Lactic acid adsorbent and method for removing lactic acid

Abstract

To reduce the adverse influence of a lactic acid adsorbent on a target from which lactic acid is removed.SOLUTION: A lactic acid adsorbent 1 contains a layered double oxide 2 and γ-alumina 4.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a lactic acid adsorbent and a method for removing lactic acid. [Background technology]

[0002] In recent years, there has been a demand for efficient artificial mass cultivation of cells and microorganisms in fields such as pharmaceutical manufacturing and regenerative medicine. Examples of cells that require mass cultivation include antibody-producing cells such as Chinese hamster ovary cells (CHO cells), stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), and differentiated cells derived from stem cells. If these cells could be stably cultured in large quantities over a long period of time, it would be possible to efficiently produce biological substances such as monoclonal antibodies and differentiated tissues derived from pluripotent stem cells.

[0003] One method for industrially mass-cultivating cells or microorganisms is suspension agitation culture using a culture tank such as a spinner flask. However, suspension agitation culture tends to require large-scale facilities. Therefore, increasing the culture density of cells is an effective way to reduce costs. However, it is known that increasing the culture density suppresses cell proliferation. This is because the concentration of waste products (metabolites) in the culture solution (liquid medium) increases as the cell density increases, which reduces the proliferation activity of cells. Lactic acid is known to be a typical waste product that affects cells.

[0004] Therefore, in order to stably grow cells at high density, it is desirable to remove lactic acid that accumulates in the culture solution. In response to this, for example, Patent Document 1 discloses a cell culture device in which a cell culture vessel and a composition adjusting solution vessel are connected by a liquid feed line equipped with a culture solution composition adjusting membrane that allows components to permeate depending on the concentration difference. In this cell culture device, waste products that accumulate in the culture solution move to the composition adjusting solution side, thereby decreasing their concentration in the culture solution. At the same time, nutrients whose concentrations decrease during culture are replenished by moving from the composition adjusting solution to the culture solution. This maintains the environment in the culture solution in a state suitable for cell culture. Note that the culture solution itself was used as the composition adjusting solution. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2015 / 122528 Summary of the Invention [Problem to be solved by the invention]

[0006] The cell culture device disclosed in Patent Document 1 removes waste products from the culture solution using the principle of dialysis. Therefore, in order to achieve sufficient removal of waste products, the volume of the composition adjusting solution tank is set to be 10 times or more the volume of the cell culture tank. This poses the problem of the enormous amount of liquid required, which is costly. In particular, when the culture solution itself is used as the composition adjusting solution, a large amount of expensive culture solution is consumed, which further increases costs. Another problem is that when waste products are removed using dialysis technology, the structure of the culture device becomes complicated.

[0007] As a method for removing lactic acid using a technique other than dialysis, a lactic acid removal method using a lactic acid adsorbent can be considered. Compared to lactic acid removal by dialysis, using a lactic acid adsorbent can reduce costs and simplify the structure of the culture device. However, when using a lactic acid adsorbent, it is desirable that the lactic acid adsorbent does not have a negative effect on the target of lactic acid removal, such as the culture medium.

[0008] The present invention has been made in view of these circumstances, and one of its objects is to provide a technique for reducing the negative impact of a lactic acid adsorbent on a target from which lactic acid is to be removed. [Means for solving the problem]

[0009] To solve the above problems, one aspect of the present invention is a lactic acid adsorbent, which contains a layered double oxide and γ-alumina.

[0010] Another embodiment of the present invention is also a lactate adsorbent, which contains γ-alumina and is used at a concentration of 0.025 g / mL or more.

[0011] Another aspect of the present invention is a method for removing lactic acid, which comprises contacting the lactic acid adsorbent according to any one of the above aspects with an aqueous solution containing lactic acid.

[0012] Any combination of the above components, or mutual substitution of the components or expressions of the present invention between methods, devices, systems, etc., are also valid aspects of the present invention. [Effects of the Invention]

[0013] According to the present invention, the negative effects of the lactic acid adsorbent on the target of lactic acid removal can be reduced. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram of a lactic acid adsorbent according to an embodiment. [Figure 2] 2(A) to 2(D) are schematic diagrams illustrating a method for removing lactic acid according to an embodiment. [Figure 3] FIG. 1 is a graph showing the lactic acid adsorption rate and glucose adsorption rate of a lactic acid adsorbent in an aqueous solution of lactic acid and glucose, and the pH of the aqueous solution after lactic acid adsorption. [Figure 4]Figure 4(A) shows the pH of the cell culture solution after lactate adsorption in the cell culture solution. Figure 4(B) shows the lactate adsorption rate of the lactate adsorbent in the cell culture solution. Figure 4(C) shows the glucose adsorption rate of the lactate adsorbent in the cell culture solution. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described below based on preferred embodiments with reference to the drawings. The embodiments are illustrative and do not limit the invention, and all features and combinations thereof described in the embodiments are not necessarily essential to the invention. Identical or equivalent components, parts, and processes shown in each drawing are designated by the same reference numerals, and redundant description will be omitted where appropriate. The scale and shape of each part shown in each drawing are set for convenience to facilitate explanation and should not be interpreted as limiting unless otherwise specified. Furthermore, when terms such as "first" and "second" are used in this specification or claims, these terms do not indicate any order or importance, but are intended to distinguish one configuration from another. Furthermore, some components that are not important for explaining the embodiments are omitted from each drawing.

