JP2010126512A - Method for producing hydroxycarboxylic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a hydroxycarboxylic acid containing a process in which impurity contained in a solution containing hydroxycarboxylic acid (except lactic acid) is removed. <P>SOLUTION: This method for producing a hydroxycarboxylic acid includes a process A in which a solution containing a hydroxycarboxylic acid such as glycolic acid, 3-hydroxybutyric acid, malic acid etc., (except lactic acid) is filtered through a nano-filtration film and the hydroxycarboxylic acid is recovered from the resultant filtrate. The solution containing a hydroxycarboxylic acid to be passed through the nano-filtration film in the process A has a pH of not lower than 2 and not higher than 5. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing hydroxycarboxylic acid, which includes a step of removing impurities from a solution containing hydroxycarboxylic acid (excluding lactic acid).

  Hydroxycarboxylic acid is widely applied to food and medical uses as well as industrial uses as a monomer raw material for biodegradable plastics, and the demand is increasing. Most of hydroxycarboxylic acids are classified into optical isomers such as (L) -form and (D) -form by the stereotype of hydroxyl group. When hydroxycarboxylic acid is used as a raw material for biodegradable plastics, the properties of the plastic obtained depend on the ratio of optical isomers (optical purity) and the amount of impurities contained. There is a need to produce quantities of hydroxycarboxylic acids. Hydroxycarboxylic acid is produced by an organic synthesis method or fermentation method. According to the production of hydroxycarboxylic acid by microbial fermentation, (L) -form or (D) -form hydroxycarboxylic acid is selected by appropriately selecting the microorganism. Alternatively, (L) -form and (D) -form mixture (racemic) hydroxycarboxylic acid can be produced.

  In general, in order to produce hydroxycarboxylic acid by microbial fermentation, an alkaline substance is added in order to maintain the culture solution at an optimum pH. Therefore, most of the hydroxycarboxylic acid, which is an acidic substance produced by fermentation, exists as a hydroxycarboxylate in the culture solution. In this case, free hydroxycarboxylic acid can be obtained by adding an acidic substance to the culture solution after the end of fermentation. As a specific example, calcium hydroxide is used as an alkaline substance to be added to the culture broth, calcium hydroxycarboxylate is accumulated in the fermentation broth, and sulfuric acid is added as an acidic substance after fermentation to separate the produced calcium sulfate. By doing so, free hydroxycarboxylic acid can be obtained.

  As a method for separating the free hydroxycarboxylic acid, in the case of a poorly soluble salt such as calcium sulfate, a method of separating with a qualitative filter paper or the like is used. However, in this method, the calcium salt precipitated as a solid can be removed, but a trace amount of calcium salt dissolved in the solution is not removed and remains as an impurity in the hydroxycarboxylic acid solution. In addition, when producing hydroxycarboxylic acid by microbial fermentation, there are various inorganic salts and sugars in addition to calcium salts as impurities, and those that are dissolved in the culture solution are filtered with qualitative filter paper. I can't separate. Therefore, when the filtrate containing this hydroxycarboxylic acid is subjected to, for example, a concentration operation in a subsequent purification step, a calcium salt or other inorganic salt is precipitated (precipitated) again in the free hydroxycarboxylic acid-containing solution. There is. When the hydroxycarboxylic acid-containing solution is heated and concentrated by an operation such as distillation while the inorganic ions are not sufficiently removed, the racemization and oligomerization of the hydroxycarboxylic acid may proceed due to the influence of the inorganic ions. Are known. Therefore, a method for effectively removing a trace amount of inorganic ion components remaining in the hydroxycarboxylic acid-containing solution is required.

  As a method for removing a trace amount of inorganic ion components or saccharides from a hydroxycarboxylic acid-containing solution, a method using an ion exchange resin is disclosed (see Patent Documents 1 and 2). However, in order to maintain the ion exchange performance of the ion exchange resin, it is necessary to periodically regenerate the ion exchange resin. In addition, regeneration of the ion exchange resin is performed using a large amount of sodium chloride aqueous solution. However, with the regeneration, a large amount of waste liquid is discharged, and there is a problem in that the waste liquid treatment is costly. Furthermore, when the ion exchange resin is repeatedly regenerated, there is a problem that the regeneration rate of the ion exchange resin is lowered, the ion exchange performance is lowered, and the removal rate of the inorganic salt is lowered.

  Also known is a method of removing trace amounts of inorganic ion components such as calcium components from a hydroxycarboxylic acid-containing solution using a bipolar membrane using an electrodialyzer (see, for example, Patent Document 3). However, the bipolar membrane used in this method is expensive and has a problem that the removal efficiency of inorganic salts such as calcium salts is not high.

  In addition, a method for separating and recovering an organic acid using a separation membrane is known, and a method for separating parapyruvic acid from a pyruvic acid solution using a reverse osmosis membrane is disclosed (see Patent Document 4). The removal rate of the inorganic salt is not disclosed, and the separation / recovery of hydroxycarboxylic acid is not mentioned.

Furthermore, a method for removing saccharides from a hydroxycarboxylic acid-containing solution using a loose reverse osmosis membrane is disclosed (see, for example, Patent Document 5). In the loose reverse osmosis membrane used in Patent Document 5, Since it cannot be said that the sugar blocking rate is sufficiently high, it is speculated that the removal rate of the inorganic salt is also insufficient, and the problem of efficiently separating the hydroxycarboxylic acid and the inorganic salt has not been solved.
JP-A-3-183487 JP-A-4-171001 JP-A-10-179183 Japanese Patent Laid-Open No. 6-306011 Japanese Patent Laid-Open No. 4-197424

  An object of the present invention is to provide a method for efficiently separating and recovering the above-described problems, that is, when producing a hydroxycarboxylic acid excluding lactic acid.

  As a result of intensive studies to solve the above problems, the present inventors have found that impurities can be efficiently removed by filtering a solution containing hydroxycarboxylic acid (excluding lactic acid) through a nanofiltration membrane. The present invention has been completed. That is, this invention is comprised from following (1)-(9).

  (1) A method for producing hydroxycarboxylic acid, comprising a step A in which a hydroxycarboxylic acid (excluding lactic acid) -containing solution is filtered through a nanofiltration membrane to recover hydroxycarboxylic acid from the permeation side.

  (2) The hydroxycarboxylic acid is one or a mixture of two or more selected from glycolic acid, hydroxybutyric acid, malic acid, tartaric acid, hydroxyvaleric acid, hydroxycaproic acid, gluconic acid and citric acid. ) A process for producing a hydroxycarboxylic acid as described above.

  (3) The method for producing a hydroxycarboxylic acid according to (1) or (2), wherein the hydroxycarboxylic acid-containing solution leading to the nanofiltration membrane in Step A has a pH of 2 or more and 5 or less.

  (4) The magnesium sulfate permeability of the nanofiltration membrane is 1.5% or less at an operating pressure of 0.5 MPa, a raw water temperature of 25 ° C., and a raw water concentration of 1000 ppm, according to any one of (1) to (3) A process for producing a hydroxycarboxylic acid.

  (5) The ratio of magnesium sulfate permeability to citric acid permeability is 3 or more at an operating pressure of 0.5 MPa, a raw water temperature of 25 ° C., and a raw water concentration of 1000 ppm, according to any one of (1) to (4) A method for producing hydroxycarboxylic acid.

  (6) The method for producing hydroxycarboxylic acid according to any one of (1) to (5), wherein the functional layer of the nanofiltration membrane contains polyamide.

  (7) The method for producing a hydroxycarboxylic acid according to (6), wherein the polyamide comprises a crosslinked piperazine polyamide as a main component and a constituent represented by Chemical Formula 1.

