Process for the manufacture of aspartic acid

Abstract

The present disclosure relates to a method for producing aspartic acid, which can reduce or remove impurities even when a crude product mixed with a considerable amount of impurities such as amino acids, organic substances, coloring substances, inorganic salts, and the like is used as a starting material. The method comprises: (q) preparing a slurry of a crystal component (X) containing a β-type crystal of aspartic acid and at least one impurity; and (r) heating the slurry to change the β-type crystal of aspartic acid to an α-type crystal, and obtaining a crystal component (Y) of aspartic acid containing the α-type crystal.

Description

Technical Field

[0001] This invention relates to a method for manufacturing aspartic acid. More specifically, this invention relates to a method for manufacturing aspartic acid having an α-type crystal morphology. Background Technology

[0002] Various amino acids are the building blocks of proteins in living organisms, and also substances capable of performing various biological or chemical functions. Therefore, they are widely used as raw materials in food, pharmaceuticals, chemicals, cosmetics, and other applications. In particular, aspartic acid and glutamic acid, known as acidic amino acids, are used as sweeteners and umami flavorings in food. Furthermore, in recent years, polyaspartic acid and polyglutamic acid, obtained by polymerizing them, have attracted considerable attention as functional raw materials that retain biodegradability or water absorption while being environmentally friendly. Against this backdrop, various methods for manufacturing or refining aspartic acid and glutamic acid have been developed.

[0003] For example, Patent Document 1 discloses a method for obtaining refined optically active β-glutamic acid crystals. In this method, crude optically active glutamic acid crystals containing α-glutamic acid crystals are placed or stirred with a crystal slurry containing an aqueous solvent at a temperature range of 50°C to 120°C, and then the glutamic acid crystals are separated. Patent Document 1 states that, compared to existing methods, the method requires only a small amount of processing material, reducing the energy and labor required. It also mentions that since no acid or alkali is needed, adverse phenomena such as the mixing of sodium chloride into the product or the racemization of optical activity can be suppressed.

[0004] Furthermore, Patent Document 2 discloses a method for purifying aspartic acid, wherein aspartic acid crystals containing at least Cl- are purified in an aqueous solution at a temperature of 50°C or higher under suspension. In Patent Document 2, the crude aspartic acid crystals produced using fermentation, enzymatic methods, and chemical synthesis contain impurities such as other amino acids, coloring substances, and inorganic salts. This suggests that, compared to existing methods, the purification method described in the document can yield high-purity aspartic acid crystals with further reduced or removed impurities such as Cl-.

[0005] Furthermore, Patent Document 3 discloses a method for crystallizing aspartic acid, which involves mixing an aqueous solution of ammonium aspartate with sulfuric acid or hydrochloric acid to crystallize aspartic acid, wherein a specific amount of malic acid coexists in the crystallization system. Patent Document 3 suggests that, with the goal of crystallizing existing columnar crystals, the cleaning effect in the cleaning process is improved, and high-purity columnar crystals of aspartic acid can be obtained.

[0006] Furthermore, in the industrial production of various amino acids, primarily aspartic acid and glutamic acid, there has been a history of using general enzymatic or extraction methods. For example, in the enzymatic production of aspartic acid, a method is employed to synthesize aspartic acid using a reversible reaction based on fumaric acid and ammonia. As a method for separating and purifying the synthesized aspartic acid, various treatments can be appropriately combined, such as adsorption using activated carbon, filtration, isoelectric point crystallization, cooling, crystal separation, drying, and immobilization using carrageenan carriers. On the other hand, in production using extraction methods, a method is employed to separate / purify the desired amino acid by decomposing a specified protein into amino acid units through a hydrolysis reaction using hydrochloric acid, etc. In this case, the separation / purification of the desired amino acid employs a combination of various separation methods, such as ion exchange chromatography or fractional crystallization based on differences in isoelectric point, adsorption, solubility, etc., among the various substances. Moreover, in recent years, the use of microbial fermentation methods has become increasingly common. For example, a method is known to cultivate Pantoea ananatis, which is endowed with the ability to produce L-glutamic acid, in a medium with pH conditions adjusted to induce L-glutamic acid precipitation, thereby generating and accumulating L-glutamic acid crystals simultaneously (Patent Document 4). In this method, L-glutamic acid is produced by presenting L-lysine in the medium when the L-glutamic acid concentration is below the concentration that causes spontaneous crystallization, thereby causing the precipitation of α-type L-glutamic acid crystals.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: Japanese Patent Publication No. 45-4730

[0010] Patent Document 2: Japanese Patent Application Publication No. 63-233958

[0011] Patent Document 3: Japanese Patent Application Publication No. 8-217733

[0012] Patent Document 4: International Publication WO2004 / 099426 Summary of the Invention

[0013] The problem that the invention aims to solve

[0014] Unlike currently used enzymatic and extraction methods, the crude product obtained from bioprocesses such as microbial fermentation to produce aspartic acid contains not only the target aspartic acid, but also a considerable amount of other amino acids, organic matter, coloring substances, inorganic salts, and other impurities derived from microorganisms or culture medium components. The inventors have discovered that establishing a separation, purification, or manufacturing technique for aspartic acid that can effectively reduce or remove these impurities from the crude product containing a considerable amount of such impurities has become a new challenge.

[0015] Technical means to solve the problem

[0016] In order to solve the aforementioned problem, the inventors conducted intensive research on the conditions for separating and purifying aspartic acid from crude product samples derived from microbial cultures and containing various impurities. As a result, they discovered that during the process of preparing β-type crystals of aspartic acid separated from the crude product sample in slurry form, heating the slurry to cause it to transform from β-type crystals to α-type crystals, and obtaining crystalline components containing the α-type crystals, various impurities are effectively reduced or removed, thereby enabling the production of high-purity aspartic acid in the form of α-type crystals. This invention was made based on the aforementioned discovery.

[0017] According to embodiments of the present invention, a method for manufacturing aspartic acid is provided below.

[0018] [1] A method for producing aspartic acid, comprising:

[0019] (q) Prepare a slurry containing β-type crystals of aspartic acid and a crystal component (X) of at least one impurity; and

[0020] (r) The slurry is heated to change the β-type crystals of aspartic acid into α-type crystals, and then a crystal component (Y) of aspartic acid containing the α-type crystals is obtained.

[0021] [2] According to the method described in [1], in step (r), the slurry is heated in a temperature range of 30°C to 190°C, preferably 60°C to 190°C, so that the β-type crystals of aspartic acid are transformed into α-type crystals.

[0022] [3] According to the method of [1] or [2], wherein, in step (r), the slurry is heated in a temperature range of 65°C to 150°C to change the β-type crystals of aspartic acid into α-type crystals.

[0023] [4] The method according to any one of [1] to [3] further comprises:

[0024] (p) In a solution (S) containing aspartic acid or its salt and at least one impurity, the pH of the solution (S) is adjusted to a predetermined pH value in the acidic region to generate β-type crystals of aspartic acid. Then, the component containing the β-type crystals is separated from the solution (S).

[0025] In step (q), the slurry of the crystal component (X) is prepared using the component containing β-type crystals.

[0026] [5] According to the method of [4], wherein in step (p), the pH of the solution (S) is adjusted to a specified value in the range of 0.50 to 6.95 to generate β-type crystals of the aspartic acid.

[0027] [6] According to the method of [4] or [5], wherein, in step (p), the pH of the solution (S) is adjusted to a specified value in the range of 1.50 to 4.50 to generate β-type crystals of the aspartic acid.

[0028] [7] The method according to any one of [4] to [6], wherein the solution (S) tested in step (p) contains seed crystals.

[0029] [8] According to the method of [7], wherein the seed crystal comprises a β-type crystal of aspartic acid.

[0030] [9] The method according to any one of [4] to [8], wherein the solution (S) tested in step (p) is a culture obtained by culturing or reacting microorganisms in a culture medium, a clarified liquid isolated from said culture, or a concentrate thereof.

[0031]

[10] The method according to any one of [4] to [9], wherein the solution (S) tested in step (p) is a solution containing the aspartic acid or its salt at a concentration of 0.1M to 5.0M.

[0032]

[11] The method according to any one of [4] to

[10] , wherein the solution (S) tested in step (p) contains at least one of the group consisting of amino acids other than aspartic acid, organic acids and their salts as said impurities.

[0033]

[12] The method according to any one of [4] to

[11] , wherein,

[0034] The solution (S) tested in process (p) contains at least the following components as said impurities:

[0035] i) Selected from at least one of the group consisting of glutamic acid, alanine, valine, and their salts; and

[0036] ii) Selected from at least one of the group consisting of pyruvate, malic acid, acetic acid, succinic acid, fumaric acid and their salts.

[0037]

[13] The method according to any one of [4] to

[12] , wherein the pH of the solution (S) tested in step (p) is in the range of 6.00 to 8.00.

[0038]

[14] According to the method of

[13] , wherein, in step (p), the pH of the solution (S) is adjusted to a predetermined value in the range of 1.00 to 6.85 by adding acid to the solution (S) to generate β-type crystals of the aspartic acid.

[0039]

[15] The method according to any one of [4] to

[14] , wherein,

[0040] In step (p), after the β-type crystals of aspartic acid are generated in the solution (S), the component containing the β-type crystals is separated from the solution (S) using a solid-liquid separation method.

[0041] In step (q), the component containing β-type crystals separated in step (p) is used to prepare a slurry of the crystal component (X).

[0042] In process (r), the slurry is heated to change the β-type crystals of aspartic acid into α-type crystals, and then the crystal component (Y) is separated from the slurry of the crystal component (X) using a solid-liquid separation method.

[0043]

[16] According to the method described in

[15] , wherein,

[0044] In step (p), after separating the crystal component containing the β-type crystal from the solution (S) using a solid-liquid separation method, the separated crystal component is washed at least once using a solvent, and then dried.

[0045] In step (q), the dried crystal component is used to prepare a slurry of the crystal component (X).

[0046] In process (r), after separating the crystal component (Y) from the slurry of the crystal component (X) using a solid-liquid separation method, the separated crystal component (Y) is washed once or more using a solvent and then dried.

[0047] The effects of the invention

[0048] According to embodiments of the present invention, even when using crude products containing a considerable amount of impurities such as amino acids other than aspartic acid, organic acids, proteins, sugars, and inorganic salts as starting materials, high-purity aspartic acid with these impurities reduced or removed can be manufactured in the form of α-type crystals. Furthermore, according to specific embodiments of the present invention, high-purity α-type crystals of aspartic acid, especially with reduced or removed coloring substances, can be manufactured. Attached Figure Description

[0049] Figure 1 This is a diagram that schematically illustrates an example of the steps that can be used in the method of the present invention.

[0050] Figure 2A This is a graph showing the results of amino acid / organic acid analysis of filtrate (A) in Experimental Example 1.

[0051] Figure 2B This is a graph showing the results of amino acid / organic acid analysis of the crude crystals (B) in Experimental Example 1.

[0052] Figure 2C This is a graph showing the results of amino acid / organic acid analysis of crystal (C) in Experimental Example 1.

[0053] Figure 3A This is a graph showing the proportion of various impurities removed from each sample through the isoelectric point crystallization process in Test Example 1.

[0054] Figure 3B This is a graph showing the proportion of various impurities removed from each sample through the hot re-slurrying process in Test Example 1.

[0055] Figure 3C This is a graph showing the removal rate of various impurities removed from each sample by the overall isoelectric point crystallization / thermal re-slurrying treatment in Test Example 1.

[0056] Figure 4A This is a photograph showing the appearance of the crystal sample obtained in Test Example 1.

[0057] Figure 4B This is a diagram showing a microscope photograph of the crystal sample obtained in Test Example 1.

[0058] Figure 4C This is a diagram showing a microscope photograph of the crystal sample obtained in Test Example 1.

[0059] Figure 4D This is a diagram showing a microscope photograph of the crystal sample obtained in Test Example 1.

