JP5564783B2 - Method for producing lactic acid - Google Patents

Description

  The present invention relates to a method for producing lactic acid, comprising a step of purifying lactic acid produced in a culture solution by fermentation culture of microorganisms. Specifically, the present invention relates to a method for producing lactic acid, including a step of obtaining a solution containing high-purity calcium lactate from the non-permeate side by passing a microorganism culture solution through a nanofilter.

  Lactic acid is widely applied to industrial uses as a monomer raw material for biodegradable plastics in addition to uses such as foods and pharmaceuticals, and the demand is increasing. 2-Hydroxypropionic acid, i.e., lactic acid, is known to be produced by fermentation by a microorganism, and the microorganism converts a substrate containing a carbohydrate typified by glucose into lactic acid. Lactic acid is classified into (L) -form and (D) -form optical isomers depending on the configuration of the hydroxyl group bonded to the carbon at the carbonyl α-position. According to microbial fermentation, (L) -form or (D) -form lactic acid is selectively selected by appropriately selecting microorganisms, or a mixture of (L) -form and (D) -form (racemic form). Of lactic acid.

  The production of lactic acid by microbial fermentation is generally carried out while maintaining an optimum pH for microbial fermentation by adding an alkaline substance to the culture solution. Since most of lactic acid, which is an acidic substance produced by microbial fermentation, is added with an alkaline substance, most of lactic acid exists as lactate in the culture solution. In this case, free lactic acid can be obtained by adding an acidic substance to the culture solution after the end of fermentation.

  Specifically, calcium hydroxide is often used as an alkaline substance added to the culture solution. In this case, lactic acid produced by microbial fermentation exists as calcium lactate in the culture solution. In addition, since calcium lactate has high calcium absorptivity, it attracts attention for food use as a good quality calcium supply source.

  As a method for obtaining calcium lactate, there is a method of concentrating a culture solution under reduced pressure to evaporate water. However, calcium lactate extracted from microbial fermentation liquor often contains impurities other than lactic acid produced in the fermentation process, such as acetic acid, and the purity of calcium lactate is low. In order to evaporate, there is a problem that heating is required and the energy cost is high.

So far, when manufacturing calcium lactate-containing foods, a technology for nanofiltering to concentrate calcium lactate has been disclosed (see Patent Document 1), but this technology is a fermented liquid containing calcium lactate. The pH of the permeate treated with a nanofilter was adjusted to obtain calcium lactate. However, it is easily predicted by those skilled in the art that the permeate contains acetic acid in addition to lactic acid. The purity of the calcium lactate to be obtained cannot be said to be high.
JP 2001-299281 A

  The present invention solves the above-mentioned problem, that is, the problem of removing organic acids other than lactic acid in the culture solution when lactic acid is produced by microbial fermentation, and efficiently recovers high-purity calcium lactate. It aims to provide a method.

As a result of intensive studies to solve the above problems, the inventors of the present invention passed a microbial fermentation broth adjusted to pH 6-11 through a nanofilter, thereby removing an aqueous solution containing calcium lactate in the broth. It was recovered from the permeate side and found that organic acids other than lactic acid could be removed from the permeate side with high efficiency, and the present invention was completed. That is, this invention is comprised from following (1)-( 4 ).

(1) A method for producing lactic acid comprising a step of purifying calcium lactate produced in a culture solution by fermentation of microorganisms in the presence of a calcium salt, wherein the pH of the culture solution is adjusted to 6 or more and 11 or less And passing the culture solution through a nanofilter to remove acetic acid in the culture solution on the permeate side, and recovering an aqueous solution containing calcium lactate from the non-permeate side.

  (2) The method for producing lactic acid according to (1), wherein the functional layer of the nanofilter contains polyamide.

  (3) The method for producing lactic acid according to (2), wherein the polyamide contains a cross-linked piperazine polyamide as a main component and a constituent represented by Chemical Formula 1.

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

( 4 ) The method for producing lactic acid according to any one of (1) to ( 3 ), wherein in adjusting the pH of the culture solution, calcium hydroxide is added to the culture solution.

The method for producing lactic acid according to the present invention can effectively remove acetic acid, which is an organic acid other than lactic acid, contained in the fermentation broth by a simple operation. Therefore, an aqueous solution containing high-purity calcium lactate can be obtained.

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

  The method for producing lactic acid according to the present invention includes a step of purifying calcium lactate produced in a culture solution by fermentation culture of a microorganism in the presence of a calcium salt, and the pH of the culture solution is 6 or more and 11 or less. And the organic medium other than lactic acid in the culture medium is removed to the permeate side through the nanofilter, and an aqueous solution containing calcium lactate is recovered from the non-permeate side.

