primary).

The separated crystals were redissolved in deionized water at an IMP concentration of 30%, activated carbon was added, and the activated carbon was separated after being maintained at a temperature of 60 ° C. for 1 hour while stirring.

1,270 ml of the filtrate from which activated carbon was removed were dried under reduced pressure at a vacuum degree of 680 mmHg and a container temperature of 55 ° C., concentrated to 68% of IMP concentration, and then charged into a crystal precipitation tube. (Secondary).

The separated crystals were re-dissolved in deionized water at an IMP concentration of 30%, activated carbon was added and maintained at a temperature of 60 ° C. for 1 hour, followed by separation of activated carbon.

 633 ml of the filtrate from which activated carbon was removed were dried under reduced pressure at a vacuum degree of 680 mmHg and a container temperature of 55 ° C., concentrated to 68% of IMP concentration, and then charged into a crystal precipitation tube. It was then dried (tertiary).

The final IMP 2 Na crystals obtained in this example were 99.9% IMP content, 16.8% moisture, pH 7.8 (5% aqueous solution), and the total weight was 179.7 g.

According to the method for purifying IMP from the microbial fermentation broth according to the present invention, it is possible to produce IMP2Na products which can be used as food additives only by the crystallization process without the ion exchange resin tower process, and there is no ion exchange resin tower process. The amount of wastewater generated and the use of chemicals such as acids and alkalis used as eluents / regenerators can be greatly reduced.

Claims (11)

Culturing the 5'-inosinic acid producing strain, and removing the cells to obtain a fermentation broth containing 5'-inosinic acid; Crystallizing disodium 5'-inosinate by adding a hydrophilic organic solvent to the fermentation broth; Redissolution of the crystals and decolorizing with activated carbon; And Removing the activated carbon and crystallizing the obtained filtrate; a method for purifying disodium 5'-inosinate from a strain fermentation broth comprising. The method of claim 1, wherein the removing the cells is performed by a method selected from the group consisting of filtration, centrifugation, and heat treatment. The method according to claim 1, wherein the pH of the fermentation broth from which the cells are removed is adjusted to 6-10. The method according to claim 3, wherein the pH of the fermentation broth from which the cells are removed is adjusted to 7.5 to 9.5. The method of claim 1, wherein the hydrophilic organic solvent is at least one solvent selected from the group consisting of methanol, ethanol, propanol, isopropanol and mixtures thereof. The method of claim 1, wherein the hydrophilic organic solvent is methanol. The method of claim 1, wherein the addition of the hydrophilic organic solvent is carried out at 50 ~ 80 ℃. The method of claim 1, further comprising the step of cooling after adding the organic solvent. The method of claim 1, wherein the decolorizing step using the activated carbon is carried out at 30 ~ 80 ℃. The method of claim 1, wherein the decolorizing using the activated carbon is performed two or more times. The method according to claim 1, wherein the step of removing the activated carbon and crystallizing the filtrate obtained is carried out using an organic solvent or using concentration.