Title of Invention: Separation method of 5'-disodium inosinate
technical field
[One]
The present invention relates to a method for isolating 5'-disodium inosinate from a microbial culture.
background
[2]
Purine derivative nucleotides such as disodium 5'-inosinate (IMP2Na) are used as raw materials for preventive treatment of metabolic disorders, growth promoters, and anticancer drugs in the pharmaceutical sector, and are important substances used as food additives such as seasonings in the food field. . Disodium inosinate is manufactured through fish meat extraction method of inosinic acid (IMP), a substance naturally present in meat and fish, RNA enzyme decomposition method, microbial fermentation method, combination of fermentation method and synthesis method, combination of RNA chemical decomposition and synthesis, etc. do.
[3]
A lot of research has been done on the preparation method of inosinic acid, which is the whole material for manufacturing disodium inosinate, and in order to obtain disodium 5'-inosinate of quality that can be used as a food additive, inosinic acid must be purified to remove impurities.
[4]
An organic solvent can be used in the crystallization step in relation to the method of culturing a microorganism to produce disodium 5'-inosinate from the culture, but a lot of organic solvent must be used, and accordingly, explosion-proof equipment and equipment for workers are required. In addition, in order to remove the organic solvent, a distillation process may be required and a corresponding equipment is required, and an alternative to this is required.
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[5]
The present invention comprises the steps of culturing 5'-inosine acid-producing microorganisms; adjusting the pH of the microbial culture solution to 7.4 to 8.0; Concentrating the pH-adjusted culture solution to form 5'-disodium inosinate crystals; separating 5'-disodium inosinate crystals from the culture solution containing the 5'-disodium inosinate crystals; and washing the separated 5'-disodium inosinate crystals by contacting them with a hydrophilic organic solvent to wash the crystals.
means of solving the problem
[6]
As used herein, "5'-inosinic acid" is a nucleoside monophosphate, also called inosine monophosphate (IMP). "5'-inosinic acid" and "salt thereof" are used interchangeably. Such salts include "5'-inosinate" or disodium 5'-inosinate.
[7]
As used herein, "culture" refers to a result obtained by culturing a microorganism. The culture may include the microbial cells, or metabolites thereof. The metabolites may exist intracellularly or extracellularly. The metabolite may be a nucleic acid metabolite. The nucleic acid metabolite may be 5'-inosine acid or a salt thereof.
[8]
As used herein, "culture broth" refers to a liquid portion from which microbial cells are removed from a culture. The culture medium may contain products discharged or derived from microorganisms. The culture medium may contain a soluble product. The culture medium may contain 5'-inosinic acid or a salt thereof.
[9]
[10]
One aspect of the present invention comprises the steps of culturing 5'-inosine acid-producing microorganisms; adjusting the pH of the microbial culture solution to 7.4 to 8.0; Concentrating the pH-adjusted culture solution to form 5'-disodium inosinate crystals; separating 5'-disodium inosinate crystals from the culture solution containing the 5'-disodium inosinate crystals; and washing the crystals by contacting the separated 5'-disodium inosinate crystals with a hydrophilic organic solvent. It provides a method for separating 5'-disodium inosinate, including.
[11]
The method may include removing the cell body from the microbial culture containing 5'-inosinic acid to obtain a culture solution.
[12]
In the step of obtaining the culture solution, the microorganism may be a microorganism producing 5'-inosine acid. The microorganism may be genetically engineered to increase the production capacity of 5'-inosine acid. The microorganism may be a bacterium. The microorganism may be a gram-positive or a gram-negative bacterium. The bacterium may be a bacterium of the genus Corynebacterium , or the genus Escherichia . The bacteria may be C. glutamicum , C. ammoniagenes , or E. coli . The microorganism may be to excrete 5'-inosinic acid to the outside of the cell.
[13]
The microbial culture may be obtained by culturing the microorganism under conditions to produce 5'-inosine acid in a medium. The microbial culture may include 5'-inosinic acid. The medium may be a known medium used for fermenting 5'-inosinic acid. The medium may contain a carbon source, a nitrogen source, and trace elements. The medium may include molasses or corn steep solution. The medium may be LB medium. The medium may contain 46 g of glucose, 30 g of fructose, 10 g of yeast extract, 18 g of KH 2 PO 4 , 42 g of K 2 HPO 4 , 6 g of urea, 10 g of MgSO 4 7H 2 O, 30 g of biotin, and 5 mg of thiamine hydrochloride per 1 L of distilled water. have.