[0060] Figure 5A This is a diagram showing the X-ray diffraction pattern of the coarse crystal (B) in Experimental Example 1.

[0061] Figure 5B This is a diagram showing the X-ray diffraction pattern of crystal (C) in Experimental Example 1.

[0062] Figure 6 This is a graph showing the analytical results of various amino acids in each sample of Experiment Example 2.

[0063] Figure 7 This is a graph showing the analytical results of various organic acids in each sample of Experiment Example 2.

[0064] Figure 8A This is a graph showing the recovery rate of aspartic acid in each sample in Experiment Example 2.

[0065] Figure 8B This is a graph showing the residual rate of various impurities in the coarse crystals (B) in Experimental Example 2.

[0066] Figure 9 This is a graph showing the residual rate of various impurities in each crystal sample in Test Example 2.

[0067] Figure 10 This is a graph showing the results of the evaluation test of the final product in Experiment Example 2.

[0068] Figure 11 This is a photograph showing the appearance of the sample without added seed crystals, taken during isoelectric point crystallization in Test Example 3.

[0069] Figure 12 This is a graph showing the residual rates of aspartic acid and other amino acids in each crystal sample in Experimental Example 3.

[0070] Figure 13 This is a graph showing the residual rates of various organic acids in each crystal sample in Experiment Example 3.

[0071] Figure 14 This is a graph showing the analytical results of various amino acids in each sample of Experimental Example 5.

[0072] Figure 15 The graph shows the analytical results of various organic acids and dihydroxyacetone (DHA) in each sample of Experimental Example 5.

[0073] Figure 16A This is a graph showing the recovery rate (residual rate) of aspartic acid in each sample of crude crystal (B) and crystal (C) obtained in Experimental Example 5.

[0074] Figure 16B This is a graph showing the residual rate of each impurity in the coarse crystal (B) obtained in Experimental Example 5.

[0075] Figure 17This is a graph showing the residual rate of each impurity in each sample of each crystal (C) obtained in Experimental Example 5.

[0076] Figure 18 This is a graph showing the results of microscopic observation of the crystal changes in each sample caused by the hot resizing treatment in Experimental Example 5. Detailed Implementation

[0077] As described above, according to the present invention, a method for manufacturing aspartic acid is provided.

[0078] The method for producing aspartic acid includes:

[0079] (q) Prepare a slurry containing β-type crystals of aspartic acid and a crystal component (X) of at least one impurity; and

[0080] (r) The slurry is heated to change the β-type crystals of aspartic acid into α-type crystals, and then a crystal component (Y) of aspartic acid containing the α-type crystals is obtained.

[0081] Furthermore, in a specific embodiment, the method of the present invention further includes the following steps before step (q):

[0082] (p) In a solution (S) containing aspartic acid or its salt and at least one impurity, the pH of the solution (S) is adjusted to a predetermined pH value in the acidic region to generate β-type crystals of aspartic acid, and then the component containing the β-type crystals is separated from the solution (S).

[0083] Here, in the subsequent process (q), the slurry of the crystal component (X) is prepared using the component containing the β-type crystal.

[0084] Hereinafter, the terms used in this invention will be explained, and the implementation of the method of this invention will be described in the order of process (p), process (q), and process (r).

[0085] <Explanation of Terminology>

[0086] In this invention, "aspartic acid" and "salts of aspartic acid" are interpreted literally. In this invention, aspartic acid can be naturally abundant in its L-form, D-form, or mixtures thereof. Furthermore, there are no particular limitations on the salts of aspartic acid that can be used in this invention; examples include ammonium salts, sodium salts, potassium salts, and calcium salts. Moreover, in this invention, aspartic acid or its salts can be in the form of anhydrous aspartic acid or its salts, or in hydrated form (e.g., monohydrate, dihydrate). Furthermore, when aspartic acid salts are produced using bioprocesses such as microbial fermentation, the solution (S) typically contains primarily L-aspartic acid or its salts. However, unless otherwise specified, the terms "aspartic acid" and "its salts (salts of aspartic acid)" in this invention are interpreted literally and are not limited to a specific structure.

[0087] In this invention, "impurities" refer to various substances other than aspartic acid or its salts, which are the target of the manufacturing process. They refer to various substances that are to be reduced or removed from the crude product in order to separate and purify aspartic acid. Examples of "impurities" include various amino acids other than aspartic acid (e.g., glycine, alanine, serine, threonine, asparagine, glutamine, lysine, arginine, histidine, valine, leucine, isoleucine, tyrosine, phenylalanine, tryptophan, proline, methionine, cysteine) and their salts, other organic acids (e.g., pyruvic acid, malic acid, acetic acid, succinic acid, fumaric acid) and their salts, proteins or peptides, carbohydrates or saccharides, glycoproteins such as peptidoglycans, inorganic salts, and SO42-. 2- or Cl - Inorganic ions, etc.

[0088] Furthermore, the object produced by the method of the present invention is aspartic acid with an α-type crystal morphology, but the starting material or crude product tested in the method may contain not only aspartic acid, but also salts of aspartic acid. In the case of aspartic acid salts that can eventually be converted into α-type crystals are of course not considered impurities.

[0089] In this invention, the terms "α-type crystal" and "β-type crystal" refer to the various crystal forms that aspartic acid can take, as is known to those skilled in the art, and can be interpreted literally. Specifically, regarding α-type crystals, when observed using a microscope or the like, plate-like crystals are observed, and when analyzed using X-ray diffraction, peaks are observed at diffraction angles near 21.65° and 23.7° in the X-ray diffraction pattern. On the other hand, regarding β-type crystals, when observed using a microscope or the like, fine columnar crystals are observed, and when analyzed using X-ray diffraction, peaks are observed at diffraction angles near 18.8°, 19.7°, and 25.0° in the X-ray diffraction pattern.

[0090] <Process(p)>

[0091] The solution (S) tested in process (p) may contain, in addition to aspartic acid or its salts, at least one of the substances described above as impurities. According to specific embodiments, as shown in the examples described later, it is particularly effective in removing impurities that cause discoloration, which are often problematic in applications such as the polymerization of aspartic acid. According to the embodiments described, it is particularly effective in reducing the high-level mixing of coloring substances into the crude aspartic acid produced using microbial fermentation.

[0092] As described above, solution (S) is a solution containing aspartic acid or its salt and at least one impurity. However, solvents for dissolving these solutes can include, for example, organic solvents such as water, ethanol, and methanol, or mixtures thereof. Furthermore, when the method of the present invention is applied to crude products or concentrates obtained using various bioprocesses such as fermentation, the culture medium or culture broth used for microorganisms typically uses water as the solvent, therefore solution (S) mainly contains water as the solvent component.

[0093] In step (p), the meaning of "solution (S) containing aspartic acid or its salt and at least one impurity" can be understood literally. Specifically, as solution (S), any solution capable of crystallizing aspartic acid into β-type crystals by adjusting the pH of solution (S) to a specified pH value in the acidic region is acceptable. Examples of solutions (S) include: cultures obtained by culturing various cultured cells such as microorganisms (e.g., bacteria, fungi, cyanobacteria, zooplankton, phytoplankton), insect cells, animal cells, and plant cells capable of producing aspartic acid or its salt; processed products obtained by physically or chemically treating said cultures (e.g., ultrasonic treatment or protease treatment); reaction products obtained by enzymatic reaction processes or chemical synthesis processes that produce aspartic acid or its salt; and supernatants obtained by removing solid components from said cultures, processed products, or reaction products by centrifugation or the like.

[0094] In recent years, a technology for producing various amino acids using fermentation or proliferation-independent bioprocesses employing transgenic strains of bacteria such as Corynebacterium glutamicum (e.g., Corynebacterium glutamicum) or Escherichia coli has been developed. In this regard, crude products recovered from bacterial fermentation or proliferation-independent bioprocesses can be advantageously tested in the method of the present invention. More specifically, cultures recovered after culturing or reacting bacteria in fermentation or proliferation-independent bioprocesses, supernatants after removing bacterial cells from said cultures, processed products of said cultures or supernatants, or concentrates thereof can be tested as solutions (S) in step (p). These biologically derived samples contain, in addition to aspartic acid or its salts as the target of purification, various amino acids, organic acids, proteins, carbohydrates, or sugars from bacterial cells or culture media. According to embodiments of the present invention, these impurities can be effectively removed, resulting in the production of high-purity aspartic acid from biologically derived samples in the form of α-crystals. However, the solutions (S) that can be used in this invention are not limited to these biologically derived samples.

[0095] In the biologically derived sample described above, it is also conceivable that the concentration of aspartic acid or its salt is not high enough to efficiently generate β-type crystals of aspartic acid via step (p). In this case, when the biologically derived sample is concentrated before step (p) to obtain a concentrate of aspartic acid or its salt in the solution sample, and this concentrate is used as the solution (S) in step (p), β-type crystals of aspartic acid can be generated efficiently and in a shorter time via step (p). Therefore, it is preferable to use this concentrate as the solution (S) in step (p). Furthermore, regarding the concentration treatment of the biologically derived sample, specifically, it can be carried out using methods such as vacuum concentration using various evaporators or vacuum pumps, adsorption using adsorbents such as activated carbon or silica, ultrafiltration, combinations of these various concentration methods, and any subsequent filtration. However, in this invention, such concentration treatment is not necessary, and it is assumed that if concentration treatment is used, the concentration treatment is not limited to the aforementioned structure.

[0096] Regarding the concentration of aspartic acid or its salt in the solution (S) tested in process (p), there is no particular limitation as long as β-type crystals of aspartic acid can be formed. For example, concentration ranges of about 0.1M to about 5.0M, about 0.2M to about 4.5M, about 0.5M to about 4.0M, and about 1.0M to about 3.5M can be listed.

[0097] Furthermore, in certain implementations, step (p) may include:

[0098] (i) Aspartic acid or its salt;

[0099] (ii) an amino acid other than aspartic acid or a salt thereof (e.g., an amino acid other than aspartic acid or a salt thereof comprising at least one selected from the group consisting of glutamic acid, alanine, valine and their salts); and

[0100] (iii) Organic acids or salts thereof (e.g., organic acids or salts thereof comprising at least one selected from the group consisting of pyruvic acid, malic acid, acetic acid, succinic acid, fumaric acid and their salts),

[0101] The solution sample (preferably a biologically derived sample or its concentrate as described above) is used as solution (S).

[0102] Here, components (ii) and (iii) are of course considered impurities, and it is preferable that their amount in the solution (S) be minimized beforehand. In embodiments of the invention, the amount of components (ii) and (iii) is inherent to the test sample and therefore should not be actively determined. However, in certain embodiments, the amounts of each component (ii) and (iii) can have the following structure in relation to the composition of the biological sample or its concentrate.

[0103] For example, in a particular embodiment, a solution sample may be used as solution (S) in step (p) that contains not only (i) aspartic acid or its salts in the various molar concentration ranges, but also (ii) amino acids other than aspartic acid or their salts in a molar concentration ranging from about 1 / 5 to about 3 / 4 of the molar concentration of the aspartic acid or its salts (e.g., an amino acid other than aspartic acid or its salts selected from the group consisting of at least one selected from glutamic acid, alanine, valine and their salts), and (iii) various organic acids or their salts in a molar concentration ranging from about 1 / 5 to about 1 / 2 of the molar concentration of the aspartic acid or its salts (e.g., an organic acid or its salts selected from the group consisting of at least one selected from pyruvate, malic acid, acetic acid, succinic acid, fumaric acid and their salts).

[0104] Furthermore, in another embodiment, step (p) may also include:

[0105] (i) Aspartic acid or its salts in the range of various molar concentrations;

[0106] (ii) Selected from at least one of the group consisting of glutamic acid, alanine, valine, and their salts (concentration, for example, about 100 μM to about 1 mM, about 1 mM to about 10 mM, about 10 mM to about 1.75 M); and

[0107] (iii) A solution sample (e.g., the biological sample or its concentrate) selected from at least one of the group consisting of pyruvate, malic acid, acetic acid, succinic acid, fumaric acid and their salts (concentration, for example, from about 100 μM to about 1 mM, from about 1 mM to about 10 mM, from about 10 mM to about 1.5 M) is used as solution (S).