  The nanofilter used in the method for producing lactic acid of the present invention is also called a nanofiltration membrane (nanofiltration membrane, NF membrane), and “a membrane that transmits monovalent ions and blocks divalent ions”. Is a film generally defined as 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.

  The material of the nanofilter may be a polymer material such as cellulose acetate polymer, polyamide, polyester, polyimide, vinyl polymer, but is not limited to the film composed of the one kind of material, It may be a film containing a film material. 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, as described in JP-A No. 62-201606, a composite membrane in which a nanofilter composed of a functional layer of polyamide 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 semipermeable membrane, a composite semipermeable membrane having a functional layer of a crosslinked polyamide obtained by polycondensation reaction of a polyfunctional amine and a polyfunctional acid halide on a support is suitable.

  In the nanofilter 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 dicarboxylic acid Examples thereof include aromatic carboxylic acids such as acid, diphenylcarboxylic acid, and pyridinecarboxylic acid, but considering solubility in a film-forming solvent, trimesic acid, isophthalic acid, terephthalic acid, and mixtures 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) Examples include secondary diamines such as primary diamines having an aromatic ring such as (nylbenzothiazole), piperazine, piperidine or derivatives thereof, among which a nanofilter having a functional layer of a crosslinked polyamide containing piperazine or piperidine as a monomer is pressure resistant. It is preferably used because of its heat resistance and chemical resistance in addition to its properties and durability. More preferably, the cross-linked piperazine polyamide or the cross-linked piperidine polyamide is a main component, and the component includes the constituent represented by the chemical formula 1. More preferably, the cross-linked piperazine polyamide is the main component, and the chemical formula 1 Polyamide containing the indicated constituents. In the above chemical formula 1, those having n = 3 are preferably used. Examples of the polyamide containing a crosslinked piperidine polyamide as a main component and containing the constituent represented by the above chemical formula 1 include those described in JP-A No. 62-201606.

  The nanofilter is generally used as a spiral membrane element, but the nanofilter used in the present invention can also be preferably used as a spiral membrane element. As a specific example of a preferable nanofilter, for example, manufactured by the same company including UTC60 manufactured by Toray Industries, Inc. having a functional layer of a polyamide containing a cross-linked piperazine polyamide as a main component and a component represented by the chemical formula (1). Nanofilter modules SU-210, SU-220, SU-600, SU-610 can also be used. In addition, NF-45, NF-90, NF-200, NF-400 made by Filmtec Co., Ltd., which uses crosslinked piperazine polyamide as a functional layer, or NF99, NF97, made by Alfa Laval Corp., which uses polyamide as a functional layer. , NF99HF, GE Sepa made by GE Osmonics, which is a cellulose acetate-based nanofilter, and the like.

  In the method for producing lactic acid of the present invention, “through a nanofilter” means that a culture solution containing lactic acid produced by fermentation of microorganisms is filtered through a nanofilter, and calcium lactate is contained on the non-permeate side. This means that the aqueous solution is recovered by filtration, and acetic acid is permeated as a filtrate to the permeate side.

  Examples of the method for evaluating the nanofilter permeability of an aqueous lactic acid solution include a method of calculating and evaluating lactic acid permeability, but the method is not limited to this method. Lactic acid permeability is determined by analysis represented by high performance liquid chromatography. Lactic acid concentration in raw water (culture solution) (raw water lactic acid concentration) and lactic acid concentration in permeated water (lactic acid solution) (permeated lactic acid concentration) ) Can be calculated by Equation 1.

Lactic acid permeability (%) = (permeated lactic acid concentration / raw water lactic acid concentration) × 100 (Formula 1).
Similar to Equation 1, the acetic acid permeability can be calculated by Equation 2.

  Acetic acid permeability (%) = (permeated water acetic acid concentration / raw water acetic acid concentration) × 100 (Equation 2).

  As a method for evaluating the membrane unit area and the permeation flow rate (membrane permeation flux) per unit pressure, the permeated water amount, the time during which the permeated water amount was collected, 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) (Equation 3).

  In the method for producing lactic acid according to the present invention, filtration of the microorganism culture solution with the nanofilter may be performed under pressure. The filtration pressure is preferably used in the range of 0.1 MPa or more and 8 MPa or less, but if it is lower than 0.1 MPa, the membrane permeation rate decreases, and if it is higher than 8 MPa, it affects the damage of the membrane. When used in the following, the membrane permeation flux is high, so that the lactic acid solution can be efficiently permeated and is less likely to affect the membrane damage, and is preferably used at 1 MPa or more and 6 MPa or less. Particularly preferred.

