JP2009142265A - Method for producing lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing lactic acid, comprising a step of removing a coloring component before occurrence of coloring in order to solve a problem in which lactic acid is colored by a Maillard reaction when heating a lactic acid solution by operation such as distillation without removing the coloring component when separating and refining lactic acid from a fermented culture solution after finishing fermentation in fermentation of lactic acid by a microorganism. <P>SOLUTION: In the method for refining lactic acid, coloring component can effectively be separated and removed by passing a culture solution of lactic acid fermented by a microorganism through a nano filter and filtering the solution. <P>COPYRIGHT: (C)2009,JPO&INPIT

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.

  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 according to the conformation of 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.

  In the production of lactic acid by microbial fermentation, a liquid medium is generally used as a fermentation nutrient source, sugars such as glucose and sucrose are used as a carbon source, and ammonia gas, ammonium sulfate and the like are used as a nitrogen source. Moreover, natural product components such as amino acids, vitamins, yeast extract, peptides and the like are added as necessary. The nutrient source contained in the fermentation broth generally has a brownish brown color. In particular, the cause of the coloring component is due to the Maillard reaction by amino groups of amino acids and peptides and carbonyl groups of sugars. It has been known. Maillard reaction is accelerated by heating conditions. When lactic acid is separated and purified from the fermentation broth after completion of fermentation, there is a problem that lactic acid is colored by the Maillard reaction if the lactic acid solution is heated by an operation such as distillation without removing the colored components. Further, when the colored lactic acid is polymerized, there is a problem that the colored component causes the polymerization to be inhibited, so that polylactic acid having a target molecular weight cannot be obtained. Furthermore, there is a problem that the polymerized polylactic acid is colored. Therefore, there is a demand for a method for effectively removing the coloring components contained in the fermentation broth.

As a method for removing the coloring component in the fermentation broth, a method using activated carbon is disclosed (for example, see Patent Document 1). However, when activated carbon is used, the coloring component to be removed is adsorbed on the activated carbon, while loss occurs due to adsorption of the target lactic acid on the activated carbon. Moreover, there was a problem that the removal of the coloring component by activated carbon is insufficient.
JP 63-264548 A (Claims)

  The present invention solves the problem as described above, that is, a method for efficiently removing lactic acid produced by microbial fermentation, and effectively purifies lactic acid. The purpose is to provide.

  As a result of intensive studies to solve the above problems, the present inventors have found that the coloring components in the culture solution can be removed with high efficiency by filtering the microbial fermentation culture solution using a nanofilter. The headline and the present invention were completed.

That is, this invention is comprised from following (1)-(7).
(1) A method for producing lactic acid comprising a step of purifying lactic acid produced in a culture solution by fermentation culture of microorganisms, wherein the culture solution is filtered through a nanofilter, and the APHA unit color number is 200 or less. A process for producing lactic acid, comprising the step A of obtaining a lactic acid solution having a coloration degree of.
(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 the pH of the culture solution in Step A is 7 or less.
(5) The method for producing lactic acid according to any one of (1) to (4), wherein the filtration pressure of the culture solution in the step A is 0.1 MPa or more and 8 MPa or less.
(6) The lactic acid solution obtained in the step A is further subjected to a step of distilling at 25 ° C. or higher and 200 ° C. or lower under a pressure of 1 Pa or higher and atmospheric pressure or lower. The manufacturing method of lactic acid as described.
(7) A separation liquid containing lactic acid obtained after Step B of adding activated carbon to the culture broth and Step C of filtering with qualitative filter paper after Step B is subjected to Step A. (1) to (6) The method for producing lactic acid according to any one of the above.

  The method for producing lactic acid according to the present invention can effectively remove the color components contained in the fermentation broth by a simple operation. Therefore, for example, even when the lactic acid solution obtained by the method for producing lactic acid according to the present invention is subjected to a heating operation thereafter, coloring of the lactic acid solution can be suppressed.

