Method for preparing disodium 5 '-guanylate crystal

Abstract

The invention relates to a method for preparing disodium 5 '-guanylate crystals, which comprises the following steps of: mixing, namely adding a sodium chloride aqueous solution into a disodium 5'-guanylate aqueous solution; a concentration step: concentrating the mixed solution of the disodium 5 '-guanylate aqueous solution and the sodium chloride aqueous solution, and precipitating the amorphous state of disodium 5'-guanylate; and a conversion step of adding a seed crystal to the mixed solution of the disodium 5 '-guanylate aqueous solution and the sodium chloride aqueous solution to convert the disodium 5'-guanylate into a columnar crystallis.According to the present application, the disodium 5 '-guanylate crystal can be produced at a high yield by an environmentally friendly method without using an organic solvent.

Description

Technical Field

[0001] This disclosure relates to a method for preparing disodium 5'-guanylate crystals in an environmentally friendly manner without using organic solvents. Background Technology

[0002] A well-known solvent crystallization method uses a hydrophilic organic solvent in the preparation of disodium 5'-guanylate derived from fermentation. The disadvantages of this method include increased costs of auxiliary materials and facilities. Furthermore, crystallization methods using hydrophilic organic solvents deviate from ESG principles; therefore, the method needs to be improved to adapt to environmental changes. Summary of the Invention

[0003] Technical issues

[0004] One object of this disclosure is to provide a technique for preparing disodium 5'-guanylate crystals in high yield without the use of organic solvents.

[0005] Technical solution

[0006] This disclosure, which aims to solve the above-mentioned technical problems, has the following structure and features:

[0007] One aspect of this disclosure provides a method for preparing disodium 5'-guanylate crystals, comprising:

[0008] In the mixing step, an aqueous solution of sodium chloride is added to an aqueous solution of disodium 5'-guanylate;

[0009] The concentration step involves concentrating a mixed solution of disodium 5'-guanylate and sodium chloride, precipitating disodium 5'-guanylate into an amorphous crystal; and

[0010] In the conversion step, seed crystals are added to a mixed solution of aqueous solution of disodium 5'-guanylate and aqueous solution of sodium chloride to convert the amorphous state of disodium 5'-guanylate into columnar crystals.

[0011] In one embodiment, the formation of the amorphous state of disodium 5'-guanylate and the transformation of the amorphous state of disodium 5'-guanylate into columnar crystals can be carried out continuously in the transformation step.

[0012] In another embodiment, the amorphous state of disodium 5'-guanylate may include the hydrate of disodium 5'-guanylate.

[0013] In another embodiment, a concentration step may be performed to concentrate the disodium 5'-guanylate to a concentration of 400 g / L to 600 g / L.

[0014] In another embodiment, the preparation method may further include adjusting the pH of the aqueous solution of 5'-guanylate disodium to 8 to 10 before performing the mixing step.

[0015] In another implementation, the pH of the 5'-guanylate disodium solution can be adjusted by adding a sodium salt.

[0016] In another embodiment, sodium chloride may be added in the mixing step at a ratio of 30% to 80% by weight of disodium 5'-guanylate.

[0017] In another embodiment, during the conversion step, seed crystals may be added at a ratio of 0.5% to 50% by weight of disodium 5'-guanylate.

[0018] In yet another embodiment, during the conversion step, seed crystals may be added when the concentration of sodium chloride in the mixed solution is about 100 g / L to about 130 g / L.

[0019] Another aspect of this disclosure provides a fermentation product comprising disodium 5'-guanylate in columnar form, wherein the disodium 5'-guanylate crystals contain chloride ions (Cl... - The concentration of ) ranges from 500 ppm to 20,000 ppm.

[0020] In one embodiment, the fermentation product may further include disodium 5'-inosinate crystals, wherein chloride ions (Cl-) are present in the mixture of disodium 5'-guanylate crystals and disodium 5'-inosinate crystals. - The concentration of ) ranges from 500 ppm to 20,000 ppm.

[0021] In another embodiment, 5'-guanylate disodium crystals may be provided in the form of 5'-guanylate disodium heptahydrate or 5'-guanylate disodium tetrahydrate.

[0022] Beneficial effects

[0023] The method for preparing disodium 5'-guanylate crystals according to one aspect of this disclosure is environmentally friendly because the disodium 5'-guanylate crystals are prepared using an aqueous solvent rather than an organic solvent. Furthermore, the yield of disodium 5'-guanylate crystals can be increased by adjusting process factors to prepare them in an amorphous state, followed by a phase transition to columnar crystals.

[0024] Furthermore, according to one aspect of this disclosure, since it contains 500 ppm to 20,000 ppm of chloride ions, it can provide 5'-guanylate disodium crystals with high storage and distribution stability. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating a method for preparing disodium 5'-guanylate according to the present disclosure.

[0026] Figure 2 This is a flowchart illustrating a method for preparing disodium 5'-guanylate crystals according to one aspect of this disclosure.

[0027] Figure 3 The XRD analysis results of the 5'-guanylate disodium crystals obtained according to Example 2 are shown.

[0028] Figure 4 The image shows a 5'-guanylate disodium crystal obtained according to Example 4, observed under a microscope.

[0029] Figure 5 The particle size analysis results of the 5'-guanylate disodium crystals obtained according to Example 4 are shown.

[0030] Figure 1 This is a flowchart illustrating a method for preparing disodium 5'-guanylate according to the present disclosure.

[0031] Figure 2 This is a flowchart illustrating a method for preparing disodium 5'-guanylate crystals according to one aspect of this disclosure.

[0032] Figure 3 The XRD analysis results of the 5'-guanylate disodium crystals obtained according to Example 2 are shown.

[0033] Figure 4 The image shows a 5'-guanylate disodium crystal obtained according to Example 4, observed under a microscope.

[0034] Figure 5 The particle size analysis results of the 5'-guanylate disodium crystals obtained according to Example 4 are shown. Detailed Implementation

[0035] This disclosure will now be described in detail. Furthermore, each description and embodiment disclosed herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific embodiments described below.