（式中、Ｒは−Ｈまたは−ＣＨ_３、ｎは０から３までの整数を表す。）。

(In the formula, R represents —H or —CH ₃ , and n represents an integer of 0 to 3).

  (8) The step of filtering the hydroxycarboxylic acid-containing solution obtained in the step A through a reverse osmosis membrane to increase the concentration of hydroxycarboxylic acid, and including the step B of (1) to (7) Production method.

  (9) The method for producing a hydroxycarboxylic acid according to any one of (1) to (8), wherein the permeate collected from Step A or the concentrate collected from Step B is subjected to Step C for recrystallization. .

  According to the present invention, impurities such as inorganic salts contained in the hydroxycarboxylic acid-containing solution can be removed, so that the hydroxycarboxylic acid can be produced with high purity and high yield.

  Hereinafter, the present invention will be described in more detail.

  In the method for producing hydroxycarboxylic acid of the present invention, a solution containing hydroxycarboxylic acid (excluding lactic acid) is filtered through a nanofiltration membrane to remove impurities such as inorganic salts in the solution containing hydroxycarboxylic acid (excluding lactic acid). It is characterized by including the process A which removes and obtains a hydroxycarboxylic acid solution.

  The hydroxycarboxylic acid (excluding lactic acid) in the present invention is a general term for compounds obtained by removing lactic acid from compounds each having one or more hydroxyl groups (OH groups) and carboxyl groups (COOH groups) in the molecule (note that Hereinafter, the term “hydroxycarboxylic acid” shall conform to this definition.) In addition, the hydroxycarboxylic acid referred to herein may be an optical isomer such as (L) -isomer or (D) -isomer, or one or a mixture of two or more. Specific examples of hydroxycarboxylic acids include glycolic acid, hydroxybutyric acid (2-hydroxybutyric acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid), malic acid, tartaric acid, hydroxyvaleric acid, hydroxycaproic acid, gluconic acid, citric acid, and the like. Linear aliphatic hydroxycarboxylic acids, branched aliphatic hydroxycarboxylic acids such as mevalonic acid and leucine acid, aromatic hydroxycarboxylic acids such as salicylic acid and mandelic acid, and the above hydroxycarboxylic acids having an unsaturated bond, In the present invention, a hydroxycarboxylic acid having 1 to 6 carbon atoms is preferable, and a linear aliphatic hydroxycarboxylic acid having 1 to 6 carbon atoms is more preferable. Glycolic acid, hydroxybutyric acid, malic acid, tartaric acid, hydroxyvaleric acid , Hydroxycaproic acid, gluconic acid and One or a mixture of two or more selected from the group consisting of citric acid is more preferable. Further, the hydroxycarboxylic acid may be a product manufactured by organic synthesis or a product manufactured by fermentation.

  The hydroxycarboxylic acid-containing solution in the present invention is an aqueous solution containing one or more hydroxycarboxylic acids, and the hydroxycarboxylic acid may be dissolved as a hydroxycarboxylic acid salt. Hydroxycarboxylic acid salts include hydroxycarboxylic acid inorganic salts such as hydroxycarboxylic acid lithium salt, hydroxycarboxylic acid sodium salt, hydroxycarboxylic acid potassium salt, hydroxycarboxylic acid magnesium salt, hydroxycarboxylic acid calcium salt, and hydroxycarboxylic acid ammonium salt. And may be a mixture thereof. When the hydroxycarboxylic acid is a product produced by fermentation, the hydroxycarboxylic acid-containing solution may be a fermentation broth.

  The nanofiltration membrane used in step A is also called a nanofilter (nanofiltration membrane, NF membrane) and is generally defined as “a membrane that transmits monovalent ions and blocks divalent ions”. It is a film. It is a membrane that is considered to have a minute gap of about several nanometers, and is mainly used to block minute particles, molecules, ions, salts, and the like in water.

  In addition, “filtering through a nanofiltration membrane” means that a hydroxycarboxylic acid-containing solution is filtered through a nanofiltration membrane to remove or prevent or filter out inorganic salts precipitated as dissolved or solid, It means that the hydroxycarboxylic acid solution is permeated as a filtrate. Here, the inorganic salt includes any form of the inorganic salt contained in the hydroxycarboxylic acid-containing solution, and is dissolved in the solution, or precipitated or precipitated in the solution. Anything included is included.

  The pH of the hydroxycarboxylic acid-containing solution used for the nanofiltration membrane in step A is preferably 2 or more and 5 or less. In the nanofiltration membrane, it is known that the ionized substance in the solution is easier to remove or prevent than the non-ionized substance. Therefore, the pH of the hydroxycarboxylic acid-containing solution is 5 or less. As a result, the proportion of the hydroxycarboxylic acid dissociated in the solution and existing as hydroxycarboxylic acid ions is reduced, and the hydroxycarboxylic acid is easily transmitted. Moreover, when pH is less than 2, there exists a possibility of affecting the damage of a nanofiltration membrane. Furthermore, when the pH is set to pKa or less of hydroxycarboxylic acid, hydroxycarboxylic acid that is not dissociated into hydroxycarboxylic acid ions and hydrogen ions is more contained in the hydroxycarboxylic acid-containing solution. Is more preferable because it can permeate through the nanofiltration membrane. In the case where the hydroxycarboxylic acid-containing solution is a product manufactured by fermentation, the pH of the culture solution may be adjusted during microbial fermentation or after microbial fermentation.

  When the hydroxycarboxylic acid is dissolved as a hydroxycarboxylate, an acidic substance may be added before permeating the nanofiltration membrane. By adding an acidic substance, the hydroxycarboxylate can be converted to a free hydroxycarboxylic acid. Examples of the acidic substance to be added include sulfuric acid, hydrochloric acid, carbonic acid, phosphoric acid, and nitric acid. Specifically, for example, sulfuric acid is added to a solution containing calcium hydroxycarboxylate to make hydroxycarboxylic acid free, and the calcium component in the solution is precipitated as calcium sulfate, which is a hardly soluble sulfate, and filtered. The calcium component can be removed or prevented more effectively by passing the filtrate (separate containing hydroxycarboxylic acid) through the nanofiltration membrane of step A. When the calcium component in the hydroxycarboxylic acid-containing solution is precipitated as a sparingly soluble sulfate and filtered, if the equivalent of sulfuric acid added to the solution exceeds the equivalent of calcium (sulfuric acid equivalent> calcium equivalent), the excess sulfuric acid If the nanofiltration membrane is partially permeated and then the permeate is exposed to heating conditions such as concentration, the permeated sulfuric acid acts as a catalyst for promoting oligomerization of hydroxycarboxylic acid, which may reduce the yield. . Therefore, when the calcium component in the hydroxycarboxylic acid-containing solution is precipitated as a poorly soluble sulfate and filtered, it is preferable to add sulfuric acid below the equivalent of the calcium component in the solution, and the addition equivalent is adjusted with pH In this case, the pH is preferably 2 or more because it is less than or equal to the calcium component equivalent.

  The nanofiltration membrane used in the present invention can be evaluated by calculating the inorganic ion removal rate (blocking rate) as a method for evaluating the degree of inorganic salt removal, blocking or filtration. Inorganic salt rejection (removal rate) is included in the concentration of inorganic salt (raw water inorganic salt concentration) and permeate (hydroxycarboxylic acid solution) contained in raw water (culture solution) by analysis typified by ion chromatography. By measuring the concentration of the inorganic salt (permeated inorganic salt concentration), it can be calculated by Equation 1.

