Method for producing disodium-5'-guanylate crystals

Abstract

The present application relates to a method for producing disodium-5'-guanylate crystals, comprising: a first step of preparing a disodium-5'-guanylate seed slurry by mixing a disodium-5'-guanylate solution, a mixed solution of sodium chloride and ethanol, and disodium-5'-guanylate seeds; and a second step of simultaneously adding a disodium-5'-guanylate solution and ethanol to the disodium-5'-guanylate seed slurry to obtain disodium-5'-guanylate crystals.

Description

Method for preparing disodium-5'-guanylate crystals

[0001] The present application relates to a method for producing high-purity disodium-5'-guanylate crystals even when impurities remain in the process solution.

[0002]

[0003] A commonly known solvent crystallization method for the production of fermentation-derived disodium-5'-guanylate heptahydrate involves the use of hydrophilic organic solvents. In this process, impurities such as guanosine, guanosine diphosphate (GDP), and guanine are removed through a resin column prior to final crystallization, after which a hydrophilic solvent is added to initiate crystallization.

[0004] However, this method uses a large amount of chemicals and water during the resin tower process, which leads to high material and utility costs, including steam, and lags behind environmentally friendly trends from an ESG perspective. Therefore, process improvements are needed to minimize the resin tower process and align with environmentally friendly trends.

[0005] Therefore, a crystallization method is needed that can produce a product in the presence of some impurities, such as guanosine diphosphate, which are major factors affecting the crystallization process, without completely removing them through the resin process.

[0006]

[0007] One purpose of the present application is to provide a technology capable of producing high-purity disodium-5'-guanylate crystals even when impurities remain in the process solution by omitting the resin tower process or reducing the impurity treatment by the resin tower process.

[0008]

[0009] One aspect of the present application provides a method for producing high purity disodium-5'-guanylate crystals.

[0010] Another aspect of the present application provides a disodium-5'-guanylate crystal comprising disodium-5'-guanylate; and 0.1 wt% to 3 wt% of guanosine diphosphate based on the total weight of the crystal.

[0011]

[0012] According to the present application, disodium-5'-guanylate crystals of excellent crystal quality can be obtained even if impurities remain in the process solution.

[0013] In addition, according to the present application, since the resin tower can be partially excluded or the disodium-5'-guanylate can be crystallized after use to a minimum, it is more environmentally friendly, the cost of using the resin tower can be reduced, and the cost of investing in the resin tower equipment when investing in new production facilities can also be reduced.

[0014]

[0015] Figure 1 is a flowchart showing a method for producing disodium-5'-guanylate according to the present application.

[0016] FIG. 2 is an image of a disodium-5'-guanylate crystal prepared according to an embodiment of the present application.

[0017] Figure 3 is an image of a disodium-5'-guanylate crystal prepared according to Comparative Example 1 of the present application.

[0018] Figure 4 is an image of a disodium-5'-guanylate crystal prepared according to Comparative Example 2 of the present application.

[0019] FIG. 5 is an image of a disodium-5'-guanylate crystal prepared according to Comparative Example 4 of the present application.

[0020]

[0021] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0022] Furthermore, those skilled in the art will recognize or be able to ascertain, using only routine experimentation, numerous equivalents to the specific embodiments of the present application described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0023] As used in the specification and appended claims of this application, the singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless the context clearly dictates otherwise, the singular terms include the plural and the plural terms include the singular. In the specification and appended claims of this application, unless the context clearly dictates otherwise, the use of "or" is intended to include "and / or."

[0024] In this application, the term "about" may be used before a specific numerical value. As used herein, the term "about" encompasses not only the exact number described after the term, but also a range that is or is nearly that number. Whether a number is or is nearly the specific number described can be determined based on the context in which it is presented. For example, the term "about" may refer to a range of -10% to +10% of a numerical value. In another example, the term "about" may refer to a range of -5% to +5% of a given numerical value. However, this is not a limitation.

[0025] In this application, the terms "first, second, third," "i), ii), iii),," or "(a), (b), (c), (d),," are used to distinguish similar configurations, and these terms do not imply that the steps are performed consecutively or in order. For example, when the terms are used in connection with steps of a method, use, or analysis, these steps may be performed simultaneously, or may be performed at intervals of seconds, minutes, hours, days, or months.

[0026] In this application, the term "comprising" means the presence of a feature, step, or component described below, and does not exclude the presence or addition of one or more features, steps, or components. The components or features described below "comprising" in this application may be essential or mandatory, but in some embodiments, other optional or non-essential components or features may be further included.