[0016] FIG. 1 is a schematic diagram of a lactic acid adsorbent 1 according to an embodiment. The lactic acid adsorbent 1 according to this embodiment contains a layered double oxide 2 (layered double oxide: LDO) and γ-alumina 4 (γ-Al2O3). The layered double oxide 2 is a calcined product of a layered double hydroxide (layered double hydroxide: LDH). The layered double hydroxide that is the source of the layered double oxide 2 has multiple metal hydroxide layers and anions and water molecules held between the metal hydroxide layers. The metal hydroxide layers are also called host layers, and contain divalent metal ions M as the constituent metals. 2+ and trivalent metal ions M 3+ Specifically, layered double hydroxides contain M(OH)2, which is a divalent metal. 2+ Part of M 3+Layered double hydroxides are composed of a host layer of octahedral layers that is positively charged due to substitution with , and a guest layer that consists of anions that compensate for the positive charge of the host layer and interlayer water. Layered double hydroxides can be obtained by known methods such as coprecipitation and sol-gel methods.

[0017] An example of a layered double hydroxide is represented by the following chemical formula: [M 2+ 1-X M 3+ x (OH)2][A n- x / n ·yH2O] In the above formula, M 2+ is Cu 2+ , Mn 2+ , Mg 2+ , Fe 2+ , Ca 2+ , Ni 2+ , Zn 2+ , Co 2+ and Cd 2+ M is a divalent metal ion selected from the group consisting of 3+ Al 3+ , Cr 3+ , Fe 3+ , Co 3+ , In 3+ , Mn 3+ and V 3+ A is a trivalent metal ion selected from the group consisting of n- is an anion in the guest layer. x is 0.20 to 0.33, n is 1 to 3, and y is 1 to 12. The anion in the guest layer is not particularly limited, but examples thereof include amino acids, dipeptides, vitamins, pH buffers, glucose metabolites, and inorganic ions. Examples of inorganic ions include CO3 2- , SO4 2- , Cl - , SiO4 4- and NO3 - is exemplified.

[0018] The layered double oxide 2 can be obtained by calcining the layered double hydroxide at, for example, 200°C to 600°C. When the layered double hydroxide is calcined, the water molecules and anions held between the host layers are removed. As a result, the structure of the layered double hydroxide collapses, and the layered double oxide 2 is formed. Note that a mixture of multiple types of layered double oxides 2, each containing different types of metal ions and anions that make up the host layers, may also be used.

[0019] By bringing a lactic acid adsorbent containing layered double oxide 2 into contact with an aqueous solution containing lactic acid, the lactic acid in the aqueous solution is adsorbed onto layered double oxide 2, and the lactic acid can be removed from the aqueous solution. When layered double oxide 2 is brought into contact with the aqueous solution, water molecules in the aqueous solution are converted into protons (H + ) and hydroxyl ion (OH - ) The protons are taken up by the host layer of layered double oxide 2. Lactic acid in the aqueous solution is also adsorbed between the host layers. This causes layered double oxide 2 to regenerate its layered double hydroxide structure.

[0020] Hydroxy ions are generated during the process of layered double oxide 2 adsorbing lactic acid, causing an increase in the pH of the aqueous solution. This increase in pH corresponds to a negative effect on the aqueous solution from which lactic acid is being removed. For example, if the aqueous solution is a culture medium for cells or microorganisms, changes such as the inactivation of proteins in the culture medium may occur. For this reason, it is desirable to prevent the increase in pH of the aqueous solution.

[0021] In contrast, the lactic acid adsorbent 1 of this embodiment contains γ-alumina 4. When γ-alumina 4 is brought into contact with an aqueous solution, water molecules in the aqueous solution react with γ-alumina 4. As a result, hydroxyl groups, i.e., surface hydroxyl groups, are generated on the surface of γ-alumina 4. The surface hydroxyl groups trap hydroxy ions in the aqueous solution. As a result, the surface hydroxyl groups become oxygen ions and water is generated. In this way, γ-alumina 4 absorbs hydroxy ions in the aqueous solution, thereby preventing an increase in the pH of the aqueous solution. Therefore, the negative effects of the lactic acid adsorbent 1 on the target for lactic acid adsorption can be reduced. In particular, when the aqueous solution is a culture solution, protein deactivation due to an increase in pH can be prevented. Furthermore, the negative effects on cells and microorganisms present in the culture solution can also be reduced.