  Inorganic salt removal rate (%) = (1− (permeate inorganic salt concentration / raw water inorganic salt concentration)) × 100 (Formula 1).

  The membrane separation performance of the nanofiltration membrane used in step A is not particularly limited, but the magnesium sulfate permeability of the nanofiltration membrane is 1.5% or less at an operating pressure of 0.5 MPa, a raw water temperature of 25 ° C., and a raw water concentration of 1000 ppm. Are preferably used. When the magnesium sulfate permeability of the nanofiltration membrane under the above conditions exceeds 1.5%, there is a possibility that an inorganic salt precipitates when the hydroxycarboxylic acid solution that has passed through the nanofiltration membrane is concentrated. The racemic and oligomerization of hydroxycarboxylic acid is likely to occur due to the influence of the permeated inorganic salt, and the yield may be reduced. More preferably, a nanofiltration membrane having a magnesium sulfate permeability of 1.0% or less is used. Here, the magnesium sulfate permeability is determined by analysis represented by ion chromatography. The magnesium sulfate concentration contained in the raw water (raw water magnesium sulfate concentration) and the magnesium sulfate concentration contained in the permeate (permeate magnesium sulfate concentration). And can be calculated by Equation 2.

  Magnesium sulfate permeability (%) = (permeate magnesium sulfate concentration) / (raw water magnesium sulfate concentration) × 100 (Equation 2).

  Moreover, the ratio of the magnesium sulfate permeability to the citric acid permeability is preferably 3 or more at an operating pressure of 0.5 MPa, a raw water temperature of 25 ° C., and a raw water concentration of 1000 ppm. If the ratio of the magnesium sulfate permeability to the citric acid permeability of the nanofiltration membrane under the above conditions is 3 or more, the inorganic salt contained in the hydroxycarboxylic acid-containing solution is removed and the hydroxycarboxylic acid is efficiently permeated. This is preferable. Here, the citric acid permeability is calculated by a method for calculating the hydroxycarboxylic acid permeability described later.

In addition, a nanofiltration membrane having a removal rate of sodium chloride (500 mg / L) of 45% or more is preferably used. The permeation performance of the nanofiltration membrane is such that the permeate flow rate (m ³ / m ² / day) of sodium chloride (500 mg / L) per unit area of the membrane is 0.5 or more and 0.3 at a filtration pressure of 0.3 MPa. A nanofiltration membrane of 8 or less is preferably used. As a method for evaluating the permeation flow rate (membrane permeation flux) per membrane unit area, the permeate amount, the time during which the permeate amount was sampled, and the membrane area can be calculated by Equation 3.

Membrane permeation flux (m ³ / m ² / day) = permeate amount / membrane area / water sampling time (formula 3).

  As a material for the nanofiltration membrane used in the present invention, a polymer material such as cellulose acetate polymer, polyamide, polyester, polyimide, vinyl polymer can be used. It is not limited to this, and a film including a plurality of film materials may be used. In addition, the membrane structure has a dense layer on at least one side of the membrane, and on the asymmetric membrane having fine pores gradually increasing from the dense layer to the inside of the membrane or the other side, or on the dense layer of the asymmetric membrane. Either a composite film having a very thin functional layer formed of another material may be used. As the composite membrane, for example, a composite membrane described in JP-A-62-201606 in which a nanofilter composed of a polyamide functional layer is formed on a support membrane made of polysulfone as a membrane material can be used.

  Among these, a composite film having a high-pressure resistance, high water permeability, and high solute removal performance and having an excellent potential and using a polyamide as a functional layer is preferable. In order to maintain durability against operating pressure, high water permeability, and blocking performance, a structure in which polyamide is used as a functional layer and is held by a support made of a porous membrane or nonwoven fabric is suitable. As the polyamide nanofiltration membrane, a composite nanofiltration membrane having a functional layer of a crosslinked polyamide obtained by a polycondensation reaction between a polyfunctional amine and a polyfunctional acid halide is suitable.

  In the nanofiltration membrane having a polyamide as a functional layer, preferable carboxylic acid components of monomers constituting the polyamide include, for example, trimesic acid, benzophenone tetracarboxylic acid, trimellitic acid, pyrometic acid, isophthalic acid, terephthalic acid, naphthalene Aromatic carboxylic acids such as dicarboxylic acid, diphenyl carboxylic acid, pyridine carboxylic acid and the like can be mentioned, but considering solubility in a film forming solvent, trimesic acid, isophthalic acid, terephthalic acid, and a mixture thereof are more preferable.

  Preferred amine components of the monomers constituting the polyamide include m-phenylenediamine, p-phenylenediamine, benzidine, methylenebisdianiline, 4,4′-diaminobiphenyl ether, dianisidine, 3,3 ′, 4- Triaminobiphenyl ether, 3,3 ′, 4,4′-tetraaminobiphenyl ether, 3,3′-dioxybenzidine, 1,8-naphthalenediamine, m (p) -monomethylphenylenediamine, 3,3′- Monomethylamino-4,4′-diaminobiphenyl ether, 4, N, N ′-(4-aminobenzoyl) -p (m) -phenylenediamine-2,2′-bis (4-aminophenylbenzimidazole), 2 , 2′-bis (4-aminophenylbenzoxazole), 2,2′-bis (4-aminophenyl) Secondary diamines such as primary diamines having an aromatic ring such as nilbenzothiazole), piperazine, piperidine or derivatives thereof, among which nanofiltration membranes having a functional layer of a crosslinked polyamide containing piperazine or piperidine as a monomer are It is preferably used because it has heat resistance and chemical resistance in addition to pressure resistance and durability. More preferably, the cross-linked piperazine polyamide or the cross-linked piperidine polyamide is a main component and a polyamide containing the constituent represented by the chemical formula (1), and more preferably the cross-linked piperazine polyamide is a main component, and the chemical formula It is a polyamide containing the structural component shown by (1). In the chemical formula (1), n = 3 is preferably used. Examples of the nanofiltration membrane having a functional layer made of a polyamide containing a crosslinked piperazine polyamide as a main component and containing the component represented by the chemical formula (1) include those described in JP-A No. 62-201606. As a specific example, a cross-linked piperazine polyamide system manufactured by Toray Industries, Inc., which has a cross-linked piperazine polyamide as a main component and a functional layer including a polyamide containing n = 3 in the chemical formula (1) as a constituent component. One example is a nanofiltration membrane UTC60.

  The nanofiltration membrane is generally used as a spiral membrane element, but the nanofiltration membrane used in the present invention is also preferably used as a spiral membrane element. Specific examples of preferred nanofiltration membrane elements include, for example, GEsepa, a nanofiltration membrane manufactured by GE Osmonics, which is a cellulose acetate-based nanofiltration membrane, NF99 or NF99HF, which is a nanofiltration membrane manufactured by Alfa Laval, whose functional layer is polyamide. NF-45, NF-90, NF-200, or NF-400, a nanofiltration membrane manufactured by Filmtec Co., Ltd. having a cross-linked piperazine polyamide as a functional layer, and having the cross-linked piperazine polyamide as a main component and represented by the chemical formula (1) The nanofiltration membrane module SU-210, SU-220, SU-600 or SU-610 made by the company including UTC60 made by Toray Industries, Inc. having a functional layer of polyamide containing a constituent component, more preferably polyamide is used. NF of Alfa Laval nanofiltration membrane as functional layer 9 or NF99HF, NF-45, NF-90, NF-200, or NF-400, a nanofiltration membrane manufactured by Filmtec Co., Ltd. having a functional layer of cross-linked piperazine polyamide and a cross-linked piperazine polyamide as a main component, and the chemical formula (1 It is a nanofiltration membrane module SU-210, SU-220, SU-600 or SU-610 manufactured by Toray Industries, Inc., which has a functional layer of a polyamide containing the structural component shown in FIG. Is a nanofiltration membrane module SU-210, SU- manufactured by the same company, including UTC60 manufactured by Toray Industries, Inc., which has a cross-linked piperazine polyamide as a main component and a functional layer of a polyamide containing the component represented by the chemical formula (1). 220, SU-600 or SU-610.