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

  The present invention is a method for producing lactic acid, comprising a step of purifying lactic acid produced in a culture solution by fermentation culture of microorganisms, wherein the culture solution is filtered through a nanofilter, and the APHA unit color number is It includes a step A for obtaining a lactic acid solution having a coloring degree of 200 or less.

  The nanofilter used in step A of the method for producing lactic acid according to the present invention is also called a nanofiltration membrane (nanofiltration membrane, NF membrane). Generally, monovalent ions permeate and divalent ions pass through. It is a film that has the property of blocking. 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.

  For the material of the nanofilter, a polymer material such as cellulose acetate polymer, polyamide, polyester, polyimide, vinyl polymer can be used. In addition, the membrane structure has a dense layer on at least one side of the membrane, and an asymmetric membrane having fine pores with gradually increasing pore diameters from the dense layer to the inside of the membrane or the other side, or a dense layer of the asymmetric membrane. In addition, any composite film having a very thin functional layer formed of another 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 containing polyamide as a membrane material, 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 aromatic rings (such as nylbenzothiazole), piperazine, piperidine or derivatives thereof, among which nanofilters having a functional layer of crosslinked polyamide containing piperazine or piperidine as a monomer are pressure resistant. It is preferably used because it has heat resistance and chemical resistance in addition to properties and durability, and is preferably an amine component containing a crosslinked piperazine polyamide as a main component and containing the structural component represented by the chemical formula 1. Moreover, in Chemical Formula 1, those having n = 3 are more preferably used. Examples of the structural component represented by 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, a nanofilter manufactured by the same company including UTC60 manufactured by Toray Industries, Inc. having a functional layer of a polyamide containing a crosslinked piperazine polyamide as a main component and the component represented by the above chemical formula 1 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 manufactured by GE Osmonics, which is a cellulose acetate-based nanofilter, and the like.

  The nanofilter used in step A of the method for producing lactic acid according to the present invention is not limited to a film composed of the above-mentioned one kind of material, and is a film including a plurality of film materials or a composite film combining a plurality of films. Also good. For example, as described in JP-A-62-201606, a composite membrane in which a nanofilter made of the above material is formed on a support membrane made of polysulfone as a membrane material can be used.

  In the step A of the method for producing lactic acid according to the present invention, “filter through a nanofilter” means that a culture solution containing lactic acid produced by microbial fermentation culture is filtered through a nanofilter to remove colored components. Alternatively, it means that permeation is blocked or filtered and the lactic acid solution is permeated as a filtrate. Here, the coloring component of the culture solution mainly includes nutrients such as sugars and peptides, or components derived from natural products such as yeast extract, etc., as long as they are colored in the culture solution. Both are included.

  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).

  As a method for evaluating the membrane unit area and the permeate flow rate per unit pressure (membrane permeation flux), the permeated water amount, the time during which the permeated water amount was sampled, and the membrane area can be calculated by Equation 2.

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

  In step A of the method for producing lactic acid of the present invention, filtration of the microorganism culture solution with a nanofilter may be performed by applying pressure. The filtration pressure is preferably performed 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 damage is affected. Therefore, the membrane permeation flux is high and the lactic acid solution is performed at 0.5 MPa or more and 7 MPa or less. Is more preferable because it can be efficiently permeated and has a low possibility of affecting the film damage, and is particularly preferably performed at 1 MPa or more and 6 MPa or less.

  In step A of the method for producing lactic acid of the present invention, the pH of the culture solution to be subjected to filtration with a nanofilter is preferably 7 or less. It is known that a nanofilter is generally easier to remove or prevent a material that is ionized in a solution than a material that is not ionized. By setting the pH of the culture solution to 7 or less, the proportion of lactic acid dissociated in the culture solution and present as lactic acid ions can be reduced, and the lactic acid can easily permeate. More preferably, the pH of the culture solution is 4 or less. Furthermore, since the pKa of lactic acid is 3.86, by setting the pH to 3.86 or less, lactic acid that is not dissociated is more contained in the culture solution than lactic acid ions. Since it can permeate | transmit, it is more preferable.

