Gas concentration regulator for bacterial culture and bacterial culture method using the same

Abstract

A gas concentration regulator for bacterial culture, comprising (a) ascorbic acid, (b) a transition metal catalyst, (c) activated carbon, (d) an alkali metal carbonate or an alkaline earth metal hydroxide, and (e) water, wherein the ascorbic acid and the water are impregnated in the activated carbon, and the average pore size of the activated carbon is greater than 2.0 nm.

Description

Technical Field

[0001] The present invention relates to a gas concentration regulator for bacterial culture and a bacterial culture method using the same. Background Art

[0002] In the cultivation of biological samples such as tissues and cells implemented in the research fields of biology, reproduction or biotechnology or in the industrial fields, a gas environment different from the atmospheric atmosphere is required. For example, as a condition for maintaining the pH of a bicarbonate buffered culture solution at pH 7.4, which is the same as the normal state of blood, it is necessary to set the atmospheric carbon dioxide concentration to about 5%. In addition, in many research fields, cell cultivation is carried out in a low-concentration oxygen atmosphere similar to that in vivo.

[0003] As a device for producing a gas environment of a high-concentration carbon dioxide atmosphere and a low-concentration oxygen atmosphere, a carbon dioxide gas incubator is known, but the burden of equipment cost, high-pressure gas management, etc. is large. Therefore, in recent years, a method using a gas concentration regulator utilizing an oxidation reaction of ascorbic acid has been widely used (see Patent Documents 1 and 2).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent No. 3818324

[0007] Patent Document 2: Japanese Patent No. 5682831 Summary of the invention

[0008] Problem that the invention aims to solve

[0009] In the culture of bacteria, it is required to create a gas environment of a high-concentration carbon dioxide atmosphere and a low-concentration oxygen atmosphere in as short a time as possible.

[0010] Therefore, the problem to be solved by the present invention is to provide a gas concentration regulator for bacteria culture which generates a large amount of carbon dioxide in the initial stage of the oxidation reaction of ascorbic acid (one hour after the start of the reaction).

[0011] Solutions for solving problems

[0012] The present inventors have conducted intensive studies and have found that the average pore size of activated carbon as a carrier of a gas concentration regulator affects the amount of carbon dioxide generated in the initial stage of the oxidation reaction of ascorbic acid. Based on this finding, the present invention has been completed.

[0013] That is, the present invention relates to the following contents.

[0014] <1>A gas concentration regulator for bacterial culture, which comprises (a) ascorbic acids, (b) transition metal catalysts, (c) activated carbon, (d) alkali metal carbonates or alkaline earth metal hydroxides, and (e) water, wherein the ascorbic acids and the water are impregnated in the activated carbon, and the average pore diameter of the activated carbon is 2.0 nm or more.

[0015] <2>The gas concentration regulator according to <1> above, wherein the average pore diameter of the activated carbon is 2.0 nm or more and 5.5 nm or less.

[0016] <3>The gas concentration regulator according to <1> or <2> above, wherein the total pore volume of the activated carbon is 0.60 cm 3 / g or more.

[0017] <4>The use of the gas concentration regulator according to any one of <1> to <3> above in bacterial culture.

[0018] <5>A method for culturing bacteria, which cultures bacteria in the presence of the gas concentration regulator according to any one of <1> to <3> above.

[0019] <6>A gas concentration regulator package, which is formed by packaging the gas concentration regulator according to any one of <1> to <3> above into a small bag with a breathable packaging material.

[0020] Effects of the Invention

[0021] For the gas concentration regulator for bacterial culture of the present invention, the oxidation reaction of ascorbic acids has a large amount of carbon dioxide generation in the initial stage of the reaction (1 hour after the start of the reaction), and a gas environment with a high concentration of carbon dioxide and a low concentration of oxygen can be produced in a short time. If the gas concentration is set to a specified value as described later, it can also be used for culturing any of anaerobic bacteria, microaerophilic bacteria, and aerobic bacteria. Detailed Embodiments

[0022] Hereinafter, an embodiment of the present invention will be described. The content of the present invention is not limited to the embodiment described below.

[0023] It should be noted that in this specification, the term "A to B" for the description of numerical values means "A or more and B or less" (when A < B) or "A or less and B or more" (when A > B). In addition, in the present invention, a combination of preferred modes is a more preferred mode.

[0024] [Gas Concentration Regulator]

[0025] The gas concentration regulator for bacterial culture of the present invention is characterized in that it comprises (a) ascorbic acid, (b) transition metal catalyst, (c) activated carbon, (d) alkali metal carbonate or alkaline earth metal hydroxide and (e) water, wherein the ascorbic acid and the water are impregnated in the activated carbon, and the average pore size of the activated carbon is 2.0 nm or more. The gas concentration regulator is preferably used in the form of packaging the composition into a package with a breathable packaging material, wherein the composition comprises (a) ascorbic acid, (b) transition metal catalyst, (c) activated carbon, (d) alkali metal carbonate or alkaline earth metal hydroxide and (e) water. The gas concentration regulator of the present invention can be used for bacterial culture.

[0026] (a) Ascorbic acid

[0027] The gas concentration regulator of the present invention contains ascorbic acid having both oxygen absorption capability and carbon dioxide gas generation capability as a main agent for oxygen absorption reaction.