  Filtration of the hydroxycarboxylic acid-containing solution in the step A with a nanofiltration membrane may be applied, and the filtration pressure is preferably used in the range of 0.1 MPa to 8 MPa. If the filtration pressure is lower than 0.1 MPa, the membrane permeation rate decreases, and if it is higher than 8 MPa, the membrane may be damaged. In addition, if the filtration pressure is 0.5 MPa or more and 7 MPa or less, the membrane permeation flux is high, so that the hydroxycarboxylic acid solution can be efficiently permeated, and the possibility of affecting the membrane damage is low. It is more preferable that it is used at 1 MPa or more and 6 MPa or less.

  The concentration of the hydroxycarboxylic acid in the hydroxycarboxylic acid-containing solution that is filtered through the nanofiltration membrane in step A is not particularly limited, but if the concentration is high, the concentration of the hydroxycarboxylic acid contained in the permeate is also high. Since the time for concentration can be shortened, it is suitable for cost reduction.

  The concentration of the inorganic salt contained in the hydroxycarboxylic acid-containing solution in step A is not particularly limited and may be equal to or higher than the saturation solubility. That is, if the inorganic salt is less than or equal to the saturation solubility, it is dissolved in the culture solution, and if the solubility is greater than or equal to the saturation solubility, it is partially precipitated. Since it is possible to remove or prevent any dissolved or dissolved in the solution, the hydroxycarboxylic acid can be used without being restricted by the concentration of the inorganic salt. It can be filtered.

  The hydroxycarboxylic acid permeability can be calculated and evaluated as an evaluation method of the hydroxycarboxylic acid nanofiltration membrane permeability when separating the hydroxycarboxylic acid from the hydroxycarboxylic acid-containing solution by the above method. The hydroxycarboxylic acid permeability is determined by analyzing the hydroxycarboxylic acid concentration (raw water hydroxycarboxylic acid concentration) contained in the raw water (culture solution) and the permeating solution (hydroxycarboxylic acid-containing solution) by analysis typified by high performance liquid chromatography. By calculating the concentration of hydroxycarboxylic acid contained (permeate hydroxycarboxylic acid concentration), it can be calculated by Equation 4.

  Hydroxycarboxylic acid permeability (%) = (permeated liquid hydroxycarboxylic acid concentration / raw water hydroxycarboxylic acid concentration) × 100 (Formula 4).

  The method for producing hydroxycarboxylic acid of the present invention is used for the step C of further recrystallizing the hydroxycarboxylic acid-containing solution from which the inorganic salt obtained by filtering the hydroxycarboxylic acid-containing solution of step A with a nanofiltration membrane is removed. Thus, it is characterized by obtaining a high purity hydroxycarboxylic acid. There is no restriction | limiting in particular in the method of recrystallization of the hydroxycarboxylic acid in process C, Known crystallization techniques, such as cooling crystallization and evaporation crystallization, are applicable. As a specific example of obtaining a hydroxycarboxylic acid crystal in the present invention, the crystal can be obtained with high purity by cooling the hydroxycarboxylic acid-containing solution recovered in Step A or Step B. Further, for the purpose of promoting crystallization, a seed crystal of hydroxycarboxylic acid may be added during the cooling process of the hydroxycarboxylic acid-containing solution. The obtained crystals can be recovered by filtration with qualitative filter paper or by centrifugation. Further, by adding a hydroxycarboxylic acid aqueous solution to the recovered crystals, stirring, and filtering again with a qualitative filter paper, the crystals can be washed, and a higher purity hydroxycarboxylic acid crystal can be obtained.

  Moreover, you may concentrate the hydroxycarboxylic acid containing solution which permeate | transmitted the nanofiltration membrane before using for the said process C. FIG. As a concentration method, the hydroxycarboxylic acid-containing solution may be concentrated using a concentrator represented by an evaporator, and the hydroxycarboxylic acid-containing solution obtained in Step A is further filtered through a reverse osmosis membrane. The step B may be used for increasing the hydroxycarboxylic acid concentration. In addition, from the viewpoint of energy reduction for concentration, Step B is preferably employed in which the concentration of hydroxycarboxylic acid is increased by filtration through a reverse osmosis membrane.

  The reverse osmosis membrane here is a filtration membrane that removes ions and low molecular weight molecules by using a pressure difference equal to or higher than the osmotic pressure of water to be treated as a driving force. For example, a cellulose-based cellulose compound such as cellulose acetate or a polyfunctional amine compound A film in which a polyamide separation functional layer is provided on a microporous support film by polycondensation with a polyfunctional acid halide can be employed. In order to suppress fouling on the reverse osmosis membrane surface, the surface of the polyamide separation functional layer is coated with an aqueous solution of a compound having at least one reactive group that reacts with an acid halide group and remains on the surface of the separation functional layer. A low fouling reverse osmosis membrane mainly for sewage treatment in which a covalent bond is formed between the acid halogen group to be reacted and the reactive group can also be preferably employed. Since most of the divalent calcium ions can be removed in the step A of the present invention, stable membrane concentration can be performed without generating scale on the reverse osmosis membrane surface. Moreover, the hydroxycarboxylic acid containing solution concentrated at the process B can be used suitably for the process C which recrystallizes a hydroxycarboxylic acid. Since most of impurities such as inorganic salts can be removed in Step A and concentrated without heating in Step B, the hydroxycarboxylic acid can be recovered with high efficiency.

  As a reverse osmosis membrane preferably used in the present invention, a composite membrane using a cellulose acetate-based polymer as a functional layer (hereinafter also referred to as a cellulose acetate-based reverse osmosis membrane) or a composite membrane using a polyamide as a functional layer (hereinafter, Polyamide-based reverse osmosis membrane). Here, as the cellulose acetate-based polymer, organic acid esters of cellulose such as cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose propionate, cellulose butyrate and the like, or a mixture thereof and those using mixed esters can be mentioned. It is done. The polyamide includes a linear polymer or a crosslinked polymer having an aliphatic and / or aromatic diamine as a monomer.

  As the membrane form, an appropriate form such as a flat membrane type, a spiral type, and a hollow fiber type can be used.

  Specific examples of the reverse osmosis membrane used in the present invention include, for example, low pressure type SU-710, SU-720, SU-720F, SU-710L, SU, which are polyamide-based reverse osmosis membrane modules manufactured by Toray Industries, Inc. -720L, SU-720LF, SU-720R, SU-710P, SU-720P, high pressure type SU-810, SU-820, SU-820L, SU-820FA, SU-820FA, cellulose acetate System reverse osmosis membrane SC-L100R, SC-L200R, SC-1100, SC-1200, SC-2100, SC-2200, SC-3100, SC-3200, SC-8100, SC-8200, manufactured by Nitto Denko Corporation NTR-759HR, NTR-729HF, NTR-70SWC, ES10-D, ES20-D, ES20 U, ES15-D, ES15-U, LF10-D, Alfa Laval RO98pHt, RO99, HR98PP, CE4040C-30D, GE GE Sepa, Filmtec BW30-4040, TW30-4040, XLE-4040, LP-4040, LE-4040, SW30-4040, SW30HRLE-4040, etc. are mentioned.