[0022] The ratio A / B of the mass proportion A (wt%) of the layered double oxide 2 to the mass proportion B (wt%) of γ-alumina 4 in the lactic acid adsorbent 1 is preferably 0.3 to 3.0, more preferably 0.33 to 2.8, and even more preferably 0.36 to 2.6. When the layered double oxide 2 is an Mg-Al LDO containing Mg and Al, the molar ratio of Mg to Al in the lactic acid adsorbent 1 (hereinafter referred to as the Mg / Al molar ratio) is preferably 0.25 to 1. In this case, Al includes both Al derived from the layered double oxide 2 and Al derived from γ-alumina 4. When the ratio A / B and the Mg / Al molar ratio satisfy the above-mentioned ranges, when an aqueous solution contains glucose as an active ingredient, the adsorbent can highly selectively adsorb lactic acid, which is the target for removal, compared to glucose, which should remain in the aqueous solution. The culture solution is an example of an aqueous solution containing glucose. The type of culture solution is not particularly limited.

[0023] The amount of lactic acid adsorbent 1 to be added to the aqueous solution, in other words, the concentration of the lactic acid adsorbent in the aqueous solution, can be appropriately set based on the designer's empirical knowledge or experiments or simulations, etc. The concentration of the lactic acid adsorbent is preferably 0.025 g / mL or more, and more preferably 0.05 g / mL or more.

[0024] Furthermore, the lactic acid adsorbent 1 containing the layered double oxide 2 and the γ-alumina 4 contains the divalent metal ions M derived from the layered double oxide 2 and the γ-alumina 4. 2+ and trivalent metal ions M 3+ Improvement of lactic acid adsorption performance is expected by adjusting the molar ratio of γ-alumina 4 to γ-alumina 4, the dispersibility of γ-alumina 4, the specific surface area of ​​γ-alumina 4, the porosity of γ-alumina 4, etc. When the layered double oxide 2 is Mg—Al LDO, the molar ratio is the Mg / Al molar ratio. For example, the molar ratio is preferably smaller than a predetermined value, and the dispersibility, specific surface area, and porosity of γ-alumina 4 are preferably higher than or greater than each of the predetermined values.

[0025] When the target for lactic acid removal is a culture medium, the cells and microorganisms cultured in this culture medium are not particularly limited. Examples of cells include pluripotent stem cells and differentiation-induced cells such as human iPS cells, human ES cells, and human Muse cells; somatic stem cells such as mesenchymal stem cells (MSCs) and nephron progenitor cells; tissue cells such as human proximal tubule epithelial cells, human distal tubule epithelial cells, and human collecting duct epithelial cells; antibody-producing cell lines such as human embryonic kidney cells (HEK293 cells); and antibody-producing cell lines derived from non-human animals such as Chinese hamster ovary cells (CHO cells) and insect cells (SF9 cells).

[0026] (Lactic acid removal method) The lactic acid removal method according to this embodiment includes contacting the above-described lactic acid adsorbent 1 with an aqueous solution containing lactic acid to allow the lactic acid adsorbent 1 to adsorb lactic acid. The method for contacting the lactic acid adsorbent with the aqueous solution is not particularly limited, but the following embodiments are exemplified. FIGS. 2(A) to 2(D) are schematic diagrams illustrating the lactic acid removal method according to this embodiment. The following description will be given taking the removal of lactic acid from a culture solution 14 as an example, but the removal of lactic acid from other aqueous solutions can also be carried out in a similar manner.

[0027] In the lactic acid adsorbent 1, the layered double oxide 2 and γ-alumina 4 are, for example, in the form of powder or pellets and are mixed together. The lactic acid adsorbent 1 shown in FIGS. 2(A) to 2(D) is, for example, a mixture of the layered double oxide 2 and γ-alumina 4 in pellet form, but is not limited to this form. For example, the lactic acid adsorbent 1 may be a mixture of the layered double oxide 2 and γ-alumina 4 in powder form and pelletized. The lactic acid adsorbent 1 is not limited to a mixture of the layered double oxide 2 and γ-alumina 4, and can also be obtained by a method in which an excess aluminum source is present in the synthesis reaction field when synthesizing a layered double hydroxide by a coprecipitation method, sol-gel method, or the like.

[0028] As shown in FIG. 2(A), in a first embodiment, an adsorption module 5 is prepared in which a column 6 is filled with a lactate adsorbent 1. The column 6 has an inlet 6a and an outlet 6b that communicate between the inside and outside of the column 6. The adsorption module 5 is connected to a culture vessel 10, such as a spinner flask, via a circulation path 8. The circulation path 8 includes an outgoing path 8a that connects the culture vessel 10 with the inlet 6a of the column 6, and a returning path 8b that connects the outlet 6b of the column 6 with the culture vessel 10. A pump 12 is connected to the outgoing path 8a. A culture solution 14 and cells 16 are accommodated in the culture vessel 10. The pump 12 may also be arranged in the returning path 8b.