[0036] This disclosure relates to a method for preparing disodium 5'-guanylate crystals. By concentrating a mixed solution of aqueous solution of disodium 5'-guanylate and aqueous solution of sodium chloride, and adding seed crystals thereto, the formation of amorphous disodium 5'-guanylate and the transformation of the amorphous state into columnar crystals can be continuously achieved. Therefore, disodium 5'-guanylate crystals can be prepared in a high-yield manner in an environmentally friendly way.

[0037] Figure 1 This is a flowchart illustrating a method for preparing disodium 5'-guanylate according to the present disclosure.

[0038] Reference Figure 1 The method for preparing 5'-guanylate disodium crystals according to this disclosure includes: a mixing step (S100), in which an aqueous sodium chloride solution is added to an aqueous 5'-guanylate disodium solution;

[0039] In the concentration step (S200), a mixed solution of disodium 5'-guanylate and sodium chloride aqueous solution is concentrated, and amorphous disodium 5'-guanylate is precipitated; and

[0040] In the conversion step (S300), seed crystals are added to a mixed solution of aqueous solution of disodium 5'-guanylate and aqueous solution of sodium chloride to convert the amorphous state of disodium 5'-guanylate into columnar crystals. Each step of the method for preparing disodium 5'-guanylate crystals of this disclosure will be evaluated below.

[0041] First, in the mixing step (S100), an aqueous solution of sodium chloride is added to an aqueous solution of disodium 5'-guanylate and mixed.

[0042] The aqueous solution of disodium 5'-guanylate used in the mixing step (S100) can be a fermentation product prepared by fermentation. As used herein, "fermentation product" can refer to a product obtained by enzymatic or metabolic decomposition of organic matter using microorganisms. For example, the fermentation product can include the culture itself obtained by culturing microorganisms in a culture medium, or a concentrate, dried product, or lyophilized product of a culture obtained by removing bacterial cells therefrom. Furthermore, in this case, the fermentation broth can include all the fermentation product containing disodium 5'-guanylate, or it can be a fermentation broth in which impurities have been removed from the fermentation product containing disodium 5'-guanylate.

[0043] The “microorganism for producing disodium 5'-guanylate” or “microorganism for producing disodium 5'-guanylate or the desired product” used in the mixing step (S100) may include all wild-type microorganisms, or naturally or artificially genetically modified microorganisms, and may be microorganisms that weaken or enhance a specific mechanism due to the insertion of exogenous genes or the enhancement or inactivation of endogenous gene activity, and may include genetically modified microorganisms that produce the desired protein or disodium 5'-guanylate.

[0044] The microorganisms disclosed herein for producing disodium 5'-guanylate can be naturally occurring microorganisms capable of producing disodium 5'-guanylate, or microorganisms to which the ability to produce disodium 5'-guanylate is imparted to a parent strain that does not possess the ability to produce disodium 5'-guanylate, but are not limited thereto. In particular, as used herein, the microorganisms for producing disodium 5'-guanylate or the desired product, or microorganisms capable of producing disodium 5'-guanylate or the desired product, can be microorganisms in which some genes in the biosynthetic pathway of the desired protein or the desired product are enhanced or weakened, or some genes in the degradation pathway of the desired protein or the desired product are enhanced or weakened. An "enhancement" or "increase" in the disodium 5'-guanylate production capacity of the microorganisms of this disclosure can mean that the disodium 5'-guanylate production capacity of the microorganisms of this disclosure is enhanced compared to the disodium 5'-guanylate production capacity of microorganisms different from those of the microorganisms, parent strains, or unmodified microorganisms of this disclosure. In one instance, the 5'-guanylate disodium production capacity of the microorganisms of this disclosure can be increased by about 1% or more, 10% or more, 100% or more, 200% or more, 500% or more, 1000% or more, 1100% or more, 1200% or more, 1300% or more, or about 1.01 times or more, 2 times or more, 5 times or more, 10 times or more, 11 times or more, 12 times or more, or 13 times or more, but not limited thereto, compared with the 5'-guanylate disodium production capacity of other microorganisms. As used herein, the term "about" means a range including all values ​​such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., including all values ​​equivalent to or approximating the value immediately following the term "about," but not limited thereto.

[0045] The microorganisms used in the mixing step (S100) may be at least one selected from the group consisting of: Candida famata as yeast, Eremothecium ashbyii and Ashbyagossypii as ascozoites, Bacillus subtilis as bacteria, and microorganisms belonging to the genus Corynebacterium.

[0046] When the microorganism used in the mixing step (S100) is a Corynebacterium, the microorganism may specifically be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, or Corynebacterium testis. Corynebacterium testudinoris, Corynebacterium crenatum, or Corynebacterium flavescens, and more particularly, Corynebacterium glutamicum, but not limited thereto.

[0047] The mixing step may also include culturing "microorganisms for the production of 5'-guanylate disodium". The cultivation of microorganisms can be carried out according to suitable culture media and culture conditions known in the art. Those skilled in the art can readily adapt such culture methods for use based on the selected strain. In particular, the culture can be batch culture, continuous culture, and / or fed-batch culture, but is not limited thereto. As used herein, the term "culture medium" refers to a mixture of substances containing nutrients required for the cultivation of microorganisms as its main component, providing nutrients and growth factors, as well as water necessary for survival and growth. Specifically, the culture medium and other culture conditions used to cultivate the microorganisms of this disclosure can be any culture medium used for the conventional cultivation of microorganisms, without any particular limitation. However, the microorganisms of this disclosure can be cultured under aerobic conditions in a conventional culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while adjusting temperature, pH, etc.