  Next, production of hydroxycarboxylic acid by fermentation culture of microorganisms that can be used in the method for producing hydroxycarboxylic acid of the present invention will be described.

  The microorganism is not particularly limited as long as it can produce hydroxycarboxylic acid. Specific examples of microorganisms capable of producing hydroxycarboxylic acid include yeast described in JP-A-10-174593, actinomycetes described in JP-A-10-174594, Escherichia coli, and JP-A-9-234091. Examples thereof include microorganisms such as Pseudomonas aeruginosa, coryneform bacteria described in JP-A-2005-27533, and Bacillus bacteria. Cultured cells such as animal cells and insect cells are also included in the microorganism used in the present invention. The microorganism to be used may be isolated from the natural environment, or may be partially modified by mutation or genetic recombination.

  The fermentation raw material used for the production of hydroxycarboxylic acid by fermentation culture of microorganisms may be any material that promotes the growth of the microorganism to be cultured and can produce the desired fermentation product hydroxycarboxylic acid satisfactorily. A normal liquid medium containing a nitrogen source, inorganic salts, and if necessary, organic micronutrients such as amino acids and vitamins is preferable. Carbon sources include sugars such as glucose, sucrose, fructose, galactose, lactose, starch saccharified solution containing these sugars, sweet potato molasses, sugar beet molasses, high test molasses, and organic acids such as acetic acid, alcohols such as ethanol And glycerin are also used. Nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein degradation products, other amino acids, vitamins, corn Steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and the like can be appropriately added. When the microorganism used in the present invention requires a specific nutrient (for example, an amino acid) for growth, the nutrient is added as such or as a natural product containing it. Moreover, you may use an antifoamer as needed. In the present invention, the culture solution refers to a solution obtained as a result of growth of microorganisms or cultured cells as fermentation raw materials. You may change suitably the composition of the fermentation raw material added to a culture solution from the fermentation raw material composition at the time of a culture | cultivation start so that the productivity of the target hydroxycarboxylic acid may become high.

  After cultivating microorganisms, batch culture or fed-batch culture is performed to increase the concentration of microorganisms, and then continuous culture (pulling) may be started. You can go. It is possible to supply the raw material culture solution and extract the culture from an appropriate time. The starting times of the supply of the raw material culture solution and the withdrawal of the culture are not necessarily the same. Further, the supply of the raw material culture solution and the withdrawal of the culture may be continuous or intermittent. What is necessary is just to add a nutrient required for microbial cell growth as shown above to a raw material culture solution so that microbial cell growth may be performed continuously. The concentration of microorganisms or cultured cells in the culture solution should be maintained at a high level so that the environment of the culture solution is not suitable for the growth of microorganisms or cultured cells and the rate of death does not increase. As an example, good production efficiency can be obtained by maintaining the dry weight at 5 g / L or more.

  The continuous culture operation performed while growing fresh cells having fermentation production ability is usually preferably performed in a single fermenter in terms of culture management. However, the number of fermenters is not limited as long as it is a continuous culture method for producing a product while growing cells. A plurality of fermenters may be used because the capacity of the fermenter is small. In this case, high productivity of the fermented product can be obtained even if continuous fermentation is performed by connecting a plurality of fermenters in parallel or in series by piping.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to a following example.

(Reference Example 1) Evaluation of magnesium sulfate rejection rate of nanofiltration membrane 10 g of magnesium sulfate (Wako Pure Chemical Industries, Ltd.) 10 g was added to 10 L of ultrapure water and stirred at 25 ° C. for 1 hour to prepare a 1000 ppm magnesium sulfate aqueous solution. Next, 10 L of the magnesium sulfate aqueous solution prepared above was injected into the raw water layer 1 of the membrane filtration apparatus shown in FIG. As the 90 φ nanofiltration membrane indicated by reference numeral 7 in FIG. 2, a crosslinked piperazine polyamide-based nanofiltration membrane “UTC60” (Nanofiltration membrane 1; manufactured by Toray), a crosslinked piperazine polyamide-based nanofiltration membrane “NF-400” (nanofiltration membrane) 2; manufactured by Filmtech), a polyamide-based nanofiltration membrane “NF99” (nanofiltration membrane 3; manufactured by Alfa Laval), and a cellulose acetate-based nanofiltration membrane “GEsepa” (nanofiltration membrane 4; manufactured by GE Osmonics) are each made of stainless steel (SUS316). The raw water temperature was adjusted to 25 ° C., the pressure of the high-pressure pump 3 was adjusted to 0.5 MPa, and the permeate 4 was collected. The concentration of magnesium sulfate contained in the raw water tank 1 and the permeate 4 was analyzed by ion chromatography (manufactured by DIONEX) under the following conditions, and the rejection rate of magnesium sulfate was calculated.

  Anion; column (AS4A-SC (manufactured by DIONEX)), eluent (1.8 mM sodium carbonate / 1.7 mM sodium bicarbonate), temperature (35 ° C.).

  Cation; column (CS12A (manufactured by DIONEX)), eluent (20 mM methanesulfonic acid), temperature (35 ° C.).

  The results are shown in Table 1.

表１の結果より、ＵＴＣ６０（ナノ濾過膜１：東レ株式会社製）が最も無機塩の阻止率が高いことが示された。

From the results in Table 1, it was shown that UTC60 (nanofiltration membrane 1: manufactured by Toray Industries, Inc.) had the highest inorganic salt rejection.

Reference Example 2 Citric Acid Permeability Evaluation of Nanofiltration Membrane 10 g of citric acid (Wako Pure Chemical Industries, Ltd.) was added to 10 L of ultrapure water and stirred at 25 ° C. for 1 hour to prepare a 1000 ppm citric acid aqueous solution. Subsequently, the permeate of the nanofiltration membranes 1 to 4 was collected under the same conditions as in Reference Example 1. The citric acid concentration contained in the raw water tank 1 and the permeate 4 is analyzed by high performance liquid chromatography (manufactured by Shimadzu Corporation) under the following conditions, and the citric acid permeability and citric acid permeability / magnesium sulfate permeability Was calculated.

  Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation), mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min), reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min), detection method: electrical conductivity, temperature: 45 ° C.

  The results are shown in Table 2.

（参考例３〜１４）ナノ濾過膜のヒドロキシカルボン酸透過性評価
超純水２０Ｌにグリコール酸（和光純薬工業株式会社製）１０ｇ、２−ヒドロキシカルボン酸（和光純薬工業株式会社製）１０ｇ、３−ヒドロキシカルボン酸（和光純薬工業株式会社製）１０ｇ、リンゴ酸（和光純薬工業株式会社製）１０ｇを添加して２５℃１時間攪拌して各ヒドロキシカルボン酸を５００ｐｐｍ含有する水溶液を調製した。次いで、図１に示す、膜濾過装置の原水槽１に上記ヒドロキシカルボン酸含有溶液２０Ｌを注入した。図２の符号７の９０φナノ濾過膜として前記ナノ濾過膜１〜４をステンレス（ＳＵＳ３１６製）製のセルにそれぞれセットし、高圧ポンプ３の圧力をそれぞれ１ＭＰａ（参考例３〜６）、３ＭＰａ（参考例７〜１０）、５ＭＰａ（参考例１１〜１４）に調整し、それぞれの圧力における透過液５を回収した。原水槽１、透過液５に含まれる各ヒドロキシカルボン酸濃度を高速液体クロマトグラフィー（ＢＥＣＫＭＡＮ ＣＯＵＬＴＥＲ社製）により分析し、ヒドロキシカルボン酸の透過率を算出した。