[0029] When the pump 12 is driven, the culture solution 14 is sucked from the culture vessel 10 and sent into the column 6 of the adsorption module 5 via the outgoing line 8a. The culture solution 14 sent into the column 6 is returned into the culture vessel 10 via the returning line 8b. As the culture solution 14 circulates between the culture vessel 10 and the adsorption module 5, it comes into contact with the lactic acid adsorbent 1 packed in the column 6. At this time, the lactic acid in the culture solution 14 is adsorbed by the layered double oxide 2. As a result, the lactic acid in the culture solution 14 is removed. Furthermore, hydroxy ions generated in the process of the layered double oxide 2 adsorbing lactic acid are adsorbed by the γ-alumina 4.

[0030] A filter (not shown) is provided at the end of the outgoing path 8a that is connected to the culture vessel 10. This prevents the cells 16 from flowing toward the adsorption module 5. In the process of circulating the culture solution 14 between the culture vessel 10 and the adsorption module 5, components such as glucose and proteins necessary for culturing the cells 16 may be replenished to the culture solution 14.

[0031] That is, in the first embodiment, lactic acid in the culture solution 14 is removed by using a culture device comprising an adsorption module 5 having a lactic acid adsorbent 1, a culture vessel 10 in which cells or microorganisms and a culture solution 14 are accommodated, and a circulation path 8 that connects the adsorption module 5 to the culture vessel 10 and circulates the culture solution 14. This culture device can also be described as a lactic acid removal device that comprises the adsorption module 5, a vessel in which an aqueous solution is accommodated, and the circulation path 8 that connects the adsorption module 5 to the vessel and circulates the aqueous solution.

[0032] As shown in FIG. 2(B), in the second embodiment, a lactic acid adsorbent 1 is supported on the inner wall surface of a culture vessel 10. A culture solution 14 and cells 16 are contained in the culture vessel 10. Therefore, the culture solution 14 comes into contact with the lactic acid adsorbent 1 exposed on the inner wall surface of the culture vessel 10. This allows lactic acid in the culture solution 14 to be adsorbed onto the layered double oxide 2. Furthermore, hydroxy ions generated in the process of the layered double oxide 2 adsorbing lactic acid can be adsorbed onto the γ-alumina 4. Examples of the culture vessel 10 include spinner flasks, petri dishes, well plates, cell culture inserts, and microspheres.

[0033] Examples of methods for supporting the lactic acid adsorbent 1 on the inner wall surface of the culture vessel 10 include a method of adhering the lactic acid adsorbent 1 to the inner wall surface of the culture vessel 10, and, if the culture vessel 10 is made of resin, a method of molding the culture vessel 10 from a resin pre-mixed with the lactic acid adsorbent 1. That is, in the second aspect, lactic acid in the culture solution 14 is removed by using a culture device comprising the culture vessel 10 and the lactic acid adsorbent 1 supported on the inner wall surface of the culture vessel 10. This culture device can also be referred to as a lactic acid removal device comprising a container for accommodating an aqueous solution and the lactic acid adsorbent 1 supported on the inner wall surface of the container.

[0034] As shown in FIG. 2(C), in a third embodiment, a culture vessel 10 has a structure in which the interior of the vessel is divided into an upper section 10a and a lower section 10b by a diaphragm 18 such as a porous membrane. An example of such a culture vessel 10 is a cell culture insert. A culture solution 14 and cells 16 are accommodated in the upper section 10a, and a culture solution 14 and a lactate adsorbent 1 are accommodated in the lower section 10b. The culture solution 14 can pass through the diaphragm 18 to move between the upper section 10a and the lower section 10b. On the other hand, the cells 16 and the lactate adsorbent 1 cannot pass through the diaphragm 18.

[0035] In this structure, the culture solution 14 comes into contact with the lactic acid adsorbent 1 contained in the lower stage 10b. This allows lactic acid in the culture solution 14 to be adsorbed by the layered double oxide 2. Furthermore, hydroxy ions generated in the process of the layered double oxide 2 adsorbing lactic acid can be adsorbed by the γ-alumina 4. That is, in the third embodiment, lactic acid in the culture solution 14 is removed by using a culture device comprising a culture vessel 10, a lactic acid adsorbent 1, and a diaphragm 18 that divides the interior of the culture vessel 10 into a first space containing the lactic acid adsorbent 1 and a second space containing cells 16. This culture device can also be described as a lactic acid removal device comprising a vessel that contains an aqueous solution, a lactic acid adsorbent 1, and a diaphragm 18 that divides the interior of the vessel 10 into a first space containing the lactic acid adsorbent 1 and a second space where entry of the lactic acid adsorbent is restricted.