  As the membrane separation performance of the nanofilter used in the method for producing lactic acid of the present invention, the salt removal rate when an aqueous sodium chloride solution (500 mg / L) adjusted to a temperature of 25 ° C. and a pH of 6.5 was evaluated at a filtration pressure of 0.75 MPa. Is preferably 45% or more. The salt removal rate referred to here can be calculated by Equation 4 by measuring the permeated water salt concentration of the sodium chloride aqueous solution.

  Salt removal rate = 100 × {1− (salt concentration in permeated water / salt concentration in feed water)} (Formula 4).

In addition, the membrane permeation performance of the nanofilter has a membrane permeation flux (m ³ / (m ² · day)) of 0.5 or more at a filtration pressure of 0.3 MPa with sodium chloride (500 mg / L). Is preferably used.

  Moreover, what has a magnesium sulfate permeability of 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 is preferably used. When the magnesium sulfate permeability of the nanofilter under the above conditions exceeds 1.5%, a part of the aqueous solution containing calcium lactate may permeate the nanofilter and the recovery rate from the non-permeation side may be reduced. More preferably, a nanofilter having a magnesium sulfate permeability of 1.0% or less is used. Magnesium sulfate permeability is determined by measuring the magnesium sulfate concentration (raw water magnesium sulfate concentration) contained in the raw water and the magnesium sulfate concentration (permeated magnesium sulfate concentration) contained in the permeate by analysis typified by ion chromatography. , Can be calculated by Equation 5.

  Magnesium sulfate permeability (%) = (permeate magnesium sulfate concentration) / (raw water magnesium sulfate concentration) × 100 (Equation 5).

  In the method for producing lactic acid according to the present invention, the pH of the culture solution leading to the nanofilter is 6 or more and 11 or less. Nanofilters are characterized by the fact that substances that are ionized (dissociated) in solution are more easily blocked than substances that are not ionized (non-dissociated). The proportion of lactic acid dissociated and present as ions in the liquid (dissociated lactic acid / non-dissociated lactic acid) is greater than the proportion of acetic acid dissociated and present as ions (dissociated acetic acid / non-dissociated acetic acid). The aqueous solution containing calcium lactate can be efficiently recovered from the non-permeate side, and organic acids other than lactic acid can be efficiently separated from the permeate side. Furthermore, if the pH of the culture solution exceeds 11, the durability of the nanofilter is adversely affected.

  In the method for producing lactic acid of the present invention, the organic acid other than lactic acid separated from the permeate side of the nanofilter is an organic acid derived from a by-product of fermentation culture or a fermentation raw material. In the present invention, acetic acid is preferred. Can be separated.

  In the method for producing lactic acid of the present invention, as a means for adjusting the pH of the culture solution, basic calcium is preferably used. For example, calcium hydroxide, calcium carbonate, calcium phosphate, calcium oxide, calcium acetate solid, or It is preferable to add an aqueous solution, and calcium hydroxide is more preferable. When the aqueous solution is added, the concentration of basic calcium is not limited, and a slurry in excess of the saturation solubility may be added.

  The concentration of lactic acid in the calcium lactate solution in the microorganism culture solution used for separation by the nanofilter is not particularly limited, but if the concentration is high, the concentration time can be shortened. 10 g / L or more and 100 g / L or less are preferable.

  As described above, high-purity free lactic acid can be obtained by adding an acidic substance to a solution containing calcium lactate collected from the non-permeating side with a nanofilter. Examples of the acidic substance to be added include sulfuric acid, hydrochloric acid, nitric acid and the like.

  Hereinafter, lactic acid production by fermentation culture of microorganisms, which is used in the method for producing lactic acid according to the present invention, will be described.

  The fermentation raw material used for lactic acid production by fermentation culture of microorganisms may be any material that promotes the growth of the microorganisms to be cultured and can produce lactic acid as the desired fermentation product, A normal liquid medium containing inorganic salts and organic micronutrients such as amino acids and vitamins as appropriate 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 productivity of the target lactic acid may become high.

  In the present invention, microorganisms are usually cultured in the range of pH 4-8 and temperature 20-40 ° C. The pH of the culture solution is adjusted to a predetermined value within the above range with an alkaline substance, specifically, a basic calcium salt. As the basic calcium salt, calcium hydroxide, calcium carbonate, calcium phosphate, calcium oxide, calcium acetate and the like are preferably used, and calcium hydroxide is more preferably used.