[0027]

[0028] The present application relates to a method for producing disodium-5'-guanylate crystals, wherein a disodium-5'-guanylate solution and ethanol are simultaneously added to a disodium-5'-guanylate seed slurry, thereby forming disodium-5'-guanylate crystals in a single step without formation and clustering of amorphous nuclei of disodium-5'-guanylate and phase transition of disodium-5'-guanylate crystals. When forming disodium-5'-guanylate crystals in two steps of clustering of amorphous nuclei and phase transition, there is a possibility of impurities being included in the stage of nuclei clustering. However, in the case of the present application, since disodium-5'-guanylate crystals are formed in a single step, high-purity crystals can be formed even when impurities are included in the fermentation liquid.

[0029] FIG. 1 is a flowchart illustrating a method for producing disodium-5'-guanylate according to the present application. The method for producing disodium-5'-guanylate crystals according to FIG. 1 includes a first step (S100) of preparing a disodium-5'-guanylate seed slurry by mixing a disodium-5'-guanylate solution, a mixed solution of sodium chloride and ethanol, and disodium-5'-guanylate seeds; and a second step (S200) of simultaneously adding a disodium-5'-guanylate solution and ethanol to the disodium-5'-guanylate seed slurry to obtain disodium-5'-guanylate crystals. Each step will be described in detail below.

[0030] In the first step (S100), a disodium-5'-guanylate solution, a mixed solution of sodium chloride and ethanol, and disodium-5'-guanylate seeds are mixed to prepare a disodium-5'-guanylate seed slurry. To this end, in the first step (S100), a task of preparing a disodium-5'-guanylate solution, a mixed solution of sodium chloride and ethanol, and a disodium-5'-guanylate seed, respectively, may be performed.

[0031] In the first step (S100), the process of preparing a disodium-5'-guanylate solution is examined. The aqueous solution can be prepared by dissolving disodium-5'-guanylate in a solvent. At this time, the use of an organic solvent can be excluded. Disodium-5'-guanylate can be prepared in a crude crystal form and then dissolved in a solvent.

[0032] The disodium-5'-guanylate utilized in step 1 (S100) may be a fermentation product prepared by fermentation. The term "fermentation product" in the present application may refer to a result of enzymatic or metabolic decomposition of an organic substance using a microorganism. For example, the fermentation product may include a culture obtained by culturing a microorganism in a culture medium, or a concentrate, dried product, or lyophilized product of the culture obtained by removing a strain therefrom. Furthermore, in this case, the fermentation liquid may include the entire fermentation product containing disodium-5'-guanylate, or may be a fermentation product containing disodium-5'-guanylate from which impurities have been removed.

[0033] The “microorganism producing disodium-5'-guanylate” or “microorganism producing disodium-5'-guanylate or the target product” used in step 1 (S100) includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may be a microorganism that has a specific mechanism weakened or strengthened due to causes such as insertion of an external gene or enhanced or inactivated activity of an endogenous gene, and may be a microorganism that includes genetic modification for production of the target protein or disodium-5'-guanylate.

[0034] The microorganism producing disodium-5'-guanylate of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce disodium-5'-guanylate, or a microorganism in which the ability to produce disodium-5'-guanylate is conferred on a parent strain that does not have the ability to produce disodium-5'-guanylate. Specifically, the microorganism producing disodium-5'-guanylate or the target product of the present application, or the microorganism having the ability to produce disodium-5'-guanylate or the target product may be a microorganism in which some of the genes in the biosynthetic pathway of the target protein or the target product are strengthened or weakened, or some of the genes in the degradation pathway of the target protein or the target product are strengthened or weakened. The "strengthening" or "increasing" of the disodium-5'-guanylate producing ability of the microorganism of the present application means that the disodium-5'-guanylate producing ability of the microorganism of the present application is improved compared to other microorganisms other than the microorganism of the present application, the parent strain, or an unmodified microorganism. For example, the microorganism of the present application may have an improved disodium-5'-guanylate production ability of 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, or 1300% or more, compared to the disodium-5'-guanylate production ability of other microorganisms, and may have an improved disodium-5'-guanylate production ability of 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 is not limited thereto.

[0035] The above-described microorganism used in the first step (S100) may be at least one selected from the group consisting of the yeast Candida famata, the ascomycetes Eremothecium ashbyii and Ashbyagossypii, the bacteria Bacillus subtilis, and the Corynebacterium sp. microorganism.

[0036] In the case where the microorganism used in step 1 (S100) is a microorganism of the genus Corynebacterium, the microorganism is specifically Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes ammoniagenes), Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium crenatum, or Corynebacterium flavescens, and more specifically, Corynebacterium glutamicum or Corynebacterium stationis, but is not limited thereto.