[0108] In step (p), in the solution (S) as described above, the pH of the solution (S) is adjusted to a predetermined pH value in the acidic region to generate β-type crystals of aspartic acid. The generation of β-type crystals of aspartic acid based on the pH adjustment of the solution (S) is based on the principle of isoelectric point crystallization of aspartic acid. Specifically, the pH adjustment of the solution (S) in step (p) can be performed as follows.

[0109] First, by adding acid or base to solution (S), the pH of solution (S) is brought close to the isoelectric point of aspartic acid, i.e., 2.77, thereby reducing the solubility of aspartic acid in solution (S) and causing aspartic acid to crystallize as β-type crystals.

[0110] Here, there is no particular limitation on whether it is an acid or a base; for example, acids such as hydrochloric acid, sulfuric acid, and acetic acid, or bases such as sodium hydroxide, potassium hydroxide, and ammonia can be used. If the pH of solution (S) is more alkaline than the isoelectric point 2.77 of aspartic acid, an acid can be used to bring the pH of solution (S) closer to the isoelectric point. On the other hand, if the pH of solution (S) is more acidic than the isoelectric point 2.77 of aspartic acid, a base can be used to bring the pH of solution (S) closer to the isoelectric point. Furthermore, when the biological sample or its concentrate described above is used as solution (S) for testing, the pH of solution (S) generally tends to be mostly neutral (pH 7.0), which is alkaline than the isoelectric point of aspartic acid. Therefore, in this case, an acid is generally used to adjust the pH of solution (S) in step (p).

[0111] In several embodiments, the pH of the solution (S) tested in step (p) is about 6.00 to about 8.00, about 6.5 to about 7.5, about 6.6 to about 7.4, about 6.7 to about 7.3, about 6.8 to about 7.2, about 6.9 to about 7.1, for example about 7.0.

[0112] Furthermore, there are no particular restrictions on the type of acid used; from the perspective of ease of operation and cost-effectiveness, sulfuric acid is the most suitable acid.

[0113] In step (p), the pH of the solution (S) can be adjusted to a value that allows the formation of β-type crystals of aspartic acid. This pH value depends on the concentration of aspartic acid or its salt in the solution (S) and is therefore not particularly limited.

[0114] For example, when a biologically derived sample or its concentrate with a pH near neutral 7.0 and a solution (S) containing a relatively high concentration (e.g., 2.3 M or higher) of aspartic acid or its salt is tested in step (p), since it also depends on other conditions, it cannot be generalized. The inventors have empirically confirmed that the formation of β-type aspartic acid crystals can begin when the pH of the solution (S) is adjusted to an acidic range (e.g., pH 4.0 to pH 6.5) closer to neutral 7.0 by adding acid. Therefore, in step (p), the pH of the solution (S) does not necessarily need to be adjusted to a value infinitely close to the isoelectric point of aspartic acid, i.e., 2.77.

[0115] That is, the term "adjusting the pH of the solution (S) to a specified pH value in the acidic region to generate the β-type crystals of aspartic acid" in step (p) means that the pH of the solution (S) can be adjusted to any pH value in the acidic region capable of generating the β-type crystals of aspartic acid, depending on the properties of the solution (S) used in step (p) or other conditions. Those skilled in the art can refer to the disclosure of this specification and appropriately determine the pH value to be adjusted for each solution (S) based on the properties of the various solutions (S) used in step (p) or other conditions.

[0116] As described above, the pH value of the solution (S) to be adjusted in step (p) is not particularly limited as long as it is a predetermined value that enables the formation of β-type crystals of aspartic acid contained in the solution (S) in the acidic region. However, in a specific embodiment, the pH of the solution (S) may be adjusted to a predetermined value, for example, within the range of about 0.50 to about 6.95, preferably about 1.0 to about 6.85, about 1.50 to about 4.50, more preferably about 2.00 to about 4.00, and particularly preferably about 2.10 to about 3.90.

[0117] Furthermore, in another embodiment, the isoelectric point of aspartic acid, 2.77, is adjusted to a range of ±2.50, preferably ±2.00, more preferably ±1.50, ±1.00, ±0.90, even more preferably ±0.80, particularly preferably ±0.70, ±0.60, ±0.50, ±0.40, ±0.30, ±0.20 or ±0.10, especially preferably ±0.09, ±0.08, ±0.07, ±0.06, ±0.05, ±0.04, ±0.03, ±0.02 or ±0.01, and most preferably the isoelectric point of aspartic acid, i.e., 2.77. According to this embodiment, since the process (p) is performed by adjusting the pH of the solution (S) based on the isoelectric point of aspartic acid (2.77), the various impurities can be effectively reduced or removed from the solution (S) while maintaining a high recovery rate of β-type crystals of aspartic acid due to the difference in isoelectric points between aspartic acid and other impurities.

[0118] The amount of acid or base added to the solution (S) is not particularly limited, as long as it is appropriately adjusted taking into account various conditions, including the initial pH value and the target pH value of the solution (S). In some embodiments, the amount of acid or base added to the solution (S) relative to approximately 100 parts by mass of aspartic acid in the solution (S) can be set in the range of approximately 50 parts by mass to approximately 200 parts by mass, or approximately 60 parts by mass to approximately 150 parts by mass.

[0119] Furthermore, in a specific embodiment, to promote the growth of β-type aspartic acid crystals, step (p) can be performed in the presence of a specified seed crystal. When step (p) is performed in the presence of a seed crystal, it is sufficient to add a specified amount of seed crystal to the solution (S) before adjusting the pH of the solution (S). Regarding the seed crystal, there is no limitation on any seed crystal that promotes the formation of β-type aspartic acid crystals, but to reliably generate the β-type crystal, a β-type crystal containing aspartic acid is preferred. In addition, in this case, regarding the β-type aspartic acid crystal used as the seed crystal, the seed crystal does not necessarily need to be purified to a high purity; a crude crystal sample containing impurities in addition to the β-type aspartic acid crystal is sufficient. For example, a crude crystal sample of aspartic acid generated by using a biologically derived sample or its concentrate as a starting material and performing isoelectric point crystallization (step (p)) without adding a seed crystal can also be used as the seed crystal.

[0120] Furthermore, the amount of seed crystals in the solution (S) is not particularly limited, as long as it is appropriately set according to other crystallization conditions. For example, in a particular embodiment, about 0.001 parts by mass to about 5.00 parts by mass, preferably about 0.001 parts by mass to about 4.00 parts by mass, relative to about 100 parts by mass of aspartic acid or its salt in the solution (S), and in another embodiment, for example, about 0.01 parts by mass to about 3.00 parts by mass, preferably about 0.01 parts by mass to about 2.50 parts by mass, and particularly preferably about 0.01 parts by mass to about 2.00 parts by mass, are added to the solution (S). However, in the present invention, adding seed crystals to the solution (S) is not necessary. As shown in the embodiments described later, even without adding seed crystals, the desired β-type crystals of aspartic acid can be generated in step (q), and the desired α-type crystals of aspartic acid can be finally produced through subsequent steps (r).

[0121] In step (p), the addition of acid or alkali to the solution (S) may sometimes generate heat of reaction such as heat of dilution or heat of dissolution. To avoid this and to achieve a uniform crystallization reaction, it is appropriate to add acid or alkali in stages. Furthermore, there are no particular limitations; if the temperature of the solution (S) rises due to the generation of the heat of reaction shortly after pH adjustment in step (p), the heat can be dissipated to room temperature, and then cooled to a temperature range of 2°C to 10°C (e.g., about 4°C). However, these heat dissipation or cooling steps are not factors that affect the formation of the crystal form of aspartic acid or its salts as specified in this invention, or the removal of impurities, and are not essential components of this invention.

[0122] Furthermore, while it is not necessarily necessary to add acid or alkali to the solution (S) in step (p), it is also possible to control the temperature of the solution (S) within a specified temperature range. In this embodiment, the temperature of the solution (S) can be controlled, for example, within the range of about 30°C to about 190°C, preferably about 35°C to about 150°C, more preferably about 40°C to about 110°C, and even more preferably about 45°C to about 105°C, and even more preferably about 50°C to about 105°C. Furthermore, when controlling the temperature of the solution (S) under normal pressure, it can be controlled within the range of about 45°C to about 100°C, preferably about 50°C to about 100°C, more preferably 60°C to about 100°C, even more preferably about 65°C to about 100°C, even more preferably about 70°C to about 100°C, particularly preferably about 75°C to about 100°C, and most preferably about 78°C to about 100°C. According to the embodiment where the temperature of the solution (S) is controlled within this specified temperature range, it is possible to produce aspartic acid of uniform quality with good reproducibility. In particular, the closer the temperature range is to 100°C, the greater the effect of reducing amino acids other than aspartic acid can be expected. Therefore, the embodiment where the temperature of the solution (S) is controlled within the specified temperature range is preferably adopted. Furthermore, when controlling the temperature of the solution (S) within the specified temperature range, embodiments in a relatively low temperature range, such as controlling it at about 30°C to about 100°C, preferably about 30°C to about 80°C, and more preferably about 40°C to about 70°C, can also be adopted. According to this embodiment, for example, when the proportion of mixed components other than aspartic acid or its salt contained in the solution (S) is relatively small, the energy input to the process can be reduced to the necessary minimum level, achieving a more efficient process. Therefore, this embodiment is preferably adopted.

[0123] As described above, when the pH of solution (S) is adjusted to a specified pH value in the acidic region to generate β-type crystals of aspartic acid, most impurities other than aspartic acid remain in the liquid component of solution (S) (i.e., the supernatant relative to the coarse crystalline solid component). Therefore, in step (p), after the β-type crystals of aspartic acid are generated in solution (S), the component containing the generated β-type crystals is separated from solution (S), thereby removing most of the impurities mixed into solution (S).

[0124] In this invention, the method for separating the component containing the generated β-type crystals from the solution (S) is not particularly limited as long as it involves removing at least a portion of the impurities remaining in the liquid portion of the solution (S). In several embodiments, for example, the following methods may be used: i) a method for separating at least a portion of the component containing the β-type crystals of aspartic acid generated in the solution (S) by means of suction or the like; ii) a method for removing the supernatant liquid portion by means of suction or the like, based on the sedimentation of the coarse crystal component (solid component) generated in the solution (S), and collecting the component containing at least a portion of the remaining β-type crystals of aspartic acid.

[0125] Furthermore, in the aforementioned case, as long as at least a portion of the impurities remaining in the liquid portion of the solution (S) is removed, it can be understood that the purification of aspartic acid has been achieved to some extent. Therefore, it is not excluded that some impurities may enter at least a portion of the component containing β-type crystals of aspartic acid obtained in step (p). Even if a considerable amount of impurities enters at least a portion of the component containing β-type crystals of said aspartic acid in step (p), further reduction or removal of impurities can be expected through subsequent steps (q).

[0126] Furthermore, in a specific embodiment, the following method can also be used: a method for separating a crude crystal component containing β-type aspartic acid crystals from a solution (S) containing β-type aspartic acid crystals by using various solid-liquid separation methods such as evaporation, filtration, suction filtration, and vacuum drying. According to this embodiment employing a solid-liquid separation method, the supernatant portion containing residual impurities can be almost completely removed, thereby effectively removing impurities. Therefore, the entry of impurities into the obtained crude crystal component containing β-type aspartic acid crystals can be significantly reduced. Therefore, this embodiment is preferably used in the present invention.

[0127] Furthermore, for the "component containing at least a portion of β-type crystals of aspartic acid" or the "crude crystal component containing β-type crystals of aspartic acid" separated from the solution (S), a cleaning process using a solvent such as water and a subsequent drying process can be performed arbitrarily, and these cleaning and drying processes can be performed repeatedly in any combination.

