Method for separating and purifying acyclic hydroxycarboxylic acids

Abstract

According to the present invention, acyclic hydroxycarboxylic acids can be recovered, purified, and processed simply, efficiently, and at low cost. Therefore, this method is extremely useful as a method for producing acyclic hydroxycarboxylic acids that can be used as raw materials for biodegradable plastics and the like. [Solution] The present invention provides a method for separating and purifying an acyclic hydroxycarboxylic acid, comprising: a water removal step of removing water from the acyclic hydroxycarboxylic acid; a heating step of heating the acyclic hydroxycarboxylic acid from which water has been removed to a temperature above its melting point and below its thermal decomposition temperature to volatilize it; and a cooling step of cooling the vapor of the volatilized acyclic hydroxycarboxylic acid.

Description

[Technical Field]

[0001] The present invention relates to a method for separating and purifying acyclic hydroxycarboxylic acid, which is a poorly volatile component produced by biosynthesis or chemical synthesis, from a mixture containing the acyclic hydroxycarboxylic acid. [Background Art]

[0002] Acyclic hydroxycarboxylic acid is a raw material for biodegradable plastics, pharmaceuticals, functional foods and the like, and establishment of an efficient production method therefor is expected. Acyclic hydroxycarboxylic acid can be produced by fermentation using microorganisms, and production methods using various raw materials and microorganisms have been studied.

[0003] For example, a method for producing lactic acid using a gramineous plant as a raw material with a bacterium of the genus Lysobacter (Non-Patent Document 1), and a method for producing 3-hydroxybutyric acid from sugars or organic acids using a bacterium of the genus Halomonas (Patent Document 1) are known.

[0004] Also, a method for producing acyclic hydroxycarboxylic acid by chemically treating a high-molecular ester compound is known (Patent Document 2).

[0005] The acyclic hydroxycarboxylic acid produced by these methods is mixed together with medium components used for microbial culture, other metabolites, catalysts used in chemical treatment, and the like, so separation and purification are required for industrial use. As methods for separating acyclic hydroxycarboxylic acid, membrane separation (Patent Document 3) and reactive distillation (Patent Document 4) are known.

[0006] Membrane separation is a method that allows a target component to pass through a membrane or removes other components by utilizing the molecular size and charge of the target component. Therefore, it is difficult to separate low-molecular compounds with similar molecular sizes or components with the same charge. Reaction distillation is a method of separating the esterified acyclic hydroxycarboxylic acid by reacting a non-volatile acyclic hydroxycarboxylic acid with an alcohol to convert it into a volatile ester compound, and then distilling the resulting compound. By hydrolyzing the distilled ester compound, the original acyclic hydroxycarboxylic acid can be obtained, but alcohol, water, and other by-reaction products are also produced and mixed in.

[0007] As described above, conventional methods have several drawbacks, including difficulty in separating acyclic hydroxycarboxylic acids from similarly low-molecular-weight, acidic compounds, and reduced yield due to the generation of by-products during the conversion and restoration process to other components by esterification. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Patent No. 6521243 [Patent Document 2] Special Publication No. 2023-533199 [Patent Document 3] Japanese Patent Publication No. 2011-172492 [Patent Document 4] Patent No. 5770162 [Non-patent literature]

[0009] [Non-Patent Document 1] Molecules, 2023, Vol. 28 (17), 6234. [Non-Patent Document 2] THE MERCK INDEX, 1976, NINTH EDITION, p.581, 583, 637, 701, 741 [Non-Patent Document 3] Chemistry Dictionary 2nd Edition (Morikita Publishing) [Non-Patent Document 4] Chemical Handbook, Basic Edition II, 4th Revised Edition (edited by the Chemical Society of Japan) [Non-Patent Document 5] TCI, (R)-3-Hydroxybutanoic Acid, [online], Tokyo Chemical Industry Co., Ltd., [Retrieved September 13, 2024], Internet<URL:https: / / www.tcichemicals.com / JP / ja / p / H1341> [Non-Patent Document 6] ChemSpider, D-(+)-Glyceric acid, [online], ROYAL SOCIETY OF CHEMISTRY, [Retrieved September 13, 2024], Internet<URL:https: / / legacy.chemspider.com / Chemical-Structure.388334.html> [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] In order to solve the above problems, the inventors of the present invention clarified the properties of acyclic hydroxycarboxylic acids through experiments and investigated various separation methods. As a result, they found that by adjusting the conditions of pH, moisture, and temperature, the amount of volatilization of acyclic hydroxycarboxylic acids could be maximized, and that they could be separated by distillation, thus completing the present invention. Therefore, the object of the present invention is to provide a simple and efficient method for separating and purifying acyclic hydroxycarboxylic acids. [Means for solving the problem]