[0037] Step 1 (S100) may further include a step of culturing a "microorganism producing disodium-5'-guanylate." The culturing of the microorganism may be performed according to an appropriate medium and culture conditions known in the art. This culturing process can be easily adjusted and used by a person skilled in the art depending on the selected strain. Specifically, the culturing may be batch, continuous, and fed-batch, but is not limited thereto. The term "medium" in the present application refers to a material mixed with nutrients as the main component necessary for culturing the microorganism, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing general microorganisms without particular limitation. However, the microorganism of the present application may be cultured under aerobic conditions in a general medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc.

[0038] In a disodium-5'-guanylate solution, disodium-5'-guanylate may exist in the form of a hydrate. For example, disodium-5'-guanylate may be provided in the form of disodium-5'-guanylate heptahydrate or disodium-5'-guanylate tetrahydrate.

[0039] The concentration of disodium-5'-guanylate in the disodium-5'-guanylate solution may be from about 150 g / L to about 350 g / L. In some cases, the concentration of disodium-5'-guanylate may be from about 200 g / L to about 350 g / L, from about 250 g / L to about 350 g / L, from about 300 g / L to about 350 g / L, from about 150 g / L to about 300 g / L, from about 200 g / L to about 300 g / L, from about 250 g / L to about 300 g / L, from about 150 g / L to about 250 g / L, or from about 200 g / L to about 250 g / L. By preparing an aqueous solution having the above-described concentration, the yield of disodium-5'-guanylate crystals can be secured in the subsequent process. When the concentration of disodium-5'-guanylate in the aqueous solution is less than about 150 g / L, the content of disodium-5'-guanylate is low, which may lower the yield of disodium-5'-guanylate crystals. In addition, when the concentration of disodium-5'-guanylate exceeds about 350 g / L, there is a concern that disodium-5'-guanylate may precipitate before the second step is performed, thereby hindering the process.

[0040] A disodium-5'-guanylate solution can be prepared without passing it through a resin column to remove impurities. Therefore, impurities contained in the crude crystal of disodium-5'-guanylate may remain in the disodium-5'-guanylate solution. Specifically, disodium-5'-guanylate-like substances such as guanosine, guanosine diphosphate, and guanine may remain in the aqueous solution. In particular, guanosine diphosphate is difficult to separate because it is similar to disodium-5'-guanylate (GMP). However, according to one embodiment of the present application, a high-purity disodium-5'-guanylate crystal can be obtained without separating guanosine diphosphate. The concentration of guanosine diphosphate in the aqueous solution may be 5 g / L or less. In some cases, some of the impurities may be removed by filtering and decolorizing disodium-5'-guanylate. However, according to the present application, high-purity disodium-5'-guanylate crystals can be obtained through a single-step crystallization process without removing impurities contained in the crude disodium-5'-guanylate crystals. Therefore, according to one embodiment of the present application, the resin tower process used in existing methods can be reduced, thereby reducing the use of chemicals and water, thereby reducing costs and establishing an environmentally friendly process.

[0041] The pH of the disodium-5'-guanylate solution can be 8 to 9. The present invention allows crystallization under near-neutral conditions. Therefore, compared to techniques for crystallizing disodium-5'-guanylate under strongly acidic conditions, the present invention offers the advantages of eliminating the need for acid-resistant equipment and ensuring safe process operation.

[0042] In the first step (S100), a step of preparing a mixed solution of sodium chloride and ethanol may be performed. The mixed solution of sodium chloride and ethanol may be prepared by dissolving sodium chloride in ethanol. The ethanol may be used in an amount corresponding to about 20 vol.% to about 40 vol.% based on the disodium-5'-guanylate solution. In addition, the ethanol may be prepared at a concentration of about 90% and then diluted to about 50% for use. In a high-concentration ethanol solution, the solubility of GMP is very low, so that disodium-5'-guanylate nuclei are rapidly generated during the crystallization process and the concentration of disodium-5'-guanylate in the solution approaches supersaturation, which may prevent disodium-5'-guanylate crystals from growing. Therefore, after preparing an ethanol solution having the above-mentioned concentration so that the nuclei can grow into crystals, the solubility of disodium-5'-guanylate may be slowly reduced.

[0043] The concentration of sodium chloride in the mixed solution of sodium chloride and ethanol can be about 5 g / L to 100 g / L. In some cases, the sodium chloride concentration is from about 10 g / L to about 100 g / L, from about 20 g / L to about 100 g / L, from about 30 g / L to about 100 g / L, from about 40 g / L to about 100 g / L, from about 50 g / L to about 100 g / L, from about 60 g / L to about 100 g / L, from about 70 g / L to about 100 g / L, from about 5 g / L to about 80 g / L, from about 10 g / L to about 80 g / L, from about 20 g / L to about 80 g / L, from about 30 g / L to about 80 g / L, from about 40 g / L to about 80 g / L, from about 50 g / L to about 80 g / L, from about 60 g / L to about 80 g / L, from about 5 g / L to about 60 g / L, about 10 g / L to about 40 g / L, or about 20 g / L to about 30 g / L.