  In the culture of microorganisms, if it is necessary to increase the oxygen supply rate, add oxygen to the air to maintain the oxygen concentration at 21% or higher, or pressurize the culture, increase the stirring speed, increase the aeration rate, etc. Can be used.

  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.

  The microorganism used in the present invention is not particularly limited, and examples thereof include yeasts such as baker's yeast often used in the fermentation industry, bacteria such as lactic acid bacteria, Escherichia coli, coryneform bacteria, filamentous fungi, actinomycetes, and the like. . Cultured cells such as animal cells and insect cells are also included in the microorganism used in the present invention. Among the microorganisms, it is preferably selected from yeast, lactic acid bacteria, and Escherichia coli. The microorganism to be used may be isolated from the natural environment, or may be partially modified by mutation or genetic recombination.

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

(Production of yeast strains capable of producing lactic acid)
Reference Example 1 Production of Yeast Strain Having Lactic Acid Production Capacity A yeast strain having a lactic acid production capacity was constructed as follows. Specifically, a yeast strain capable of producing L-lactic acid was constructed by linking a human-derived LDH gene downstream of the PDC1 promoter on the yeast genome. For polymerase chain reaction (PCR), La-Taq (manufactured by Takara Shuzo) 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 (manufactured by TAKARA), and then ligated to a pUC118 vector (cut with the restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver.2 (manufactured by TAKARA). Escherichia coli DH5α was transformed with the ligation plasmid product, and plasmid DNA was collected to obtain a plasmid in which various L-ldh genes (accession number; AY009108, SEQ ID NO: 3) were subcloned. The obtained pUC118 plasmid into which the L-ldh gene was inserted was digested with restriction enzymes XhoI and NotI, and each of the obtained DNA fragments was inserted into the XhoI / NotI cleavage site of the yeast expression vector pTRS11. In this way, a human-derived L-ldh gene expression plasmid pL-ldh5 (L-ldh gene) was obtained.

  By PCR using the plasmid pL-ldh5 containing the human-derived LDH gene as an amplification template and the oligonucleotides represented by SEQ ID NO: 4 and SEQ ID NO: 5 as primer sets, a 1.3-kb human-derived LDH gene and Saccharomyces cerevisiae derived A DNA fragment containing the terminator sequence of the TDH3 gene 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 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.

  The optical purity of L-lactic 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.

  Moreover, the optical purity of L-lactic acid is calculated by Formula 4. Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid.

  Optical purity (%) = 100 × (LD) / (L + D) (formula 6).

  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 cells were used in subsequent examples as yeast strain SW-1.

Reference Example 2 Production of L-lactic acid 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 conducted using the lactic acid fermentation medium shown in Table 1. 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) was used. The operating conditions of Reference Example 2 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. The results of batch fermentation are shown in Table 2.

Reference Example 3 Evaluation of inorganic filter (magnesium sulfate) rejection rate of nanofilter 10 g of magnesium sulfate (manufactured by 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 magnesium sulfate aqueous solution. Next, 10 L of the magnesium sulfate aqueous solution prepared above was injected into the raw water tank 1 of the membrane filtration apparatus shown in FIG. As the 90φ nanofilter indicated by reference numeral 7 in FIG. 2, a crosslinked piperazine polyamide-based nanofilter “UTC60” (Nanofilter 1; manufactured by Toray Industries, Inc.), a crosslinked piperazine polyamide-based nanofilter “NF-400” (nanofilter 2; film) Tech), polyamide-based nanofilter “NF99” (Nanofilter 3; manufactured by Alfa Laval), and cellulose acetate-based nanofilter “GEsepa” (Nanofilter 4; manufactured by GE Osmonics) are each set in a cell made of stainless steel (manufactured by SUS316). The raw water temperature was adjusted to 25 ° C. and the pressure of the high-pressure pump 3 was adjusted to 0.5 MPa, and the permeate 5 was recovered. The concentration of magnesium sulfate contained in the raw water tank 1 and the permeate 5 was analyzed by ion chromatography (manufactured by DIONEX) under the following conditions, and the transmittance 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 3.

Examples 1-3
(Preparation of culture solution to be separated by nano filter module)
The culture solution (2 L) fermented with lactic acid in Reference Examples 1 and 2 until pH 7.5 (Example 1), pH 9.0 (Example 2), and pH 10.0 (Example 3). Calcium hydroxide (Wako Pure Chemical Industries) was added.