[0011] The method for separating and purifying acyclic hydroxycarboxylic acids according to the present invention is: A water removal step to remove water from an acyclic hydroxycarboxylic acid, A heating step involves heating the acyclic hydroxycarboxylic acid, from which water has been removed, at a temperature above its melting point but below its thermal decomposition point to volatilize it. A cooling step to cool the vapor of the volatile acyclic hydroxycarboxylic acid, It is characterized by consisting of the following.

[0012] Furthermore, the separation and purification method for acyclic hydroxycarboxylic acids according to the present invention is: Before the water removing step, a pH adjusting step of adjusting the acyclic hydroxycarboxylic acid to a pH not higher than the pH value of the acid dissociation constant, characterized by passing through the step.

[0013] Furthermore, the method for separating and purifying an acyclic hydroxycarboxylic acid according to the present invention is characterized in that: the heating step is a heating step of heating the acyclic hydroxycarboxylic acid from which water has been removed at a temperature not lower than the melting point and lower than the boiling point to volatilize the acyclic hydroxycarboxylic acid, what is claimed is:

[0014] These methods for separating and purifying an acyclic hydroxycarboxylic acid according to the present invention are characterized in that: in the cooling step, a transferring step of moving the volatilized vapor of the acyclic hydroxycarboxylic acid from a heating site in the heating step to a cooling site in the cooling step by an air flow, characterized by comprising the step.

[0015] In addition, each of the aforementioned separation and purification methods is characterized in that: the acyclic hydroxycarboxylic acid is constituted by a basic skeleton having 2 to 4 carbon atoms, characterized by that, and in addition to this, in addition to this, characterized by comprising one or more selected from the group consisting of glycolic acid, lactic acid, glyceric acid, 3-hydroxybutyric acid and malic acid. what is claimed is:

[0016] The apparatus for separating and purifying an acyclic hydroxycarboxylic acid according to the present invention comprises: a reaction vessel for holding an acyclic hydroxycarboxylic acid; a heating unit that heats the reaction vessel to evaporate water from the acyclic hydroxycarboxylic acid and volatilize the acyclic hydroxycarboxylic acid; and a cooling pipe for cooling the volatilized vapor of the acyclic hydroxycarboxylic acid, characterized by consisting of the above components.

[0017] Furthermore, the separation and purification apparatus is The reaction vessel is connected to the cooling tube, and an inlet is provided for introducing gas into the apparatus in order to transfer the vapor of the volatile acyclic hydroxycarboxylic acid by airflow, The reaction vessel is connected to the cooling tube, and an outlet is provided for releasing gas into the apparatus in order to transfer the vapor of the volatile acyclic hydroxycarboxylic acid by gasflow. It is characterized by having the following features. [Effects of the Invention]

[0018] According to the present invention, acyclic hydroxycarboxylic acids can be separated and purified inexpensively and efficiently from a mixture containing acyclic hydroxycarboxylic acids. Unlike membrane separation methods, which require expensive consumables such as separation membranes, high-purity acyclic hydroxycarboxylic acids can be obtained using sublimation or distillation equipment. In particular, by generating an airflow within the apparatus and moving the vaporized acyclic hydroxycarboxylic acid from the heating area to the cooling area, the acyclic hydroxycarboxylic acid can be recovered efficiently. [Brief explanation of the drawing]

[0019] [Figure 1] Schematic diagram of the apparatus used in Example 1 [Figure 2] Schematic diagram of the apparatus used in Example 2 [Figure 3] Schematic diagram of the apparatus used in Examples 3 and 4 [Modes for carrying out the invention]

[0020] This invention relates to a method for separating and purifying acyclic hydroxycarboxylic acids and an apparatus used therefor. The acyclic hydroxycarboxylic acids that can be separated by the present invention are non-volatile and have a basic chemical structure with 2 to 4 carbon atoms. This is because if the basic skeleton has five or more carbon atoms, it will thermally decompose at temperatures above its melting point, making it unsuitable.