[0128] The above describes in detail the process (p) that can be performed before process (q) in a specific embodiment of the present invention. However, in the case where the embodiment in which process (p) is performed before process (q) is adopted, in process (q) described in detail below, the component or crude crystal component containing the β-type crystals of aspartic acid generated in process (p) can be used to prepare a "slurry containing the crystal component (X) of the β-type crystals of aspartic acid".

[0129] <Process (q)>

[0130] Next, the process (q) will be explained.

[0131] As described above, step (q) is "preparing a slurry containing β-type crystals of aspartic acid and at least one impurity crystal component (X)".

[0132] Here, "crystal component (X) containing β-type crystals of aspartic acid and at least one impurity" can be interpreted literally, and the terms "β-type crystals of aspartic acid" and "impurity" have the meanings explained above. However, "crystal component (X) containing β-type crystals of aspartic acid and at least one impurity" is not necessarily limited to "a component containing at least a portion of β-type crystals of aspartic acid" or "a crude crystal component containing β-type crystals of aspartic acid" obtained by process (p), or a component obtained by performing a prescribed treatment on it. Crystal samples obtained in other processes may also be tested in process (q) without limitation.

[0133] Furthermore, regarding the "crystal component (X) containing β-type crystals of aspartic acid and at least one impurity" in step (q), if the specified sample obtained in step (p) or other processes already has the form of a slurry and can be tested in subsequent steps (r), it is prepared directly as the "slurry of crystal component (X)" without any treatment and is directly tested in step (r). This is also included in the "preparation of slurry of crystal component (X)" in step (q).

[0134] On the other hand, if the specified sample obtained through process (p) or other procedures is in the form of a suspension or slurry of coarse crystals at the time of acquisition, it can be separated into a supernatant and a coarse crystal component using various solid-liquid separation methods such as evaporation, filtration, suction filtration, and vacuum drying. The separated coarse crystal component can be arbitrarily washed and dried using a solvent such as water, and the obtained coarse crystal component can be resuspended in a solvent such as water to prepare a "slurry of crystal component (X)". Furthermore, if the sample obtained in advance is in the form of a coarse crystalline solid or semi-solid substance and is not in the form of a slurry, the preparation of a "slurry of crystal component (X)" by suspending the sample in any solvent such as water is also naturally included in process (q). Furthermore, even if the pre-obtained sample is already in slurry form, step (q) also includes preparing a "slurry of crystal component (X)" by resuspending coarse crystals in a solvent such as water. The coarse crystals are obtained by separating the solid component (coarse crystal component) using various solid-liquid separation methods, followed by arbitrary washing or drying of the separated solid component (coarse crystal component). Alternatively, a substance obtained by further diluting the pre-obtained coarse crystal slurry sample with a solvent such as water can also be prepared as a "slurry of crystal component (X)," and this process is also included in step (q).

[0135] That is, the term "slurry of crystal component (X)" in step (q) can be interpreted literally, meaning a mixture in which the crystal component exists in excess in the solvent beyond its saturation solubility, relative to a crystal solution formed by the complete dissolution of the crystal component, and the crystal particles are suspended in the solvent. The saturation solubility of the crystal component also depends on factors such as temperature, and therefore cannot be generalized. The concentration of crystal component (X) in the slurry can be set, for example, to about 10 w / v% to 70 w / v%, preferably about 15 w / v% to 60 w / v%, more preferably about 20 w / v% to 50 w / v%. However, it is not limited to these ranges. Furthermore, the type of solvent is not particularly limited, but from the viewpoint of ease of operation, water (e.g., ion-exchanged water, pure water, ultrapure water) is preferred.

[0136] In the embodiment where a crude crystalline solid material containing β-type aspartic acid is used to prepare a slurry of crystal component (X) in step (q), the crude crystalline solid material may also be suspended in a predetermined amount of a solvent such as water to prepare crude crystalline slurries of various concentration ranges, thereby preparing a slurry of crystal component (X). In this case, it is preferable to prepare crystal component (X) by suspending the crude crystalline solid material in a solvent that substantially contains water. "A solvent that substantially contains water" means that the unavoidable contamination of solvent substances other than water is not excluded.

[0137] In step (q), the "slurry of crystal component (X)" can contain not only β-type crystals of aspartic acid, but also impurities that should be removed or reduced by subsequent steps (r). The impurities that the "slurry of crystal component (X)" can contain are substances inherently mixed into the crude crystal sample that is to be refined in step (r), and therefore their type or amount is not limited.

[0138] Furthermore, as explained for the solution (S), it is preferable to have a smaller amount of impurities mixed into the crystal component (X) or its slurry. However, if the crystal component (X) or its slurry is derived from a biological sample or its concentrate, it is conceivable that various amino acids other than aspartic acid from bacterial cells or culture media, or their salts, various organic acids or their salts, proteins, carbohydrates or sugars, etc., may be mixed in.

[0139] Regarding the aforementioned aspect, in a particular embodiment, the "slurry of crystal component (X)" in step (q) is derived from a biological sample or its concentrate, and more specifically, may contain the following components.

[0140] (i) Aspartic acid at concentrations (e.g., about 0.05 M to about 4.5 M, about 0.8 M to about 4.0 M, about 1.0 M to about 3.5 M) capable of forming coarse crystal slurries;

[0141] (ii) An amino acid other than aspartic acid or a salt thereof, comprising, for example, at a concentration of about 0.05 mM to about 1.0 M, about 0.1 mM to about 1.0 M, about 1 mM to about 1.0 M, about 1 mM to about 1.0 M, about 1 mM to about 800 mM, about 1 mM to about 500 mM, or about 1 mM to about 100 mM; and

[0142] (iii) An organic acid or its salt comprising at least one selected from the group consisting of pyruvate, malic acid, acetic acid, succinic acid, fumaric acid and their salts (concentration, for example, about 0.05 mM to about 1.0 M, about 0.1 mM to about 1.0 M, about 1 mM to about 1.0 M, about 1 mM to about 800 mM, about 1 mM to about 500 mM, about 1 mM to about 100 mM).

[0143] As described above, several embodiments of step (q) are illustrated, but in step (q), it is sufficient to prepare a "slurry of crystal component (X)" that can be tested in subsequent steps (r), and its specific form is not limited within the scope of the meaning of the term.

[0144] <Process(r)>

[0145] Step (r) involves heating the "slurry of crystal component (X)" prepared in step (q) to change the β-type crystals of aspartic acid contained in the slurry into α-type crystals, and then obtaining the crystal component (Y) of aspartic acid containing the α-type crystals.

[0146] Furthermore, the term "heating the slurry to change the β-type crystals of aspartic acid into α-type crystals" in process (r) is a term for the crystal change of aspartic acid in the slurry from β-type crystals to α-type crystals caused by the heat treatment of the slurry of crystal component (X). It is a concept that includes not only the form of the crystal change that occurs during the heat treatment of the slurry, but also the form of the crystal change that occurs after the heat treatment (e.g., during or after cooling or refrigeration).

[0147] In process (r), the heating temperature, heating time, and pressurization conditions when heating the slurry of crystal component (X) are not particularly limited as long as the desired crystal change is produced. The heating temperature can be set, for example, to a range of about 60°C to about 190°C, about 61°C to about 190°C, about 62°C to about 190°C, about 63°C to about 190°C, about 64°C to about 190°C, about 65°C to about 190°C, preferably about 65°C to about 150°C, more preferably about 65°C to about 110°C, about 66°C to about 110°C, about 67°C to about 110°C, and even more preferably to a range of about 68°C to about 110°C or about 69°C to about 110°C. Furthermore, when heating is performed under normal pressure, the heating temperature can be set to, for example, about 60°C to about 100°C, about 61°C to about 100°C, about 62°C to about 100°C, about 63°C to about 100°C, about 64°C to about 100°C, more preferably about 65°C to about 100°C, about 66°C to about 100°C, about 67°C to about 100°C, and even more preferably in the range of about 68°C to about 100°C, about 69°C to about 100°C.

[0148] Furthermore, in another embodiment, a relatively low temperature range for heating can also be used, such as approximately 30°C to approximately 190°C, approximately 35°C to approximately 190°C, approximately 36°C to approximately 190°C, approximately 37°C to approximately 190°C, approximately 38°C to approximately 190°C, approximately 39°C to approximately 190°C, approximately 40°C to approximately 190°C, preferably approximately 30°C to approximately 150°C, approximately 35°C to approximately 150°C, approximately 36°C to approximately 150°C, approximately 37°C to approximately 150°C, approximately 38°C to approximately 150°C, approximately 39°C to approximately 150°C, approximately 40°C to approximately 150°C, or approximately 60°C to approximately 150°C. More preferably, the temperature range is about 30°C to about 110°C, about 35°C to about 110°C, about 36°C to about 110°C, about 37°C to about 110°C, about 38°C to about 110°C, about 39°C to about 110°C, about 40°C to about 110°C, or about 60°C to about 110°C. Furthermore, when heating is performed under normal pressure, the temperature range may also be about 30°C to about 100°C, about 35°C to about 100°C, about 36°C to about 100°C, about 37°C to about 100°C, about 38°C to about 100°C, about 39°C to about 100°C, or about 40°C to about 100°C.

[0149] Furthermore, in several embodiments, the heating temperature may be set to a range of approximately 70°C to approximately 100°C, approximately 75°C to approximately 100°C, approximately 78°C to approximately 100°C, approximately 80°C to approximately 100°C, approximately 85°C to approximately 100°C, approximately 88°C to approximately 100°C, approximately 90°C to approximately 100°C, or approximately 95°C to approximately 100°C. When heating is performed under normal pressure, the closer the temperature range is to 100°C, the higher the level of reduction or removal of impurities such as glutamine or alanine (other than aspartic acid) can be. Therefore, embodiments based on these temperature ranges are preferred.

[0150] Furthermore, the heating time can be appropriately set within the range that produces the desired crystal change, depending on the properties of the slurry of crystal component (X) or the heating conditions, and is not particularly limited. Generally speaking, when heating under normal pressure, the closer the heating temperature is to 100°C, the easier it is to generate α-type crystals from β-type crystals in a relatively short time. On the other hand, when using a relatively low heating temperature, there is a tendency for a relatively long heating time to be required from β-type crystals to the generation of α-type crystals. For example, as a lower limit value for the heating time, it can be set to, for example, 5 minutes or more after the sample temperature reaches the specified heating temperature, preferably about 10 minutes or more, about 15 minutes or more, or about 30 minutes or more. In another embodiment, the heating time can also be set to, for example, about 1 hour or more, about 2 hours or more, or about 3 hours or more after the sample temperature reaches the specified heating temperature, in order to reliably obtain α-type crystals of aspartic acid. Furthermore, there is no particular limitation on the upper limit of the heating time, as long as it is set to produce the desired amount of α-type aspartic acid crystals according to various conditions. For example, it can be set to about 20 hours, about 15 hours, about 10 hours, or about 5 hours.

[0151] Furthermore, the ranges of values ​​obtained by arbitrarily combining the lower and upper limits of the heating time described above are ranges of heating times that can be used in specific embodiments, and are explicitly stated in this specification as embodiments. Additionally, the heating of the slurry of crystal component (X) in process (r) can also be carried out under pressure as long as it can generate α-type crystals from β-type crystals.

[0152] As described above, there are no particular limitations on the heat treatment of the slurry of crystal component (X) in process (r), but the sample can be placed at room temperature and then cooled to a temperature range of 2°C to 10°C (for example, about 4°C). Furthermore, as described above, not only the morphology of crystal changes that occur during the heat treatment process, but also the morphology of crystal changes that occur during the cooling or refrigeration of the crystal sample after the heat treatment can be included in the present invention.

