Method for producing disodium 5'-guanylate crystals

Abstract

This application relates to a method for producing disodium 5'-guanylate crystals, comprising a mixing step of adding an aqueous sodium chloride solution to an aqueous solution of disodium 5'-guanylate; a concentration step of concentrating the mixed solution of the aqueous solution of disodium 5'-guanylate and the aqueous sodium chloride solution to precipitate amorphous crystals of disodium 5'-guanylate; and a transition step of adding seeds to the mixed solution of the aqueous solution of disodium 5'-guanylate and the aqueous sodium chloride solution to transform the amorphous crystals of disodium 5'-guanylate into columnar crystals. According to this application, disodium 5'-guanylate crystals can be produced in high yield by an environmentally friendly method without using organic solvents.

Description

Technical Field

[0001] This application relates to a method for producing disodium 5'-guanylate crystals in an environmentally friendly manner without using an organic solvent.

Background Art

[0002] In the production of disodium 5'-guanylate derived from fermentation, the generally known solvent crystallization method is a method using a hydrophilic organic solvent. In such a process, there is a drawback that the use of a hydrophilic organic solvent increases the cost of by-products and utility costs. In addition, since the crystallization method using a hydrophilic organic solvent deviates from the perspective of ESG, it is required to improve the process in accordance with the trend of environmental change.

Summary of the Invention

Problems to be Solved by the Invention

[0003] An object of this application is to provide a technique capable of producing disodium 5'-guanylate crystals in a high yield without using an organic solvent.

Means for Solving the Problems

[0004] This application aimed at solving the above problems has the following constitution and features.

[0005] One aspect of this application is a method for producing disodium 5'-guanylate crystals, which includes a mixing step of adding an aqueous sodium chloride solution to an aqueous disodium 5'-guanylate solution, a concentration step of concentrating the mixed solution of the aqueous disodium 5'-guanylate solution and the aqueous sodium chloride solution to precipitate amorphous crystals of disodium 5'-guanylate, and a transfer step of adding seeds to the mixed solution of the aqueous disodium 5'-guanylate solution and the aqueous sodium chloride solution to transfer the amorphous crystals of disodium 5'-guanylate into columnar crystals.

[0006] In one specific example, the formation of amorphous crystals of disodium 5'-guanylate and the transformation of the amorphous crystals of disodium 5'-guanylate into columnar crystals may occur sequentially in the transition step.

[0007] According to other specific examples, the amorphous crystals of disodium 5'-guanylate may contain hydrate of disodium 5'-guanylate.

[0008] In other specific examples, the concentration step may be carried out to concentrate the concentration of 5'-disodium guanylate to 400 g / L to 600 g / L.

[0009] In further specific examples, the method may further include adjusting the pH of the 5'-disodium guanylate aqueous solution to 8-10 before performing the mixing step.

[0010] Furthermore, according to another specific example, the pH of the 5'-disodium guanylate aqueous solution may be adjusted by adding a sodium salt.

[0011] In further specific examples, in the mixing step, sodium chloride may be added in a proportion of 30% to 80% of the weight of disodium 5'-guanylate.

[0012] In further specific examples, in the transfer step, the seed may be added in a proportion of 0.5% to 50% relative to the weight of the disodium 5'-guanylate.

[0013] In another specific example, in the transition step, the seed may be added when the concentration of sodium chloride in the mixed solution is at a level of approximately 100 g / L to approximately 130 g / L, where the concentration of disodium 5'-guanylate is approximately 100 g / L to 130 g / L.

[0014] Another aspect of this application provides a fermented product comprising columnar disodium 5'-guanylate crystals, wherein the concentration of chloride ions (Cl-) in the disodium 5'-guanylate crystals is 500 ppm to 20,000 ppm.

[0015] In one specific example, the fermented product further contains disodium 5'-inosinate crystals, and the concentration of chloride ions (Cl-) in the mixture of the disodium 5'-guanylate crystals and the disodium 5'-inosinate crystals may be 500 ppm to 20,000 ppm.

[0016] In other specific examples, the fermented product may be provided in which the 5'-guanylate disodium crystals are in the form of 5'-guanylate disodium heptahydrate or 5'-guanylate disodium tetrahydrate. [Effects of the Invention]

[0017] A method for producing disodium 5'-guanylate crystals according to one aspect of this application is an environmentally friendly method because it produces disodium 5'-guanylate crystals using an aqueous solvent instead of an organic solvent. Furthermore, by adjusting the process factors so that disodium 5'-guanylate forms amorphous crystals and then undergoes a phase transition to columnar crystals, the yield of disodium 5'-guanylate crystals can be increased.

[0018] Furthermore, according to one aspect of this application, since the chloride ion content is 500 ppm to 20,000 ppm, it is possible to provide disodium 5'-guanylate crystals with high stability in storage and distribution. [Brief explanation of the drawing]

[0019] [Figure 1] This is a flowchart showing the method for producing disodium 5'-guanylate according to this application. [Figure 2] This is a flowchart showing a method for producing disodium 5'-guanylate crystals according to one aspect of this application. [Figure 3]It is a diagram showing the XRD analysis results of the disodium 5'-guanylate crystals obtained in Example 2. [Figure 4] It is an image of the disodium 5'-guanylate crystals obtained in Example 4 observed under a microscope. [Figure 5] It is a diagram showing the particle size analysis results of the disodium 5'-guanylate crystals obtained in Example 4.

Modes for Carrying Out the Invention

[0020] Hereinafter, these will be specifically described. Note that each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application are included in this application. Also, this application is not limited to the following specific descriptions.