[14]
[15]
The microbial culture is 0.5 to 20% by weight, 1.0 to 20% by weight, 2.5 to 20% by weight, 5 to 20% by weight based on the total weight of the culture. Or 5 to 10% by weight of 5'-inosine acid may be included. The 5'-inosinic acid may be present in an extracellular medium. The microbial culture may have a pH of 6 to 9. The microbial culture may be one that has not been subjected to ion exchange chromatography or an organic solvent, for example, methanol crystallization.
[16]
[17]
Removing the cell body may be removing cells or cell debris from the liquid component in the culture. Removal of the cell body may be performed by a known method. Removal of the cell body may be performing filtration, centrifugation, or a combination thereof.
[18]
[19]
The method may include adjusting the pH of the culture medium to 7.4 to 8.0. Adjusting the pH may be performed by adding an acid or a base to the culture solution. The acid may be hydrochloric acid. The base may be NaOH. The pH may be pH 7.4 to 8.0, pH 7.6 to 8.0, pH 7.8 to 8.0, pH 7.4 to 7.8, or pH 7.4 to 7.6.
[20]
[21]
The method may include concentrating the pH-adjusted culture solution to form 5'-disodium inosinate crystals. This step may be a crystallization process in which 5'-inosine acid is converted to 5'-inosodium disodium crystal by concentrating the culture medium. This crystallization process is performed by simply concentrating the culture solution to increase the concentration of 5'-inosine acid in the culture solution, and may not use an organic solvent. The organic solvent not used may be a hydrophilic organic solvent. The hydrophilic organic solvent not used may 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. can The hydrophilic organic solvent not used may be ethanol. The concentration of the ethanol may be 50 to 70% in water based on the volume.
[22]
[23]
Concentrating the pH-adjusted culture solution may be to reduce the moisture content of the culture solution. The step of concentrating may be to evaporate moisture by applying heat to the culture solution. The step of concentrating the culture solution is 50 ℃ or more, 60 ℃ or more, 70 ℃ or more, 80 ℃ or more, 90 ℃ or more, 50 to 90 ℃, 50 to 80 ℃, 50 to 70 ℃, 50 to 60 ℃, 60 to 90 It may be carried out at ℃, 60 to 80 ℃, 60 to 70 ℃, or 60 to 65 ℃. The concentration step may be to perform concentration under reduced pressure. The concentration step may be performed by increasing the concentration of 5'-inosine acid in the culture medium until 5'-disodium inosinate crystals are formed. The concentration step is carried out until the concentration of 5'-inosinic acid in the culture medium is 380 to 600 g / L, 400 to 550 g / L, 400 to 500 g / L, 400 to 475 g / L, or 425 to 475 g / L. it could be The concentration step may be performed until 5'-disodium inosinate crystals are formed and the culture solution has a slurry shape containing crystals. Hereinafter, a slurry solution containing crystals is also referred to as "crystal slurry". As used herein, "slurry" refers to a mixture of solids, eg, crystals, with a specific gravity greater than 1 suspended in a liquid generally water. The size of the crystal may have a particle size of 20 to 400 um.
[24]
[25]
The method may further include the step of concentrating the culture solution before the step of adjusting the pH of the culture solution. In this concentration step, 5'-disodium inosinate crystals in the culture medium may not be formed or substantially not formed. The concentration step may be to reduce the moisture content of the culture medium. The step of concentrating may be to evaporate moisture by applying heat to the culture solution. The concentration step may be performed until the concentration of 5'-inosinic acid in the culture medium becomes 150 to 360 g/L. In the concentration step, the concentration of 5'-inosinic acid may be 150 to 360 g / L, 200 to 300 g / L, 220 to 280 g / L, or 240 to 280 g / L. The step of concentrating the culture solution is 50 ℃ or more, 60 ℃ or more, 70 ℃ or more, 80 ℃ or more, 90 ℃ or more, 50 to 90 ℃, 50 to 80 ℃, 50 to 70 ℃, 50 to 60 ℃, 60 to 90 It may be carried out at ℃, 60 to 80 ℃, 60 to 70 ℃, or 60 to 65 ℃. The concentration step may be to perform concentration under reduced pressure.