[0048] The aqueous solution of disodium 5'-guanylate used in the mixing step (S100) can be prepared by dissolving a fermentation product containing disodium 5'-guanylate prepared by the above method in an aqueous solvent. For example, the aqueous solution of disodium 5'-guanylate can be prepared by dissolving disodium 5'-guanylate in crystalline form obtained from the fermentation product in water. In the aqueous solution of disodium 5'-guanylate, disodium 5'-guanylate can be present in the form of a hydrate. For example, disodium 5'-guanylate can be provided in the form of disodium 5'-guanylate heptahydrate or disodium 5'-guanylate tetrahydrate. In this disclosure, an aqueous solvent is used instead of an organic solvent. Therefore, the preparation method of this disclosure can be carried out in an environmentally friendly manner by excluding the use of organic solvents.

[0049] The aqueous solution of disodium 5'-guanylate used in the mixing step (S100) can have a concentration of about 50 g / L to about 600 g / L of disodium 5'-guanylate. In some cases, the concentration of disodium 5'-guanylate can be about 50 g / L to about 550 g / L, about 50 g / L to about 500 g / L, about 50 g / L to about 450 g / L, about 50 g / L to about 400 g / L, about 50 g / L to about 350 g / L, about 50 g / L to about 300 g / L, about 50 g / L to about 250 g / L, about 50 g / L to about 200 g / L, about 50 g / L to about 150 g / L, about 50 g / L to about 100 g / L, about 100 g / L to about 450 g / L, about 150 g / L to about 450 g / L, about 200 g / L to about 450 g / L, about 250 g / L to about 450 g / L, about 300 g / L to about 450 g / L, about 350 g / L to about 450 g / L. The concentrations are approximately 400 g / L to approximately 450 g / L. Aqueous solutions of disodium 5'-guanylate are prepared within the above concentration ranges. When an aqueous solution of disodium 5'-guanylate is prepared within the above concentration range and mixed with sodium chloride, amorphous disodium 5'-guanylate can be sufficiently produced.

[0050] In the mixing step (S100), an aqueous solution of sodium chloride is mixed with an aqueous solution of disodium 5'-guanylate prepared as described above. The aqueous solution of sodium chloride can be a solution in which sodium chloride (NaCl) is uniformly dissolved in an aqueous solvent. The aqueous solvent used to prepare the aqueous solution of sodium chloride and the aqueous solvent used to prepare the above-mentioned aqueous solution of disodium 5'-guanylate can be of the same or different types. For example, both aqueous solutions can be prepared using water as a solvent. In this disclosure, as described above, no organic solvent is used in the method for preparing disodium 5'-guanylate crystals. According to the prior art, the solubility of disodium 5'-guanylate is reduced by treating the precipitated crystals with an organic solvent. When a hydrophilic solvent is used instead of an organic solvent, an additive that can reduce the solubility of the solvent is required. According to this disclosure, sodium chloride, which is approved as a food additive, is added to reduce the solubility of disodium 5'-guanylate in a hydrophilic solvent.

[0051] The sodium chloride aqueous solution used in the mixing step (S100) can be in a state before supersaturation, wherein no sodium chloride salt precipitates. The sodium chloride aqueous solution can be a solution with a sodium chloride concentration of about 10 g / L to 300 g / L. In some cases, the sodium chloride concentration can be about 10 g / L to 200 g / L, about 10 g / L to 100 g / L, about 50 g / L to 300 g / L, about 100 g / L to 300 g / L, and about 200 g / L to 300 g / L. The concentration of the sodium chloride aqueous solution can be determined based on the concentration and amount of the 5'-guanylate disodium aqueous solution used in the mixing step (S100).

[0052] In the mixing step (S100), sodium chloride can be added at a ratio of 30% to 80% by weight of disodium 5'-guanylate. In some cases, sodium chloride can be added at a ratio of about 40% to about 70% or about 40% to about 60% by weight of disodium 5'-guanylate. This differs from techniques that induce a phase transition by treating sodium chloride to a level of 90% to 120% by weight of disodium 5'-guanylate. The method of adding excess sodium chloride increases cost with increasing salt content, and there is a risk of reduced content due to NaCl precipitation and the formation of fine particles when the salt concentration rises to the supersaturation point of the salt during subsequent concentration. In contrast, according to one aspect of this disclosure, a small amount of sodium chloride is used, and the salt in the solution is not supersaturated, thus no precipitation occurs, and therefore disodium 5'-guanylate crystals can be obtained in high yield.

[0053] There are no limitations on the method of mixing the aqueous solutions of disodium 5'-guanylate and sodium chloride in the mixing step (S100). For example, the aqueous solutions of disodium 5'-guanylate and sodium chloride can be mixed in various ways, such as by adding the aqueous solution of sodium chloride dropwise to the aqueous solution of disodium 5'-guanylate.

[0054] Next, a concentration step (S200) is performed, in which a mixed solution of disodium 5'-guanylate and sodium chloride is concentrated and amorphous disodium 5'-guanylate is precipitated.

[0055] In the concentration step (S200), disodium 5'-guanylate is precipitated in an amorphous state. When mixed with an aqueous sodium chloride solution, disodium 5'-guanylate can be provided in hydrate form, and when the solubility reaches the supersaturation point, the hydrated form of disodium 5'-guanylate forms nuclei. Subsequently, the nuclei of disodium 5'-guanylate aggregate together and precipitate in an amorphous state.

[0056] A concentration step (S200) is performed to increase the concentration of disodium 5'-guanylate. The concentration step (S200) can be performed to concentrate the disodium 5'-guanylate to a concentration of 400 g / L to 600 g / L. In some cases, during the concentration step (S200), the disodium 5'-guanylate can be concentrated to concentrations of 400 g / L to 550 g / L, 400 g / L to 500 g / L, 400 g / L to 450 g / L, 450 g / L to 600 g / L, 500 g / L to 600 g / L, and 550 g / L to 600 g / L. By concentrating the mixed solution to the above concentration range, a phase transition occurs continuously as seed crystals are added in the conversion step (S300), thus continuously forming amorphous tetrahydrate or heptahydrate columnar crystals during the concentration process.

[0057] There are no restrictions on the method for performing the concentration step (S200). Concentration can be performed using a conventional concentrator (e.g., a forced circulation concentrator, a thin film concentrator, or a rotary concentrator, etc.) selected according to those skilled in the art.