[0021] This application relates to a method for producing disodium 5'-guanylate. By concentrating a mixed solution of an aqueous disodium 5'-guanylate solution and sodium chloride and adding seeds, the formation of amorphous crystals of disodium 5'-guanylate and the subsequent transformation of the amorphous crystals into columnar crystals can occur continuously. Therefore, disodium 5'-guanylate crystals can be produced in an environmentally friendly manner and with a high yield.

[0022] Figure 1 is a flowchart showing the method for producing disodium 5'-guanylate according to this application.

[0023] As shown in Figure 1, the method for producing disodium 5'-guanylate crystals according to this application includes a mixing step S100 of adding an aqueous sodium chloride solution to an aqueous solution of disodium 5'-guanylate, a concentration step S200 of concentrating the mixed solution of the aqueous solution of disodium 5'-guanylate and the aqueous sodium chloride solution to precipitate amorphous crystals of disodium 5'-guanylate, and a transition step S300 of adding seeds to the mixed solution of the aqueous solution of disodium 5'-guanylate and the aqueous sodium chloride solution to transform the amorphous crystals of disodium 5'-guanylate into columnar crystals. The steps of the method for producing disodium 5'-guanylate crystals according to this application will be described below.

[0024] First, in mixing step S100, an aqueous solution of sodium chloride is added to an aqueous solution of disodium 5'-guanylate and mixed.

[0025] The aqueous solution of disodium 5'-guanylate used in mixing step S100 is a fermented product prepared by fermentation. In this application, "fermented product" means the result of the enzymatic or metabolic decomposition of organic matter using microorganisms. For example, the fermented product includes the culture itself obtained by culturing microorganisms in a culture medium, or a concentrated, dried, or freeze-dried product of the culture obtained by removing the microbial strain therefrom. Furthermore, the fermented liquid may include the entire fermented product containing disodium 5'-guanylate, or it may be a fermented product containing disodium 5'-guanylate from which impurities have been removed.

[0026] The "microorganisms producing 5'-disodium guanylate" or "microorganisms producing 5'-disodium guanylate or the target product" used in mixing step S100 include all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially. These are microorganisms in which a specific mechanism has been weakened or strengthened due to reasons such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and which have been genetically modified for the production of the target protein or 5'-disodium guanylate.

[0027] The microorganisms that produce 5'-disodium guanylate in this application may be microorganisms that naturally possess the ability to produce 5'-disodium guanylate, or microorganisms in which the ability to produce 5'-disodium guanylate has been conferred to a parent strain that does not possess the ability to produce 5'-disodium guanylate, but are not limited to these. Specifically, in this application, microorganisms that produce 5'-disodium guanylate or the target product, or microorganisms that possess the ability to produce 5'-disodium guanylate or the target product, may be microorganisms in which a portion of the genes in the biosynthetic pathway of the target protein or target product has been strengthened or weakened, or microorganisms in which a portion of the genes in the degradation pathway of the target protein or target product has been strengthened or weakened. "Strengthened" or "improved" 5'-disodium guanylate production ability of the microorganisms in this application means that the 5'-disodium guanylate production ability of the microorganisms in this application is improved compared to other microorganisms, parent strains, or unmodified microorganisms. For example, the microorganism of this application may have a production capacity of approximately 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 production capacity of other microorganisms, or it may have an improvement of approximately 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 to these. The term "approximately" includes a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and any numerical value that is equivalent to or in a similar range to the numerical value following the term "approximately" is acceptable, but is not limited to these.

[0028] The microorganism used in mixing step S100 is at least one selected from the group consisting of the yeast Candida famata, the ascomycetes Eremothecium ashbyii and Ashbya gossypii, the bacteria Bacillus subtilis, and microorganisms of the genus Corynebacterium sp.

[0029] If the microorganism used in mixing step S100 is a Corynebacterium species, then the microorganism is specifically Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. These include Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium crenatum, or Corynebacterium flavescens, and more specifically, Corynebacterium glutamicum, but are not limited to these.

[0030] The mixing step S100 may further include a step of culturing a "microorganism that produces disodium 5'-guanylate." The culture of the microorganism can be carried out in a suitable culture medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be batch, continuous, or fed-batch culture, but is not limited to these. In this application, "culture medium" means a substance mixed mainly with nutrients necessary for culturing the microorganism, supplying nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganism in this application may be any culture medium commonly used for culturing microorganisms, and the microorganism can be cultured in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions with the temperature, pH, etc. adjusted.

[0031] The aqueous solution of disodium 5'-guanylate used in mixing step S100 is obtained by dissolving the ferment product containing disodium 5'-guanylate, prepared in the process described above, in an aqueous solvent. For example, the aqueous solution of disodium 5'-guanylate is prepared by dissolving the crystalline form of disodium 5'-guanylate obtained from the ferment product in water. Disodium 5'-guanylate in the aqueous solution exists in hydrate form. For example, disodium 5'-guanylate is provided in the form of disodium 5'-guanylate heptahydrate or disodium 5'-guanylate tetrahydrate. In this application, an aqueous solvent is used and no organic solvent is used. Since the use of organic solvents is eliminated, the manufacturing method of this application can be carried out in an environmentally friendly manner.