[26]
[27]
The method may further comprise cooling the culture solution containing the crystals formed after the step of concentrating. The cooling may be 25 to 45 ℃, 25 to 40 ℃, 25 to 35 ℃, or 25 to 30 ℃ cooling. The cooling step may be cooling the crystal slurry at a constant rate. The cooling may be performed at the cooling temperature for 1.0 to 3.0 hours, for example, 1.5 to 2.5 hours, or 2 hours. The constant cooling rate may be 8.0 to 17.0 °C/hr, for example, 10.0 to 15.0 °C/hr, 1.0 to 14.0 °C/hr, 2.0 to 13.0 °C/hr, or about 12.5 °C/hr. The cooling may be performed while injecting the formed crystal slurry into a jacketed glass crystal tube and stirring. When cooled to a lower temperature, the solubility is lowered and more crystals can be recovered. In addition, it is possible to obtain a harder and higher quality crystal when cooling at a uniform rate.
[28]
The method may further include aging the crystals by incubating the slurry at the cooling temperature after cooling is complete. The incubation may be performed for 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1.5 to 2.5 hours, or about 2 hours. The incubation may be performed while stirring the crystal slurry into a jacketed glass crystal tube.
[29]
[30]
The method may further include separating 5'-disodium inosinate crystals from the culture solution containing the formed 5'-disodium inosinate crystals.
[31]
The step of separating the crystal may be to perform a known crystal separation method within the scope not departing from the object of the present invention. The step of separating the crystals may be, for example, centrifugation, filtration, or a combination thereof. The centrifugation may be performed 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. The centrifugation may be performed for 10 to 30 minutes of rotation time.
[32]
[33]
The method may include washing the crystals by contacting the separated 5'-disodium inosinate crystals with a hydrophilic organic solvent.
[34]
In the washing step, the contacting may include mixing the crystals with a hydrophilic organic solvent. The washing may be to reduce the content 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% in water by volume. The hydrophilic organic solvent may be ethanol or a mixture of water and ethanol. The ethanol may be advantageous in that it is inexpensive and has low toxicity. The hydrophilic organic solvent is 10 to 50%, for example, 20 to 50%, 15 to 40%, 20 to 40%, 25 to 40%, 25 to 35% or 25 to 50% of the crystal-containing sample formed by volume. It can be %.
[35]
In the method, the step of separating the crystal and the step of washing the crystal may be performed simultaneously. In addition, the step of separating the crystal and the step of washing the crystal may be performed separately.
[36]
[37]
The method of the present invention may further include the step of removing the coloring material by contacting the washed crystals with activated carbon in an aqueous solvent. The contacting may be mixing and incubating the washed crystals with activated carbon in an aqueous solvent. The aqueous solvent may be water.
[38]
The step of removing the color developing material comprises dissolving the washed crystals in water to a concentration of 250 to 500 g/L, and adding 0.1 to 10% of activated carbon based on the weight of 5'-inosinic acid to an aqueous solution containing crystals. may include. The contacting may be performed at 40 to 80°C.
Effects of the Invention
[39]
According to the separation method of 5'-disodium inosinate according to the present invention, 5'-disodium inosinate crystals can be efficiently separated. In particular, according to the method, the organic solvent is used only in the washing step, so the amount used is small. According to the method, the separated 5'-disodium inosinate crystals have a high separation yield and high purity.
[40]
Specifically, by adjusting the pH of the culture medium, it is possible to significantly increase the separation yield of 5'-disodium inosinate crystals. In addition, by contacting the separated 5'-disodium disodium inosinate crystals with a hydrophilic organic solvent to wash the crystals, the yield is prevented from being lowered compared to washing with water, the color of the formed crystals is improved, and harmful to the crystals Since the organic solvent content is remarkably reduced, it is possible to secure economic feasibility.
Modes for carrying out the invention
[41]
Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
[42]
[43]
Example: IMP Fermentation and IMP Isolation from Culture
[44]
[45]
1. IMP production and culture of microorganisms
[46]
Corynebacterium glutamicum ( Corynebacterium glutamicum ) was inoculated in a 30L fermentation incubator containing 18L of medium and cultured at 31° C. for 140 hours to generate 5'-inosine acid (IMP) in the culture. The 5'-inosinic acid was secreted out of the cell and was present in the culture medium. The medium has an initial pH of 7.2, and per 1 L of distilled water, glucose 46 g, fructose 30 g, yeast extract 10 g, KH 2 PO 4 18 g, K 2 HPO 4 42 g, urea 6 g, MgSO 4 7H 2 O 10 g, biotin 30 g, and thiamine 5 mg of hydrochloride was included. The pH of the final culture was 6.8.