[0021] Furthermore, the number of hydroxyl groups and carboxyl groups only needs to be one or more, and either or both may be present in multiples. For example, acyclic hydroxycarboxylic acids having one hydroxyl group and one carboxyl group include glycolic acid, lactic acid, 3-hydroxypropionic acid, tartonic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, and 4-hydroxybutyric acid. Examples of hydroxycarboxylic acids having one carboxyl group and multiple hydroxyl groups include dihydroacetic acid and glyceric acid. An example of an acyclic hydroxycarboxylic acid having one hydroxyl group and multiple carboxyl groups is malic acid. Tartaric acid is an example of an acyclic hydroxycarboxylic acid that has multiple carboxyl groups and multiple hydroxyl groups.

[0022] An acyclic hydroxycarboxylic acid having a chemical structure that satisfies these conditions, or a mixture containing an acyclic hydroxycarboxylic acid, is used as the acyclic hydroxycarboxylic acid-containing sample. The acyclic hydroxycarboxylic acid-containing sample used in this embodiment consists of an acyclic hydroxycarboxylic acid, a pH adjuster, and, if a fermentation broth is used, other components in the fermentation broth. pH adjusters can include, for example, acids or sodium hydroxide. Other components in the fermentation liquid include, for example, components of the nutrient medium (organic compounds such as sucrose, inorganic salts such as sodium nitrate) and components produced by the metabolism of microorganisms (organic compounds such as amino acids).

[0023] The pH of the sample containing acyclic hydroxycarboxylic acids is adjusted to be below the value of the acid dissociation constant specific to each acyclic hydroxycarboxylic acid. At this time, since volatilization efficiency decreases at pH values ​​close to the acid dissociation constant, it is desirable to adjust to a lower pH. Therefore, a pH 0.5 or more lower than the acid dissociation constant is preferable, and a pH 1.0 or more lower than the acid dissociation constant is optimal. On the other hand, if the pH is too low, the yield will decrease due to the decomposition of acyclic hydroxycarboxylic acids, so a pH of 1.0 or higher is preferable.

[0024] The type of acid used for pH adjustment should be one that alters the pH of the acyclic hydroxycarboxylic acid-containing sample and has low volatility within the distillation temperature range. For example, sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, boric acid, sulfonic acids, etc., can be used. Furthermore, organic acids exhibiting lower acid dissociation constants than acyclic hydroxycarboxylic acids can also be used, such as citric acid, fumaric acid, tartaric acid, oxalic acid, and p-toluenesulfonic acid.

[0025] These acids can be used individually or in combination to adjust the pH. If the pH of the sample containing an acyclic hydroxycarboxylic acid is too low (below 1.0), an alkaline agent or the above-mentioned organic acid can be used. The alkaline agent is not particularly limited, and any agent that produces ammonium ions or hydroxide ions in aqueous solution, or ammonia, amines, alkali metal salts, etc., can be used.

[0026] If a sample containing an acyclic hydroxycarboxylic acid contains water, the acyclic hydroxycarboxylic acid hardly volatilizes. Therefore, after pH adjustment, the water is removed from the sample containing the acyclic hydroxycarboxylic acid. The method for removing water is not particularly limited, but examples include heating evaporation, freeze-drying, and reverse osmosis membranes, and these can be used individually or in combination of two or more types.

[0027] In this embodiment, heating evaporation is used as the method for removing water. For example, when heating and evaporating water, the apparatus shown in Figures 1 to 3 can be used. Furthermore, the apparatus shown in Figure 1 can remove water by heating it with the cooling tube 2 removed.

[0028] Figures 1 to 3 show schematic diagrams of the apparatus used for separating and purifying acyclic hydroxycarboxylic acids.