[0032] The aqueous solution of disodium 5'-guanylate used in mixing step S100 has a concentration of disodium 5'-guanylate of approximately 50 g / L to approximately 600 g / L. In some cases, the concentration of disodium 5'-guanylate can be approximately 50 g / L to 550 g / L, approximately 50 g / L to 500 g / L, approximately 50 g / L to 450 g / L, approximately 50 g / L to 400 g / L, approximately 50 g / L to 350 g / L, approximately 50 g / L to 300 g / L, approximately 50 g / L to 250 g / L, or approximately 50 g / L to 200 g / L. The concentration ranges are approximately 50 g / L to 150 g / L, 50 g / L to 100 g / L, 100 g / L to 450 g / L, 150 g / L to 450 g / L, 200 g / L to 450 g / L, 250 g / L to 450 g / L, 300 g / L to 450 g / L, 350 g / L to 450 g / L, and 400 g / L to 450 g / L. When aqueous solutions of disodium 5'-guanylate within the aforementioned concentration ranges are prepared and mixed with sodium chloride, amorphous crystals of disodium 5'-guanylate are sufficiently formed.

[0033] In mixing step S100, the aqueous solution of disodium 5'-guanylate prepared as described above is mixed with an aqueous solution of sodium chloride. The aqueous solution of sodium chloride is a solution in which sodium chloride (NaCl) is homogeneously 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 aqueous solution of disodium 5'-guanylate described above may be of the same type or of different types. For example, both aqueous solutions may be prepared using water as the solvent. In this application, as described above, no organic solvent is used in the process of producing disodium 5'-guanylate crystals. In the prior art, disodium 5'-guanylate was treated with an organic solvent to reduce its solubility and precipitate crystals. When an organic solvent is replaced with a hydrophilic solvent, an additive is needed to lower the solubility of the solvent. According to this application, sodium chloride, which is permitted as a food additive, is added to lower the solubility of the hydrophilic solvent in relation to disodium 5'-guanylate.

[0034] The sodium chloride aqueous solution used in mixing step S100 is in a pre-supersaturated state where no sodium chloride salts precipitate. The sodium chloride aqueous solution is a solution with a sodium chloride concentration of approximately 10 g / L to 300 g / L. In some cases, the sodium chloride concentration is approximately 10 g / L to 200 g / L, approximately 10 g / L to 100 g / L, approximately 50 g / L to 300 g / L, approximately 100 g / L to 300 g / L, or approximately 200 g / L to 300 g / L. The concentration of the sodium chloride aqueous solution is determined by considering the concentration and amount of the 5'-disodium guanylate aqueous solution used in mixing step S100.

[0035] In mixing step S100, sodium chloride is added in a proportion of 30% to 80% relative to the weight of disodium 5'-guanylate. In some cases, sodium chloride is added in a proportion of approximately 40% to 70% or approximately 40% to 60% relative to the weight of disodium 5'-guanylate. This is distinct from techniques that induce a phase transition by treating with sodium chloride at a level of 90% to 120% relative to the weight of disodium 5'-guanylate. Methods that add an excess amount of sodium chloride increase costs as the amount of salt used increases, and in the subsequent concentration process, when the salt concentration rises to the supersaturation point of the salt, the NaCl salt precipitates, which may lead to a decrease in content and finer crystal formation. In contrast, according to one aspect of this application, a small amount of sodium chloride is used, and the salt in the solution does not supersaturate and precipitate, so disodium 5'-guanylate crystals can be obtained in high yield.

[0036] Any method can be used to mix the 5'-disodium guanylate aqueous solution and the sodium chloride aqueous solution in mixing step S100. For example, the 5'-disodium guanylate aqueous solution and the sodium chloride aqueous solution can be mixed by various methods, such as adding the sodium chloride aqueous solution dropwise to the 5'-disodium guanylate aqueous solution.

[0037] Next, a concentration step S200 is performed in which the mixed solution of disodium 5'-guanylate and sodium chloride is concentrated to precipitate amorphous crystals of disodium 5'-guanylate.

[0038] In the concentration step S200, amorphous crystals of disodium 5'-guanylate precipitate. When mixed with an aqueous sodium chloride solution, disodium 5'-guanylate is provided in hydrate form, and when the hydrate form of disodium 5'-guanylate reaches the supersaturation point, it forms nuclei. Subsequently, the nuclei of disodium 5'-guanylate aggregate and precipitate as amorphous crystals.

[0039] The concentration step S200 is performed to increase the concentration of disodium 5'-guanylate. The concentration step S200 is performed to concentrate the concentration of disodium 5'-guanylate to 400 g / L to 600 g / L. In some cases, the concentration of disodium 5'-guanylate is concentrated in the concentration step S200 to 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, or 550 g / L to 600 g / L. When the mixed solution is concentrated within the aforementioned concentration range, seeds are introduced in the transition step S300, resulting in a transition from amorphous crystals to columnar heptahydrate crystals. During the concentration process, amorphous crystals that continuously form undergo a phase transition due to the introduction of seeds, forming tetrahydrate or heptahydrate columnar crystals.

[0040] Any method may be used to carry out the concentration step S200. Concentration can be carried out in a conventional concentrator (e.g., a forced-circulation concentrator, a thin-film concentrator, a rotary concentrator, etc.) as appropriate by those skilled in the art.

[0041] The concentration step S200 is performed for approximately 2 to 15 hours. In some cases, the concentration may be performed for approximately 2 to 10 hours, 2 to 5 hours, 5 to 15 hours, or 10 to 15 hours. The concentration time is determined by considering the concentration of disodium 5'-guanylate, the amount of disodium 5'-guanylate produced, etc.