[0044] According to the present application, sodium chloride, which is approved as a food additive, can be added to lower the solubility of disodium-5'-guanylate in a hydrophilic solvent. Therefore, the addition of sodium chloride can prevent disodium-5'-guanylate seeds from dissolving within the seed slurry. Accordingly, the number of seeds within the seed slurry can be increased, and thus the yield of disodium-5'-guanylate crystals obtained in the subsequent crystallization step can be improved. When the concentration of sodium chloride in the mixed solution of sodium chloride and ethanol is less than about 5 g / L, the effect of sodium chloride in preventing disodium-5'-guanylate seed dissolution may be minimal. In addition, when the concentration of sodium chloride in the mixed solution of sodium chloride and ethanol exceeds about 80 g / L, sodium chloride may be precipitated in a solid form in the ethanol solution. When sodium chloride is precipitated, disodium-5'-guanylate crystallization may not be uniform. This is because the precipitated sodium chloride solid may move around in the ethanol solution, and the composition may vary depending on the location in the solution as disodium-5'-guanylate crystallization progresses.

[0045] In relation to disodium-5'-guanylate seeds, the seed, also called seed crystal or seed crystal, may refer to a substance used as a catalyst for crystallization or granulation of a liquid. Specifically, the seed in the present application may be a crystal of disodium-5'-guanylate.

[0046] The disodium-5'-guanylate in the disodium-5'-guanylate seed may be provided in the form of a hydrate. For example, the seed may be provided in the form of a disodium-5'-guanylate heptahydrate or a disodium-5'-guanylate tetrahydrate. In the first step (S100), the disodium-5'-guanylate seed may be introduced in a ratio of about 5 wt% to about 10 wt% based on the total weight of the seed slurry. By introducing the disodium-5'-guanylate seed in the above-described content range, the formation of disodium-5'-guanylate crystals can be optimized.

[0047] In the first step (S100), a disodium-5'-guanylate solution prepared as described above, a mixed solution of sodium chloride and ethanol, and disodium-5'-guanylate seeds are mixed to prepare a disodium-5'-guanylate seed slurry. There is no limitation on the mixing method for preparing the seed slurry. For example, the disodium-5'-guanylate solution, the mixed solution of sodium chloride and ethanol, and the disodium-5'-guanylate seeds can be mixed using a commonly used stirring method. There is also no limitation on the order in which the disodium-5'-guanylate solution, the mixed solution of sodium chloride and ethanol, and the disodium-5'-guanylate seeds are introduced. For example, the above-described elements can be introduced sequentially or simultaneously.

[0048] The concentration of ethanol in the seed slurry may be 30 wt% to 50 wt%. In some cases, the concentration of ethanol may be about 30 wt% to about 50 wt%, about 35 wt% to about 50 wt%, about 40 wt% to about 50 wt%, about 45 wt% to about 50 wt%, about 30 wt% to about 45 wt%, about 35 wt% to about 45 wt%, about 40 wt% to about 45 wt%, about 30 wt% to about 40 wt%, about 35 wt% to about 40 wt%, or about 30 wt% to about 35 wt%. The reason why the ethanol content in the slurry is adjusted to the above-described range is because crystal formation is easy when the ethanol content in the slurry is provided in the above-described content range. In addition, by adjusting the ethanol content in the mixed solution at the time of completion of crystallization in the second stage (S200) to a similar level as the ethanol content in the seed slurry prepared in the first stage (S100), the influence of crystallization due to changes in solvent content during the crystallization process can be minimized.

[0049] Next, a second step (S200) is performed in which a disodium-5'-guanylate solution and ethanol are simultaneously added to the disodium-5'-guanylate seed slurry to obtain disodium-5'-guanylate crystals.

[0050] The disodium-5'-guanylate seed slurry mixed in the second step (S200) is the one prepared in the first step (S100). In addition, the disodium-5'-guanylate solution mixed in the second step (S200) may also be the one prepared in the first step (S100). Therefore, the disodium-5'-guanylate solution prepared in the first step (S100) may be divided into portions, and some may be used to prepare the seed slurry in the first step (S100) and the other portion may be used to perform crystallization in the second step (S200). However, in some cases, for example, when performing crystallization in the second step (S200), if a disodium-5'-guanylate solution having a different concentration from the disodium-5'-guanylate solution used in preparing the seed slurry is required, the disodium-5'-guanylate solution may be prepared again in the second step (S200).