(Separation experiment with nano filter module)
Next, 2 L of the culture solution obtained above was injected into the raw water tank 1 of the membrane filtration apparatus shown in FIG. A 4-inch nanofilter module 2 (crosslinked piperazine polyamide-based nanofilter module SU-610, manufactured by Toray Industries, Inc.) is set in a dedicated vessel, and the pressure of the high-pressure pump 3 is adjusted to 2 MPa. Liquid 4 and permeate 5 were collected. The concentration of lactic acid and acetic acid contained in the non-permeate 4 and the permeate 5 was analyzed by high performance liquid chromatography (manufactured by Shimadzu Corporation). The results are shown in Table 4.

  As shown in Table 4, at all pH values of 7.5, 9.0, and 10.0, acetic acid is efficiently removed on the permeate side, and an aqueous solution containing high-purity calcium lactate is removed on the non-permeate side. From the above, it was found that the recovery was possible with high efficiency.

  In Examples 1 to 3, the nanofilter module was replaced with a new membrane without using a new membrane, but acetic acid was removed with high efficiency at the filtration pressure.

Examples 4-15
(Preparation of culture solution to be separated by 90φ nano filter)
Calcium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the culture solution (2 L) fermented with lactic acid in Reference Examples 1 and 2 until the pH was 7.5, 9.0, 10.0.

(Separation experiment with nanofiltration membrane)
Next, 2 L of the filtrate obtained in the above example was injected into the raw water tank 1 of the membrane filtration apparatus shown in FIG. As the 90φ nanofilter denoted by reference numeral 7 in FIG. 2, the nanofilters 1 to 4 are set in cells made of stainless steel (manufactured by SUS316), and the pressure of the high-pressure pump 3 is adjusted to 2 MPa. Permeate 4 and permeate 5 were collected. The concentration of lactic acid and acetic acid contained in the non-permeate 4 and the permeate 5 was analyzed by high performance liquid chromatography (manufactured by Shimadzu Corporation). The results are shown in Table 5.

  As shown in Table 5, it was found that at all pHs, acetic acid was removed with high efficiency from the permeate side, and an aqueous solution containing high-purity calcium lactate could be recovered with high efficiency from the non-permeate side. In Examples 4 to 15, acetic acid was removed with high efficiency at the above filtration pressure without replacing the nanofilter with a new membrane.

Comparative Example 1
(Preparation of culture solution to be separated by nano filter module)
The culture solution (2 L) fermented with lactic acid in Reference Examples 1 and 2 was used (pH 5.0).

(Separation experiment with nano filter module)
Next, 2 L of the culture solution obtained above was injected into the raw water tank 1 of the membrane filtration apparatus shown in FIG. The nanofilter module 2 (piperazine polyamide semipermeable membrane SU-610, manufactured by Toray Industries, Inc.) is set in a dedicated vessel, the pressure of the high-pressure pump 3 is adjusted to 2 MPa, and the non-permeate 4 and permeate at pH 4.5 are operated. 5 was recovered. The concentration of lactic acid and acetic acid contained in the non-permeate 4 and the permeate 5 was analyzed by high performance liquid chromatography (manufactured by Shimadzu Corporation). The results are shown in Table 6.

  As shown in Table 6, at pH 5.0, acetic acid is removed with high efficiency on the permeate side, but since lactate is also contained on the permeate side, the recovery rate of calcium lactate is low and efficient. It was found that they were not separated.

  From the results of the above Examples and Comparative Examples, by passing the microbial fermentation broth adjusted to pH 6-11 through the nanofilter, the aqueous solution containing calcium lactate in the broth was collected from the non-permeate side and permeated. It was revealed that acetic acid can be removed from the liquid side with high efficiency.

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

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Raw water tank 2 Nano filter module 3 High pressure pump 4 Flow of non-permeate 5 Flow of permeate 6 Flow of culture solution sent by high pressure pump 7 90φ nano filter 8 Support plate

Claims (4)

A method for producing lactic acid comprising a step of purifying calcium lactate produced in a culture solution by fermentation culture of a microorganism in the presence of a calcium salt, the pH of the culture solution being adjusted to 6 or more and 11 or less, A method for producing lactic acid, comprising: passing a culture solution through a nanofilter to remove acetic acid in the culture solution on the permeate side, and collecting an aqueous solution containing calcium lactate from the non-permeate side.   The method for producing lactic acid according to claim 1, wherein the functional layer of the nanofilter contains polyamide. The method for producing lactic acid according to claim 2, 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 lactic acid according to any one of claims 1 to 3 , wherein in adjusting the pH of the culture solution, calcium hydroxide is added to the culture solution.

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