[0042] In the enrichment step S200, the temperature inside the enricher is adjusted to be in the range of approximately 25°C to approximately 85°C. In some cases, the internal temperature of the enricher may be adjusted to be in the range of approximately 25°C to approximately 75°C, approximately 25°C to approximately 65°C, approximately 25°C to approximately 55°C, approximately 25°C to approximately 45°C, approximately 25°C to approximately 35°C, approximately 35°C to approximately 85°C, approximately 35°C to approximately 75°C, approximately 35°C to approximately 65°C, approximately 35°C to approximately 55°C, approximately 35°C to approximately 45°C, approximately 45°C to approximately 85°C, approximately 45°C to approximately 75°C, approximately 45°C to approximately 65°C, approximately 45°C to approximately 55°C, approximately 55°C to approximately 85°C, approximately 55°C to approximately 75°C, approximately 65°C to approximately 75°C, or approximately 75°C to approximately 85°C. By maintaining the temperature range of the concentration process within the above range, 5'-disodium guanylate crystals can be obtained without the denaturation of the 5'-disodium guanylate to be produced. If necessary, the vacuum level inside the concentration apparatus is adjusted so that the temperature does not fall outside the above range while concentration is being performed.

[0043] During the concentration step S200, a transition step S300 is performed in which seeds are added to transform the amorphous crystals of 5'-disodium guanylate into columnar crystals. Here, the fact that the transition step S300 is performed during the concentration step S200 means that the solution is concentrated to a certain level of concentration, and then seeds are added. However, while adding seeds, the solution is further concentrated as needed to maintain the concentration of 5'-disodium guanylate in the solution within a predetermined range. In some cases, if the concentration of 5'-disodium guanylate in the solution obtained after mixing in the mixing step S100 is at a level in which amorphous crystals precipitate, the transition step S300, in which seeds are added, is performed simultaneously with the start of the concentration step S200. Therefore, the concentration step S200 does not necessarily have to precede the transition step S300.

[0044] The seed added in the transition step S300 is a crystal of 5'-disodium guanylate. After seeding, the amorphous crystals of 5'-disodium guanylate transform into columnar crystals. The columnar crystals have a more distinct morphology and higher crystallinity compared to the amorphous crystals. Therefore, the columnar crystals can be separated from the mother liquor relatively easily, thereby increasing the yield of 5'-disodium guanylate. The columnar crystals of 5'-disodium guanylate contain 5'-disodium guanylate in the form of 5'-disodium guanylate heptahydrate.

[0045] In the transition step S300, the seed is added when the concentration of sodium chloride in the mixed solution is at a level of approximately 100 g / L to approximately 130 g / L. By continuing the concentration after adding the seed, the formation of amorphous crystals of 5'-disodium guanylate and the transition of 5'-disodium guanylate to columnar crystals due to the addition of the seed occur simultaneously.

[0046] In the transition step S300, the size of the columnar 5'-disodium guanylate crystals formed changes depending on the seed addition conditions. Specifically, as mentioned above, when seeds are added when the sodium chloride concentration in the mixed solution is approximately 100 g / L to approximately 130 g / L, the formation of amorphous 5'-disodium guanylate crystals and the transition of 5'-disodium guanylate to columnar morphology crystals due to seed addition occur simultaneously, resulting in 5'-disodium guanylate crystals with excellent crystallinity. In contrast, when seeds are added outside the aforementioned concentration range, especially when the sodium chloride concentration is higher than the above range, the crystal transition rate slows down, and the crystals are formed in a fine-grained form rather than a columnar form. In order to obtain columnar morphology crystals in high yield in the transition step S300, it is necessary to adjust process factors such as the concentration of 5'-disodium guanylate in the solution, the temperature of the solution, and the concentration of sodium chloride, as mentioned above. Various factors influence the crystallization phase transition process, including the crystallization solvent, temperature, concentration of disodium 5'-guanylate, cation concentration, and other derivatives, all of which can inhibit the phase transition. If an incorrect element is present in the elements that create the crystallization environment, the crystal phase transition will not proceed smoothly, and even if the phase transition occurs, it will be obtained in the form of small, fine-grained crystals, resulting in a decrease in process yield.

[0047] The execution temperature of the transition step S300 and the concentration step S200 may be changed considering the form of disodium 5'-guanylate. For example, if disodium 5'-guanylate is 5'-guanylate heptahydrate, adjusting the process temperature to approximately 25°C to approximately 42°C allows for efficient concentration to produce amorphous crystals and transition to columnar crystals. Also, if disodium 5'-guanylate is 5'-guanylate tetrahydrate, adjusting the process temperature to approximately 42°C to approximately 85°C allows for efficient concentration to produce amorphous crystals and transition to columnar crystals.

[0048] After the transfer step S300, the columnar crystals of 5'-disodium guanylate that have been formed are separated from the mother liquor to obtain the final product, columnar crystals of 5'-disodium guanylate. Various methods can be used to separate the crystals, such as vacuum membrane filtration, pressure membrane filtration, centrifugation, and basket separation.

[0049] As mentioned above, according to this application, 5'-disodium guanylate crystals are produced using an aqueous solvent instead of an organic solvent, making the process environmentally friendly. Furthermore, by adjusting the process factors so that 5'-disodium guanylate forms amorphous crystals and then undergoes a phase transition to columnar crystals, the yield of 5'-disodium guanylate crystals can be increased.

[0050] The method for producing disodium 5'-guanylate crystals according to one aspect of this application has been described above. The following describes a more specific embodiment of the method for producing disodium 5'-guanylate.

[0051] Figure 2 is a flowchart showing a method for producing disodium 5'-guanylate crystals according to one aspect of this application.

[0052] In the explanation of Figure 2, to avoid duplication of content, parts that are the same as those explained in Figure 1 will be omitted.