[0051] In the second step (S200), ethanol may be introduced in an amount of 60 vol.% to 80 vol.% relative to the amount of the disodium-5'-guanylate solution introduced in the second step (S200). Additionally, the ethanol may be a solution having an ethanol concentration of 70% to 99.9%. By introducing ethanol in the above-described concentration and amount in the second step (S200), the ethanol content in the mixed solution upon completion of crystallization in the second step (S200) and the ethanol content in the seed slurry prepared in the first step (S100) can be adjusted to a similar level. Accordingly, the influence of crystallization due to changes in solvent content during the crystallization process can be minimized. Specifically, the concentration of ethanol in the mixed solution in which disodium-5'-guanylate seed slurry, disodium-5'-guanylate solution, and ethanol are simultaneously introduced may be 30 wt% to 50 wt%, which may be similar to the concentration of ethanol in the seed slurry in the first step (S100) described above.

[0052] In the second step (S200), a disodium-5'-guanylate solution and ethanol can be simultaneously introduced into the disodium-5'-guanylate seed slurry described above. After the above substances are introduced, disodium-5'-guanylate crystals can be formed in a single step within the reactor without formation and clustering of disodium-5'-guanylate amorphous nuclei or phase transition of disodium-5'-guanylate crystals.

[0053] According to the prior art, when disodium-5'-guanylate in a solution reaches the supersaturation point, nuclei are generated and the nuclei gather to form amorphous clusters. Through the phase transition from the amorphous clusters to disodium-5'-guanylate hydrate crystals, columnar crystal-like disodium-5'-guanylate hydrate crystals are produced. The generated columnar crystal-like disodium-5'-guanylate hydrate crystals can be disodium-5'-guanylate heptahydrate crystals or disodium-5'-guanylate tetrahydrate crystals. Here, after the nuclei gather to form an amorphous cluster and the phase transition to a crystal occurs, various factors such as solvent, temperature, and disodium-5'-guanylate concentration affect the phase transition, and in particular, impurities with a similar structure to disodium-5'-guanylate, such as guanosine diphosphate, can interfere with the phase transition. As a result, the crystal transition may not occur and the product may remain amorphous, or even if crystal transition occurs, the product quality may be significantly reduced. Therefore, according to the conventional technology, an appropriate crystallization environment must be created for disodium-5'-guanylate hydrate crystallization, and in particular, interfering impurities must be removed prior to crystallization. To this end, impurities such as guanosine, guanosine diphosphate, and guanine were generally removed through a resin tower process.

[0054] However, according to the present application, by simultaneously introducing a disodium-5'-guanylate solution and ethanol into a seed slurry, crystallization is achieved in a single-step process in which the seeds in the seed slurry control nucleation and crystals are continuously formed. Therefore, compared to the existing two-step crystallization method, since there is no amorphous nucleation or clustering of amorphous nuclei, it is relatively free from the problems of impurities being caught between nuclei to form crystals containing impurities or impurities restricting crystal growth. Accordingly, even if impurities remain in the process solution, disodium-5'-guanylate crystals of excellent crystal quality can be obtained.

[0055] After performing the second step (S200), an additional step of separating and drying the obtained disodium-5'-guanylate crystals from the mother liquor may be performed. The step of separating and drying the crystals may be performed using a conventional method. For example, a solid-liquid separator such as a pressure-reducing membrane filtration device, a pressure-reducing membrane filtration device, or a centrifugal separator may be used to separate the crystals, but is not limited thereto.

[0056] In the second step (S200), the crystallization process can be performed at a temperature of about 30°C to about 50°C. Uniform crystallization of disodium-5'-guanylate can be achieved within the above-described temperature range. The crystallization process can be performed for about 4 hours to about 10 hours.

[0057] The crystal manufactured after the second step (S200) may be a disodium-5'-guanylate hydrate crystal, specifically a disodium-5'-guanylate heptahydrate crystal or a disodium-5'-guanylate tetrahydrate crystal.

[0058] Guanosine diphosphate may remain in the disodium-5'-guanylate crystals manufactured after the second step (S200). Guanosine diphosphate may remain in the crystals at a ratio of about 0.1 wt% to about 3 wt% of the total crystal weight. However, since the crystallization method according to the present application has a small effect on crystallization even if guanosine diphosphate remains, compared to the existing crystallization method in which a phase transition occurs after the formation of amorphous nuclei, even if residual guanosine diphosphate exists in the crystals, the shape, size, purity, and moisture content of the crystals can satisfy the quality of the product to be manufactured.

[0059]

[0060] Next, according to one aspect of the present application, a disodium-5'-guanylate crystal is provided, comprising disodium-5'-guanylate; and 0.1 wt% to 3 wt% of guanosine diphosphate based on the total weight of the crystal.