[0153] As described above, in step (r), the "slurry of crystal component (X)" prepared in step (q) is heated to change the β-type crystals of aspartic acid contained in the slurry into α-type crystals.

[0154] In the slurry of crystal component (X), after generating α-type crystals of aspartic acid using the heat treatment, a crude crystal component (Y) containing the α-type crystals is obtained.

[0155] Here, the meaning of “obtaining the crude crystal component (Y) containing α-type crystals of aspartic acid” is as follows.

[0156] As described above, in the slurry of crystal component (X), when the β-type crystals of aspartic acid are transformed into α-type crystals by heat treatment, at least a portion of the impurities mixed into the slurry are dissolved in the liquid component (i.e., the supernatant relative to the coarse crystalline solid component).

[0157] Therefore, in process (r), by separating the crystal component containing at least the generated α-type crystal from the whole crystal slurry in which α-type crystals of aspartic acid are generated using the heat treatment, most of the impurities mixed into the crystal component (X) or its slurry can be removed.

[0158] Here, the method for separating the crystalline component containing the generated α-type crystals from the slurry that has undergone heat treatment is not particularly limited as long as it removes at least a portion of the impurities remaining in the liquid portion of the slurry. In several embodiments, for example, methods can be used to separate at least a portion of the α-type crystals of aspartic acid generated in the slurry using methods such as suction; or methods can be used to remove the supernatant liquid portion after sedimentation or centrifugation of the crystalline component (solid component), and to collect the remaining component containing at least a portion of the α-type crystals of aspartic acid. Furthermore, in such cases, as long as at least a portion of the impurities remaining in the supernatant liquid portion is removed, the purification of aspartic acid can be considered to be achieved to a certain extent; therefore, it is not excluded that some impurities may enter the obtained component containing at least a portion of the α-type crystals of aspartic acid.

[0159] Furthermore, in another embodiment, a method can be used to separate the crystalline components containing α-type aspartic acid crystals from the crystal slurry containing α-type aspartic acid crystals using various solid-liquid separation methods such as evaporation, filtration, suction filtration, and vacuum drying. According to this embodiment employing solid-liquid separation, the supernatant portion containing residual impurities can be almost completely removed, thereby effectively removing impurities and significantly reducing the ingress of impurities into the obtained crystalline components containing α-type aspartic acid crystals. Therefore, this embodiment is preferably used in the present invention.

[0160] As described above, in process (r), α-type crystals of aspartic acid, which is the target, are manufactured.

[0161] <Other processes or conditions, etc.>

[0162] At least some or all of steps (p), (q), and (r) can be performed using appropriate apparatus or devices, depending on the amount of α-type aspartic acid crystals to be produced. For example, isoelectric point crystallization in step (p) and heat treatment in step (r) can be performed using any heating device, as long as it is suitably selected. Specifically, the choice is appropriate depending on the target manufacturing scale. For example, in the case of laboratory-scale manufacturing, there is no particular limitation, and commercially available beakers or heated stirrers, which are also used in the embodiments described later, can be used. On the other hand, in the case of industrial-scale manufacturing, general-purpose and special-purpose reactors, or reaction tanks or heating devices designed to constitute equipment, can be used to perform at least some or all of steps (p), (q), and (r). That is, the method of the present invention also includes implementations using various combinations of laboratory-scale devices, combinations of various reactors or heating devices, large-scale manufacturing equipment, and other various structures.

[0163] Furthermore, although not necessarily required, the method of the present invention may arbitrarily include the following step: confirming the formation of β-type columnar crystals from the intermediate products such as the solution (S) after step (p) and the crystal component (X) obtained from step (p) by visual observation or microscopic observation and / or X-ray diffraction. Furthermore, the method of the present invention may also arbitrarily include the following step: confirming the formation of α-type plate-like crystals from the coarse crystal slurry after heat treatment in step (r) and / or the crystal component (Y) obtained from step (r) by the method described above.

[0164] Furthermore, the method of the present invention is not necessarily required, but in all or part of steps (p) to (r), as shown in the embodiments described later, a step may be included to monitor the residual amount, residual rate, removal rate, etc. of impurities in the sample at any time point using various chemical analysis methods such as high performance liquid chromatography (HPLC).

[0165] The specific embodiments of the present invention have been described in detail above, but of course, the present invention is not limited to the described embodiments. Various changes, modifications, and combinations can be made to the structures, elements, and features without departing from the spirit of the present invention.

[0166] Furthermore, in this invention, the terms “comprising,” “containing,” and “having” are used interchangeably unless otherwise specified, and do not exclude the existence of elements other than those mentioned as objects.

[0167] Furthermore, in this specification, "~" means a value above the value preceding the description of "~" and below the value following the description of "~". Additionally, in the numerical ranges and values ​​described in this specification, where the word "approximately" is used, the numerical ranges and values ​​other than that word are also explicitly stated in this specification as elements that can constitute embodiments of the present invention.

[0168] The following examples and comparative examples are shown to illustrate the present invention in more detail, but the present invention is not limited to the examples.

[0169] Example

[0170] [Experimental Example 1]

[0171] This experimental example involves culturing a transgenic Corynebacterium glutamicum capable of producing aspartic acid (hereinafter sometimes referred to as a Corynebacterium producing Aeromonas sobria serine protease (Asp)) in a specified reaction medium, using the fermentation broth of the resulting culture as the starting material, and roughly following the... Figure 1 The process shown is an example of producing α-type crystals of aspartic acid. The process is described in detail below.

[0172] (1) Concentration / Activated Carbon Treatment

[0173] A recombinant strain of *Corynebacterium glutamicum*, with an enzyme gene capable of participating in the L-aspartate metabolic pathway introduced and altered, was cultured in a specified reaction medium to generate aspartate in the reaction medium. 5 L of the clarified fermentation broth (S) after cell removal from the obtained culture was used in the following process flow.

[0174] The fermentation clarified broth (S) of 5 L was concentrated under reduced pressure using a flash evaporator (Tokyo Rika & Co., Ltd., model MF-10B), a diaphragm vacuum pump (Tokyo Rika & Co., Ltd., model EVP-1200), and a vacuum controller (Tokyo Rika & Co., Ltd., model NVC-2200). Next, powdered activated carbon (CARBORAFFIN, manufactured by Osaka Gas Chemical Co., Ltd.) at a concentration of 4 g per 100 g of aspartic acid was added to the concentrate. After stirring at room temperature for 70 minutes, the concentrate was separated into activated carbon and filtrate (A) of 1400 mL using a suction filtration method. The filtrate (A) of 1400 mL was then subjected to isoelectric point crystallization as described later.

[0175] Furthermore, since 5 L of the fermentation clarification broth (S) was concentrated in 1400 mL of filtrate (A), the final concentration of aspartic acid in the filtrate (A) was calculated to be 2.5 M.

[0176] (2) Isoelectric point crystallization

[0177] 0.3 g of crude crystals obtained by the method described later were added as seed crystals to the filtrate (A) beforehand. 300 g of sulfuric acid was then slowly added to the resulting solution at room temperature with stirring, thereby adjusting the pH of the solution to approximately 2.77, which corresponds to the isoelectric point of aspartic acid. Furthermore, a pH meter (manufactured by Horiba Manufacturing Co., Ltd., model D-71) was used to measure the pH of the solution. As a result of the isoelectric point crystallization treatment using the pH adjustment of the solution, crystalline components were generated in the solution. Additionally, during the pH adjustment, the temperature rose to approximately 70°C due to the heat generated by the neutralization reaction; therefore, the solution was cooled to room temperature while stirring, and then the stirring was stopped and the solution was cooled at 4°C.

[0178] Furthermore, the coarse crystals added to the filtrate (A) as seed crystals are obtained in advance as follows: that is, for the concentrate (filtrate) from which the fermentation clarification broth is obtained in the same way, except that no seed crystals are added, the same isoelectric point crystallization is performed to generate coarse crystals, and a sample that generates the largest possible columnar crystals is selected in advance and used as the seed crystals.

[0179] (3) Crystal separation

[0180] The crystal product obtained by isoelectric point crystallization was subjected to solid-liquid separation using a suction filtration method to obtain a solid crystal component. This solid crystal component was then washed by spraying 1750 mL of ultrapure water from above to remove impurities adhering to the crystal surface. This washing process was repeated four times to obtain wet coarse crystals. The obtained wet coarse crystals were transferred to a stainless steel square tank and placed in a constant temperature dryer (manufactured by AS ONE Co., Ltd., model OFW-300B) at 55°C for drying. The dried crystal sample was then pulverized using a mixer (manufactured by Hanwa Co., Ltd., model BKE-07) and collected in a plastic container. The obtained coarse crystal sample (B) weighed 460 g.

[0181] (4) Heat treatment (heat re-slurrying treatment)

[0182] 90.0 g of the coarse crystal (B) sample obtained in item (3) was measured using an electronic balance (Shimadzu Corporation, model UW6200H) and suspended in ultrapure water to a final volume of 300 mL to prepare a 30% coarse crystal slurry. The coarse crystal slurry was heated in a beaker while being stirred using a hot stirrer (AS ONE Corporation, model HS-360H). Heating was stopped 10 minutes after the sample temperature reached 100°C, and the sample was cooled to room temperature while being stirred. Then, stirring was stopped, and the sample was cooled at 4°C.

[0183] (5) Crystal separation

[0184] The hot reslurry liquid obtained as described above was subjected to solid-liquid separation using a suction filtration method, separating it into a supernatant and a solid crystal component. For the obtained solid crystal component, 100 mL of ultrapure water was sprayed from above to wash the crystals and remove impurities adhering to the surface. The washed wet coarse crystals were transferred to a stainless steel square tank and placed in a constant temperature dryer (manufactured by AS ONE Co., Ltd., model OFW-300B) for drying at 55°C. Subsequently, the dried crystal sample was pulverized using a mixer (manufactured by Hanwa Co., Ltd., model BKE-07), and the crystal sample was recycled into a plastic container. The weight of the obtained crystal (C) was 71.4 g.

[0185] (6) Various analyses

[0186] In each of the aforementioned processes, a portion is collected from the fermentation clarified broth (S), concentrated broth, filtrate (A), crude crystals (B), and crystals (C), and tested in various amino acid analyses and various organic acid analyses. Furthermore, for crude crystals (B) and crystals (C), an analytical sample is prepared by dissolving a portion of them in an aqueous solution of sodium hydroxide (manufactured by Fujifilm and Koko Pure Chemical Industries, Ltd.) at a concentration of 100 g / L.

[0187] Specifically, in the analysis of various amino acids, sodium citrate buffer (manufactured by Fujifilm and Koichi Pure Chemical Industries Co., Ltd.) at pH 2.2 was used. The fermentation broth (S) was diluted 1000-fold, the concentrate and filtrate (A) were diluted 2500-4000-fold, and the crude crystals (B) and crystals (C) were diluted 1000-fold. Each diluted sample was analyzed using a high-performance liquid chromatography (HPLC) system (manufactured by Shimadzu Corporation, Prominence). On the other hand, in the analysis of various organic acids, 0.75 mM sulfuric acid (manufactured by Fujifilm and Koichi Pure Chemical Industries Co., Ltd.) was used. The fermentation broth (S) was diluted 100-fold, the concentrate and filtrate (A) were diluted 250-400-fold, and the crude crystals (B) and crystals (C) were diluted 20-fold. Each diluted sample was analyzed using an HPLC system (manufactured by Shimadzu Corporation, Prominence).

[0188] In addition, during the formal heat treatment (thermal reslurrying process) and at various time points from (b) to (d) below, a portion of the samples were collected and observed using a microscope (manufactured by Olympus Corporation, model CX41LF).

[0189] (b) The point at which the sample temperature reaches 70°C after heating begins (before crystal change).

[0190] (c) The point at which the sample temperature reaches 77°C after heating begins (during crystal change).

[0191] (d) The point at which the sample temperature reaches 100°C after heating begins (after crystal change).