[0053] As shown in Figure 2, in mixing step S100, the wet crude crystals of disodium 5'-guanylate prepared by fermentation are dissolved to prepare an aqueous solution of disodium 5'-guanylate. The dissolution of the wet crude crystals of disodium 5'-guanylate can be carried out by a conventional method, such as adding the wet crude crystals of disodium 5'-guanylate to an aqueous solvent and then stirring.

[0054] Alternatively, before performing mixing step S100, a step to adjust the pH of the 5'-disodium guanylate aqueous solution may be performed. Specifically, the pH of the 5'-disodium guanylate aqueous solution is adjusted to 8-10. Within the aforementioned pH range, the transition of 5'-disodium guanylate to columnar crystalline form occurs rapidly. The pH adjustment of the 5'-disodium guanylate aqueous solution is performed by adding a sodium salt. This prevents the addition of cations other than sodium ions (Na+) to the mixed solution, thereby preventing the inhibition of crystallization of 5'-disodium guanylate. Examples of sodium salts used for pH adjustment include sodium hydroxide, monosodium citrate, disodium citrate, trisodium citrate, monosodium phosphate, disodium phosphate, trisodium phosphate, sodium carbonate, and sodium bicarbonate.

[0055] In mixing step S100, a decolorization step may be further performed to remove impurities. The decolorization step is for removing residual pigment substances from the fermentation liquid and is carried out by an adsorption decolorization method using activated carbon or clay (activated clay, acid clay) as an adsorbent. However, the above is an example, and the method of performing the decolorization step should be appropriately selected considering the physical properties of the fermentation liquid containing L-citrulline, the characteristics of the subsequent process, etc. The order of pH adjustment, cell filtration, and decolorization steps may be changed as needed. For example, cell filtration and decolorization may be completed first, and then the pH may be adjusted, or decolorization may be performed before cell filtration.

[0056] After the transition step S300, once columnar crystals of disodium 5'-guanylate have formed, the solution may be cooled, the columnar crystals separated, and the separated columnar crystals may be dried. The drying of the crystals may be carried out at room temperature.

[0057] According to one aspect of this application, the columnar disodium 5'-guanylate crystals produced by the above method are provided in the form of a product mixed with disodium 5'-inosinate crystals. Specifically, according to one aspect of this application, a fermented product is provided comprising columnar disodium 5'-guanylate crystals and disodium 5'-inosinate crystals, wherein the concentration of chloride ions (Cl-) in the mixture of the disodium 5'-guanylate crystals and the disodium 5'-inosinate crystals is 500 ppm to 20,000 ppm.

[0058] Alternatively, according to one aspect of this application, a crystal or fermented product is provided comprising disodium 5'-guanylate crystals, wherein the disodium 5'-guanylate crystals have a chloride ion concentration of 500 ppm to 20,000 ppm.

[0059] 5'-disodium guanylate crystals according to one aspect of this application may have a chloride ion (Cl-) concentration in the crystal ranging from 500 ppm to 20,000 ppm. 5'-disodium guanylate crystals having the above-mentioned chloride ion concentration are provided in a columnar crystalline form. Since columnar crystalline 5'-disodium guanylate crystals are easily separated during the manufacturing process, they contain fewer impurities and are provided with high crystalline purity.

[0060] Furthermore, if the chloride ion concentration is within the above range, microbial growth can be controlled, and solidification due to the distribution or storage environment of the manufactured product can be prevented. Therefore, 5'-disodium guanylate crystals containing the aforementioned chloride ion concentration are not only highly pure but also advantageous in terms of storage and distribution. In addition, if chloride ions remain in the final product at a concentration of 500 ppm to 20,000 ppm, microbial growth is controlled and solidification under pressure is reduced, making product management easier, resulting in a low rate of microbial contamination and safe consumption.

[0061] The 5'-disodium guanylate having the aforementioned chloride ion concentration is produced by the method for producing 5'-disodium guanylate crystals according to one aspect of this application described above. According to the method for producing 5'-disodium guanylate crystals according to one aspect of this application, if the concentration of sodium chloride in the mixed solution is at a level of approximately 100 g / L to approximately 130 g / L for the concentration of 5'-disodium guanylate, then when a seed is added, the formation of amorphous 5'-disodium guanylate crystals and the transformation of 5'-disodium guanylate to columnar crystalline form due to the addition of the seed occur simultaneously. The 5'-disodium guanylate crystals prepared by this method contain a specific concentration of chloride ions in the crystal because sodium chloride is added at a specific concentration in the transformation step. Specifically, the crystal is provided with 500 ppm to 20,000 ppm of chloride ions.

[0062] In contrast, experiments have confirmed that disodium 5'-guanylate crystals produced by prior art methods, such as ethanol crystallization, rather than by the method described in this application, provide chloride ions in concentrations of less than 100 ppm. [Examples]

[0063] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the application is not limited thereto. Technical matters not described herein can be fully understood and readily implemented by a skilled technician in the art of this application or a similar art.

[0064] The method for producing disodium 5'-guanylate crystals according to one aspect of this application has been described above. The advantageous effects of this application will now be explained based on the experimental results of the examples and comparative examples.

[0065] Comparative Example 1. Crystallization using a hydrophilic organic solvent (conventional crystallization method) Crude disodium 5'-guanylate crystals with a purity of 90% derived from microbial fermentation liquid were dissolved to prepare 0.5 L of a 30 wt.% disodium 5'-guanylate solution. Then, 0.55 L of ethanol was added at room temperature to generate amorphous crystals. During the ethanol addition, 10 wt.% of 5'-guanylate crystal heptahydrate seed was added by weight, and then ethanol was continuously added to induce a phase transition from amorphous 5'-guanylate crystals to 5'-guanylate heptahydrate crystals. The crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated by centrifugation, and then dried at room temperature to obtain the product. The quality of the obtained product was 100% content, 23% moisture content, and 95% yield. Disodium 5'-guanylate heptahydrate crystals were slurryed in 99.5% ethanol solvent, and particle size analysis was performed using a wet particle size analyzer. The average particle size was at the level of 150 μm. The residual Cl content in the final crystals, analyzed by chromatography, was at the level of 39.2 ppm.