[0061] According to the present application, disodium-5'-guanylate crystals can be produced in a single step process as previously reviewed, and disodium-5'-guanylate crystals of excellent crystal quality can be obtained even if impurities remain in the process solution. Generally, impurities such as guanosine, guanosine diphosphate, and guanine remain in the crystal solution for producing disodium-5'-guanylate crystals, and since these have similar physical properties to disodium-5'-guanylate, it is difficult to separate them from the process solution.

[0062] According to the process of the present invention, disodium-5'-guanylate crystals can be produced even when the above-described difficult-to-separate impurities, particularly guanosine diphosphate, remain. The disodium-5'-guanylate crystals thus produced contain 0.1 to 3 wt% of guanosine diphosphate based on the total weight of the crystals.

[0063] The above-described content of guanosine diphosphate cannot appear in disodium-5'-guanylate crystals manufactured through a two-step process of nucleation and crystal phase transition according to the prior art. As will be seen in the experimental examples below, when the two-step process according to the prior art is performed, it is difficult to obtain crystals even if the guanosine diphosphate concentration is only 0.9 wt%. In addition, when the resin tower process is performed prior to crystal acquisition as in the prior art, the guanosine diphosphate in the process solution is almost completely removed, and likewise, the guanosine diphosphate concentration in the crystal cannot appear even if it is 0.1 wt% to 3 wt%.

[0064] Additionally, the disodium-5'-guanylate crystals described above may exhibit an oval shape.

[0065] In addition, the disodium-5'-guanylate crystals described above may be crystals in the form of a hydrate, and in some cases, may be disodium-5'-guanylate heptahydrate crystals or disodium-5'-guanylate tetrahydrate crystals.

[0066] Accordingly, according to one aspect of the present application, there is provided a disodium-5'-guanylate crystal comprising disodium-5'-guanylate and 0.1 to 3 wt% of guanosine diphosphate based on the total weight of the crystal. The crystal is easy to separate because it has an excellent crystal shape while containing impurities, and is suitable for use because it has a high disodium-5'-guanylate content.

[0067]

[0068] The present application will be described in more detail below through examples. However, the following examples are merely preferred embodiments intended to illustrate the present application and are therefore not intended to limit the scope of the present application. Furthermore, technical details not described herein can be readily understood and implemented by those skilled in the technical field of the present application or similar fields.

[0069] The above has examined a method for producing disodium-5'-guanylate (GMP) crystals according to one embodiment of the present application. Below, the beneficial effects mentioned in the present application will be examined through experimental results from examples and comparative examples.

[0070]

[0071] Example 1. Preparation of disodium-5'-guanylate crystals through single-step crystallization.

[0072] In order to evaluate the feasibility of the disodium-5'-guanylate crystallization method according to the present application in a state where guanosine diphosphate remains in the fermentation broth, crude disodium-5'-guanylate crystals with a purity of 90% derived from a microbial fermentation broth were dissolved to a concentration of 260 g / L, and guanosine diphosphate was artificially added to adjust the guanosine diphosphate to 0.9 wt.% relative to the mass of disodium-5'-guanylate.

[0073] 90% ethanol corresponding to 30 vol.% of this solution was diluted with 50% ethanol, sodium chloride was dissolved in the diluted ethanol to a level of 80 g / L, and 10 wt.% of disodium-5'-guanylate seed was added to prepare a seed slurry.

[0074] Crystallization was performed by simultaneously adding a disodium-5'-guanylate solution and 90% ethanol (80 vol.% of the disodium-5'-guanylate solution) to the seed slurry, thereby continuously forming crystals.

[0075] The crystallization process was carried out at 42°C for 6 hours. After cooling to 25°C, the crystals and mother liquor were separated using a centrifuge, and the recovered crystals were dried.

[0076] The obtained crystals were confirmed to have a disodium-5'-guanylate content of 99.1% and a moisture content of 23.0%, confirming that they were of a quality suitable for commercialization. The manufactured disodium-5'-guanylate crystals were confirmed to contain 0.3% residual guanosine diphosphate.

[0077] Here, the disodium-5'-guanylate content can be calculated as follows.

[0078] GMP content = Mass of GMP in dried sample (Disodium-5-guanylate + 7 parts of H2O water) / Mass of dried (surface water removed) sample (Disodium-5-guanylate + 7 parts of H2O water + mass of some dried impurities) * 100

[0079] Disodium-5'-guanylate is a heptahydrate product in which seven H2O atoms exist within the crystal. Since surface water exists on the crystal surface before drying, the content of the final product can be expressed as the content of disodium-5'-guanylate (including crystal water) present in the total mass of the product after removing surface water through drying. Referring to Fig. 2, it can be confirmed that the obtained disodium-5'-guanylate crystals exhibit an oval shape rather than a columnar shape.