[0192] Furthermore, for each sample of coarse crystal (B) and crystal (C), the crystal structure of each sample was analyzed using X-ray diffraction (manufactured by Rigaku Corporation, SmartLab X-ray diffraction equipment) and in accordance with conventional methods.

[0193] <Results>

[0194] The results of amino acid and organic acid analyses are shown in Tables 1 and 2, and Figures 2A to 2C and Figures 3A-3C middle.

[0195] [Table 1]

[0196]

[0197] [Table 2]

[0198]

[0199] Fermentation clarified liquid (S) Concentrate Filtrate (A) Coarse crystals (B) Crystal (C) Total solids 187.79 (g / L) - - 99.76％ 100.00％ Total Asp (mmol) 655.40 634.35 632.92 527.19 530.13 Asp concentration 89.04 (g / L) 307.80 (g / L) 314.98 (g / L) 0.78 (g / g) 0.99 (g / g) <![CDATA[Asp purity (%) #1 > 47.42 - - 78.19 99.00 <![CDATA[Asp recovery rate (%) #2 > 100 96.79 96.57 80.44 80.89

[0200] #1: Values ​​of each Asp concentration / percentage of each total solids

[0201] #2: Percentage of total Asp (mmol / s) in fermentation broth (S) (655.40 mmol / s)

[0202] like Figures 2A to 2C As shown, relative to the total amount of aspartic acid (632.92 mmol) in the filtrate (A) tested at the isoelectric point crystallization, aspartic acid was retained in a high proportion through each purification step of isoelectric point crystallization and thermal reslurry treatment, and aspartic acid crystals could be produced with a recovery rate of 83.76% (i.e., a loss of about 16%) (Table 2). Figure 2C On the other hand, regarding the certain amounts of various amino acids other than aspartic acid mixed in the filtrate (A) tested in isoelectric point crystallization, namely glutamic acid (57.57 mmol), alanine (187.21 mmol), and valine (32.81 mmol), relative to the amounts of each component in filtrate (A), more than 97% were removed by the purification process of isoelectric point crystallization, and further, by the purification process of thermal re-slurrying treatment, the proportions were finally removed to 97.85%, 99.12%, and 97.62%, respectively (Table 2). Figures 3A-3C ).

[0203] Furthermore, the various organic acids mixed in the filtrate (A), namely pyruvic acid (1.32 mmol), malic acid (76.07 mmol), acetic acid (90.9 mmol), succinic acid (18.34 mmol), and fumaric acid (8.19 mmol), were effectively removed by the isoelectric point crystallization purification process described below. Specifically, pyruvic acid and acetic acid were removed by 100% via the isoelectric point crystallization purification process, fumaric acid by 99.88%, and malic acid and succinic acid by over 90% (Table 2). Figure 3A Furthermore, the small amounts of fumaric acid, malic acid, and succinic acid remaining in the crude crystals (B) obtained by isoelectric point crystallization are removed by a further refining process involving thermal reslurry treatment, resulting in the removal of 100% of acetic acid, succinic acid, and fumaric acid. For malic acid, only 0.94 mmol (1.24%) remains, meaning that 98.76% of the total amount (76.07 mmol) in the filtrate (A) is removed. In the final product crystals (C), the purity of aspartic acid increases to 99.00%, thus obtaining high-purity aspartic acid crystals (Table 2). Figures 2A to 2C , Figures 3A-3C ).

[0204] Furthermore, when a 100 g / L solution of crude aspartic acid crystals (B) was mixed with an equal volume of 100 mM barium chloride (manufactured by Wako Pure Chemical Industries, Ltd.), turbidity occurred, indicating the presence of sulfate ions in the sample. In contrast, when a 100 g / L solution of crystals (C) was similarly mixed with an equal volume of 100 mM barium chloride (manufactured by Wako Pure Chemical Industries, Ltd.), no turbidity occurred. Therefore, it was determined that a considerable amount of sulfate ions remaining in the crude crystals (B) immediately following the isoelectric point were effectively removed by the thermal reslurry treatment, resulting in crystals (C) as the final product containing almost no sulfate ions.

[0205] Furthermore, photographs of each coarse crystal (B) slurry sample taken during the heat treatment (thermal reslurrying) of the coarse crystal (B) slurry sample are shown below. Figure 4A In the image, the sample in the beaker on the left is a slurry of coarse crystals (B) before heat treatment, while the sample in the beaker on the right is a slurry of coarse crystals (B) after heat treatment at a specified time. Both slurry samples have the appearance of slurry with the specified turbidity, as seen in the photograph. However, the coarse crystal (B) slurry sample before heat treatment (beaker on the left) has a cloudy white appearance, while the coarse crystal (B) slurry sample after heat treatment (beaker on the right) has a cloudy yellow-white appearance. These differences in appearance are due to the change from a cloudy white appearance to a cloudy yellow-white appearance over time during heat treatment.

[0206] Furthermore, at specified time points during the hot re-slurry treatment, each crystal sample was observed using a microscope (manufactured by Olympus Corporation, model CX41LF). The results showed that at the point when the sample temperature reached 70°C after the start of heating, such as... Figure 4B As shown in the photograph, fine columnar coarse crystals were observed. However, later, from the point when the sample temperature reached 77°C after the start of heating, as shown in the photograph... Figure 4C As shown in the photograph, larger plate-like crystals initially form from coarse columnar crystals. This occurs when the sample temperature reaches 100°C after heating begins. Figure 4D As shown in the photograph, almost all the columnar coarse crystals transform into plate-like crystals.

[0207] The microscopic observations showed that aspartic acid existed in the β-type crystal form in the coarse crystal (B). It was speculated that subsequent heat treatment would cause the crystal form in crystal (C) to change to the α-type. As described above, X-ray diffraction was used to analyze the coarse crystal (B) and crystal (C). The results showed that... Figure 5AIn the X-ray diffraction pattern shown, the peak values ​​of the diffracted X-rays were confirmed at diffraction angles of 18.8°, 19.7°, and 25.0°, confirming that the aspartic acid crystallized from the coarse crystal (B) is of the β-type crystal type. Figure 5B In the X-ray diffraction pattern shown, when the peak values ​​of the diffracted X-rays were confirmed at diffraction angles of 21.65° and 23.7°, it was confirmed that the crystal form of aspartic acid crystallized in crystal (C) was α-type crystal.

[0208] As described above, according to embodiments of the present invention, even when using crude product obtained by fermentation using microorganisms as starting material, it is shown that while maintaining a high recovery rate of aspartic acid, other amino acids, organic acids, sulfate ions, etc., which are equivalent to impurities, can be efficiently removed, and finally, high-purity aspartic acid can be produced in the useful α-type crystal form.

[0209] [Experimental Example 2] Study on the temperature of hot re-slurrying

[0210] In Test Example 1, the number of test specimens was set to four, and the test conditions for each test specimen were changed as follows. Otherwise, the test was carried out using the same method as in Test Example 1. That is, relative to the procedure in Test Example 1, in this test example, for each specimen, the heating temperature in item (4) "Heat Treatment (Hot Re-slurrying Method)" was changed to 70°C, 80°C, 90°C, and 100°C. In addition, the heating time used for each specimen is as described in the results below.

[0211] <Results>

[0212] Microscopic observations during the refining process of hot resizing revealed a change from β-type crystals to α-type crystals in any of the samples that were heated at 70°C, 80°C, 90°C, and 100°C.

[0213] More specifically, in the samples heated to 70°C, no crystal change was observed one hour after the sample temperature reached 70°C, but crystal change was subsequently observed during cooling to room temperature. Furthermore, in the samples heated to 80°C, the crystal change was observed approximately 16 minutes after the sample temperature reached 80°C. Moreover, in the samples heated to 90°C and 100°C respectively, the crystal change was observed approximately 10 minutes after each sample reached its respective heating temperature. Thus, based on the results of samples heated to 70°C, 80°C, 90°C, and 100°C, crystal change was observed in every sample, and the following trend was observed: when the heating temperature is higher, the heating time required to produce crystal change is shorter; conversely, as the heating temperature decreases, the heating time required to produce crystal change is longer.

[0214] Next, the results of amino acid and organic acid analysis are shown in Tables 3 and 4, and... Figure 6 , Figure 7 and Figure 8A , Figure 8B middle.

[0215] [Table 3]

[0216]

[0217]

[0218] [Table 4]

[0219]

[0220] * Residual amount relative to the total amount of each component in the fermentation clarified broth (S)

[0221] Rate.

[0222] As shown in Table 3 and Figure 6 As shown, the amount of aspartic acid shifted at a high level with little loss in the fermentation clarified broth (S), crude crystals (B), and crystals (C). A recovery rate of 85.32% was observed in the crude crystals (B), and aspartic acid could ultimately be recovered in the crystals (C) at a rate of 77%–80% (Table 4). Figure 8A ).

[0223] On the other hand, in the fermentation clarified broth (S), various amino acids other than aspartic acid, namely glutamic acid, alanine, and valine, are as shown in Table 3 and Figure 6 That's understandable. While the amount is less than the total amount of aspartic acid, a considerable amount is still mixed in. This is removed by isoelectric point crystallization, thus reducing the residue in the crude crystals (B) to less than 6% (Table 4). Figure 8B Furthermore, the residual percentages of these amino acids other than aspartic acid in the crystal (C) were reduced to less than 2%, especially for valine, which was 100% removed in all samples at all heat treatment temperatures, with a residual percentage of 0% (Table 4). Figure 9 ).

[0224] Furthermore, as shown in Table 3 and Figure 7 It is understood that the fermentation broth (S) contained a considerable amount of organic acids, namely pyruvic acid, malic acid, acetic acid, succinic acid, and fumaric acid, but a considerable amount was removed by isoelectric point crystallization. More specifically, in the crude crystals (B), although the residual rate of malic acid was relatively high, it was reduced to 13.71%, pyruvic acid and succinic acid were reduced to less than 10%, and fumaric acid was reduced to 0.20%, while acetic acid was completely removed (Table 4). Figure 8B ).

[0225] Furthermore, as an aspect of concern, in any crystal (C) obtained through hot re-slurrying treatment at 70°C, 80°C, 90°C, and 100°C, acetic acid, succinic acid, and fumaric acid were completely removed, and malic acid was found to remain, but the residual rate was only about 2.2% to 2.4%. This confirms that the samples of crystal (C) are high-quality as refined aspartic acid (Table 4). Figure 9 ).

[0226] As described above, Experimental Example 2 also showed a high recovery rate of α-crystals of aspartic acid. In particular, it was shown that when the refining process in the hot reslurry treatment was carried out at a relatively high temperature, α-crystals of aspartic acid could be generated in a short time, and amino acids other than aspartic acid and various organic acids could be effectively removed.

[0227] (Evaluation Tests: Polymerization and Coloring Tests)

[0228] Polymerization coloring tests were conducted using 2g of β-type coarse crystals (after isoelectric point crystallization) obtained in Experiment 2, α-type crystals obtained by heat treatment at 100°C (thermal reslurry method), and commercially available aspartic acid powder (manufactured by Kyowa Hakko Bio Co., Ltd., high purity grade) as a control, as described below.

[0229] 2g of each sample was mixed with 2mL of 85% phosphoric acid (manufactured by Fujifilm and Koichi Chemical Co., Ltd.) in a 60mm glass petri dish. The mixture was then placed in a constant temperature desiccator (manufactured by AS ONE Co., Ltd., model OFW-300B) and heated at 160°C for 16-20 hours, thereby polymerizing the aspartic acid. The heat-treated samples were then visually observed. Figure 10 As shown, strong brown staining was observed in the β-type coarse crystal sample after isoelectric point crystallization, but the staining was suppressed in the α-type crystal sample obtained by hot re-slurrying at 100°C, maintaining a quality that was in no way inferior to that of high-purity aspartic acid powder used as a control.