[0066] Comparative Example 2. Crystallization in a non-concentrated aqueous system Crude disodium 5'-guanylate crystals were dissolved to prepare 0.5 L of a 30 wt.% solution of disodium 5'-guanylate. 0.55 L of a 30 wt.% solution of sodium chloride dissolved in water was also prepared. The amount of sodium chloride relative to the disodium 5'-guanylate solution was approximately 90% by mass. Sodium chloride solution was dripped into the disodium 5'-guanylate solution at approximately 42°C to induce a phase transition from amorphous disodium 5'-guanylate crystals to disodium 5'-guanylate heptahydrate crystals. The solution was gradually cooled to approximately 25°C, and the crystals separated using a basket centrifuge had a content of 98%, a water content of 25%, and a yield of 69%.

[0067] Comparative Example 3. Crystallization in a non-concentrated aqueous system Crude disodium 5'-guanylate crystals were dissolved to prepare 0.2 L of a 30 wt.% solution of disodium 5'-guanylate, and then 50 wt.% NaOH (soldium hydroxide solution) was added and the solution was titrated to a pH of 9.0. Sodium chloride was dissolved in water to prepare 0.2 L of a 30 wt.% solution. The amount of sodium chloride relative to the disodium 5'-guanylate solution was 40% by mass. The two solutions were mixed, and the temperature of the homogeneous solution containing precipitated amorphous crystals was monitored and maintained to ensure it did not exceed approximately 25°C to 42°C. A 1 wt.% seed of disodium 5'-guanylate heptahydrate was added to induce a phase transition, but no phase transition occurred. The amorphous crystals were centrifuged using a basket separator, but the crystals could not be separated.

[0068] Experimental Example 1 - Synthesis of disodium 5'-guanylate at various amounts of sodium chloride [Examples]

[0069] Crude disodium 5'-guanylate crystals were dissolved to prepare 0.5 L of a 30 wt.% solution of disodium 5'-guanylate, and then 50 wt.% NaOH (soldium hydroxide solution) was added and the solution was titrated to a pH of 9.0. Sodium chloride was dissolved in water to prepare 0.2 L of a 30 wt.% solution. The amount of sodium chloride relative to the mass of disodium 5'-guanylate in the disodium 5'-guanylate solution was 40% by mass. The two solutions were mixed, and concentration and crystallization were started in a homogeneous solution in which amorphous crystals precipitated. The vacuum level was adjusted, and the internal temperature was measured with a thermometer to ensure that it did not exceed 25°C to 42°C during concentration. At a concentration ratio of 2.1 times, 1 wt.% of disodium 5'-guanylate heptahydrate seed was added, and a phase transition was induced continuously. After seeding, amorphous 5'-disodium guanylate crystals underwent a phase transition to columnar 5'-disodium guanylate heptahydrate crystals. The slurry was concentrated until the concentration of 5'-disodium guanylate heptahydrate reached a minimum of 48 wt.% during the phase transition. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated by centrifugation, and then dried at room temperature. The obtained 5'-disodium guanylate crystals had a content of 100%, a moisture content of 23%, and a yield of 93%. The 5'-disodium guanylate heptahydrate crystals were slurryed with 99.5% ethanol, and wet particle size analysis was performed using a particle size analyzer. The average particle size was found to be 250 μm. The amount of residual anions in the final crystals analyzed by HPLC was Cl. - The reading was 1474.9 ppm. [Examples]

[0070] Crude disodium 5'-guanylate crystals were dissolved to prepare 0.5 L of a 30 wt.% solution of disodium 5'-guanylate. Then, 50 wt.% NaOH (soldium hydroxide solution) was added and the solution was titrated to a pH of 9.0. Next, anhydrous sodium carbonate anhydrous was added at a ratio of 5% by weight of disodium 5'-guanylate. Sodium chloride was dissolved in water to prepare 0.33 L of a 30 wt.% solution. The amount of sodium chloride relative to the mass of disodium 5'-guanylate in the disodium 5'-guanylate solution was approximately 60% by mass. The two solutions were mixed, and concentration and crystallization were started in a homogeneous solution where amorphous crystals precipitated. Concentration was performed while monitoring the internal temperature to ensure it did not exceed 25°C to 42°C. 1 wt.% of 5'-disodium guanylate heptahydrate seed was added at a concentration of 1.05 times, and a phase transition was induced to occur continuously. After seed addition, amorphous 5'-disodium guanylate crystals continuously underwent a phase transition to columnar 5'-disodium guanylate heptahydrate crystals. The mixture was concentrated until the concentration of 5'-disodium guanylate heptahydrate reached a minimum of 44%. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated by centrifugation, and then dried at room temperature. The obtained 5'-disodium guanylate crystals had a content of 100.7%, a moisture content of 23%, and a yield of 85%. The 5'-disodium guanylate heptahydrate crystals were slurryed with 99.5% ethanol, and wet particle size analysis was performed using a particle size analyzer. The average particle size was 290 μm. The amount of residual anions in the final crystals analyzed by HPLC was Cl. - The reading was 4723 ppm.

[0071] Figure 3 shows the results of the XRD analysis of the 5'-disodium guanylate crystals obtained in Example 2.