As the membrane separation performance of the nanofilter used in Step A of the method for producing lactic acid of the present invention, those having a removal rate of sodium chloride (500 mg / L) of 45% or more are preferably used. As the permeation performance of the membrane of the nanofilter, a permeated water amount (m ³ / day) of 4 or more with sodium chloride (500 mg / L) at a filtration pressure of 0.3 MPa is preferably used.

  The concentration of lactic acid in the lactic acid solution in the microorganism culture solution used for filtration through the nanofilter is not particularly limited. However, if the concentration is high, the concentration of lactic acid contained in the permeate is also high, so the concentration time is shortened. Therefore, it is suitable for cost reduction.

  The concentration of lactic acid in the lactic acid solution in the microorganism culture solution used for separation by the nanofilter is not particularly limited, but if the concentration is high, the concentration of lactic acid contained in the permeate is also high, so the concentration time is shortened. Therefore, it is suitable for cost reduction. For example, the concentration of lactic acid in the culture solution is preferably 10 g / L or more and 100 g / L or less.

  The APHA unit color number can be used as an index indicating the degree of coloration of the lactic acid solution that has passed through the culture solution or the nanofilter. The APHA unit color number (Hazen unit color number) is a value obtained according to the measurement method of JISK0071-1 (established on October 20, 1998). As an index indicating the coloring degree other than the APHA unit color number (Hazen unit color number), the Gardner color number (JISK0071-2, established on October 20, 1998) can also be used.

  The coloring degree of the culture solution before purification is 500 or less in terms of the APHA unit color number and 18 or less in the Gardner color number under normal culture conditions. According to the present invention, a lactic acid solution having a coloration degree with an APHA unit color number of 200 or less and a Gardner color number of 8 or less can be obtained by the operation of Step A, that is, by filtering the culture solution with a nanofilter. .

  In the method for producing lactic acid according to the present invention, the lactic acid solution obtained after Step B of adding activated carbon to the culture solution and Step C of filtering with qualitative filter paper after Step B may be subjected to Step A. Addition of activated carbon is preferable because the coloring component can be reduced to about 300 as the APHA unit color number and to about 11 as the Gardner color number. The shape of the activated carbon may be powder or granular, and may be in the form of a sheet or cylinder. The activated carbon may be added to the culture solution or the culture solution may be passed through the activated carbon. Can be used.

  In the method for producing lactic acid according to the present invention, high-purity lactic acid can be obtained by subjecting the lactic acid solution obtained by filtering the culture solution with a nanofilter in step A to a further distillation step. The distillation step is preferably performed under a reduced pressure of 1 Pa or more and atmospheric pressure (normal pressure, about 101 kPa) or less. The distillation temperature when performing under reduced pressure is preferably 25 ° C. or more and 200 ° C. or less. However, when distillation is performed at 180 ° C. or more, lactic acid may be racemized due to the influence of impurities. If it is more than 180 degreeC, the distillation of lactic acid can be performed suitably. Before being subjected to this distillation step, the lactic acid solution that has permeated the nanofilter may be once concentrated using a concentrating device represented by an evaporator. When distilling a lactic acid solution having an APHA unit color number of 200 or more and a Gardner color number of 8 or more, the obtained lactic acid may be further colored due to heating during distillation. When the polymerization reaction is carried out using the colored lactic acid as a raw material, the resulting polylactic acid is also colored. Furthermore, the coloring component may act as a polymerization inhibitor during the polymerization reaction, and there is a possibility that polylactic acid having the target molecular weight cannot be obtained.