[0029] The apparatus shown in Figure 1 consists of a reaction vessel 1 (for example, a sublimation vessel manufactured by CosmoSpeed) for heating a sample containing an acyclic hydroxycarboxylic acid, a cooling tube 2 for cooling the vapor of the acyclic hydroxycarboxylic acid that has been heated and volatilized, and a heating unit 3 for heating the sample containing the acyclic hydroxycarboxylic acid in the reaction vessel 1. When the acyclic hydroxycarboxylic acid is heated, the vapor of the volatile acyclic hydroxycarboxylic acid adheres to the cooling tube 2. The acyclic hydroxycarboxylic acid can be recovered by removing the condenser 2 from the reaction vessel 1.

[0030] The apparatus shown in Figures 2 and 3 consists of a reaction vessel 1 for heating a sample containing acyclic hydroxycarboxylic acid (for example, a branched reaction vessel from Tokyo Rikakikai Co., Ltd. or a 200 mL two-necked flask from Cosmosbead Co., Ltd. can be used), a condenser 2 for cooling the vapor of the heated and volatile acyclic hydroxycarboxylic acid (for example, a Liebig condenser from Beadrex Co., Ltd. can be used), a heating unit 3 for heating the sample containing acyclic hydroxycarboxylic acid in the reaction vessel 1, and a trap 4 for recovering the vapor of the acyclic hydroxycarboxylic acid (for example, a Dean-Stark trap from Tokyo Rikakikai Co., Ltd. or a round-bottom flask from Shibata Scientific Co., Ltd. can be used). 5 indicates the air inlet, which is the opening of the reaction vessel 1 for supplying air into the reaction vessel 1, and 6 indicates the air outlet, which is the opening from which air is discharged from the trap 4. The vapor of the acyclic hydroxycarboxylic acid that has evaporated by heating above its melting point and remains in the reaction vessel 1 can be moved to the trap 4 by air supplied from the inlet 5, where it can be precipitated and recovered. To move the acyclic hydroxycarboxylic acid vapor remaining in reaction vessel 1 to trap 4, air can be blown through the apparatus to move the vapor from the heating area to the cooling area. For example, air can be introduced through inlet 5 and discharged through outlet 6, or air can be drawn in through outlet 6 with inlet 5 open.

[0031] In all of the apparatuses shown in Figures 1 to 3, a pH-adjusted acyclic hydroxycarboxylic acid-containing sample is placed in reaction vessel 1 and heated at a temperature of 100°C or lower. In this case, with the apparatus shown in Figures 2 and 3, water can be removed from the acyclic hydroxycarboxylic acid-containing sample by supplying air from the inlet 5 and discharging the water vapor generated in the reaction vessel 1 to the outside of the reaction vessel 1 (outside the outlet 6). When using the apparatus shown in Figure 1, the cooling tube 2 can be removed and the mixture heated to generate steam, which can then be discharged outside the reaction vessel 1 to remove water.

[0032] After removing the water, the condenser 2 and the container from which the acyclic hydroxycarboxylic acid is recovered from the mixture containing the acyclic hydroxycarboxylic acid (meaning the non-heated section of the reaction vessel 1 in the apparatus of Figure 1, and meaning trap 4 in the apparatus of Figures 2 and 3) are dried, and any remaining water adhering to the inside of the apparatus is removed, or the condenser 2 and the container are replaced with ones that do not have water adhering to them, and then the acyclic hydroxycarboxylic acid-containing sample in the reaction vessel 1 is heated to distill the acyclic hydroxycarboxylic acid.

[0033] With the apparatus shown in Figures 2 and 3, the acyclic hydroxycarboxylic acid can be distilled by generating an airflow from the inlet 5 to the outlet 6 while heating the acyclic hydroxycarboxylic acid-containing sample in the reaction vessel 1. When heating a sample containing acyclic hydroxycarboxylic acids, the temperature must be kept above the melting point to efficiently volatilize the acyclic hydroxycarboxylic acids, but temperatures that lead to thermal decomposition are unsuitable. Acyclic hydroxycarboxylic acids are relatively easy to decompose with heat, and some are known to decompose before reaching their boiling point. The melting points of acyclic hydroxycarboxylic acids vary, but are generally in the range of 25°C to 200°C, and the heating temperature must be kept within this range. To ensure efficient distillation and avoid thermal decomposition, the heating temperature is preferably between 50°C and 150°C.