(Reference Examples 3 to 14) Hydroxycarboxylic acid permeability evaluation of nanofiltration membrane 10 g of glycolic acid (manufactured by Wako Pure Chemical Industries, Ltd.) and 20 g of 2-hydroxycarboxylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) in 20 L of ultrapure water 10 g of 3-hydroxycarboxylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) and 10 g of malic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were added and stirred at 25 ° C. for 1 hour to prepare an aqueous solution containing 500 ppm of each hydroxycarboxylic acid. Prepared. Next, the hydroxycarboxylic acid-containing solution 20L was injected into the raw water tank 1 of the membrane filtration apparatus shown in FIG. As the 90φ nanofiltration membrane of reference numeral 7 in FIG. 2, the nanofiltration membranes 1 to 4 are set in cells made of stainless steel (manufactured by SUS316), and the pressure of the high-pressure pump 3 is 1 MPa (Reference Examples 3 to 6), 3 MPa ( Reference Examples 7 to 10) were adjusted to 5 MPa (Reference Examples 11 to 14), and the permeate 5 at each pressure was recovered. Each hydroxycarboxylic acid concentration contained in the raw water tank 1 and the permeate 5 was analyzed by high performance liquid chromatography (manufactured by BECKMAN COULTER), and the hydroxycarboxylic acid permeability was calculated.

Column: Inertsil ODS-3 (manufactured by GL Science), ZORBAX SB-CN (manufactured by Agilent), mobile phase: acetonitrile / 0.1% phosphoric acid aqueous solution = 5/95 (flow rate 1 mL / min), detection method: UV 210 nm , Temperature: 40 ° C
The results are shown in Table 3.

（参考例１５）ナノ濾過膜で濾過する発酵培養液の準備
ヒドロキシカルボン酸の分離精製実験に供するヒドロキシカルボン酸含有溶液として、乳酸発酵培養液に各種ヒドロキシカルボン酸を添加したヒドロキシカルボン酸含有モデル培養液を調製した。以下に乳酸発酵培養液の作製方法を示す。

(Reference Example 15) Preparation of fermentation broth to be filtered through nanofiltration membrane Hydroxycarboxylic acid-containing model culture in which various hydroxycarboxylic acids are added to lactic acid fermentation broth as a hydroxycarboxylic acid-containing solution for separation and purification experiment of hydroxycarboxylic acid A liquid was prepared. A method for preparing a lactic acid fermentation broth is shown below.

(Production of yeast strains capable of producing lactic acid)
Yeast strains capable of producing lactic acid were constructed as follows. Specifically, a yeast strain having L-lactic acid production ability was constructed by linking a human-derived L-LDH gene downstream of the PDC1 promoter on the yeast genome. For polymerase chain reaction (PCR), La-Taq (manufactured by Takara Shuzo Co., Ltd.) or KOD-Plus-polymerase (manufactured by Toyobo Co., Ltd.) was used according to the attached instruction manual.

  After culturing and recovering human breast cancer cell line (MCF-7), total RNA was extracted using TRIZOL Reagent (Invitrogen), and SuperScript Choice System (Invitrogen) was used with the obtained total RNA as a template. CDNA was synthesized by reverse transcription reaction. Details of these operations followed the attached protocol. The obtained cDNA was used as an amplification template for subsequent PCR.

  The L-LDH gene was cloned by PCR using KOD-Plus-polymerase using the cDNA obtained by the above operation as an amplification template and the oligonucleotides represented by SEQ ID NO: 1 and SEQ ID NO: 2 as a primer set. Each PCR amplified fragment was purified, and the end was phosphorylated with T4 Polynucleotide Kinase (Takara Shuzo Co., Ltd.), and then ligated to pUC118 vector (cut with restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver.2 (Takara Shuzo Co., Ltd.). Escherichia coli DH5α was transformed with the ligation plasmid product, and the plasmid DNA was recovered to obtain a plasmid in which the human-derived L-LDH gene (accession number; AY009108, SEQ ID NO: 3) was subcloned. The obtained pUC118 plasmid into which the L-LDH gene was inserted was digested with restriction enzymes XhoI and NotI, and the resulting DNA fragments were inserted into the XhoI / NotI cleavage sites of the yeast expression vector pTRS11. In this way, a human-derived L-LDH gene expression plasmid pL-LDH5 was obtained.

  1.3 kb human-derived L-LDH gene by PCR using plasmid pL-LDH5 containing human-derived L-LDH gene as an amplification template and oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 5 as a primer set, and Saccharomyces -A DNA fragment containing the terminator sequence of the TDH3 gene derived from cerevisiae was amplified. In addition, a DNA fragment containing the TRP1 gene derived from Saccharomyces cerevisiae of 1.2 kb was amplified by PCR using the plasmid pRS424 as an amplification template and the oligonucleotides represented by SEQ ID NO: 6 and SEQ ID NO: 7 as a primer set. Each DNA fragment was separated by 1.5% agarose gel electrophoresis and purified according to a conventional method. A product obtained by the PCR method using a mixture of the 1.3 kb fragment and the 1.2 kb fragment obtained here as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 7 as a primer set is 1 2.5% agarose gel electrophoresis was carried out to prepare a 2.5 kb DNA fragment linked with human-derived LDH gene and TRP1 gene according to a conventional method. Saccharomyces cerevisiae strain NBRC10505 was transformed with this 2.5 kb DNA fragment in a conventional manner so as not to require tryptophan.

  Confirmation that the obtained transformed cells were cells in which the human-derived L-LDH gene was linked downstream of the PDC1 promoter on the yeast genome was performed as follows. First, genomic DNA of a transformed cell was prepared according to a conventional method, and a 0.7 kb amplified DNA fragment was obtained by PCR using the oligonucleotides represented by SEQ ID NO: 8 and SEQ ID NO: 9 as a primer set. Confirmed by being. In addition, whether or not the transformed cell has lactic acid production ability is shown below that lactic acid is contained in the culture supernatant obtained by culturing the transformed cell in SC medium (METHODS IN YEAS GENETIC 2000 EDITION, CSHL PRESS). It confirmed by measuring the amount of lactic acid by HPLC method on conditions.

  Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation), mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min), reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min), detection method: electrical conductivity, temperature: 45 ° C.

  As a result of HPLC analysis, 4 g / L of L-lactic acid was detected, and D-lactic acid was below the detection limit. From the above examination, it was confirmed that this transformant has L-lactic acid production ability. The obtained transformed cell was used for subsequent batch fermentation as yeast strain SW-1.

(Production of L-lactic acid fermentation broth by batch fermentation)
The most typical batch fermentation was performed as a fermentation form using microorganisms, and the lactic acid productivity was evaluated. A batch fermentation test was performed using the lactic acid fermentation medium shown in Table 4. The medium was used after autoclaving (121 ° C., 15 minutes). The yeast SW-1 strain constructed in Reference Example 1 was used as a microorganism, and the concentration of lactic acid as a product was evaluated using the HPLC shown in Reference Example 1, and the glucose concentration was measured using a glucose test Wako. C (Wako Pure Chemical Industries, Ltd.) was used. The operating conditions for fermentation culture are shown below.

  Reaction tank capacity (lactic acid fermentation medium amount): 2 (L), temperature adjustment: 30 (° C.), reaction tank aeration rate: 0.2 (L / min), reaction tank stirring speed: 400 (rpm), pH adjustment: Adjust to pH 5 with 1N calcium hydroxide.

  First, the SW-1 strain was cultured with shaking in a 5 ml lactic acid fermentation medium overnight in a test tube (pre-culture). The culture solution was inoculated into 100 ml of fresh lactic acid fermentation medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (pre-culture). Temperature adjustment and pH adjustment were performed, and fermentation culture was performed. The amount of bacterial cell growth at this time was 15 in terms of absorbance at 600 nm.