[0072] Analysis of the peaks obtained by XRD analysis confirmed that the results were consistent with the XRD values ​​obtained from the columnar morphology of 5'-disodium guanylate heptahydrate reported in conventional literature. Therefore, the results in Figure 3 confirm that columnar morphology 5'-disodium guanylate heptahydrate crystals were synthesized according to one embodiment of this application. [Examples]

[0073] Crude disodium 5'-guanylate crystals were dissolved to prepare 0.5 L of a 30 wt.% solution of disodium 5'-guanylate, and then anhydrous sodium phosphate anhydrous was added at a ratio of 5% by weight of disodium 5'-guanylate. Sodium chloride was dissolved in water to prepare 0.25 L of a 30 wt.% solution. The amount of sodium chloride relative to the mass of disodium 5'-guanylate in the disodium 5'-guanylate solution was at the level of 50% by mass. The two solutions were mixed, and concentration and crystallization were started in a homogeneous solution in which amorphous crystals precipitated. Concentration was carried out while monitoring the temperature with a thermometer to ensure that the internal temperature did not exceed 25°C to 42°C. At a concentration ratio of 1.39, 1 wt.% of disodium 5'-guanylate heptahydrate seed was added, and a phase transition was induced continuously. After seeding, amorphous 5'-disodium guanylate crystals underwent a phase transition to columnar 5'-disodium guanylate heptahydrate crystals. The slurry was concentrated until the concentration of 5'-disodium guanylate heptahydrate reached a minimum of 45 wt.%. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated by centrifugation, and then dried at room temperature. The obtained 5'-disodium guanylate crystals had a content of 100.4%, a moisture content of 23%, and a yield of 87%. The 5'-disodium guanylate heptahydrate crystals were slurryed in 99.5% ethanol solvent, and wet particle size analysis was performed using a particle size analyzer. The average particle size was 397 μm. The amount of residual anions in the final crystals analyzed by HPLC was Cl. - The reading was 2660 ppm. [Examples]

[0074] Crude disodium 5'-guanylate crystals were dissolved to prepare 0.5 L of a 30 wt.% solution of disodium 5'-guanylate, and then 50 wt.% NaOH (soldium hydroxide solution) was added and the solution was titrated to a pH of 9.0. Sodium chloride was dissolved in water to prepare 0.3 L of a 30 wt.% solution. The amount of sodium chloride relative to the disodium 5'-guanylate solution was 60% by mass. The two solutions were mixed, and concentration and crystallization were started in a homogeneous solution in which amorphous crystals precipitated. Concentration was carried out while monitoring the internal temperature with a thermometer to ensure it did not exceed 25°C to 42°C. At a concentration ratio of 1.12, 1 wt.% of disodium 5'-guanylate heptahydrate seed was added, and a phase transition was induced continuously. After seeding, amorphous 5'-disodium guanylate crystals underwent a phase transition to columnar 5'-disodium guanylate heptahydrate crystals. The slurry was concentrated until the concentration of 5'-disodium guanylate heptahydrate reached a minimum of 44 wt.%. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated by centrifugation, and then dried at room temperature. The obtained 5'-disodium guanylate crystals had a content of 102%, a moisture content of 22.5%, and a yield of 88%. The 5'-disodium guanylate heptahydrate crystals were slurryed in 99.5% ethanol solvent, and wet particle size analysis was performed using a particle size analyzer. The average particle size was 329 μm (Figure 2). The amount of residual anions in the final crystals analyzed by HPLC was Cl. - The concentration was 1874 ppm. [Examples]

[0075] Crude disodium 5'-guanylate crystals were dissolved to prepare 0.5 L of a 30 wt.% solution of disodium 5'-guanylate, and then 50 wt.% NaOH (soldium hydroxide solution) was added and the solution was titrated to a pH of 9.0. Sodium chloride was dissolved in water to prepare 0.25 L of a 30 wt.% solution. The amount of sodium chloride relative to the mass of disodium 5'-guanylate in the disodium 5'-guanylate solution was 50% by mass. The two solutions were mixed, and concentration and crystallization were started in a homogeneous solution in which amorphous crystals precipitated. Concentration was carried out while monitoring the internal temperature with a thermometer to ensure it did not exceed 25°C to 85°C. At a concentration ratio of 1.4, 1 wt.% of disodium 5'-guanylate heptahydrate seed was added, and a phase transition was induced continuously. After seeding, amorphous 5'-disodium guanylate crystals underwent a phase transition to thin columnar 5'-disodium guanylate tetrahydrate crystals. The slurry was concentrated until the concentration of 5'-disodium guanylate tetrahydrate reached a minimum of 40 wt.%. The concentrated crystal slurry was cooled to an internal temperature of 25°C, the crystals were separated by centrifugation, and then dried at room temperature. The obtained 5'-disodium guanylate crystals had a content of 111.5%, a moisture content of 17.6%, and a yield of 88.4%. The 5'-disodium guanylate tetrahydrate crystals were slurryed in 99.5% ethanol solvent, and wet particle size analysis was performed using a particle size analyzer. The average particle size was found to be 130 μm. The amount of residual anions in the final crystals analyzed by HPLC was Cl. - The reading was 2478 ppm.

[0076] Figure 4 shows a microscopic image of the 5'-disodium guanylate crystals obtained in Example 4. As can be seen in the figure, the obtained crystals exhibit a columnar morphology, and no amorphous crystals were observed. Therefore, it was confirmed that, due to a continuous phase transition of the crystal, almost all of the 5'-disodium guanylate in the solution is obtained in the form of columnar crystals, and that 5'-disodium guanylate crystals can be obtained in high yield.