[0034] The speed and direction of the airflow generated from the inlet 5 to the outlet 6 should be such that it gently circulates throughout the entire heated reaction vessel 1, and should be adjusted so that the acyclic hydroxycarboxylic acid-containing sample introduced into the reaction vessel 1 does not scatter or the temperature inside the reaction vessel 1 fluctuates significantly. For example, depending on the shape and size of the reaction vessel 1, in the apparatus shown in Figure 2, if the outer diameter of the reaction vessel 1 is 3 cm and the distance from the inlet 5 to the bottom surface of the reaction vessel 1 is 10.5 cm, then the airflow velocity should be between 0.5 and 4.0 liters per minute, and more preferably between 1.5 and 2.5 liters per minute. Furthermore, in the apparatus shown in Figure 3, if a flask with a capacity of 200 milliliters is used for reaction vessel 1, the airflow velocity should be between 0.5 and 4.0 liters per minute, similar to the apparatus in Figure 2, with a preferred airflow of 1.5 to 2.5 liters per minute.

[0035] If a larger reaction vessel 1 is used, the airflow velocity should be increased and adjusted to a level that does not cause the acyclic hydroxycarboxylic acid-containing sample to scatter or the temperature to drop. In addition to air, any gas that does not alter the acyclic hydroxycarboxylic acid can be used for the airflow, such as nitrogen or carbon dioxide. In the following embodiment, air is used as the gas used for the airflow. The heating unit 3 only needs to be able to maintain a constant temperature, and can use, for example, an aluminum block constant temperature bath, an oil bath, or a ribbon heater, which can be used individually or in combination.

[0036] The present invention will be described in detail below based on examples using the apparatus shown in Figures 1 to 3, but the present invention is not limited to these examples.

[0037] (Example 1: Investigation of volatilization conditions for acyclic hydroxycarboxylic acids) In this example, DL-lactic acid, (R)-3-hydroxybutyrate, DL-malic acid, DL-calcium lactate, (R)-3-hydroxybutyrate sodium salt, and DL-malate sodium salt, purchased from a reagent manufacturer, were used as acyclic hydroxycarboxylic acid-containing samples, respectively. Note that pH adjustment was not performed in this example. 10 to 50 mg of each of these samples were placed at the bottom of the reaction vessel 1 (CosmoSpeed, Type A, outer diameter 35 mm, height 190 mm) of the apparatus shown in Figure 1. Under atmospheric pressure, the heating section 3 was heated to a constant temperature using an oil bath while cooling water (20°C) was circulated through the cooling tube 2. After one hour, the condenser tube 2 was washed, and each acyclic hydroxycarboxylic acid-containing sample was recovered and quantified by high-performance liquid chromatography to calculate the recovery rate of each sample. Table 1 shows the temperature conditions and recovery rates for each sample.

[0038] [Table 1]

[0039] The heating temperatures were compared at temperatures lower and higher than the melting point of each sample (lactic acid: 53°C, 3-hydroxybutyric acid: 45.5-48°C, malic acid: 100°C). The results in Table 1 suggest that the acyclic hydroxycarboxylic acids were not recovered and did not volatilize at temperatures below the melting point of each sample. On the other hand, at temperatures higher than the melting point of each sample, the salt was not recovered, but it was recovered in the free acid state, indicating that the acyclic hydroxycarboxylic acid vapor was being released. These findings indicate that acyclic hydroxycarboxylic acids can be volatilized in their free acid state by raising them to a temperature higher than the melting point of each sample, and that the volatilized vapor can be recovered by cooling it.

[0040] (Example 2: Effect of distillation temperature on free acid) In this example, glycolic acid, DL-3-hydroxybutyric acid, and L-malic acid, purchased from a reagent manufacturer, were used as acyclic hydroxycarboxylic acid-containing samples, respectively. Note that pH adjustment was not performed in this example. Ten mg of each of these samples was placed at the bottom of the reaction vessel 1 of the apparatus shown in Figure 2, and distillation was carried out by supplying air at a flow rate of 2 L / min through the inlet 5 while maintaining the heating unit 3 at the respective set temperatures. Cooling tube 2 was set to 10°C. After 30 minutes, the acyclic hydroxycarboxylic acid precipitated in condenser 2 and trap 4 was collected and quantified by high-performance liquid chromatography, and the recovery rate relative to the amount of acyclic hydroxycarboxylic acid-containing sample added to reaction vessel 1 was calculated. The conditions and results for each sample are shown in Table 2.