[0058] The concentration step (S200) can be carried out for approximately 2 hours to approximately 15 hours. In some cases, concentration can be carried out for approximately 2 hours to approximately 10 hours, approximately 2 hours to approximately 5 hours, approximately 5 hours to approximately 15 hours, or approximately 10 hours to approximately 15 hours. The concentration time can be determined based on the concentration of disodium 5'-guanylate, the yield of disodium 5'-guanylate, etc.

[0059] In the concentration step (S200), the temperature inside the concentrator can be adjusted to a range of about 25°C to about 85°C. In some cases, the temperature inside the concentrator can be adjusted to a range of about 25°C to about 75°C, about 25°C to about 65°C, about 25°C to about 55°C, about 25°C to about 45°C, about 25°C to about 35°C, about 35°C to about 85°C, about 35°C to about 75°C, about 35°C to about 65°C, about 35°C to about 55°C, about 35°C to about 45°C, about 45°C to about 85°C, about 45°C to about 75°C, about 45°C to about 65°C, about 45°C to about 55°C, about 55°C to about 85°C, about 55°C to about 75°C, about 55°C to about 65°C, about 65°C to about 85°C, about 65°C to about 75°C, or about 75°C to about 85°C. By maintaining the temperature range of the concentration process within the aforementioned range, 5'-guanylate disodium crystals can be obtained without denaturing the 5'-guanylate disodium to be produced. If necessary, the vacuum level inside the concentration apparatus can be adjusted so that the temperature does not fluctuate outside the aforementioned range during concentration.

[0060] During the concentration step (S200), a conversion step (S300) is performed, in which seed crystals are added to convert the amorphous state of disodium 5'-guanylate into columnar crystals. Specifically, performing the conversion step (S300) concurrently with the concentration step (S200) can mean concentrating the solution to a specific concentration level before adding the seed crystals. However, when adding the seed crystals, the concentration of disodium 5'-guanylate in the solution can be maintained within a specific range by continuously concentrating the solution as needed. Furthermore, in some cases, if the concentration of disodium 5'-guanylate in the solution provided after mixing in the mixing step (S100) is at a level sufficient to precipitate the amorphous state, the conversion step of adding seed crystals can be performed concurrently with the start of the concentration step (S200). Therefore, the concentration step (S200) does not necessarily have to be performed before the conversion step (S300).

[0061] The seed crystal added in the conversion step (S300) can be a crystal of disodium 5'-guanylate. After the seed crystal is injected, the amorphous state of disodium 5'-guanylate can be converted into columnar crystals. Compared with the amorphous state, the columnar crystals have a clearer shape and higher crystallinity. Therefore, the columnar crystals can be separated from the mother liquor relatively easily, thereby increasing the yield of disodium 5'-guanylate. The columnar crystals of disodium 5'-guanylate can include disodium 5'-guanylate in the form of disodium 5'-guanylate heptahydrate.

[0062] In the conversion step (S300), when the concentration of sodium chloride in the mixed solution is at a level of about 100 g / L to about 130 g / L, seed crystals may be added. Since concentration continues even after the addition of seed crystals, the formation of amorphous disodium 5'-guanylate and the conversion to columnar disodium 5'-guanylate based on the addition of seed crystals can occur simultaneously.

[0063] In the conversion step (S300), the size of the formed columnar crystals of disodium 5'-guanylate can vary depending on the conditions of seed crystal injection. In particular, as mentioned above, if the seed crystal is added when the concentration of sodium chloride in the mixed solution is about 100 g / L to about 130 g / L, the formation of the amorphous state of disodium 5'-guanylate and the conversion to columnar crystals of disodium 5'-guanylate according to the addition of the seed crystal can occur simultaneously, and disodium 5'-guanylate crystals with excellent crystallinity can be obtained.

[0064] Conversely, when seed crystals are added outside the aforementioned concentration range, especially when the added seed crystals result in a sodium chloride concentration higher than the range, the crystal transformation rate can be slowed down, and the crystals may form fine particles instead of columnar shapes. To obtain columnar crystals in high yield during the transformation step (S300), it is important to adjust process factors (e.g., the concentration of disodium 5'-guanylate in the solution, the temperature of the solution, and the concentration of sodium chloride, as previously described). This is because various factors (e.g., crystallization solvent, temperature, concentration of disodium 5'-guanylate, cation concentration, other derivatives, etc.) can influence the crystal phase transformation process and interfere with it. If any element constituting the crystallization environment is not handled correctly, the crystal phase transformation may not occur smoothly, or even if it does, it may be obtained in the form of fine crystals, which can reduce the process yield.

[0065] The temperatures of the conversion step (S300) and the concentration step (S200) can be varied depending on the form of disodium 5'-guanylate. For example, when disodium 5'-guanylate is provided as a heptahydrate, the process temperature can be adjusted to a range of approximately 25°C to approximately 42°C to effectively induce the formation of an amorphous state through concentration and conversion into columnar crystals. Furthermore, when disodium 5'-guanylate is provided as a tetrahydrate, the process temperature can be adjusted to a range of approximately 42°C to approximately 85°C to effectively induce the formation of an amorphous state through concentration and conversion into columnar crystals.

[0066] The final product, disodium 5'-guanylate, can be obtained by separating columnar crystals of 5'-guanylate formed after the conversion step (S300) from the mother liquor. Various methods can be used to separate the crystals (e.g., vacuum membrane filtration, pressure-driven membrane filtration, centrifugation, basket separation, etc.).

[0067] As described above, according to this disclosure, the preparation of disodium 5'-guanylate crystals uses an aqueous solvent instead of an organic solvent, which is therefore environmentally friendly. Furthermore, the yield of disodium 5'-guanylate crystals can be increased by adjusting process factors to prepare disodium 5'-guanylate in an amorphous state, followed by a phase transition to columnar crystals.

[0068] In the foregoing, a method for preparing disodium 5'-guanylate crystals according to one aspect of this disclosure was evaluated. Below, more specific aspects of the method for preparing disodium 5'-guanylate will be evaluated.