[47]
[48]
After adjusting the pH to 10.5 by adding NaOH of 50w/w% to the resulting culture, the cells were removed by filtration through a microfilter, and a supernatant was obtained. The concentration of 5'-IMP in the supernatant was measured using HPLC. The supernatant contained 140 g/L of 5'-IMP.
[49]
[50]
2. Formation of 5'-disodium inosinate crystals
[51]
2L of the filtrate was put into a rotary evaporator (N-1110V: EYELA, Japan), and moisture in the filtrate was evaporated, and the filtrate was concentrated until the concentration of 5'-IMP reached 260 g/L. Evaporation was performed using a vacuum controller NVC-2200 at an evaporator internal pressure of 120 mmHg, a vessel temperature of 65° C., and an evaporation rate of 1 L/hr. At this time, the temperature of the filtrate inside the vessel was 55°C. This concentration process is hereinafter referred to as "primary concentration".
[52]
[53]
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 seems to be due to the removal of ammonia during the evaporation process.
[54]
[55]
Next, to 1077 mL of the obtained concentrated filtrate, 4.5 mL of 35% (v/v) hydrochloric acid per 1 L was added to adjust the pH to 7.4, and the temperature was maintained at 55°C. Hereinafter, this process is referred to as "pH adjustment process".
[56]
[57]
1082 mL of the concentrated filtrate, whose pH has been adjusted, is placed in the rotary evaporator and under the same conditions as described above, a supersaturated solution in which crystals and liquids with a concentration of 460 g/L of 5'-IMP in the filtrate are mixed, that is, a slurry state. concentrated until Here, the concentration "460 g/L" of 5'-IMP means the amount of both 5'-IMP and 5'-IMP 2Na crystals dissolved. As a result, 5'-IMP and 2Na + ions in 608 mL of the supersaturated solution are precipitated as 5'- disodium inosinate (hereinafter also referred to as "5'-IMP2Na") crystals, and the concentrated filtrate is a solid crystal and a liquid phase of the filtrate became a mixed viscous suspension (hereinafter also referred to as "slurry"). Immediately after the crystallization was finished, the temperature of the obtained slurry was 55° C. and the pH was 8.0. This process is hereinafter referred to as "second concentration and crystallization process". That is, 5'-IMP2Na crystals were formed by the secondary concentration and crystallization process, indicating that 5'-IMP can be converted into 5'-IMP2Na crystals by concentration without using an organic solvent. This is a significant effect that is unexpected for a person skilled in the art.
[58]
[59]
608 mL of the slurry was poured into a jacketed glass crystal tube, and an overhead stirrer was installed on the top (eyela, zz-2121) and cooled at a cooling rate of 12.5° C./hr to 30° C. for 2 hours while stirring at 200 rpm. A jacketed glass crystal tube was used to cool the slurry at a constant rate. After cooling, the jacketed glass crystal tube containing the slurry was incubated at 30° C. for 2 hours while stirring. This process corresponds to the aging process to allow the crystal growth to continue. Hereinafter, this process is referred to as "cooling and aging process".
[60]
[61]
3. Recovery of 5'-disodium inosinate crystals
[62]
5'-IMP2Na crystals were isolated from the obtained aged slurry. Specifically, 608 mL of the obtained aged slurry was placed in a basket separator H-110F (KOKUSAN Co. Ltd., Japan) and centrifuged at 340xg for 20 minutes. The H-110F centrifuge has a perforated basket installed therein, and the basket is connected to an external rotation supply. The perforated basket is made of polyamide multifilament fiber filter fabric, and the air permeability of the filter is 250 L/m 2 /s at 2 mbar. As a result of the centrifugation, 300 g of a cake containing IMP2Na crystals from which the liquid was removed from the slurry was obtained.
[63]
[64]
Next, IMP2Na crystals were washed by spraying 150 ml of a 60% (v/v) aqueous ethanol solution thereto (hereinafter also referred to as "ethanol washing"). As a result of washing, 272 g of 5'-IMP2Na crystals were recovered and dried at room temperature for 24 hours to obtain 265 g of dried columnar 5'-IMP2Na crystals. This 5'-IMP2Na crystal is also called 5'-IMP2Na crystal 7.5 hydrate.