[0230] [Experimental Example 3]

[0231] (1) Concentration / Activated Carbon Treatment

[0232] First, similarly to the method described in Example 1(1), 5 L of the fermentation broth of the recombinant Corynebacterium glutamicum was concentrated under reduced pressure to obtain a concentrate with an estimated aspartic acid concentration of 2.5 M. Next, 4 g of powdered activated carbon (CARBORAFFIN manufactured by Osaka Gas Chemical Co., Ltd.) per 100 g of aspartic acid was added to the obtained concentrate, and after stirring at room temperature for 60 minutes, the activated carbon and filtrate (A) were separated by suction filtration.

[0233] (2) Isoelectric point crystallization

[0234] The filtrate (A) was dispensed into three beakers, each containing 350 mL, to serve as sample (i) without seed crystals, sample (ii) with seed crystals, and sample (iii) with seed crystals, respectively, and subjected to isoelectric point crystallization. Specifically, for each sample, sulfuric acid was slowly added at room temperature with stirring, and the pH of each sample solution was adjusted to approximately the isoelectric point of aspartic acid, 2.77, using a pH meter (D-71 manufactured by Horiba Corporation), thereby causing aspartic acid to crystallize in each sample. Furthermore, the amount of sulfuric acid added was 86.00 g for sample (i) without seed crystals, 86.69 g for sample (ii) with seed crystals, and 88.90 g for sample (iii) with seed crystals, in that order. In addition, for samples (ii) and (iii) with seed crystals, approximately 0.1 g of crude aspartic acid crystals were added when the pH of each solution reached 5.5. Furthermore, during isoelectric point crystallization, the temperature of each sample solution rises to about 70°C. Therefore, the solution is cooled to room temperature while being stirred, and then the stirring is stopped and each sample is cooled to 4°C.

[0235] When the solutions of samples (i) to (iii) were subjected to isoelectric point crystallization as described above, it was observed that the solutions were transparent and sugar-colored before isoelectric point crystallization, but changed to pale yellow-white turbid solutions after isoelectric point crystallization, in which coarse crystals crystallized. Furthermore, Figure 11 The images show the appearance of sample (i) before isoelectric point crystallization (left side) and after isoelectric point crystallization (right side).

[0236] For each sample containing coarse crystals, the supernatant and coarse crystals (solid component) were separated by suction filtration. For each coarse crystal sample obtained in this manner, impurities adhering to the crystal surface were removed by spraying 450 mL of ultrapure water from above. The cleaned wet coarse crystal samples were transferred to a stainless steel square tank and placed in a constant temperature dryer (AS ONE Co., Ltd.'s "OFW-300B"), where they were dried at 55°C. Subsequently, the dried coarse crystal samples were pulverized using a mixer (Hanwa Co., Ltd.'s "BKE-07") and collected into separate plastic containers.

[0237] 116.17 g of coarse crystals (B1) from sample (i) without added seed crystals, 122.31 g of coarse crystals (B2) from sample (ii) with added seed crystals, and 116.24 g of coarse crystal samples (B3) from sample (iii) with added seed crystals were obtained in the manner described above.

[0238] In addition, a portion of the crude crystals (B1), (B2) and (B3) were tested in amino acid analysis and organic acid analysis in the same manner as in Test Example 1.

[0239] (3) Heat treatment (heat re-slurrying treatment)

[0240] Next, 100.0 g of each of the coarse crystals (B1), (B2), and (B3) was measured using an electronic balance (Shimadzu Corporation, "UW6200H"). Each coarse crystal sample was suspended in ultrapure water to a final volume of 334 mL to prepare a 30% coarse crystal slurry. These 30% coarse crystal slurries were heated in beakers using a heated stirrer (AS ONE Corporation, "HS-360H"). Heating was stopped 10 minutes after each sample reached 100°C, and then the samples were cooled to room temperature while stirring. Finally, stirring was stopped, and each sample was cooled to 4°C.

[0241] Next, each sample was separated into a supernatant and a crystalline component (solid component) by suction filtration. For each crystalline sample obtained in this way, impurities adhering to the crystal surface were removed by spraying 100 mL of ultrapure water from above. The cleaned wet crystalline samples were transferred to a stainless steel square tank and placed in a constant temperature dryer (ASONE Corporation's "OFW-300B"), and dried overnight at 55°C. Then, each crystalline sample was ground into powder using a spatula and collected in a plastic container.

[0242] Thus, 93.05 g of crystals (C1) from sample (i) without seed crystals, 88.98 g of crystals (C2) from sample (ii) with seed crystals, and 93.22 g of crystal sample (C3) from sample (iii) with seed crystals were obtained. A portion of the crystal samples (C1), (C2), and (C3) were tested in amino acid analysis and organic acid analysis in the same manner as in Test Example 1.

[0243] <Results>

[0244] The results of various amino acid and organic acid analyses are shown in Tables 5-7, and... Figure 12 and Figure 13 In detail, Table 5 shows the concentrations and total amounts of various components in the fermentation clarified broth (S), the concentrated fermentation clarified broth (S) obtained by vacuum concentration, the filtrate after activated carbon treatment and suction filtration (A), the coarse crystals (B1) to (B3) obtained by isoelectric point treatment, and the crystals (C1) to (C3) obtained by hot re-slurrying treatment. Furthermore, Table 6 and... Figure 12 and Figure 13 The table shows the residual rates of various components in each of the coarse crystals (B1) to (B3) and the crystals (C1) to (C3). Furthermore, the residual rate corresponds to the percentage (%) of the total amount of each component in each coarse crystal sample or crystal sample relative to the total amount of each component in the filtrate (A) tested during isoelectric point crystallization. Table 7 also shows the total Asp content, purity (%), and recovery rate (%) in each sample.

[0245] [Table 5]

[0246]

[0247]

[0248]

[0249] As shown in Table 6 and Figure 12As shown, relative to the total amount of aspartic acid (955.78 mmol) in the filtrate (A) tested in isoelectric point crystallization, aspartic acid was retained in a high proportion through each refining process of isoelectric point crystallization and thermal reslurry treatment, and aspartic acid crystals could be finally produced in crystals (C1) to crystals (C3) with a recovery rate of about 67% to 71%. On the other hand, for various amino acids other than aspartic acid mixed in a certain amount in the filtrate (A) tested in isoelectric point crystallization, through the refining process of isoelectric point crystallization, the residual rate of glutamic acid (70.25 mmol) was reduced to about 3%, the residual rate of alanine (161.42 mmol) was reduced to about 2%, and valine (22.62 mmol) was completely removed in any of the crude crystals (B1) to crude crystals (B3) (Table 6). Figure 12 Furthermore, the small amounts of glutamic acid and alanine remaining in the crude crystals (B1) to (B3) are reduced to approximately 1.8% and approximately 0.8% respectively in the crystals (C1) to (C3) through a refining process involving thermal resizing. Figure 12 ).

[0250] Furthermore, the amounts of various organic acids mixed in the filtrate (A), namely pyruvic acid (0.56 mmol), malic acid (84.65 mmol), acetic acid (114.52 mmol), succinic acid (49.31 mmol), and fumaric acid (12.58 mmol), are also as shown in Table 6. Figure 13 The removal is effective as shown. Specifically, pyruvic acid and acetic acid are 100% removed through a purification process involving isoelectric point crystallization. Figure 13 Furthermore, although succinic acid and fumaric acid remain in certain amounts in the crude crystals (B), they are completely removed in the crystals (C1) to (C3). Figure 13 Furthermore, small amounts of malic acid remain in the crude crystals (B) and crystals (C1) to (C3), but in the final products (C1) to (C3), the residue rate is less than 2%. Figure 13 In any crystalline sample, the purity of aspartic acid showed a high value of about 96% to 97%, thus obtaining high-purity α-type crystals of aspartic acid (Table 7).

[0251] As one of the aspects that should be focused on in this test example, in the sample (i) without any added seed crystals, as in the samples (ii) and (iii) with added seed crystals, according to an embodiment of the invention, it is shown that various amino acids or organic acids other than aspartic acid mixed in with the crude product obtained by fermentation can be effectively removed while refining the desired crystal form of aspartic acid.

[0252] [Experimental Example 4] Study on the isoelectric point crystallization temperature

[0253] In Test Example 1, the temperature of the solution in the "isoelectric point crystallization" step (2) was maintained at 30°C, 50°C and 80°C respectively using a water bath or oil bath. Otherwise, the test was conducted using the same methods as in Test Example 1 (1) to (6).

[0254] <Results>

[0255] Analysis of crude crystals (B) prepared by isoelectric point crystallization at controlled temperatures of 30°C, 50°C, and 80°C confirmed that each sample contained a high proportion of aspartic acid. Furthermore, it was confirmed that each sample contained various amino acids other than aspartic acid, namely glutamic acid, alanine, and valine, and also contained organic acids other than aspartic acid. In the X-ray diffraction patterns of the crude crystals (B), the samples treated at any temperature showed similar characteristics to... Figure 5A The figures shown also display the peak values ​​of the diffracted X-rays at diffraction angles of 18.8°, 19.7°, and 25.0°. Therefore, it is confirmed that the aspartic acid crystallized from each sample of the coarse crystal (B) is of the β-type crystal form.

[0256] For each sample of coarse crystals (B) prepared in the manner described above, when heat-treated (heat re-slurrying) according to the method described in (4) of Test Example 1, similar to Test Example 1, a change in crystal shape was confirmed in each sample. That is, when the appearance of the slurry sample before heat treatment was observed using a microscope, fine columnar crystals were observed. In contrast, after heat treatment, at the latest when the sample temperature reached 100°C after the start of heating, almost all the columnar crystals were observed to change into plate-like crystals.

[0257] Furthermore, analysis of each sample of crystals (C) obtained after crystal separation following heat treatment confirmed that the purity of aspartic acid in each sample was above 99.00%, thus obtaining high-purity aspartic acid crystals. Additionally, it was confirmed that each sample of crystals (C) as the final product after heat treatment contained almost no sulfate ions. Moreover, when X-ray diffraction was used to analyze each sample, the X-ray diffraction pattern of each sample showed... Figure 5B Similarly, the peak values ​​of the diffracted X-rays were confirmed at diffraction angles of 21.65° and 23.7° in the figure shown. Therefore, it was determined that the aspartic acid in all samples of crystal (C) was of the α-type crystal form.

[0258] As described above, it has been confirmed that even when the isoelectric point crystallization process is controlled and implemented within a wide temperature range such as 30°C, 50°C, and 80°C, β-type crystalline aspartic acid can be obtained. In summary, it is also possible to combine the isoelectric point crystallization process implemented within this wide temperature range with a subsequent hot re-slurrying process. According to one embodiment of the present invention, it is shown that crude products obtained by microbial fermentation can be used as starting materials, and high-purity aspartic acid can be ultimately produced from said starting materials in the form of useful α-type crystals.

[0259] [Experimental Example 5] Study on the temperature of hot re-slurrying

[0260] (1) Concentration / Activated Carbon Treatment

[0261] As the starting material for the concentration treatment, 3 L of the fermentation broth of the Asp-producing Corynebacterium culture used in Test Example 1 was used. The sample with added powdered activated carbon was heated to 60°C and stirred for at least 60 minutes to perform activated carbon treatment. Otherwise, the concentration / activated carbon treatment was performed using the same method as in Test Example 1. Furthermore, the volume of the obtained concentrate (filtrate (A)) was 720 mL.

[0262] (2) Isoelectric point crystallization

[0263] First, the concentrated solution obtained as described above was heated to 50°C using a microbial culture apparatus (manufactured by ABLE Corporation, model BMJ-01NC). Then, the heater was stopped, and 55g of sulfuric acid was added to the sample over 50 minutes while stirring, adjusting the pH to approximately 2.73 so that the pH of the sample at room temperature after cooling would be approximately 2.77, thereby crystallizing aspartic acid. Furthermore, during the pH adjustment, 0.05g of crude aspartic acid crystals were added to the sample as seed crystals at pH 5.0 and 4.7, respectively. When the seed crystals were added to the sample, the sample temperature rose to approximately 60°C; therefore, the sample was cooled to room temperature while stirring, and the crystals were then removed into a beaker and cooled at 4°C.