[0069] Figure 2 This is a flowchart illustrating a method for preparing disodium 5'-guanylate crystals according to one aspect of this disclosure.

[0070] In description Figure 2 In the middle, the previous ones will be omitted. Figure 1 To avoid content duplication, the description should not repeat itself.

[0071] Reference Figure 2 In the mixing step (S100), an aqueous solution of 5'-guanylate disodium can be prepared by dissolving the wet crude crystals of 5'-guanylate prepared by fermentation. The dissolution of the wet crude crystals of 5'-guanylate disodium can be carried out by conventional methods, such as adding the wet crude crystals of 5'-guanylate disodium to an aqueous solvent and then stirring.

[0072] Additionally, before the mixing step (S100), the pH of the 5'-guanylate disodium aqueous solution can be adjusted. Specifically, the pH of the 5'-guanylate disodium aqueous solution can be adjusted to 8 to 10. Within this pH range, the conversion of 5'-guanylate disodium to columnar crystals can proceed rapidly. The pH adjustment of the 5'-guanylate disodium aqueous solution can be achieved by adding sodium salt. Therefore, the removal of sodium ions (Na+) can be prevented. + Other cations besides sodium guanylate are added to the mixed solution and interfere with the crystallization of disodium 5'-guanylate. Examples of sodium salts that can be used to adjust pH include sodium hydroxide, monosodium citrate, disodium citrate, trisodium citrate, monosodium phosphate, disodium phosphate, trisodium phosphate, sodium carbonate, sodium bicarbonate, etc.

[0073] In the mixing step (S100), an additional decolorization process can be performed to remove impurities. The decolorization process aims to remove residual coloring substances from the fermentation broth and can be carried out using an adsorption decolorization method employing activated carbon or white clay (activated clay, acidic clay) as adsorbents. However, the above description is for illustrative purposes, and considering the physical properties of the fermentation broth containing L-citrulline and the characteristics of subsequent processes, the method for performing the decolorization process can be appropriately selected. The order of pH adjustment, bacterial filtration, and decolorization can be varied as needed. For example, pH can be adjusted after bacterial filtration and decolorization, or decolorization can be performed first followed by bacterial cell filtration.

[0074] After the conversion step (S300) and the formation of 5'-guanylate disodium columnar crystals, the steps of cooling the solution, separating the columnar crystals, and drying the separated columnar crystals can be performed. The drying of the crystals can be carried out at room temperature.

[0075] According to one aspect of this disclosure, the columnar crystals of disodium 5'-guanylate prepared by the above method can be provided in the form of a mixture with disodium 5'-inosinate crystals. In particular, according to one aspect of this disclosure, a fermentation product comprising columnar disodium 5'-guanylate crystals and disodium 5'-inosinate crystals, wherein chloride ions (Cl...) are present in the mixture of the disodium 5'-guanylate crystals and the disodium 5'-inosinate crystals. - The concentration of ) ranges from 500 ppm to 20,000 ppm.

[0076] Furthermore, according to one aspect of this disclosure, a crystal or fermentation product comprising disodium 5'-guanylate crystals, wherein the disodium 5'-guanylate crystals have a chloride ion concentration of 500 ppm to 20,000 ppm.

[0077] According to one aspect of this disclosure, 5'-guanylate disodium crystals contain chloride ions (Cl...) - The concentration can range from 500 ppm to 20,000 ppm. Disodium 5'-guanylate crystals with chloride ion concentrations within this range can be provided in columnar crystal form. Because columnar disodium 5'-guanylate crystals are easily separated during preparation, they can contain small amounts of impurities and can have high crystal purity.

[0078] Furthermore, when the chloride ion concentration is within the aforementioned range, microbial growth can be controlled, and solidification caused by the environment in which the manufactured product is sold or stored can be prevented. Therefore, 5'-guanylate disodium crystals containing the aforementioned chloride ion concentration are not only highly pure but also advantageous in terms of storage and sales. Moreover, when chloride ions remain in the final product at a concentration of 500 ppm to 20,000 ppm, microbial growth can be controlled, and solidification due to stress can be reduced. This makes product management easier, and the rate of microbial contamination can be reduced, thus ensuring the product's safe consumption.

[0079] Disodium 5'-guanylate, having the aforementioned chloride ion concentration, can be prepared by the method described above for preparing disodium 5'-guanylate crystals according to one aspect of this disclosure. According to the method for preparing disodium 5'-guanylate crystals according to one aspect of this disclosure, when the concentration of sodium chloride in the mixed solution is about 100 g / L to about 130 g / L of disodium 5'-guanylate, the formation of an amorphous state of disodium 5'-guanylate and the conversion to columnar crystals of disodium 5'-guanylate based on the addition of seed crystals can occur simultaneously. The disodium 5'-guanylate crystals prepared according to this method can contain a specific concentration of chloride ions in the crystals because a specific concentration of sodium chloride is added in the conversion step. Specifically, the chloride ion concentration in the crystals is from 500 ppm to 20,000 ppm.

[0080] In contrast, experiments have shown that 5'-guanylate disodium crystals prepared by methods according to existing techniques (e.g., ethanol crystallization) rather than those according to this disclosure contain less than 100 ppm of chloride ions.

[0081] Embodiments of the present invention

[0082] The present disclosure will be described in detail below by way of embodiments. However, these embodiments are merely preferred embodiments given for illustrative purposes, and therefore the scope of the present disclosure is not intended to be limited to or restricted by these embodiments. Furthermore, those skilled in the art within the scope of this disclosure or similar technical fields will fully understand and readily implement technical features not described herein.

[0083] The method for preparing disodium 5'-guanylate crystals according to one aspect of this disclosure has been evaluated above. The beneficial effects mentioned in this disclosure will be described below through examples and comparative experimental results.