[65]
[66]
Purity and transmittance were measured for the obtained dried 5'-IMP2Na crystal. As a result, the dried 5'-IMP2Na crystals had a yield of 94.6%, a purity of 95.7%, and a transmittance of 86.10%. The yield and purity were calculated according to the following formula.
[67]
[Equation 1]
[68]
Yield = Weight of the obtained 5'-IMP2Na crystal / Weight of 5'-IMP2Na crystal in 2L of the filtrate x 100
[69]
[70]
In order to determine the purity, 1.0 g of the dried 5'-IMP2Na crystal and standard 5'-IMP2Na crystal (Sigma, ≥9.0% (HPLC)) were dissolved in 1L of tertiary distilled water, and each 1.0g/L concentration of experimental group and standard product A solution was prepared. 5 uL of experimental and standard solutions were loaded onto the column in an Agilent 1260 Infinity Quaternary LC (Agilent Technology Inc.) system. The column was a Shiseido CAPCELL PAK C18 ACR (150mm x 4.6mm, 3um). Next, the absorbance at 254 nm of the eluate flowing out while flowing acetonitrile 2% (v/v)/phosphate buffer (pH 2.4) 98% (v/v) to the column at a flow rate of 1 ml/min was measured. did. The phosphate buffer contained 2 g/L of ammonium phosphate, 0.2 g/L of tetrabutyl ammonium phosphate, and 0.82 g/L of phosphoric acid. At this time, the temperature was 35 °C. This HPLC condition was also used to measure the concentration of 5'-IMP in the filtrate. As a result, the purity was calculated according to the following formula.
[71]
[Equation 2]
[72]
Purity = 5'-IMP weight/weight of solids x 100
[73]
[74]
In addition, for transmittance measurement, a solution obtained by dissolving the experimental group in water at a concentration of 5 (w/v)% was placed in a square cell of CARY 100 UV-VIS (Agilent Technology Inc.) and transmittance was measured at 420 nm.
[75]
[76]
In addition, in the "recovery of 5'-disodium inosinate crystals" as a control 1 experiment, except that 60% (v/v) aqueous ethanol solution was not used in the washing process (hereinafter also referred to as "no washing"), the same 5'-IMP2Na crystals were recovered by this method. As a result, the obtained purified crystal of 5'-IMP2Na had a dry weight of 266 g, a yield of 95%, a purity of 86.40%, and a transmittance of 40.48%.
[77]
[78]
In addition, as a control 2 experiment, in the "recovery of 5'-disodium inosinate crystals", in the washing process, instead of a 60% (v/v) aqueous ethanol solution, deionized water at a content of 25% relative to the volume of the 5'-IMP2Na crystal slurry was used. 5'-IMP2Na crystals were recovered in the same manner except that they were used (hereinafter also referred to as "washing with water"). As a result, the obtained purified crystal of 5'-IMP2Na had a dry weight of 240 g, a yield of 85.7%, a purity of 94.64%, and a transmittance of 72.72%.
[79]
[Table 1]
dry weight (g) transference number(%) water(%) Transmittance (%)
experimental group 265 94.6 95.70 86.10
control group 1 266 95.0 86.40 40.48
control group 2 240 85.7 94.64 72.72
[80]
Table 1 shows the dry weight, yield, purity and permeability of 5'-IMP2Na crystals obtained in the experimental group, control group 1 and control group 2. As shown in Table 1, when a 60% (v/v) aqueous ethanol solution was used in the crystal separation process, the purity and permeability of the crystals were improved at the same time compared to the case where no washing was performed or the case of washing with water. Specifically, the experimental group increased purity by 10.8% and 1.1%, and transmittance by 112.7% and 18.4%, compared to Control 1 and Control 2. This increase in purity and permeability is a significant effect unexpected from the prior art. In particular, the fact that the transmittance was increased by 112.7% and 18.4% has an advantage that the produced 5'-IMP2Na crystal can easily satisfy food standards. Specifically, in order to meet the standards for food, the transmittance must be at a level of 98%, whereas the control groups 1 and 2 need to perform recrystallization two or more times, whereas the experimental group can achieve the standard for food even by performing one additional time. In addition, in order to adjust the control groups 1 and 2 to the level of the experimental group, an increase in the amount of washing is required, and the recovery rate decreases.
[81]
[82]
4. Influence of pH in the crystallization step
[83]
In this section, the effect of the pH of the solution on the crystallization of 5'-IMP in the crystallization step was confirmed.