[0041] [Table 2]

[0042] The results in Table 2 show that glycolic acid, which has a melting point of 78-80°C, did not volatilize at 60°C (below its melting point) and was not distilled even with an airflow. However, distillation became possible at 80°C, and 22% of the added glycolic acid was distilled and recovered. 3-hydroxybutyric acid was not distilled at 30°C, which is below its melting point, but 2.7% was distilled and recovered at 50°C, which is above its melting point. Malic acid did not volatilize or undergo distillation at 90°C, which is below its melting point, but it did volatilize at 130°C, which is above its melting point, and 2.6% was distilled and recovered. Furthermore, all three samples could not be recovered at all under conditions without airflow, even at temperatures above their melting points. From the above, it was found that a sample containing acyclic hydroxycarboxylic acid in a free acid state can be partially volatilized by heating it above its melting point, and by supplying air from the inlet 5 into the reaction vessel 1 to generate an airflow within the apparatus, the vapor of the volatilized acyclic hydroxycarboxylic acid moves from the reaction vessel 1 to the trap 4, allowing the acyclic hydroxycarboxylic acid to be recovered.

[0043] (Example 3: Effects of pH and temperature conditions) In this example, glycolic acid, L-lactic acid, DL-glyceric acid, (R)-3-hydroxybutyric acid, and L-malic acid, purchased from a reagent manufacturer, were used as acyclic hydroxycarboxylic acid-containing samples. To each sample (concentration 1-4%), a 10N sodium hydroxide aqueous solution was added to adjust the pH of each sample (aqueous solution). Then, 5 mL of each sample (aqueous solution) was placed in the reaction vessel 1 (two-necked round-bottom flask) of the apparatus shown in Figure 3, and the heating section 3 was heated to 90-100°C to remove the water. After removing the water and drying the inside of the apparatus, distillation was performed by blowing air through the inlet 5 at a flow rate of 2 L / min while maintaining the heating section 3 at each set temperature. Cooling tube 5 was set to 15°C. After 2 hours, the acyclic hydroxycarboxylic acid precipitated in the condenser 5 and trap 4 (round-bottom flask) was collected and quantified by high-performance liquid chromatography, and the recovery rate relative to the amount of acyclic hydroxycarboxylic acid-containing sample added to reaction vessel 1 (two-necked round-bottom flask) was calculated. The conditions and results for each sample are shown in Table 3.

[0044] The properties of each sample in Table 3 (pKa, melting point, boiling point, decomposition point) were taken from Non-Patent Documents 5-9. However, since the sample decomposes thermally before reaching its boiling point under normal pressure, the boiling point (decomposition point) shown is the decomposition point during thermal decomposition, the boiling point under reduced pressure, or an estimated boiling point calculated from the molecular structure.

[0045] [Table 3]

[0046] The results in Table 3 show that all five acyclic hydroxycarboxylic acids could be distilled by raising the pH to a level lower than their respective acid dissociation constants and the temperature to a level above their melting point. On the other hand, when the pH was adjusted to a level higher than the acid dissociation constant, distillation did not occur even when the temperature was raised above the melting point. Furthermore, even at pH levels significantly lower than the acid dissociation constant, distillation did not occur at temperatures below the melting point. Malic acid, which has two carboxyl groups, could be distilled by adjusting the pH to a level lower than the lower of its acid dissociation constants. Furthermore, for all five samples, even when the pH was set lower than the acid dissociation constant and the temperature was above the melting point, no acyclic hydroxycarboxylic acids could be recovered under conditions where no airflow was generated inside the apparatus.

[0047] From the above, it was found that acyclic hydroxycarboxylic acids, which have a basic structure with 2 to 4 carbon atoms, can be partially volatilized even at temperatures below their boiling point (and below thermal decomposition) by adjusting the pH to be lower than the acid dissociation constant, removing water, and heating them above their melting point. Furthermore, it was found that by generating an airflow within the apparatus and moving the vaporized acyclic hydroxycarboxylic acid from the heating area to the cooling area, the acyclic hydroxycarboxylic acid can be efficiently recovered.