[0036] As shown in Figure 2(D), in a fourth embodiment, a particulate lactic acid adsorbent 1 is dispersed, precipitated, or suspended in a culture solution 14. This allows lactic acid in the culture solution 14 to be adsorbed onto the layered double oxide 2. Furthermore, hydroxy ions generated in the process of the layered double oxide 2 adsorbing lactic acid can be adsorbed onto the γ-alumina 4. Note that the lactic acid adsorbent 1 preferably has a predetermined size or larger, for example, 10 µm or larger, to prevent it from being phagocytosed by cells 16. When the layered double oxide 2 and γ-alumina 4 are each in the form of pellets as shown in the figure, it is preferable that each of the layered double oxide 2 and γ-alumina 4 has a predetermined size or larger.

[0037] That is, in the fourth aspect, lactic acid in the culture solution 14 is removed by using a culture device including a culture vessel 10 and a lactic acid adsorbent 1 that is added to the culture solution 14 in the culture vessel 10. This culture device can also be described as a lactic acid removal device that includes a vessel that contains an aqueous solution and a lactic acid adsorbent 1 that is added to the aqueous solution in the vessel.

[0038] Preferably, the lactic acid adsorbent 1 is coated with a resin such as polyvinyl alcohol, or a bio-derived gel such as collagen, alginic acid, or gelatin. This prevents microparticles that may affect the cells 16 from leaking out of the lactic acid adsorbent 1 into the culture solution 14. Alternatively, the lactic acid adsorbent 1 is formed by kneading a ceramic binder, a resin binder, a bio-derived gel, or the like with the layered double oxide 2 and / or γ-alumina 4. This also prevents microparticles from leaking out. Examples of ceramic binders include alumina binders and colloidal silica. Examples of resin binders include polyvinyl alcohol and carboxymethyl cellulose. Examples of bio-derived gels include collagen, alginic acid, and gelatin.

[0039] The method for detecting the lactic acid concentration in an aqueous solution is not particularly limited, but it is preferable to use a medium component analyzer. Alternatively, the lactic acid concentration can be detected by a colorimetric method using a predetermined measurement reagent, an enzyme electrode method utilizing the substrate specificity of an enzyme, or high performance liquid chromatography (HPLC).

[0040] As described above, the lactic acid adsorbent 1 according to this embodiment contains the layered double oxide 2 and γ-alumina 4. This allows the γ-alumina 4 to trap hydroxy ions that are generated when the layered double oxide 2 adsorbs lactic acid. This makes it possible to suppress an increase in the pH of an aqueous solution to which the lactic acid adsorbent 1 has been added. This reduces the negative impact of the lactic acid adsorbent 1 on the target of lactic acid removal.

[0041] Furthermore, according to this embodiment, unlike the case of removing lactic acid using conventional dialysis techniques, lactic acid can be removed without using a huge amount of aqueous solution. Therefore, lactic acid can be removed at low cost. In particular, when the aqueous solution is a culture solution for cells or the like, the amount of culture solution used can be reduced compared to conventional dialysis techniques. Since culture solutions are generally expensive, further cost reductions are possible. Furthermore, since lactic acid can be removed simply by contacting a lactic acid adsorbent with a lactic acid-containing aqueous solution, this embodiment allows for simplification of the structure of the culture device and the lactic acid removal device.

[0042] Furthermore, the removal of lactic acid allows for the mass cultivation of cells at high densities. Furthermore, the decrease in pH of the culture medium caused by lactic acid itself can be suppressed, which also makes it possible to mass cultivate cells at high densities. Furthermore, when the cells are pluripotent stem cells, the removal of lactic acid not only enables the mass cultivation of cells at high densities, but also maintains the cells in an undifferentiated state, i.e., their pluripotency (multipotency). Therefore, it is possible to obtain large quantities of cells suitable for the production of biological materials and the creation of differentiation-induced tissues. This reduces the costs required for pharmaceutical manufacturing and regenerative medicine.

[0043] Preferably, the ratio A / B of the mass proportion A of the layered double oxide 2 to the mass proportion B of the γ-alumina 4 is 0.3 or more and 3.0 or less. This allows highly selective adsorption of lactic acid, which is the target for removal, compared to glucose that should remain in the aqueous solution, when the aqueous solution contains glucose as an active ingredient. Therefore, the lactic acid adsorbent 1 of this embodiment is particularly useful for removing lactic acid from a culture solution. The lactic acid adsorbent 1 can also be used in combination with other adsorbents for cellular waste products.

[0044] The embodiments of the present invention have been described in detail above. The above-described embodiments merely illustrate specific examples of implementing the present invention. The content of the embodiments does not limit the technical scope of the present invention, and many design modifications, such as changing, adding, or deleting components, are possible within the scope of the inventive concept defined in the claims. A new embodiment with design modifications will combine the effects of the combined embodiments and modifications. In the above-described embodiments, content that allows such design modifications is emphasized by using notations such as "in this embodiment" or "in this embodiment," but design modifications are also permitted even in content without such notation. Any combination of the above-described components is also valid as an aspect of the present invention.