[0077] Figure 5 shows the grain size analysis results of the 5'-disodium guanylate crystals obtained in Example 4, confirming that the crystal grain sizes are distributed relatively uniformly.

[0078] Experimental Example 2. Investigation of microbial growth and solidification phenomena at various chloride ion concentrations in 5'-disodium guanylate crystals. (1) Confirmation of the effect on controlling microbial growth The amounts of residual chloride ions in the crystals produced by the manufacturing process of Comparative Example 1 (crystallization using a hydrophilic organic solvent) and the crystals produced by the manufacturing process of Example 1 are as follows:

[0079] [Table 1]

[0080] When microorganisms were applied to the crystals and the subsequent reduction rate of microorganisms was compared, it was confirmed that a microbial growth control effect was observed in crystals prepared by the manufacturing method of the example, where chloride ion concentrations ranged from 5,703 ppm to 20,000 ppm. In particular, the microbial reduction effect in crystals containing chloride ions at a chloride ion concentration of 20,000 ppm was confirmed to be equivalent to that of sodium chloride. In contrast, the residual chlorine content of crystals produced by the hydrophilic organic solvent method (Comparative Example 1) was at the level of 34 ppm, and a tendency for a large amount of microorganisms to grow over time was observed. The microbial growth control rates over time for each crystal are as follows.

[0081] [Table 2]

[0082] (2) Confirmation of the effect of reducing solidification Fermented products containing disodium 5'-guanylate may solidify depending on the distribution or storage environment. However, it has been shown that the solidification rate decreases in chlorine-containing crystals, making them more advantageous for storage compared to crystals produced by conventional hydrophilic organic solvent methods.

[0083] To confirm this, the degree of solidification was measured using a ring shear tester. Crystals produced by the manufacturing process of Comparative Example 1 (crystallization using a hydrophilic organic solvent) and crystals produced by the manufacturing process of Example 1 were placed into the equipment, and the following are the results of measuring SIGMA [Pa]: the pressure value required to solidify the substance, FC [Pa]: (yield strength) the pressure value required to crush the solidified substance, and FFC: (flowability) fluidity.

[0084] [Table 3]

[0085] In the crystals (chloride ion residual crystals) produced by the method of Example 1, it was demonstrated that the FC (pressure value required to crush the solidified material) was low, the FFC (Frequency Fluid Value) was high, and the fluidity was high. Specifically, in the crystals produced in Example 1 with a chloride ion residual content of 5,703 ppm to 20,000 ppm, the FC value was 961 Pa to 1,129 Pa, which was lower than the FC value of 1,461 Pa for the crystals produced in Comparative Example 1 with a chloride ion residual content of 34 ppm. This means that the crystals with a high chloride ion residual content produced in the example had relatively little solidification, and therefore the pressure required to crush the solidified material was low.

[0086] Furthermore, the FFC value, an indicator of fluidity, was higher in the crystals produced in Example 1, where the chloride ion residue was 5,703 ppm to 20,000 ppm, than in the crystals produced in the Comparative Example, where the chloride ion residue was 34 ppm, suggesting less solidification.

[0087] Thus, it was confirmed that the disodium 5'-guanylate crystals produced by the manufacturing method according to one embodiment of this application have a chloride ion concentration in the range of 500 ppm to 20,000 ppm, resulting in less solidification and the ability to control microbial growth.

[0088] From the above explanation, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A mixing step in which an aqueous solution of sodium chloride is added to an aqueous solution of disodium 5'-guanylate, A concentration step is to concentrate the mixed solution of the aqueous solution of 5'-disodium guanylate and the aqueous solution of sodium chloride to precipitate amorphous crystals of 5'-disodium guanylate, A method for producing 5'-disodium guanylate crystals, comprising a transition step of adding seeds to a mixed solution of the aqueous 5'-disodium guanylate solution and the aqueous sodium chloride solution to transform the amorphous crystals of 5'-disodium guanylate into columnar crystals.

2. The method for producing disodium 5'-guanylate crystals according to claim 1, wherein in the transition step, the formation of amorphous crystals of disodium 5'-guanylate and the transition of the amorphous crystals of disodium 5'-guanylate to columnar crystals are carried out in succession.

3. The method for producing disodium 5'-guanylate crystals according to claim 1, wherein the amorphous crystals of disodium 5'-guanylate contain hydrate of disodium 5'-guanylate.

4. The method for producing 5'-disodium guanylate crystals according to claim 1, wherein the concentration step is performed to concentrate the concentration of 5'-disodium guanylate to 400 g / L to 600 g / L.

5. A method for producing disodium 5'-guanylate crystals according to claim 1, further comprising the step of adjusting the pH of the aqueous solution of disodium 5'-guanylate to 8 to 10 before performing the mixing step.

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

7. The method for producing disodium 5'-guanylate crystals according to claim 1, wherein in the mixing step, sodium chloride is added in a proportion of 30% to 80% of the weight of disodium 5'-guanylate.

8. The method for producing disodium 5'-guanylate crystals according to claim 1, wherein in the transition step, the seed is added in a proportion of 0.5% to 50% relative to the weight of the disodium 5'-guanylate.

9. A method for producing 5'-disodium guanylate crystals according to claim 1, wherein in the transition step, a seed is added when the concentration of sodium chloride in the mixed solution is at a level of about 100 g / L to about 130 g / L of the concentration of 5'-disodium guanylate.

10. It contains columnar 5'-disodium guanylate crystals, A fermented product in which the concentration of chloride ions (Cl-) in the 5'-disodium guanylate crystals is 500 ppm to 20,000 ppm.

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

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

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