[0080]

[0081] Comparative Example 1. Preparation of disodium-5'-guanylate crystals using nucleation and crystal phase transition (GDP concentration of 0.1 wt.% or less)

[0082] Disodium-5'-guanylate with a purity of 90% derived from microbial fermentation broth was dissolved in crude crystals to a concentration of 260 g / L. At this time, guanosine diphosphate was present in the solution in an amount of 0.1 wt.% or less relative to the mass of disodium-5'-guanylate.

[0083] This solution was placed in a crystallization tube, the temperature was maintained at 34°C, and 90% ethanol (110 vol.% of the solution) was added for 6 hours. When ethanol was added, disodium-5'-guanylate nuclei and clustering occurred, precipitating amorphous disodium-5'-guanylate crystals.

[0084] About 1 hour after adding ethanol, 10 wt.% of disodium-5'-guanylate seeds are added and ethanol is continuously added, resulting in the formation of additional disodium-5'-guanylate nuclei, the formation of disodium-5'-guanylate amorphous crystals by clustering of previously created nuclei, and the transition of amorphous crystals into columnar crystals, thereby resulting in disodium-5'-guanylate crystallization.

[0085] After crystallization was completed, the disodium-5'-guanylate crystals were cooled to 25°C, the crystals and mother liquor were separated using a centrifuge, the crystals were recovered, and the final product was obtained by drying at room temperature.

[0086] The quality of the obtained product was confirmed to be 101.1% disodium-5'-guanylate content, 19.3% moisture content, and 0% guanosine diphosphate content within the crystal.

[0087] Here, the disodium-5'-guanylate content refers to the ratio of the disodium-5'-guanylate mass to the mass of the entire sample excluding moisture (mass of disodium-5'-guanylate including moisture / mass of dried sample (excluding moisture) * 100).

[0088] Additionally, moisture content refers to the ratio of the mass of water, including crystal water and surface water, to the mass of the sample solid matter.

[0089] As can be seen in Fig. 3, the shape of the obtained crystal is a columnar shape.

[0090] Therefore, it was confirmed from the experiment of Comparative Example 1 that the two-step (amorphous nucleus formation and columnar phase transition) crystallization process can be performed normally when the concentration of guanosine diphosphate in the process solution is 0.1 wt.% or less.

[0091]

[0092] Comparative Example 2. Preparation of disodium-5'-guanylate crystals using nucleation and crystal phase transition (GDP concentration of 0.5 wt.%)

[0093] Disodium-5'-guanylate with a purity of 90% derived from microbial fermentation broth was dissolved in crude crystals to a concentration of 275 g / L, and guanosine diphosphate was added to adjust the guanosine diphosphate to 0.5 wt.% relative to the mass of disodium-5'-guanylate.

[0094] This solution was placed in a crystallization tube, and the temperature was maintained at 34°C. Then, 90% ethanol (110 vol.% based on the solution) was added for 6 hours. Approximately 1 hour after adding ethanol, 10 wt.% of disodium-5'-guanylate seed was added, and ethanol was added for the remaining 5 hours to proceed with crystallization.

[0095] After crystallization was completed, the disodium-5'-guanylate crystals were cooled to 25°C, the crystals and mother liquor were separated using a centrifuge, the crystals were recovered, and the final product was obtained by drying at room temperature.

[0096] The quality of the product obtained at this time was good, with a disodium-5'-guanylate content of 99.8% and a moisture content of 21.0%. However, as can be seen in Fig. 4, the crystal shape was oval, not columnar, and the content of guanosine diphosphate within the crystal was confirmed to be 0.3%.

[0097] Therefore, referring to Comparative Example 2, it was confirmed that disodium-5'-guanylate crystals could be manufactured using the conventional two-step (amorphous nucleation and columnar phase transition) crystallization process. However, as the amount of the impurity guanosine diphosphate increased, oval crystals rather than columnar crystals were obtained compared to when the guanosine diphosphate impurity was contained in a very small amount of 0.1 wt.% or less (Comparative Example 1), indicating that guanosine diphosphate affected the two-step crystal manufacturing. In addition, it was confirmed that 0.3 wt.%, which is a considerably large amount compared to the amount of added guanosine diphosphate (0.5 wt.%), remained in the crystals, indicating that a relatively large amount of impurities remained in the crystals during the two-step crystallization. This is in contrast to the example where only 0.3 wt.% of guanosine diphosphate remained in the crystal even when 0.9 wt.% of guanosine diphosphate was added. Therefore, it can be seen that the two-step disodium-5'-guanylate crystal preparation method is more susceptible to impurities than the crystal preparation method according to the example of the present application.

[0098]

[0099] Comparative Example 3. Preparation of disodium-5'-guanylate crystals using nucleation and crystal phase transition (GDP concentration of 0.9 wt.%)

[0100] Disodium-5'-guanylate with a purity of 90% derived from microbial fermentation broth was dissolved in crude crystals to a concentration of 260 g / L, and guanosine diphosphate was added to adjust the guanosine diphosphate to 0.9 wt.% relative to the mass of disodium-5'-guanylate.