[0084] Comparative Example 1. Crystallization using hydrophilic organic solvents (conventional crystallization method)

[0085] A 0.5 L solution of 30 wt.% disodium 5'-guanylate was prepared by dissolving crude disodium 5'-guanylate crystals of 90% purity derived from microbial fermentation broth. Subsequently, 0.55 L of ethanol was added at room temperature to form an amorphous state. Simultaneously with the addition of ethanol, 10 wt.% disodium 5'-guanylate heptahydrate seed crystals were added, followed by continuous addition of ethanol to induce a phase transition from the amorphous state to the disodium 5'-guanylate heptahydrate crystals. After cooling the crystal slurry to an internal temperature of 25°C, the crystals were separated using a centrifuge and dried at room temperature to obtain the product. The obtained product showed the following mass: purity 100%, moisture 23%, yield 95%. The disodium 5'-guanylate heptahydrate crystals were slurried using 99.5% ethanol, and wet particle size analysis was performed using a particle size analyzer. The average particle size was at the 150 μm level. Chromatographic analysis of the final crystals showed that the Cl content... - The residual level was 39.2 ppm.

[0086] Comparative Example 2. Crystallization of non-concentrated aqueous solution

[0087] A 0.5 L solution of 30 wt.% disodium 5'-guanylate was prepared by dissolving crude disodium 5'-guanylate crystals. A 0.55 L solution of 30 wt.% was prepared by dissolving sodium chloride in water. The amount of sodium chloride was approximately 90% by mass relative to the disodium 5'-guanylate solution. The sodium chloride solution was added dropwise to the disodium 5'-guanylate solution at approximately 42°C to induce a phase transition from the amorphous state of disodium 5'-guanylate to the heptahydrate crystals of disodium 5'-guanylate. After slow cooling to approximately 25°C, the crystals separated using a basket centrifuge had a purity of 98%, a moisture content of 25%, and a yield of 69%.

[0088] Comparative Example 3. Crystallization of non-concentrated aqueous solution

[0089] A 0.2 L solution of 30 wt.% disodium 5'-guanylate was prepared by dissolving crude disodium 5'-guanylate crystals, followed by the addition of 50 wt.% NaOH (sodium hydroxide solution) to adjust the pH to 9.0. A 0.2 L solution of 30 wt.% was prepared by dissolving sodium chloride in water. The amount of sodium chloride was approximately 40% by mass relative to the disodium 5'-guanylate solution. The temperature at which the homogeneous amorphous solution precipitated by mixing the two solutions was measured using a thermometer and maintained at a temperature not exceeding approximately 25°C to approximately 42°C. 1 wt.% seed crystal of disodium 5'-guanylate heptahydrate was added to induce a phase transition, but no phase transition occurred. The amorphous state was centrifuged using a basket separator, but the crystals could not be separated.

[0090] Experimental Example 1 - Synthesis of disodium 5'-guanylate based on the amount of sodium chloride added

[0091] Example 1

[0092] A 0.5 L solution of 30 wt.% disodium 5'-guanylate was prepared by dissolving crude disodium 5'-guanylate crystals, followed by the addition of 50 wt.% NaOH (sodium hydroxide solution) to adjust the pH to 9.0. A 0.2 L solution of 30 wt.% was prepared by dissolving sodium chloride in water. The amount of sodium chloride was approximately 40% by mass relative to the mass of disodium 5'-guanylate in the disodium 5'-guanylate solution. Concentration and crystallization were initiated in a homogeneous solution, with the amorphous state precipitated from the homogeneous solution by mixing the two solutions. Concentration was carried out by adjusting the vacuum and measuring the internal temperature with a thermometer to ensure the temperature did not exceed 25°C to 42°C. At a concentration level of 2.1 times, 1 wt.% of disodium 5'-guanylate heptahydrate seed crystals were added to continuously induce a phase transition. After the seed crystals were injected, the amorphous state of disodium 5'-guanylate continuously transformed into columnar disodium 5'-guanylate heptahydrate crystals. During the phase transition, the disodium 5'-guanylate heptahydrate was concentrated until a concentration of at least 48 wt.% was reached. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated using a centrifuge, and dried at room temperature. The obtained disodium 5'-guanylate crystals had a purity of 100%, a moisture content of 23%, and a yield of 93%. The disodium 5'-guanylate heptahydrate crystals were slurried using 99.5% ethanol, and wet particle size analysis was performed using a particle size analyzer. The results showed an average particle size of 250 μm. HPLC analysis revealed residual anions (Cl-) in the final crystals. - The amount of ) was 1474.9 ppm.

[0093] Example 2

[0094] A 0.5 L solution of 30 wt.% disodium 5'-guanylate was prepared by dissolving crude disodium 5'-guanylate crystals, followed by the addition of 50 wt.% NaOH (sodium hydroxide solution) to adjust the pH to 9.0. Anhydrous sodium carbonate was then added at a ratio of 5% by weight of disodium 5'-guanylate. A 0.33 L solution of 30 wt.% was prepared by dissolving sodium chloride in water. The amount of sodium chloride was approximately 60% by mass relative to the mass of disodium 5'-guanylate in the disodium 5'-guanylate solution. Concentration and crystallization were initiated in a homogeneous solution, with the amorphous state precipitated from the homogeneous solution by mixing the two solutions. Concentration was carried out by measuring the internal temperature with a thermometer to ensure the temperature did not exceed 25°C to 42°C. At a concentration level of 1.05, 1 wt.% of disodium 5'-guanylate heptahydrate seed crystals was added to continuously induce a phase transition. After seed crystal injection, the amorphous state of disodium 5'-guanylate continuously transformed into columnar disodium 5'-guanylate heptahydrate crystals. The disodium 5'-guanylate heptahydrate was concentrated until a concentration of at least 44 wt.% was achieved. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated using a centrifuge, and dried at room temperature. The obtained disodium 5'-guanylate crystals had a purity of 100.7%, a moisture content of 23%, and a yield of 85%. The disodium 5'-guanylate heptahydrate crystals were slurried using 99.5% ethanol, and wet particle size analysis was performed using a particle size analyzer. The results showed an average particle size of 290 μm. HPLC analysis revealed residual anions (Cl-) in the final crystals. - The amount of ) was 4723 ppm.