[0048] (Example 4: Separation of acyclic hydroxycarboxylic acids from fermentation broth) Acyclic hydroxycarboxylic acids can be produced by fermentation using microorganisms. In fermentation production, microorganisms are cultured using a culture medium to which various nutrients have been added. The fermentation liquid after cultivation contains not only the produced acyclic hydroxycarboxylic acid, but also culture medium components, other metabolites, and microbial cells. Therefore, it is necessary to recover acyclic hydroxycarboxylic acids from the fermentation liquid.

[0049] Therefore, in this example, we attempted to recover 3-hydroxybutyric acid from the fermentation liquid using the following method. The fermentation liquid of 3-hydroxybutyric acid was prepared as follows, in accordance with the method described in Patent Document 1 (Japanese Patent Publication No. 6521243).

[0050] The 3-hydroxybutyrate-producing bacterium used was Halomonas sp. strain OITC1261. Halomonas sp. strain OITC1261 is deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture) under accession number NITE P-02027. The nutrient medium consisted of sucrose, sodium nitrate, sodium chloride, potassium sulfate, and other trace inorganic salts. Sucrose was added in three separate additions during the incubation period. First, Halomonas sp. strain OITC1261 was inoculated into 100 mL of nutrient medium in an Erlenmeyer flask and pre-cultured overnight (35°C, 200 rpm). The entire volume of the pre-culture medium was inoculated into 2 L of separately prepared nutrient medium, and the main culture was carried out for 65 hours using a benchtop culture apparatus under constant culture conditions (temperature: 35°C, stirring speed: 700 rpm, pH 9.0, aeration rate: 5 L / min) to obtain 3-hydroxybutyric acid fermentation liquid.

[0051] The fermentation liquid was centrifuged to remove insoluble components such as microbial cells, and the resulting solution was used as the sample. The dry solid content of this sample (solution) is 11%, and of that, 57% is 3-hydroxybutyric acid. After adjusting the pH of the sample (solution) using hydrochloric acid, 5 mL was placed in reaction vessel 1 (two-necked round-bottom flask) of the apparatus shown in Figure 3, and the heating section 3 was heated to 90-100°C to remove water. After removing the water and drying the inside of the apparatus, distillation was performed by blowing air through the inlet 5 at a flow rate of 2 L / min while maintaining the heating section 3 at each set temperature. Cooling tube 2 was set to 15°C. After 2 hours, the acyclic hydroxycarboxylic acid precipitated in condenser 2 and trap 4 (round-bottom flask) was collected and quantified by high-performance liquid chromatography. The recovery rate relative to the amount of 3-hydroxybutyric acid contained in the acyclic hydroxycarboxylic acid-containing sample added to reaction vessel 1 (two-necked round-bottom flask) was calculated. The experimental conditions and results are shown in Table 4.

[0052] [Table 4]

[0053] The acid dissociation constant of 3-hydroxybutyric acid is 4.29, and its melting point is around 48°C. As shown in Table 4, when the pH is higher than the acid dissociation constant (pH 5.5), 3-hydroxybutyric acid does not volatilize and cannot be recovered, even at a distillation temperature of 100°C. If the pH is lower than the acid dissociation constant, some of the 3-hydroxybutyric acid will volatilize and be carried away by the airflow, allowing it to precipitate and be recovered in the condenser 2 and trap 4 (round-bottom flask).

[0054] The recovery rate improved by increasing the heating temperature, reaching 43% at 100°C. Furthermore, the distilled 3-hydroxybutyric acid was of high purity (over 90%) in all cases. The 3-hydroxybutyric acid produced through fermentation is mixed with components of the culture medium and microbial metabolites.