[84]
[85]
Specifically, the pH was adjusted to 7.2 by adding 35% (v/v) hydrochloric acid to 2000 mL of the concentrated filtrate (pH 9.0) obtained after "primary concentration" in "2. 5'-disodium inosinate crystal formation" The temperature was kept at 55°C.
[86]
[87]
2000 mL of the concentrated filtrate whose pH has been adjusted in this way is put into the rotary evaporator, and under the same conditions as described above, a supersaturated solution in which crystals and liquids with a concentration of 400 g/L of 5'-IMP in the filtrate are mixed, that is, a slurry state. concentrated until Here, the concentration "400 g/L" of 5'-IMP means the amount of both 5'-IMP and 5'-IMP 2Na crystals dissolved. As a result, 5'-IMP and 2Na + ions in 608 mL of the supersaturated solution were precipitated as 5'-IMP2Na crystals, and the concentrated filtrate became a viscous suspension in which solid crystals and liquid filtrate were mixed (hereinafter Also called "slurry"). Immediately after the crystallization was finished, the temperature of the obtained slurry was 55° C. and the pH was 8.0.
[88]
[89]
608 mL of the slurry was poured into a jacketed glass crystal tube, and an overhead stirrer was installed on the top (eyela, zz-2121) and cooled at a cooling rate of 12.5° C./hr to 30° C. for 2 hours while stirring at 200 rpm. A jacketed glass crystal tube was used to cool the slurry at a constant rate. After cooling, the jacketed glass crystal tube containing the slurry was incubated at 30° C. for 2 hours while stirring. This process corresponds to the aging process to allow the crystal growth to continue. Hereinafter, this process is referred to as "cooling and aging process".
[90]
[91]
608 mL of the cooled filtrate was brought to pH 7.4-9.0 using 35% (v/v) hydrochloric acid and 50% NaOH solution. The supernatant of the filtrate at each pH was loaded onto HPLC as described above and eluted to measure the concentration of 5'-IMP in the filtrate. Table 2 shows the concentration of 5'-IMP in the filtrate according to pH.
[92]
[Table 2]
pH 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0
IMP concentration (g/L) 60.2 52.7 48.3 49.0 52.4 55.4 60.6 73.7 86.0 113.7
[93]
As shown in Table 2, the concentration of 5'-IMP was the lowest at pH 7.4 to 8.0. In Table 2, the low solubility at pH 7.4 to 8.0 means that 5'-IMP2Na crystals are well formed in this pH range.
[94]
[95]
5. Effect of organic solvent, concentration and content used in the separation of 5'-disodium inosinate crystals
[96]
In this section, when other organic solvents, concentrations and contents are changed instead of 60% (v/v) ethanol aqueous solution in "3. Recovery of 5'-disodium inosinate crystals", the solubility of 5'-IMP, 5' The effect on the color of -IMP2Na crystals and the purity, water content and absorbance of 5'-IMP2Na crystals was confirmed.
[97]
[98]
(1) Solubility of 5'-IMP in organic solvents and absorbance of solutions in which crystals are dissolved
[99]
608 ml of the aged slurry obtained in "2. 5'-disodium inosinate crystal formation" was added to 1000 ml of the organic solvent shown in Table 3 and stirred at 25° C. for 1 hour so that the 5'-IMP in the slurry was dissolved in the organic solvent. did This solution was centrifuged at 1500 rpm for 10 minutes to obtain a supernatant.
[100]
[101]
Thereafter, the concentration of 5'-IMP in the supernatant was measured using HPLC. The crystals were separated as described in "3. Recovery of 5'-disodium inosinate crystals" and dissolved in water to prepare a 5 (w/v)% aqueous solution, and absorbance at 420 nm was measured. Since the main impurities in the crystal are yellow-brown, absorbance of 420 nm was selected to measure the content of these color substances. Absorbance was measured using a CARY 100 UV-VIS (Agilent Technology Inc.) instrument.
[102]
Table 3 shows the solubility of 5'-IMP according to the type and concentration of the organic solvent. In Tables 3 and 4, % is on a volume/volume basis.
[103]
[Table 3]
division 10% 30% 50% 60% 70% 90% 100%
water - - - - - - 178.86
methanol 100.71 29.76 4.91 2.51 1.50 0.80 1.14
ethanol 93.32 12.59 1.97 1.23 0.12 0.00 0.93
isopropanol 96.02 15.63 2.39 1.76 0.11 0.00 0.00
[104]
Table 4 shows the absorbance of a solution obtained by dissolving 5'-IMP2Na crystals obtained by different types and concentrations of organic solvents in water.