[0095] Figure 3 The XRD analysis results of the 5'-guanylate disodium crystals obtained according to Example 2 are shown.

[0096] When the peaks appearing in the XRD analysis results were analyzed, these results were confirmed to be consistent with the XRD values ​​previously reported in the literature for columnar form of 5'-guanylate disodium heptahydrate. Therefore, by Figure 3 The results confirmed the synthesis of columnar 5'-guanylate disodium heptahydrate crystals according to one aspect of this disclosure.

[0097] Example 3

[0098] A 0.5 L solution of 30 wt.% disodium 5'-guanylate was prepared by dissolving crude disodium 5'-guanylate crystals, followed by the addition of anhydrous sodium carbonate at a ratio of 5% by weight of disodium 5'-guanylate. A 0.25 L solution of 30 wt.% was prepared by dissolving sodium chloride in water. The amount of sodium chloride was approximately 50% by mass relative to the mass of disodium 5'-guanylate in the disodium 5'-guanylate solution. Concentration and crystallization were initiated in a homogeneous solution, with the amorphous state precipitated from the homogeneous solution by mixing the two solutions. Concentration was carried out by measuring the internal temperature with a thermometer to ensure that the temperature did not exceed 25°C to 42°C. At a concentration level of 1.39, 1 wt.% of disodium 5'-guanylate heptahydrate seed crystals were added to continuously induce a phase transition. After seed crystal injection, the amorphous state of disodium 5'-guanylate continuously transformed into columnar disodium 5'-guanylate heptahydrate crystals. The disodium 5'-guanylate heptahydrate was concentrated until a concentration of at least 45 wt.% was achieved. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated using a centrifuge, and dried at room temperature. The obtained disodium 5'-guanylate crystals had a purity of 100.4%, a moisture content of 23%, and a yield of 87%. The disodium 5'-guanylate heptahydrate crystals were slurried using 99.5% ethanol, and wet particle size analysis was performed using a particle size analyzer. The results showed an average particle size of 397 μm. HPLC analysis revealed residual anions (Cl-) in the final crystals. - The amount of ) was 2660 ppm.

[0099] Example 4

[0100] A 0.5 L solution of 30 wt.% disodium 5'-guanylate was prepared by dissolving crude disodium 5'-guanylate crystals, followed by the addition of 50 wt.% NaOH (sodium hydroxide solution) to adjust the pH to 9.0. A 0.3 L solution of 30 wt.% was prepared by dissolving sodium chloride in water. The amount of sodium chloride was approximately 60% by mass relative to the disodium 5'-guanylate solution. Concentration and crystallization were initiated in a homogeneous solution, with the amorphous state precipitated from the homogeneous solution by mixing the two solutions. Concentration was carried out by adjusting the vacuum and measuring the internal temperature with a thermometer to ensure the temperature did not exceed 25°C to 42°C. At a concentration level of 1.12, 1 wt.% of disodium 5'-guanylate heptahydrate seed crystals were added to continuously induce a phase transition. After the seed crystals were injected, the amorphous state of disodium 5'-guanylate continuously transformed into columnar disodium 5'-guanylate heptahydrate crystals. The disodium 5'-guanylate heptahydrate was concentrated until the concentration reached at least 44 wt.%. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated using a centrifuge, and then dried at room temperature. The obtained disodium 5'-guanylate crystals had a purity of 102%, a moisture content of 22.5%, and a yield of 88%. The disodium 5'-guanylate heptahydrate crystals were slurried using 99.5% ethanol, and wet particle size analysis was performed using a particle size analyzer. The results showed an average particle size of 329 μm. HPLC analysis revealed residual anions (Cl-) in the final crystals. - The level was 1874 ppm. Figure 2 ).

[0101] Example 5

[0102] A 0.5 L solution of 30 wt.% disodium 5'-guanylate was prepared by dissolving crude disodium 5'-guanylate crystals, followed by the addition of 50 wt.% NaOH (sodium hydroxide solution) to adjust the pH to 9.0. A 0.25 L solution of 30 wt.% was prepared by dissolving sodium chloride in water. The amount of sodium chloride was approximately 50% by mass relative to the mass of disodium 5'-guanylate in the disodium 5'-guanylate solution. Concentration and crystallization were initiated in a homogeneous solution, with the amorphous state precipitated from the homogeneous solution by mixing the two solutions. Concentration was carried out by adjusting the vacuum and measuring the internal temperature with a thermometer to ensure the temperature did not exceed 25°C to 42°C. At a concentration level of 1.4 times, 1 wt.% of disodium 5'-guanylate heptahydrate seed crystals were added to continuously induce a phase transition. After seed crystal injection, the amorphous state of disodium 5'-guanylate continuously transformed into fine columnar disodium 5'-guanylate heptahydrate crystals. The disodium 5'-guanylate heptahydrate was concentrated until a concentration of at least 40 wt.% was achieved. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated using a centrifuge, and dried at room temperature. The obtained disodium 5'-guanylate crystals had a purity of 111.5%, a moisture content of 17.6%, and a yield of 88.4%. The disodium 5'-guanylate heptahydrate crystals were slurried using 99.5% ethanol, and wet particle size analysis was performed using a particle size analyzer. The results showed an average particle size of 130 μm. HPLC analysis revealed residual anions (Cl-) in the final crystals. - The amount of ) was 2478 ppm.

[0103] Figure 4 The image shows disodium 5'-guanylate crystals obtained according to Example 4, observed under a microscope. As can be seen from the accompanying image, the obtained crystals are columnar in form, and no amorphous state was observed. Therefore, it is confirmed that most of the disodium 5'-guanylate in solution is obtained in columnar crystal form through a continuous phase transition, thus enabling the high-yield acquisition of disodium 5'-guanylate crystals.

[0104] Figure 5 The particle size analysis results of the 5'-guanylate disodium crystals obtained according to Example 4 are shown, and it can be confirmed that the particle size distribution of the crystals is relatively uniform.