[0264] In addition, similar to Example 1, various analyses were performed on the samples at each stage, but not only on various amino acids / organic acids, but also on the contamination of dihydroxyacetone (DHA), which can be a coloring substance, using conventional HPLC methods.

[0265] In addition, the test conditions other than these are the same as those in Test Example 1.

[0266] (3) Crystal separation

[0267] In the crystal separation operation of Experimental Example 1, the crystal product obtained by isoelectric point crystallization was washed with 400 mL of ultrapure water five times from above to remove impurities adhering to its surface. Otherwise, a crude crystal sample was obtained under the same conditions as in Experimental Example 1. Furthermore, the amount of the crude crystal sample obtained was 87.91 g.

[0268] (4) Heat treatment (heat re-slurrying treatment)

[0269] 30.0 g of dried coarse crystals were obtained from the coarse crystal sample obtained in step (3) using an electronic balance (Shimadzu Corporation, model UW6200H). 70 g of ultrapure water was added to prepare a 30% (w / w%) coarse crystal slurry sample. Using a water bath (Taitec Corporation, model SM-05N) and a stirrer (Nishin Rika Co., Ltd., model SW-501J), these coarse crystal slurry samples were kept at 40°C, 45°C, 60°C, and 70°C respectively and stirred. The crystal changes of each sample were observed using a microscope (Olympus Corporation, model CX41LF). In addition, regarding the heating time, after the start of heating, the heating was stopped when the crystal change in the sample was confirmed. Once the crystal change was confirmed, the sample was cooled to room temperature while stirring. Then, stirring was stopped and each sample was cooled at 4°C.

[0270] After cooling, solid-liquid separation was performed using a suction filtration method. For each solid sample, the crystals were washed with 30 mL of ultrapure water flowing from above to remove impurities adhering to the surface. The washed wet crystals were transferred to an aluminum square tank and dried at 55°C using a constant temperature dryer (manufactured by AS ONE Co., Ltd., model OFW-300B). The dried crystals were then crushed using a spatula and collected in a plastic container. Each sample was then tested in an amino acid / organic acid analysis to calculate the residual impurity rate.

[0271] <Results>

[0272] The analytical results of amino acids and various organic acids / dihydroxyacetone (DHA) are shown in Tables 8-10. Figure 14 , Figure 15 , Figure 16A and Figure 16B , Figure 17 middle.

[0273] [Table 8]

[0274]

[0275]

[0276] [Table 9]

[0277]

[0278] * Residual percentage relative to the total amount of each component in the fermentation clarified liquid (S).

[0279] [Table 10]

[0280]

[0281] In any sample treated with 40℃, 45℃, 60℃, and 70℃ as the heat re-slurry processing temperature, the amount of aspartic acid increased at a high level without significant loss in the fermentation clarification broth (S), crude crystals (B), and crystals (C) (Table 8). Figure 14 The aspartic acid content was 84.06% in the coarse crystals (B) and approximately 80% in the final crystals (C) (Table 9). Figure 16A Furthermore, as can be seen from Table 10, regarding the recovery rate of aspartic acid in crystal (C) relative to crude crystal (B), a high recovery rate of approximately 95% can be achieved in any sample using the aforementioned thermal reslurry treatment temperature.

[0282] On the other hand, various amino acids other than aspartic acid, as well as various organic acids and dihydroxyacetone (DHA), were mixed into the fermentation clarification broth (S), which were present in small but considerable amounts compared to the total amount of aspartic acid. However, similar to Example 2, it was found that through isoelectric point crystallization and subsequent thermal re-slurrying processes, a considerable amount of each impurity was removed (Table 8). Figure 14 , Figure 15 In detail, it was found that, regarding various amino acids other than aspartic acid, at the time point of crude crystals (B), glutamic acid and alanine showed a residual rate of about 1%, and valine showed a residual rate of only 0.25%, which were almost removed during the isoelectric point crystallization process. Furthermore, a further reduction in the residual rates was confirmed in any crystal (C) sample treated with various thermal reslurry treatment temperatures (Table 9). Figure 14 Furthermore, when observing the residual rates of various organic acids in each sample of the coarse crystals (B), although a considerable amount of malic acid of about 20% was confirmed to have been mixed in, the other organic acids showed a value of 0% and were not detected (Table 9 and...). Figure 16B Furthermore, it was confirmed that, through a process involving thermal reslurrying at the respective processing temperatures, the malic acid content in each sample of the resulting crystals (C) was reduced to approximately 1% (Table 9). Figure 17 Furthermore, regarding dihydroxyacetone (DHA), which is the causative agent of coloring, it was confirmed to be mixed in with the fermentation broth (S) (Table 8). Figure 15 The value of 0% was observed at the time point of the coarse crystals (B) obtained by isoelectric point crystallization, indicating that the removal of certain substances was highly achieved by isoelectric point crystallization (Tables 8 and 9). Figure 15 , Figure 16A and Figure 16B ).

[0283] Next, the results of microscopic observations over time during the hot re-slurrying process for each sample subjected to the aforementioned hot re-slurrying temperatures (40°C, 45°C, 60°C, and 70°C) are presented. Figure 18 In addition, the numbers at the top of the micrographs at each heat re-slurrying temperature indicate the elapsed time from the start of heating, in units of time [time (h): minutes (min)].

[0284] In each of the crystal (C) samples obtained by using the aforementioned hot resizing treatment temperatures, fine columnar crystals were observed in the very early stages of the hot resizing treatment. However, when the heating treatment began, it was observed that the columnar crystals (β-type crystals) changed into plate-like crystals (α-type crystals) over time. Specifically, in the samples where relatively high temperatures of 60°C and 70°C were used as the hot resizing treatment temperatures, the crystal particles almost completely changed into plate-like crystals (α-type crystals) after approximately 1 hour and 30 minutes, respectively. Furthermore, in the samples where relatively high temperatures of 45°C and 40°C were used as the hot resizing treatment temperatures, the crystal particles almost completely changed into plate-like crystals (α-type crystals) after 21 hours and 91 hours, respectively.

[0285] Furthermore, in this experimental example, similar to Experimental Example 1, the crystal structure of each sample, both coarse crystal (B) and crystal (C), was analyzed using X-ray diffraction. The results were consistent with... Figure 5A Similarly, as shown in the figure, for each sample of coarse crystal (B), when the peak values ​​of the diffraction X-rays were confirmed at diffraction angles of 18.8°, 19.7°, and 25.0°, it was determined that the crystal form of aspartic acid crystallized in each sample was β-type. Furthermore, for each sample of crystal (C), the same applies... Figure 5B Similarly, as shown in the figure, when the peak values ​​of the diffracted X-rays were confirmed at each diffraction angle of 21.65° and 23.7°, it was determined that the crystal form of aspartic acid in each sample was α-type crystal.

[0286] According to this experimental example, it is shown that even when the thermal re-slurrying process is performed not only in a high temperature range above 70°C, but also in a relatively low temperature range including 40°C, 45°C, 60°C, etc., by setting an appropriate heating treatment time, β-type crystals can be converted into α-type crystals. Furthermore, through the process specified in this invention, α-type crystals of aspartic acid can be recovered with high recovery rate and high purity.

[0287] As described above, according to embodiments of the present invention, it is reproducibly shown that even when using crude product obtained by utilizing microbial fermentation as starting material, regardless of whether seed crystals are added, other amino acids, organic acids, sulfate ions, etc., which are equivalent to impurities, can be efficiently removed while maintaining a high recovery rate of aspartic acid, and aspartic acid can ultimately be produced in the form of useful α-type crystals.

[0288] Industrial availability

[0289] Aspartic acid can be used as a raw material in the manufacture of chemically synthesized materials such as food, cosmetics, pharmaceuticals, and water-absorbing / biodegradable amino acid polymers, thus the present invention has high industrial applicability.

Claims

1. A method for producing aspartic acid, comprising: (q) Prepare a slurry containing β-type crystals of aspartic acid and a crystal component (X) of at least one impurity; and (r) The slurry is heated to change the β-type crystals of aspartic acid into α-type crystals, and then a crystal component (Y) of aspartic acid containing the α-type crystals is obtained. In step (q), a slurry of the crystal component (X) is prepared using the component containing β-type crystals obtained through step (p) described below. (p) In a solution (S) containing aspartic acid or its salt and at least one impurity, the pH of the solution (S) is adjusted to a predetermined pH value in the acidic region, and β-type crystals of aspartic acid are generated based on the principle of isoelectric point crystallization of aspartic acid. Then, the component containing the β-type crystals is separated from the solution (S). in, The solution (S) used in process (p) is derived from one or a combination of the following: A culture obtained by culturing or reacting microorganisms or cultured cells that can produce aspartic acid or its salts using sugars as raw materials; The treated product obtained by subjecting the culture to physical or chemical treatment; The supernatant obtained by removing solid components from the culture or the treated material, or the culture obtained by culturing or reacting microorganisms in a culture medium, and the clarified liquid separated from the culture; Alternatively, at least one concentrate formed from the aforementioned, As one of the impurities, it is at least one of the group consisting of amino acids selected from aspartic acid, organic acids and their salts, and contains the following components: i) Selected from at least one of the group consisting of glutamic acid, alanine, valine, and their salts, and ii) Selected from at least one of the group consisting of pyruvate, malic acid, acetic acid, succinic acid, fumaric acid and their salts.

2. The method according to claim 1, wherein, In process (r), the slurry is heated in a temperature range of 30°C to 190°C to change the β-type crystals of aspartic acid into α-type crystals.

3. The method according to claim 1, wherein, In process (r), the slurry is heated in a temperature range of 60°C to 150°C to change the β-type crystals of aspartic acid into α-type crystals.

4. The method according to claim 1, wherein, Before process (q), there is also process (p).

5. The method according to claim 4, wherein, In step (p), the pH of the solution (S) is adjusted to a specified value within the range of 0.50 to 6.95 to generate β-type crystals of the aspartic acid.

6. The method according to claim 4, wherein, In step (p), the pH of the solution (S) is adjusted to a specified value in the range of 1.50 to 4.50 to generate β-type crystals of the aspartic acid.

7. The method according to claim 4, wherein, The solution (S) tested in process (p) contains seed crystals.

8. The method according to claim 7, wherein, The seed crystal contains a β-type crystal of aspartic acid.

9. The method according to claim 4, wherein, The solution (S) tested in process (p) is a solution containing the aspartic acid or its salt at a concentration of 0.1 M to 5.0 M.

10. The method according to claim 4, wherein, The pH of the solution (S) tested in process (p) is in the range of 6.00 to 8.

00.

11. The method according to claim 10, wherein, In step (p), the pH of the solution (S) is adjusted to a specified value in the range of 1.00 to 6.85 by adding acid to the solution (S) to generate β-type crystals of the aspartic acid.

12. The method according to claim 4, wherein, In step (p), after the β-type crystals of aspartic acid are generated in the solution (S), the component containing the β-type crystals is separated from the solution (S) using a solid-liquid separation method. In step (q), the component containing β-type crystals separated in step (p) is used to prepare a slurry of the crystal component (X). In process (r), the slurry is heated to change the β-type crystals of aspartic acid into α-type crystals, and then the crystal component (Y) is separated from the slurry of the crystal component (X) by solid-liquid separation.

13. The method according to claim 12, wherein, In step (p), after separating the crystal component containing the β-type crystal from the solution (S) using a solid-liquid separation method, the separated crystal component is washed at least once using a solvent, and then dried. In step (q), the dried crystal component is used to prepare a slurry of the crystal component (X). In process (r), after separating the crystal component (Y) from the slurry of the crystal component (X) using a solid-liquid separation method, the separated crystal component (Y) is washed once or more using a solvent and then dried.

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