（実施例１〜１７）ナノ濾過膜によるグリコール酸の分離実験
（ナノ濾過膜で濾過する培養液の準備）
参考例１５で発酵した培養液２Ｌに、グリコール酸８０ｇを添加し、グリコール酸を４０ｇ／Ｌ含有するモデル培養液を調製した。

Examples 1 to 17 Glycolic acid separation experiment using nanofiltration membrane (Preparation of culture solution to be filtered through nanofiltration membrane)
Glycolic acid 80 g was added to 2 L of the culture broth fermented in Reference Example 15 to prepare a model culture solution containing 40 g / L of glycolic acid.

(Generation of sparingly soluble calcium salt with sulfuric acid)
Concentrated sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to 2 L of the glycolic acid-containing culture solution prepared above until the pH was 1.9, 2.0, 2.2, 2.6, 4.0, It stirred at 25 degreeC for 1 hour, and produced | generated glycolic acid and calcium sulfate from the calcium glycolate in a culture solution. Subsequently, the precipitated calcium sulfate was suction filtered with qualitative filter paper No. 2 (manufactured by Advantech Co., Ltd.), the precipitate was filtered off, and 2 L of filtrate was collected. In addition, a separation experiment was also performed on a culture solution (2 L) having a pH of 5 to which no concentrated sulfuric acid was added (Example 17).

(Separation of glycolic acid by nanofiltration membrane)
Next, 2 L of the filtrate obtained above was injected into the raw water tank 1 of the membrane filtration apparatus shown in FIG. As the 90φ nanofiltration membrane of reference numeral 7 in FIG. 2, the nanofiltration membranes 1 to 4 are respectively set in cells made of stainless steel (made of SUS316), and the pressure of the high-pressure pump 3 is adjusted to 1 MPa, 3 MPa, and 5 MPa, respectively. The permeate 4 at the pressure of The concentration of sulfate ion and calcium ion contained in raw water tank 1 and permeate 4 is the same as in Reference Example 1, ion chromatography (manufactured by DIONEX), and the concentration of glycolic acid is the same as in Reference Examples 3-14. And analyzed by high performance liquid chromatography (manufactured by BECKMAN COULTER). The results are shown in Table 5.

表５に示すように、すべてのｐＨ、濾過圧力において、硫酸カルシウムが高効率で除去されたことがわかった。また、上記実施例１〜１７において、ナノ濾過膜を新しい膜に取り換えることなく、上記の濾加圧において、硫酸カルシウムが高効率で除去された。

As shown in Table 5, it was found that calcium sulfate was removed with high efficiency at all pH and filtration pressures. In Examples 1 to 17, calcium sulfate was removed with high efficiency in the filtration and pressurization without replacing the nanofiltration membrane with a new membrane.

(Examples 18 to 34) Separation experiment of 3-hydroxybutyric acid by nanofiltration membrane Model culture solution containing 80 g of 3-hydroxybutyric acid in 2 L of the culture solution fermented in Reference Example 15 and containing 40 g / L of 3-hydroxybutyric acid The sample was subjected to a separation experiment of 3-hydroxybutyric acid using a nanofiltration membrane in the same manner as in Examples 1 to 17. The 3-hydroxybutyric acid concentration in the permeate was analyzed by high performance liquid chromatography (manufactured by BECKMAN COULTER) under the same conditions as in Reference Examples 3-14. The results are shown in Table 6.

（実施例３５〜５１）ナノ濾過膜によるリンゴ酸の分離実験
参考例１５で発酵した培養液２Ｌにリンゴ酸８０ｇを添加してリンゴ酸を４０ｇ／Ｌ含有するモデル培養液を調製し、実施例１〜１７と同様の操作によりナノ濾過膜によるリンゴ酸の分離実験を行った。透過液中のリンゴ酸濃度は参考例３〜１４と同様の条件で高速液体クロマトグラフィー（ＢＥＣＫＭＡＮ ＣＯＵＬＴＥＲ社製）により分析した。結果を表７に示す。

(Examples 35 to 51) Separation experiment of malic acid with nanofiltration membrane A model culture solution containing 40 g / L of malic acid was prepared by adding 80 g of malic acid to 2 L of the culture solution fermented in Reference Example 15, and Examples A separation experiment of malic acid using a nanofiltration membrane was performed in the same manner as in 1-17. The malic acid concentration in the permeate was analyzed by high performance liquid chromatography (manufactured by BECKMAN COULTER) under the same conditions as in Reference Examples 3-14. The results are shown in Table 7.

（比較例１〜３）定性濾紙を用いた培養液からの難溶性硫酸塩の除去実験
実施例９、２６、４３と同様の方法で調製したグリコール酸、３−ヒドロキシ酪酸、リンゴ酸含有培養液（各２Ｌ）をｐＨが２．６になるまで濃硫酸を滴下後、１時間２５℃で攪拌し、培養液中のグリコール酸カルシウム、３−ヒドロキシ酪酸カルシウム、リンゴ酸カルシウムをそれぞれグリコール酸、３−ヒドロキシ酪酸、リンゴ酸、硫酸カルシウムに変換した。次いで、沈殿した硫酸カルシウムを定性濾紙Ｎｏ．２を用いて吸引濾過により、沈殿物を濾別し、濾液（各２Ｌ）を回収した。得られた濾液中の硫酸イオン、カルシウムイオン、グリコール酸（比較例１）、３−ヒドロキシ酪酸（比較例２）、リンゴ酸（比較例３）の濃度を上記と同様に測定した。結果を表８に示す。

(Comparative Examples 1 to 3) Removal experiment of hardly soluble sulfate from culture solution using qualitative filter paper Glycolic acid, 3-hydroxybutyric acid, malic acid-containing culture solution prepared in the same manner as in Examples 9, 26 and 43 Concentrated sulfuric acid was added dropwise until pH of 2.6 (each 2 L) was stirred for 1 hour at 25 ° C., and calcium glycolate, calcium 3-hydroxybutyrate, and calcium malate in the culture solution were glycolic acid, 3 -Converted to hydroxybutyric acid, malic acid, calcium sulfate. Subsequently, the precipitated calcium sulfate was added to the qualitative filter paper No. The precipitate was filtered off by suction filtration using 2 and the filtrate (2 L each) was recovered. The concentrations of sulfate ion, calcium ion, glycolic acid (Comparative Example 1), 3-hydroxybutyric acid (Comparative Example 2), and malic acid (Comparative Example 3) in the obtained filtrate were measured in the same manner as described above. The results are shown in Table 8.

表８に示すように、定性濾紙を用いて濾過した濾液中のカルシウムイオンは、ナノ濾過膜を用いて濾過した濾液中のカルシウムイオンの約１０００倍もの残存が検出された。このことから、ナノ濾過膜を用いることで難溶性塩の効率的な除去が可能であることが示され、さらにはヒドロキシカルボン酸結晶の純度向上につながることが示唆された。

As shown in Table 8, about 1000 times as many calcium ions in the filtrate filtered using the qualitative filter paper as the calcium ions in the filtrate filtered using the nanofiltration membrane were detected. From this, it was shown that the use of the nanofiltration membrane enables efficient removal of the hardly soluble salt, and further suggests that the purity of the hydroxycarboxylic acid crystal is improved.