  According to the method for producing lactic acid of the present invention, the color of the obtained lactic acid solution is 200 or less as the APHA unit color number and 8 or less as the Gardner color number by filtering the culture solution with a nanofilter in step A. Can do. Further, by subjecting the obtained lactic acid solution to a distillation step, a lactic acid solution having a coloration degree of APHA unit color number of 100 or less and Gardner color number of 4 or less can be obtained. Furthermore, by subjecting the distillation residue of the lactic acid solution to step A again, the loss of distillation can be prevented and the isolated yield of lactic acid can be improved. Furthermore, by carrying out a polymerization reaction using lactic acid obtained by the above distillation as a raw material, polylactic acid having a target molecular weight and a low coloring degree can be obtained.

  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 a microorganism used for fermentation culture in the present invention requires a specific nutrient (for example, 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. 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 lactic acid may become high.

  The culture of microorganisms in the present invention can be usually carried out at a pH of 4-8 and a temperature of 20-40 ° C. The pH of the culture solution can be adjusted to a predetermined value within the above range by 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.

  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 in the culture solution is preferably maintained in a high state within a range where the ratio of the culture solution environment becomes inappropriate for the growth of microorganisms and the ratio of killing is not high, in order to obtain efficient productivity. As a result, 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.

  Although there is no restriction | limiting in particular about the microorganisms used by this invention, For example, yeasts, such as baker's yeast often used in fermentation industry, bacteria, such as colon_bacillus | E._coli and coryneform bacteria, filamentous fungi, actinomycetes, etc. are mentioned. 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.

  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 a primer set, 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. Whether or not the transformed cells have lactic acid production ability is determined by the fact that lactic acid is contained in the culture supernatant obtained by culturing the transformed cells in SC medium ("METHODS IN YEAS GENETIC 2000 EDITION", CSHL PRESS). It confirmed by measuring the amount of lactic acid by HPLC method on the conditions shown in.

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 aqueous copper sulfate flow rate: 1.0 ml / min
Detection method: UV254 nm
Temperature: 30 ° C.

  Further, the optical purity of L-lactic acid is calculated by Equation 3. 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 3).

  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: adjusted to pH 7 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.

Examples 1-16
(Preparation of culture solution to be purified with nanofilter)
Concentrated sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) until the pH of the culture solution (2 L) fermented by lactic acid fermentation in Reference Examples 1 and 2 reached 1.9, 2.6, 4.0, and 6.0, respectively. Was added at 25 ° C. for 1 hour to convert calcium lactate in the culture solution into lactic acid and calcium sulfate. Next, the precipitated calcium sulfate was filtered by suction filtration using qualitative filter paper No. 2 (manufactured by Advantech), and 2 L of filtrate was collected.

(Separation experiment with nano filter)
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 nanofilter indicated by reference numeral 7 in FIG. 2, a crosslinked piperazine polyamide semipermeable membrane “UTC60” (Nanofilter 1; manufactured by Toray Industries, Inc.), a crosslinked piperazine polyamide semipermeable membrane “NF-400” (nanofilter 2; film) Tech), polyamide semipermeable membrane “NF99” (Nanofilter 3; manufactured by Alfa Laval), and cellulose acetate semipermeable membrane “GEsepa” (Nanofilter 4; manufactured by GE Osmonics) are each set in a cell made of stainless steel (SUS316). The pressure of the high pressure pump 3 was adjusted to 4 MPa, and the permeated water 4 at each pH was recovered. The concentration of lactic acid contained in the raw water tank 1 and the permeated water 4 was analyzed as the number of APHA unit colors by high performance liquid chromatography (manufactured by Shimadzu Corporation) and the degree of coloration by a colorimeter (manufactured by Nippon Denshoku). The results are shown in Table 3.

  As shown in Table 3, it was found that the coloring components were removed with high efficiency in all Examples conducted at pH 1.9, 2.6, 4.0, and 6.0, respectively.

  Moreover, in the said Example 1- Example 16, it implemented using the same film | membrane all the time without replacing | exchanging a nano filter by a new film on the way, but in the range of said filtration pressurization, a coloring component is highly efficient. Removed.