[0045] In the lactic acid adsorbent 1 according to the embodiment, γ-alumina 4 is used mainly as a pH rise inhibitor. However, after extensive investigation, the present inventors have found that γ-alumina 4 also has lactic acid adsorption performance. Therefore, examples of the lactic acid adsorbent 1 according to the modified example include a lactic acid adsorbent 1 containing γ-alumina 4, a lactic acid adsorbent 1 containing γ-alumina 4 without containing a layered double oxide 2, and a lactic acid adsorbent 1 consisting only of γ-alumina 4. The present inventors have also found that a good lactic acid adsorption effect can be obtained by using γ-alumina 4 at a concentration of 0.025 g / mL or more, more preferably at a concentration of 0.05 g / mL or more.

[0046] Furthermore, the solid acid contained in the lactic acid adsorbent 1 is not limited to γ-alumina 4. That is, other forms of the lactic acid adsorbent 1 include lactic acid adsorbents 1 containing a layered double oxide 2 and a solid acid other than γ-alumina 4, such as silica, titania, zirconia, or zeolite. The lactic acid adsorbent 1 can also be used in combination with a plurality of different solid acids. Thus, one example of the lactic acid adsorbent 1 contains a layered double oxide 2 and at least one solid acid selected from the group consisting of γ-alumina 4, silica, titania, zirconia, and zeolite.

[0047] The embodiments may be specified by the following items. [1st item] A layered double oxide (2), γ-alumina (4), Lactic acid absorbent (1). [Second item] In the lactic acid adsorbent (1), the ratio A / B of the mass proportion A of the layered double oxide (2) to the mass proportion B of the γ-alumina (4) is 0.3 or more and 3.0 or less. The lactate adsorbent (1) described in item 1. [3rd item] contacting an aqueous solution containing lactic acid to adsorb lactic acid in the aqueous solution; A lactate adsorbent (1) according to item 1 or 2. [4th item] The aqueous solution contains glucose, The lactate adsorbent (1) described in item 3. [Item 5] The aqueous solution is a cell or microbial culture medium (14); A lactate adsorbent (1) according to item 4. [Item 6] Contains γ-alumina (4), Used at concentrations of 0.025 g / mL or higher, Lactic acid absorbent (1). [Item 7] A method for producing a lactic acid adsorbent (1) according to any one of items 1 to 6, comprising contacting the lactic acid adsorbent (1) with an aqueous solution containing lactic acid. Lactic acid removal method. [Example]

[0048] Examples of the present invention will be described below, but these examples are merely illustrative examples for suitably explaining the present invention and do not limit the present invention in any way.

[0049] [Synthesis of lactic acid adsorbent] (synthesis of gamma-alumina) 170 mL of pure water was placed in a flask and stirred at 70°C and 300 rpm. 250 mL of an aqueous solution of Al(NO3)3·9H2O and 250 mL of an aqueous solution of NH4HCO3 were simultaneously added to the stirred pure water, and the mixture was allowed to react at 70°C for 3 hours while maintaining the pH at 8.0 with a 2M NaOH solution. The amount of Al(NO3)3·9H2O in the 250 mL of aqueous solution was 10.42 g. The amount of NH4HCO3 in the 250 mL of aqueous solution of NH4HCO3 was 2.5 g. The resulting suspension was centrifuged to precipitate the precipitate. The precipitate was collected and washed three times with pure water, followed by three times with ethanol. The precipitate was then dried under reduced pressure at 40°C for 40 hours. The resulting powder was calcined at 550°C for 2 hours to obtain a white powder. The obtained white powder was confirmed to be γ-alumina by powder X-ray diffraction (PXRD). Furthermore, the white powder was confirmed to be nanoparticles with an average primary particle diameter of 5.72 nm by observation with a scanning electron microscope (SEM). Furthermore, the white powder had a BET specific surface area of ​​276 m2 by the BET method. 2 / g.

[0050] (Synthesis of Mg-Al LDO) 500 mL of Na2CO3 aqueous solution was placed in a five-neck round-bottom flask and stirred at 300 rpm at 30 °C. 500 mL of Mg(NO3)2-Al(NO3)3 mixed solution (Mg / Al molar ratio = 2.0) was added dropwise to the stirred solution at a rate of 15 mL / min. A 1.25 M NaOH aqueous solution was added dropwise to the solution to adjust the pH of the solution to 10.5 ± 0.1. Stirring was continued for 1 hour after the addition of the Mg(NO3)2-Al(NO3)3 mixed solution was completed. The suspension obtained by this process was filtered under suction to obtain the product. The obtained product was dried under reduced pressure at 40 °C for 40 hours and then crushed in an agate mortar. It was then calcined in an electric furnace at 500 °C for 2 hours to obtain a white powder. PXRD analysis of the obtained white powder revealed that the divalent metal ions constituting the host layer were Mg. 2+ The trivalent metal ions that make up the host layer are Al 3+ The white powder was confirmed to be a layered double oxide, i.e., Mg-Al LDO. Analysis using an ICP optical emission spectrometer (iCAP6500 Duo, manufactured by ThermoFisher Scientific (registered trademark)) confirmed that the Mg / Al molar ratio of the white powder was 2.0.