(Examples 52 to 54) Recrystallization purification from hydroxycarboxylic acid solution using reverse osmosis membrane Permeate of Example 9 (glycolic acid-containing solution, nanofiltration membrane 1, pH 2.6), Example 26 (3- The permeate of hydroxybutyric acid-containing solution, nanofiltration membrane 1, pH 2.6), the permeate of Example 43 (malic acid-containing solution, nanofiltration membrane 1, pH 2.6), 2 L of each, membrane filtration shown in FIG. It poured into the raw water tank 1 of the apparatus. As a 90φ reverse osmosis membrane of reference numeral 7 in FIG. 2, polyamide reverse osmosis membrane (UTC-70, manufactured by Toray Industries, Inc.) is set in a cell made of stainless steel (SUS316), and the pressure of the high pressure pump 3 is set to 3 MPa. After adjustment, the reverse osmosis membrane concentrate 5 was recovered. The hydroxycarboxylic acid concentration contained in the reverse osmosis membrane concentrate was analyzed by high performance liquid chromatography (manufactured by BECKMAN COULTER) under the same conditions as in Reference Examples 3-14. As a result, the glycolic acid concentration was 130.3 g / L, the 3-hydroxybutyric acid concentration was 124 g / L, and the malic acid concentration was 102.2 g / L. This concentrated liquid was cooled to 10 ° C. to precipitate glycolic acid crystals, 2-hydroxybutyric acid crystals, and malic acid crystals. Qualitative filter paper No. Suction filtration was performed using 2 (manufactured by Advantech Co., Ltd.) to separate each hydroxycarboxylic acid crystal. Each obtained hydroxycarboxylic acid crystal was stirred in a hydroxycarboxylic acid aqueous solution of 15 times the weight of the crystal in order to wash the crystal, and collected again by suction filtration. The optical purity before and after recrystallization of each hydroxycarboxylic acid was measured by the HPLC method under the following conditions.

  Column: TSK-gel Enantio L1 (manufactured by Tosoh Corporation), mobile phase: 1 mM copper sulfate aqueous solution, flow rate: 1.0 ml / min, detection method: UV254 nm, temperature: 30 ° C.

  Further, the weight of each hydroxycarboxylic acid crystal after recrystallization was measured, and the recrystallization yield was calculated according to Formula 5.

  Recrystallization yield (%) = 100 × hydroxycarboxylic acid weight in reverse osmosis membrane concentrated liquid / hydroxycarboxylic acid crystal weight (formula 5).

  The results are shown in Table 9.

表９に示すように、ナノ濾過膜による無機塩などの不純物の除去、逆浸透膜濃縮によるラセミ化やオリゴマー化の抑制により、高純度のヒドロキシカルボン酸結晶が得られた。

As shown in Table 9, high-purity hydroxycarboxylic acid crystals were obtained by removing impurities such as inorganic salts with a nanofiltration membrane, and suppressing racemization and oligomerization by reverse osmosis membrane concentration.

(Comparative Examples 4 to 6) Recrystallization purification from hydroxycarboxylic acid solution from which inorganic salts were removed with qualitative filter paper Permeate of Comparative Example 1 (glycolic acid-containing solution, qualitative filter paper No. 2, pH 2.6), Comparative Example 2 The permeate of (3-hydroxybutyric acid-containing solution, qualitative filter paper No. 2, pH 2.6), the permeate of Comparative Example 3 (malic acid-containing solution, qualitative filter paper No. 2, pH 2.6), 2 L each It injected into the raw | natural water tank 1 of the membrane filtration apparatus shown in 1, and reverse osmosis membrane concentration and recrystallization refinement | purification were performed by the same operation as Examples 52-54. The hydroxycarboxylic acid concentration contained in the reverse osmosis membrane concentrate was analyzed by high performance liquid chromatography (manufactured by BECKMAN COULTER) under the same conditions as in Reference Examples 3-14. In addition, the optical purity of each hydroxycarboxylic acid before and after recrystallization was analyzed by HPLC method, and the recrystallization yield was calculated by Formula 5. The results are shown in Table 10.

表１０に示したように、実施例５２〜５４と比較して、ヒドロキシカルボン酸結晶収率が２０％程度低い結果が得られ、定性濾紙で除去しきれなかった不純物の影響により再結晶が阻害されたことが示唆された。

As shown in Table 10, as compared with Examples 52 to 54, a result of about 20% lower hydroxycarboxylic acid crystal yield was obtained, and recrystallization was inhibited by the influence of impurities that could not be removed by qualitative filter paper. It was suggested that

  From the results of the above Examples and Comparative Examples, it was revealed that calcium sulfate in the culture solution can be removed with high efficiency by the nanofiltration membrane. That is, according to the present invention, the calcium component in the hydroxycarboxylic acid-containing culture solution can be removed with high efficiency by the nanofiltration membrane, and the calcium component does not precipitate even when concentrated, and further recrystallized to achieve high purity. It was revealed that a hydroxycarboxylic acid can be produced. Also, by concentrating with a reverse osmosis membrane, it is possible to avoid heating of the hydroxycarboxylic acid-containing solution and suppress the racemization and oligomerization of the hydroxycarboxylic acid to obtain a hydroxycarboxylic acid crystal at a high yield and low cost. It has become possible.

It is a schematic diagram showing one embodiment of the nanofiltration membrane separation device used by the present invention. It is a schematic diagram which shows one embodiment of cell sectional drawing with which the nanofiltration membrane of the nanofiltration membrane separation apparatus used by this invention was mounted | worn.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Raw water tank 2 Cell equipped with nanofiltration membrane or reverse osmosis membrane 3 High pressure pump 4 Flow of membrane permeate 5 Flow of membrane concentrate 6 Flow of culture solution sent by high pressure pump 7 Nanofiltration membrane 8 Support plate

Claims (9)

  A method for producing hydroxycarboxylic acid, comprising a step A in which a hydroxycarboxylic acid (excluding lactic acid) -containing solution is filtered through a nanofiltration membrane to recover hydroxycarboxylic acid from the permeation side.   The hydroxycarboxylic acid is one or a mixture of two or more selected from glycolic acid, hydroxybutyric acid, malic acid, tartaric acid, hydroxyvaleric acid, hydroxycaproic acid, gluconic acid and citric acid. A process for producing a hydroxycarboxylic acid.   The method for producing hydroxycarboxylic acid according to claim 1 or 2, wherein the pH of the hydroxycarboxylic acid-containing solution leading to the nanofiltration membrane in Step A is 2 or more and 5 or less.   Magnesium sulfate permeability of the nanofiltration membrane is 1.5% or less at an operating pressure of 0.5 MPa, a raw water temperature of 25 ° C., and a raw water concentration of 1000 ppm. Production method.   The production of hydroxycarboxylic acid according to any one of claims 1 to 4, wherein the ratio of the magnesium sulfate permeability to the citric acid permeability is 3 or more at an operating pressure of 0.5 MPa, a raw water temperature of 25 ° C, and a raw water concentration of 1000 ppm. Method.   The method for producing a hydroxycarboxylic acid according to any one of claims 1 to 5, wherein the functional layer of the nanofiltration membrane contains polyamide. The method for producing a hydroxycarboxylic acid according to claim 6, wherein the polyamide comprises a crosslinked piperazine polyamide as a main component and a constituent represented by the chemical formula 1.

(In the formula, R represents —H or —CH ₃ , and n represents an integer of 0 to 3.)   The method for producing a hydroxycarboxylic acid according to any one of claims 1 to 7, comprising a step B in which the hydroxycarboxylic acid-containing solution obtained in the step A is filtered through a reverse osmosis membrane to increase the hydroxycarboxylic acid concentration.   The method for producing hydroxycarboxylic acid according to claim 1, wherein the permeate collected from Step A or the concentrate collected from Step B is subjected to Step C for recrystallization.

Images