Example 5
(Distillation of lactic acid solution)
The permeate 1L (lactic acid concentration: 23 g / L, APHA unit color number 140) adjusted to pH 1.9 in Example 1 and purified with a nanofilter was reduced under reduced pressure using a rotary evaporator (manufactured by Tokyo Rika Co., Ltd.). The water was evaporated and concentrated at 50 hPa).

  Subsequently, vacuum distillation was performed at 133 Pa and 130 ° C. Moreover, when the coloring degree of distilled lactic acid was measured with the colorimeter, the increase in APHA unit color number was not seen. Table 4 shows the results.

Comparative Example 1
(Colored component removal experiment using activated carbon)
In the same manner as in Example 1, 1 g of granular activated carbon (Shirakaba, manufactured by Nihon Enviro Chemicals) was added to the culture solution (2 L) obtained by lactic acid fermentation in Reference Examples 1 and 2, and stirred at 25 ° C. for 2 hours. did. Next, concentrated sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise until the pH reached 1.9, and the mixture was stirred at 25 ° C. for 1 hour. Precipitated calcium sulfate and granular activated carbon were separated by suction filtration using qualitative filter paper No. 2 (manufactured by Advantech). The coloration degree of the filtrate was 300 in APHA unit color. From this, it was found that the coloring component was not sufficiently removed.

Comparative Example 2
(Distillation of lactic acid)
Water of 1 L of the filtrate obtained in Comparative Example 1 (lactic acid concentration: 23 g / L, APHA unit color 300) was evaporated under reduced pressure (50 hPa) using a rotary evaporator (manufactured by Tokyo Rika).

  Subsequently, when vacuum distillation was performed at 133 Pa and 130 ° C., the distilled lactic acid was colored. In addition, partially oligomerized lactic acid was confirmed in the distillation residue. Table 5 shows the results.

  From the results of the above Examples and Comparative Examples, it was revealed that the coloring components in the culture solution can be removed with high efficiency by using the nanofilter for the filtration of the culture solution. That is, it has been clarified that the coloring component in the lactic acid solution in the microorganism culture solution can be removed with high efficiency by the present invention. Moreover, even when the lactic acid solution obtained by filtration with a nanofilter was further subjected to a distillation step, it became clear that oligomerization did not proceed even under heating conditions, and lactic acid was not colored.

It is a schematic diagram which shows one embodiment of the nano filter membrane separation apparatus used with the purification method of this invention. It is a schematic diagram which shows one embodiment of cell sectional drawing with which the nano filter of the nano filter membrane separation apparatus used with the purification method of this invention was mounted | worn.

Explanation of symbols

1 Raw Water Tank 2 Cell with Nano Filter Membrane 3 High Pressure Pump 4 Flow of Membrane Permeate 5 Flow of Membrane Concentrate 6 Flow of Culture Solution Pumped by High Pressure Pump 7 Nano Filter 8 Support Plate

Claims (7)

  A method for producing lactic acid comprising a step of purifying lactic acid produced in a culture solution by fermentation culture of microorganisms, wherein the culture solution is filtered through a nanofilter to obtain a coloration degree of APHA unit color number of 200 or less. The manufacturing method of lactic acid including the process A which obtains the lactic acid solution which is.   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 the culture solution in step A has a pH of 7 or less.   The method for producing lactic acid according to any one of claims 1 to 4, wherein the filtration pressure of the culture solution in the step A is 0.1 MPa or more and 8 MPa or less.   The lactic acid solution according to any one of claims 1 to 5, wherein the lactic acid solution obtained in the step A is further subjected to a step of distilling at 25 ° C or more and 200 ° C or less under a pressure of 1 Pa or more and atmospheric pressure or less. Method.   The separation liquid containing lactic acid obtained after the process B of adding activated carbon to the culture solution and the process C of filtering with a qualitative filter paper after the process B is supplied to the process A. Of producing lactic acid.

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