[0051] (Preparation of several test examples with different mixing ratios of Mg-Al LDO and γ-alumina) (Test Example 1: Ex.1) The synthesized Mg-Al LDO and γ-alumina were mixed at a mass ratio (wt%) of 100:0 to prepare a lactic acid adsorbent according to Test Example 1. In other words, Test Example 1 is a lactic acid adsorbent that does not contain γ-alumina. The Mg / Al molar ratio of Test Example 1 is 2.0, and the ratio A / B of the mass ratio A (wt%) of Mg-Al LDO to the mass ratio B (wt%) of γ-alumina is impossible.

[0052] (Test Example 2: Ex.2) The synthesized Mg-Al LDO and γ-alumina were mixed in a mass ratio (wt%) of 88.6:11.4 to prepare a lactic acid adsorbent according to Test Example 2. The Mg / Al molar ratio of Test Example 2 was 1.5, and the ratio A / B was 7.77.

[0053] (Test Example 3: Ex.3) The synthesized Mg-Al LDO and γ-alumina were mixed in a mass ratio (wt%) of 72.1:27.9 to prepare a lactic acid adsorbent according to Test Example 3. The Mg / Al molar ratio of Test Example 3 was 1.0, and the ratio A / B was 2.58.

[0054] (Test Example 4: Ex.4) The synthesized Mg—Al LDO and γ-alumina were mixed in a mass ratio (wt%) of 46.2:53.7 to prepare a lactic acid adsorbent according to Test Example 4. The Mg / Al molar ratio of Test Example 4 was 0.5, and the ratio A / B was 0.86.

[0055] (Test Example 5: Ex.5) The synthesized Mg—Al LDO and γ-alumina were mixed in a mass ratio (wt%) of 26.9:73.1 to prepare a lactic acid adsorbent according to Test Example 5. The Mg / Al molar ratio of Test Example 5 was 0.25, and the ratio A / B was 0.37.

[0056] (Test Example 6: Ex.6) The synthesized Mg-Al LDO and γ-alumina were mixed in a mass ratio (wt%) of 0:100 to prepare a lactic acid adsorbent according to Test Example 6. In other words, Test Example 6 is a lactic acid adsorbent that does not contain a layered double oxide. The Mg / Al molar ratio of Test Example 6 is 0, and the ratio A / B is 0.

[0057] [Analysis of adsorption performance and pH change in aqueous solutions of lactic acid and glucose] Lithium lactate (Kanto Chemical Co., Ltd.) and glucose (Kanto Chemical Co., Ltd.) were added to pure water to prepare an aqueous solution with a glucose concentration of 1000 mg / L and an aqueous solution with a lactic acid concentration of 10 mM. A 1 M aqueous solution of sodium hydroxide was added to each aqueous solution to adjust the pH of the aqueous solution to 7.2. Then, 10 mL of each aqueous solution was dispensed into multiple 50 mL Erlenmeyer flasks. Additionally, 0.125 g of the lactic acid adsorbent for each test example was added to the aqueous solution in each flask. Therefore, the concentration of the lactic acid adsorbent was 0.0125 g / mL.

[0058] Each aqueous solution was shaken at 37°C at 150 rpm for 24 hours. After 24 hours of shaking, the lactic acid adsorbent and the aqueous solution were separated using a 0.1 μm filter. The lactic acid concentration in the lactic acid aqueous solution and the glucose concentration in the glucose aqueous solution were measured using HPLC (JASCO Corporation). The pH of the lactic acid aqueous solution was also measured using a glass electrode pH measuring device (MM-43X: manufactured by DKK-TOA Corporation) and a pH electrode (GST-5841C(S): manufactured by DKK-TOA Corporation). The adsorption rates of lactic acid and glucose for each lactic acid adsorbent were calculated using the following formula: Adsorption rate (%) = {(concentration before adsorption - concentration after adsorption) / concentration before adsorption} × 100

[0059] The results are shown in Figure 3. Figure 3 shows the lactic acid adsorption rate and glucose adsorption rate of the lactic acid adsorbent in an aqueous solution of lactic acid and glucose, and the pH of the aqueous solution after lactic acid adsorption. As shown in Figure 3, in Test Example 1, which did not contain γ-alumina, the pH of the aqueous solution increased from 7.2 to 11.39 due to the adsorption of lactic acid by the lactic acid adsorbent. On the other hand, in Test Examples 2-6, which contained γ-alumina, the increase in pH of the aqueous solution was suppressed compared to Test Example 1. Furthermore, as the mixing ratio of γ-alumina in the lactic acid adsorbent increased, the increase in pH decreased. This confirmed that the combination of a layered double oxide and γ-alumina can suppress the increase in pH of the aqueous solution caused by the lactic acid adsorbent. The inventors confirmed that the pH of the glucose aqueous solution hardly changed after lactic acid adsorption, specifically, was in the range of 8.68 to 9.75.