[105]
[106]
[Table 4]
division 10% 30% 50% 60% 70% 90% 100%
water - - - - - - 0.03
methanol 0.03 0.04 0.04 0.05 0.06 0.14 0.23
ethanol 0.04 0.04 0.04 0.08 0.12 0.35 0.44
isopropanol 0.03 0.03 0.04 0.17 0.22 0.44 0.50
[107]
As shown in Tables 3 and 4, when the concentration of the hydrophilic organic solvent was 50 to 70%, it was confirmed that the concentration of 5'-IMP in the mother solution was low and the absorbance of the washed crystals was low.
[108]
[109]
(2) Characteristics of crystals according to the organic solvent content
[110]
The same procedure except that the amount of 60% (v/v) aqueous ethanol solution used in "3. Recovery of 5'-disodium inosinate crystals" was changed to 0 to 40% (v/v) relative to the volume of the slurry. 5'-IMP2Na crystals were separated, and crystal purity, crystal moisture, and absorbance were measured. Crystal moisture was measured by the dry oven method, which is a method of measuring the weight before and after drying at 125° C. in an oven for 3 hours. Crystal purity, and absorbance were determined as described above.
[111]
[Table 5]
division 0% 5% 15% 25% 35% 40%
Crystalline purity (%) 86.4 91.18 92.60 95.7 96.00 96.01
Crystal Moisture (%) 29.46 30.40 30.89 30.83 30.62 30.56
absorbance 0.42 0.24 0.17 0.12 0.09 0.09
[112]
As shown in Table 5, when a 60% (v/v) aqueous ethanol solution, which is a washing solution, is used in 25% or more, for example, 25% to 35% of the volume of the crystal slurry, crystal purity, crystal moisture and absorbance are simultaneously improved. became
Claims
[Claim 1]
Culturing a 5'-inosinic acid producing microorganism; adjusting the pH of the microbial culture solution to 7.4 to 8.0; Concentrating the pH-adjusted culture solution to form 5'-disodium inosinate crystals; separating 5'-disodium inosinate crystals from the culture solution containing the 5'-disodium inosinate crystals; and washing the crystals by contacting the 5'-disodium inosinate crystals with a hydrophilic organic solvent.
[Claim 2]
The method according to claim 1, wherein the microbial culture solution comprises 5 to 20% by weight of 5'-inosinic acid based on the total weight of the culture solution.
[Claim 3]
The method according to claim 1, wherein the microorganism is a bacterium of the genus Corynebacterium or Escherichia .
[Claim 4]
The method according to claim 1, wherein the step of concentrating is to perform concentration under reduced pressure.
[Claim 5]
The method according to claim 1, wherein the concentration step is performed until the concentration of 5'-inosinic acid in the culture medium becomes 400 to 550 g/L.
[Claim 6]
The method according to claim 1, further comprising the step of concentrating the culture solution before the step of adjusting the pH.
[Claim 7]
The method according to claim 6, wherein the step of concentrating the culture solution before adjusting the pH is performed until the concentration of 5'-inosine acid in the culture solution becomes 150 to 360 g/L.
[Claim 8]
The method according to claim 6, wherein the step of concentrating the culture solution before the step of adjusting the pH is to perform concentration under reduced pressure.
[Claim 9]
The method of claim 1 , comprising cooling the crystals formed after the concentrating step.
[Claim 10]
10. The method of claim 9, wherein the cooling is cooling to 25-30 °C.
[Claim 11]
The method according to claim 1, wherein the hydrophilic organic solvent is a C1-C5 alcohol.
[Claim 12]
The method according to claim 11, wherein the alcohol is at least one selected from the group consisting of methanol, ethanol, propanol and isopropanol.
[Claim 13]
The method according to claim 1, wherein the hydrophilic organic solvent is ethanol.
[Claim 14]
The method according to claim 1, wherein the concentration of the hydrophilic organic solvent is 50 to 70% (v/v).
[Claim 15]
The method according to claim 1, wherein the volume of the hydrophilic organic solvent is 20 to 50% of the total volume of the crystal-containing sample formed.
[Claim 16]
The method of claim 1 , further comprising the step of contacting the washed crystals with activated carbon in an aqueous solvent to remove the chromogen.