[0055] In the sample used in this study, 3-hydroxybutyric acid accounted for 57% of the dry solid content, indicating a high level of impurities, making it unsuitable for industrial use. On the other hand, 3-hydroxybutyric acid recovered by distillation shows a high recovery rate and significantly improved purity, making it fully suitable for industrial use. Based on the above, the method of the present invention allows for the efficient separation, purification, and recovery of acyclic hydroxycarboxylic acids produced through fermentation. [Explanation of symbols]

[0056] 1. Reaction vessel 2 Cooling pipe 3 Heating section 4 Traps 5 Inlet 6 Outlet

Claims

1. A water removal step to remove water from an acyclic hydroxycarboxylic acid, A heating step involves heating the acyclic hydroxycarboxylic acid, from which water has been removed, at a temperature above its melting point but below its thermal decomposition point to volatilize it. A cooling step to cool the vapor of the volatile acyclic hydroxycarboxylic acid, A method for separating and purifying acyclic hydroxycarboxylic acids, characterized by comprising the following.

2. A pH adjustment step to adjust the pH of an acyclic hydroxycarboxylic acid to a value below the acid dissociation constant, A water removal step to remove water from the acyclic hydroxycarboxylic acid after pH adjustment, A heating step involves heating the acyclic hydroxycarboxylic acid, from which water has been removed, at a temperature above its melting point but below its thermal decomposition point to volatilize it. A cooling step to cool the vapor of the volatile acyclic hydroxycarboxylic acid, A method for separating and purifying acyclic hydroxycarboxylic acids, characterized by comprising the following.

3. A pH adjustment step to adjust the pH of an acyclic hydroxycarboxylic acid to a value below the acid dissociation constant, A water removal step to remove water from the acyclic hydroxycarboxylic acid after pH adjustment, A heating step involves heating the acyclic hydroxycarboxylic acid, from which water has been removed, to a temperature above its melting point and below its boiling point to volatilize it. A cooling step to cool the vapor of the volatile acyclic hydroxycarboxylic acid, A method for separating and purifying acyclic hydroxycarboxylic acids, characterized by comprising the following.

4. A pH adjustment step to adjust the pH of an acyclic hydroxycarboxylic acid to a value below the acid dissociation constant, A water removal step to remove water from the acyclic hydroxycarboxylic acid after pH adjustment, A heating step involves heating the acyclic hydroxycarboxylic acid, from which water has been removed, to a temperature above its melting point and below its boiling point to volatilize it. A cooling step to cool the vapor of the volatile acyclic hydroxycarboxylic acid, In the aforementioned cooling process, A transfer step involves moving the vaporized acyclic hydroxycarboxylic acid from the heating location in the heating step to the cooling location in the cooling step by airflow. A method for separating and purifying acyclic hydroxycarboxylic acids, characterized by comprising the following.

5. The aforementioned acyclic hydroxycarboxylic acid, Composed of a basic skeleton with 2 to 4 carbon atoms, A method for separating and purifying an acyclic hydroxycarboxylic acid according to any one of claims 1 to 4.

6. The aforementioned acyclic hydroxycarboxylic acid, It is composed of a basic skeleton with 2 to 4 carbon atoms. It contains one or more selected from glycolic acid, lactic acid, glyceric acid, 3-hydroxybutyric acid, and malic acid. A method for separating and purifying an acyclic hydroxycarboxylic acid according to any one of claims 1 to 4.

7. A reaction vessel for acyclic hydroxycarboxylic acids, A heating section is provided to heat the reaction vessel to evaporate water from the acyclic hydroxycarboxylic acid and volatilize the acyclic hydroxycarboxylic acid. A cooling tube for cooling the vapor of the volatile acyclic hydroxycarboxylic acid, A separation and purification apparatus for acyclic hydroxycarboxylic acids, characterized by comprising the following:

8. A reaction vessel for acyclic hydroxycarboxylic acids, A heating section is provided to heat the reaction vessel to evaporate water from the acyclic hydroxycarboxylic acid and volatilize the acyclic hydroxycarboxylic acid. A cooling tube for cooling the vapor of the volatile acyclic hydroxycarboxylic acid, The reaction vessel is connected to the cooling tube, and an inlet is provided for introducing gas into the apparatus in order to transfer the vapor of the volatile acyclic hydroxycarboxylic acid by airflow, The reaction vessel is connected to the cooling tube, and an outlet is provided for releasing gas into the apparatus in order to transfer the vapor of the volatile acyclic hydroxycarboxylic acid by gasflow. A separation and purification apparatus for acyclic hydroxycarboxylic acids, characterized by comprising the following:

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