[0060] Furthermore, in Test Examples 3-5, lactic acid was highly selectively adsorbed compared to glucose. This confirmed that when the ratio A / B, where A is the mass proportion of the layered double oxide and B is the mass proportion of γ-alumina, is 0.3 or more and 3.0 or less, lactic acid can be highly selectively adsorbed. Furthermore, Test Example 6, which does not contain a layered double oxide and consists only of γ-alumina, was also able to adsorb lactic acid. This confirmed that γ-alumina alone can be used as a lactic acid adsorbent. Furthermore, a comparison between Test Example 1 and Test Example 6 confirmed that γ-alumina has a higher lactic acid adsorption rate and a lower glucose adsorption rate than layered double oxides.

[0061] [Analysis of adsorption performance and pH fluctuations in cell culture medium] Lithium lactate (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to pluripotent stem cell medium (StemFit® AK02N: Ajinomoto Co., Inc.) to prepare a culture medium with a glucose concentration of 250 mg / dL and a lactate concentration of 10 mM. 20 mL of this culture medium was dispensed into multiple 50 mL tubes (ThermoFisher Scientific®). Various amounts of lactate adsorbent (Test Examples 1, 4, 6) were added to the culture medium in each tube. The amounts added were 2.0 g, 1.0 g, 0.5 g, and 0.1 g. The concentrations of the lactate adsorbent were therefore 0.1 g / mL, 0.05 g / mL, 0.025 g / mL, and 0.005 g / mL.

[0062] The culture medium containing the lactate adsorbent was shaken at 37°C at 60 rpm for 24 hours. After 24 hours, the culture medium and the lactate adsorbent were separated using a 0.22 μm filter. The lactate concentration, glucose concentration, and pH of the culture medium were measured using a blood gas analyzer (ABL800 FLEX: Radiometer). The pH of the culture medium without the lactate adsorbent was also measured. The lactate adsorption rate and glucose adsorption rate for each lactate adsorbent were calculated based on the above formula.

[0063] The results are shown in Figures 4(A) to 4(C). Figure 4(A) shows the pH of the cell culture medium after lactic acid adsorption in the cell culture medium. Figure 4(B) shows the lactic acid adsorption rate of the lactic acid adsorbent in the cell culture medium. Figure 4(C) shows the glucose adsorption rate of the lactic acid adsorbent in the cell culture medium.

[0064] As shown in Figure 4(A), Test Examples 4-6, which contained γ-alumina, tended to be able to suppress the increase in pH of the culture solution compared to Test Example 1, which did not contain γ-alumina. Furthermore, the amount of increase in pH tended to decrease as the mixing ratio of γ-alumina in the lactic acid adsorbent increased. This tendency was particularly pronounced in Test Examples 5 and 6, where the ratio A / B of the layered double oxide to γ-alumina was 0.5 or less. This confirmed that the increase in pH of the culture solution caused by the lactic acid adsorbent can be suppressed by combining the layered double oxide with γ-alumina.

[0065] Furthermore, as shown in Figures 4(B) and 4(C), Test Examples 4-6 tended to adsorb lactic acid more selectively than glucose. This tendency was particularly pronounced at adsorbent concentrations of 0.025 g / mL or higher, and was even stronger at adsorbent concentrations of 0.05 g / mL or higher. Furthermore, at adsorbent concentrations of 0.05 g / mL or higher, Test Examples 4 and 5, which contained both layered double oxide and γ-alumina, had higher lactic acid adsorption rates than Test Example 6, which contained only γ-alumina. Furthermore, a comparison of Test Example 1 and Test Example 6 confirmed that, in the case of a culture solution, unlike in the case of an aqueous solution, the layered double oxide had a higher lactic acid adsorption rate than γ-alumina. [Explanation of symbols]

[0066] 1 Lactic acid adsorbent, 2 Layered double oxide, 4 γ-alumina, 14 Culture medium, 16 Cells.

Claims

1. a layered double oxide; γ-alumina, Lactic acid absorbent.

2. a ratio A / B of a mass proportion A of the layered double oxide to a mass proportion B of the γ-alumina in the lactic acid adsorbent is 0.3 or more and 3.0 or less; The lactic acid adsorbent according to claim 1.

3. contacting an aqueous solution containing lactic acid to adsorb lactic acid in the aqueous solution; The lactic acid adsorbent according to claim 1 or 2.

4. The aqueous solution contains glucose. The lactic acid adsorbent according to claim 3.

5. The aqueous solution is a cell or microbial culture medium. The lactic acid adsorbent according to claim 4.

6. Contains γ-alumina, Used at a concentration of 0.025 g / mL or greater Lactic acid absorbent.

7. A method for producing a lactic acid adsorbent according to any one of claims 1, 2 and 6, comprising contacting the lactic acid adsorbent with an aqueous solution containing lactic acid. Lactic acid removal method.

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