[0105] Experimental Example 2. Evaluation of microbial growth and solidification phenomena based on chloride ion concentration in disodium 5'-guanylate crystals.

[0106] (1) Confirmation of the effectiveness of microbial growth control

[0107] The amounts of residual chloride ions in the crystals prepared by the preparation method of Comparative Example 1 (crystallization with hydrophilic organic solvent) and the crystals prepared by the preparation method of Example 1 are as follows.

[0108] [Table 1]

[0109]

[0110] As a result of comparing the rate of microbial reduction after applying microorganisms to crystals, a microbial growth control effect was confirmed in crystals prepared by the preparation method according to the examples, which contained chloride ion concentrations ranging from 5,703 ppm to 20,000 ppm. Specifically, in the case of crystals containing a chloride ion concentration of 20,000 ppm, a microbial reduction effect similar to the microbial proliferation control effect of salt was confirmed. In contrast, the amount of residual chlorine in crystals prepared by the hydrophilic organic solvent method (Comparative Example 1) was 34 ppm, and microorganisms tended to proliferate significantly over time. The microbial growth control rate over time for each type of crystal is as follows.

[0111] [Table 2]

[0112]

[0113] (2) Confirmation of curing reduction effect

[0114] Products containing the prepared disodium 5'-guanylate may solidify due to the product's sales or storage environment. However, in the case of chlorine-containing crystals, the solidification rate appears to be reduced, which is more favorable for storage compared to crystals prepared using existing hydrophilic organic solvent methods.

[0115] To confirm the reduction effect, the degree of curing was measured using a ring shear tester. After the crystals prepared by the preparation method of Comparative Example 1 (crystallization with hydrophilic organic solvent) and the crystals prepared by the preparation method of Example 1 were injected into the corresponding testers, σ[Pa]: the pressure value required for curing the material; FC[Pa]: yield strength, the pressure value required for breaking the cured material; and FFC: measuring fluidity, the results are shown below.

[0116] [Table 3]

[0117]

[0118] The crystals (chloride ion residual crystals) prepared by the method of Example 1 have low FC values ​​and high FFC values. FC value is the pressure required to break the curing of the material, and a high FFC value indicates that the crystals exhibit high fluidity. In particular, the FC values ​​of crystals with chloride ion residual amounts of 5,703 ppm to 20,000 ppm prepared according to Example 1 were confirmed to be 961 Pa to 1,129 Pa, which is lower than the FC value of 1,461 Pa obtained by crystals with chloride ion residual amounts of 34 ppm prepared according to Comparative Example 1. This means that the crystals with high chloride ion residual amounts prepared according to the examples cure less, and therefore have lower pressure values ​​that break the curing.

[0119] Furthermore, the crystals prepared according to Example 1 with a chloride ion residue of 5,703 ppm to 20,000 ppm had a higher FFC value (FFC value is an indicator of fluidity) than the crystals prepared according to Comparative Example 1 with a chloride ion residue of 34 ppm, indicating reduced curing.

[0120] Therefore, it was confirmed that the chloride ion concentration of the 5'-guanylate disodium crystals prepared by the preparation method according to one embodiment of the present disclosure ranges from 500 ppm to 20,000 ppm, and thus solidification is reduced and microbial growth is controlled.

[0121] Based on the foregoing, those skilled in the art will understand that this disclosure can be implemented in other specific forms without modifying the technical concept or essential features of this disclosure. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of this disclosure. Rather, this disclosure is intended to cover not only the exemplary embodiments but also a variety of alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of this disclosure as defined by the appended claims.

Claims

1. A method for preparing disodium 5'-guanylate crystals, comprising: In the mixing step, an aqueous solution of sodium chloride is added to an aqueous solution of disodium 5'-guanylate; The concentration step involves concentrating the mixed solution of the 5'-guanylate disodium aqueous solution and the sodium chloride aqueous solution, and precipitating the 5'-guanylate disodium in amorphous state. as well as The conversion step involves adding seed crystals to the mixed solution of the aqueous solution of disodium 5'-guanylate and the aqueous solution of sodium chloride to convert the amorphous state of disodium 5'-guanylate into columnar crystals.

2. The method according to claim 1, wherein, In the transformation step, the formation of the amorphous state of disodium 5'-guanylate and the transformation of the amorphous state of disodium 5'-guanylate into columnar crystals are carried out continuously.

3. The method according to claim 1, wherein the amorphous state of 5'-guanylate disodium comprises 5'-guanylate disodium hydrate.

4. The method according to claim 1, wherein the concentration step is performed such that disodium 5'-guanylate is concentrated to a concentration of 400 g / L to 600 g / L.

5. The method of claim 1, further comprising adjusting the pH of the aqueous solution of 5'-guanylate disodium to 8 to 10 prior to performing the mixing step.

6. The method of claim 5, wherein the pH of the aqueous solution of disodium 5'-guanylate is adjusted by adding a sodium salt.

7. The method according to claim 1, wherein, In the mixing step, sodium chloride is added at a ratio of 30% to 80% by weight of disodium 5'-guanylate.

8. The method according to claim 1, wherein, In the conversion step, seed crystals are added at a ratio of 0.5% to 50% by weight of disodium 5'-guanylate.

9. The method according to claim 1, wherein, In the conversion step, the seed crystal is added when the concentration of sodium chloride in the mixed solution is about 100 g / L to about 130 g / L.

10. A fermentation product comprising disodium 5'-guanylate in columnar form, wherein the disodium 5'-guanylate crystals contain chloride ions (Cl... - The concentration of ) ranges from 500 ppm to 20,000 ppm.

11. The fermentation product according to claim 10, further comprising disodium 5'-inosinate crystals, wherein the mixture of the disodium 5'-guanylate crystals and the disodium 5'-inosinate crystals contains chloride ions (Cl... - The concentration of ) ranges from 500 ppm to 20,000 ppm.

12. The fermentation product according to claim 10, wherein the disodium 5'-guanylate crystals are provided in the form of disodium 5'-guanylate heptahydrate or disodium 5'-guanylate tetrahydrate.

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