[0101] This solution was placed in a crystallization tube, the temperature was maintained at 34°C, and 110 vol.% of 90% ethanol was added relative to the solution for 6 hours. One hour after adding ethanol, 10 wt.% of disodium-5'-guanylate seed was added, and ethanol was added for the remaining 5 hours to proceed with crystallization.

[0102] However, in Comparative Example 3, crystal transition did not occur properly, so after the disodium-5'-guanylate nucleus was precipitated, the crystal shape remained amorphous and crystal separation was impossible.

[0103] Therefore, in the case of the conventional two-step disodium-5'-guanylate crystal manufacturing process, it was confirmed that the process could not proceed when the same amount of guanosine diphosphate impurity (0.9 wt.%) as in the example was added. That is, as previously reviewed, it can be seen that the conventional two-step disodium-5'-guanylate crystal manufacturing method is more vulnerable to impurities than the crystal manufacturing method according to the example of the present application.

[0104]

[0105] Comparative Example 4. Preparation of disodium-5'-guanylate crystals using nucleation and crystal phase transition (GDP concentration level of 1.2 wt.%)

[0106] Disodium-5'-guanylate with a purity of 90% derived from microbial fermentation broth was dissolved to a concentration of 272 g / L from crude crystals, and guanosine diphosphate was artificially added to adjust the guanosine diphosphate to 1.2 wt.% relative to the mass of disodium-5'-guanylate.

[0107] This solution was placed in a crystallization tube, the temperature was maintained at 34°C, and 90% ethanol (110 vol.% of the solution) was added for 6 hours. Approximately 1 hour after adding ethanol, 10 wt.% of disodium-5'-guanylate seed was added, and ethanol was added for the remaining 5 hours to proceed with crystallization.

[0108] After crystallization was completed, the disodium-5'-guanylate crystals were cooled to 25°C, the crystals and mother liquor were separated using a centrifuge, the crystals were recovered, and the final product was obtained by drying at room temperature.

[0109] The product obtained at this time had a disodium-5'-guanylate content of 84.8% and a moisture content of 22.0%, which did not meet the quality standards.

[0110] As can be seen in Fig. 5, the obtained disodium-5'-guanylate crystal shape is oval, and the content of guanosine diphosphate in the crystal is 0.3 wt.%.

[0111]

[0112] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. A first step of preparing a disodium-5'-guanylate seed slurry by mixing a disodium-5'-guanylate solution, a mixed solution of sodium chloride and ethanol, and disodium-5'-guanylate seeds; and A method for producing disodium-5'-guanylate crystals, comprising a second step of simultaneously adding a disodium-5'-guanylate solution and ethanol to the disodium-5'-guanylate seed slurry to obtain disodium-5'-guanylate crystals.

2. In paragraph 1, A method for producing disodium-5'-guanylate crystals, wherein the above sodium chloride and ethanol mixed solution contains sodium chloride at a concentration of 5 g / L to 100 g / L.

3. In paragraph 1, A method for producing disodium-5'-guanylate crystals, wherein the concentration of ethanol in the disodium-5'-guanylate seed slurry is 30 wt% to 50 wt%.

4. In paragraph 1, A method for producing disodium-5'-guanylate crystals, wherein the concentration of ethanol in the mixed solution, in which the disodium-5'-guanylate solution and ethanol are simultaneously added to the disodium-5'-guanylate seed slurry in the second step, is 30 wt% to 50 wt%.

5. In paragraph 1, A method for producing disodium-5'-guanylate crystals, wherein in the second step, disodium-5'-guanylate crystals are formed in a single step without formation and clustering of amorphous nuclei of disodium-5'-guanylate or phase transition of disodium-5'-guanylate crystals.

6. In paragraph 1, A method for preparing disodium-5'-guanylate crystals, further comprising the step of dissolving disodium-5'-guanylate crystals to prepare the disodium-5'-guanylate solution.

7. In paragraph 1, A method for producing disodium-5'-guanylate crystals, further comprising a step of separating and drying disodium-5'-guanylate crystals obtained after performing the second step from the mother liquor.

8. In paragraph 1, A method for producing disodium-5'-guanylate crystals, wherein the pH of the disodium-5'-guanylate solution is 8 to 9.

9. Disodium-5'-guanylate; and Disodium-5'-guanylate crystals comprising 0.1% to 3% by weight of guanosine diphosphate (GDP) based on the total weight of the crystal.

10. In paragraph 9, The above disodium-5'-guanylate crystal is an oval-shaped disodium-5'-guanylate crystal.

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