[0028] Ascorbic acid refers to L-ascorbic acid and its stereoisomers and salts and hydrates thereof. As L-ascorbic acid salts, sodium L-ascorbate, potassium L-ascorbate, calcium L-ascorbate, etc. can be listed. As stereoisomers of L-ascorbic acid, isoascorbic acid (D-isoascorbic acid) can be listed. As isoascorbic acid salts, sodium isoascorbate, potassium isoascorbate, calcium isoascorbate, etc. can be listed. Ascorbic acid can be a single type, or two or more types can be used in combination.

[0029] In the gas concentration regulator of the present invention, from the viewpoint of oxygen absorption performance, ascorbic acid is impregnated in activated carbon together with water. Specifically, an aqueous solution of ascorbic acid obtained by dissolving ascorbic acid in water is impregnated in activated carbon as a porous carrier. At this time, a high concentration of the aqueous solution of ascorbic acid can reduce the amount of porous carrier used, so the concentration of ascorbic acid is preferably set to a concentration as close to the saturated solubility as possible. Therefore, as ascorbic acid, it is preferred to select a compound with high solubility in water, specifically, L-sodium ascorbate is preferred. When using sodium L-ascorbate, it is appropriate to set the concentration of the aqueous solution to 40 to 55% by mass.

[0030] In the gas concentration regulator, the oxidation reaction of ascorbic acid is used to absorb oxygen in the atmosphere to adjust its concentration, and the generated carbon dioxide is used to adjust the carbon dioxide concentration in the atmosphere. It should be noted that in this oxidation reaction, carbon dioxide equimolar to the consumed oxygen molecules will be generated in theory. Due to the above principle, if the oxygen concentration decreases, the carbon dioxide concentration will also increase with it, but when a compound with carbon dioxide absorption capacity, such as an alkaline earth metal hydroxide, is mixed in the gas concentration regulator, the increase of carbon dioxide can also be suppressed. On the contrary, if a compound with carbon dioxide generation capacity is mixed in the gas concentration regulator, more carbon dioxide than the oxygen reduction amount can also be generated. In this way, in the gas concentration regulator of the present invention, by appropriately selecting the compounding components and / or the compounding amount of the composition constituting the gas concentration regulator, the degree of oxidation reaction and the amount of carbon dioxide generated are adjusted, so that the desired oxygen concentration and carbon dioxide concentration can be adjusted under a specific atmosphere. By adjusting to the desired oxygen concentration and carbon dioxide concentration, the culture environment of various bacteria suitable for anaerobic bacteria, microaerobic bacteria or aerobic bacteria can be adjusted.

[0031] (b) Transition Metal Catalysts

[0032] The gas concentration regulator of the present invention contains a transition metal catalyst that promotes the oxidation reaction of ascorbic acid.

[0033] The transition metal catalyst is a catalyst of a metal compound such as a salt or oxide of a transition metal. As the transition metal, iron, manganese, zinc, copper, and cobalt are suitable. As the salt of the transition metal, a halide and an inorganic acid salt of the transition metal are included, such as a chloride or sulfate of the transition metal. As a representative example, an anhydrous salt or a hydrated salt of ferrous chloride, ferric chloride, ferrous sulfate, ferrous sulfate, manganese chloride, zinc sulfate, copper sulfate, copper chloride, and cobalt sulfate can be listed, wherein preferably ferrous sulfate heptahydrate with good solubility in water and good compatibility.

[0034] From the viewpoint of promoting the oxidation reaction of ascorbic acid, the content of the transition metal catalyst in the gas concentration adjuster is preferably 5 to 25 parts by mass, more preferably 10 to 20 parts by mass, based on 100 parts by mass of ascorbic acid.

[0035] (c) Activated carbon

[0036] The gas concentration regulator of the present invention contains activated carbon. The activated carbon has a function as a carrier for the aqueous solution impregnated with ascorbic acid, and has a function of promoting oxidation reaction due to its large specific surface area and large contact area with air.

[0037] As activated carbon, for example, activated carbon produced by various methods such as activation by steam using sawdust, coal, coconut shell, etc. as raw materials, activation using drugs such as zinc chloride, etc. can be used. In addition, for activated carbon, granular activated carbon is preferred in order to load an aqueous solution of ascorbic acid on the activated carbon and fill it in a small bag in a granular form. From the viewpoint of oxygen absorption performance and filling property (fluidity) in a package, the particle size of the granular activated carbon is preferably 0.1 to 2 mm, more preferably 0.5 to 1 mm.

[0038] In the present invention, the average pore size of the activated carbon is more than 2.0nm. By making the average pore size of the activated carbon more than 2.0nm, the oxidation reaction of the ascorbic acid of the gas concentration regulator for bacterial culture of the present invention produces a large amount of carbon dioxide in the initial stage of the reaction (1 hour after the reaction starts), and a gas environment of a high-concentration carbon dioxide atmosphere and a low-concentration oxygen atmosphere can be produced in a short time. From the viewpoint of the amount of carbon dioxide produced in the oxidation reaction of ascorbic acid in the initial stage of the reaction (1 hour after the reaction starts), the average pore size of the activated carbon is preferably more than 2.2nm, more preferably more than 2.5nm. The upper limit of the average pore size of the activated carbon can be less than 5.5nm, less than 5.0nm, less than 4.5nm, or less than 4.0nm. It should be noted that the average pore size of the activated carbon in the present invention can be measured by the method described in the embodiments described later.

[0039] In the present invention, from the viewpoint of the amount of carbon dioxide generated in the initial stage of the oxidation reaction of ascorbic acid (one hour after the start of the reaction), the total pore volume of the activated carbon is preferably 0.60 cm 3 / g or more, more preferably 0.65cm 3 / g or more, more preferably 0.68cm 3 The upper limit of the total pore volume of activated carbon can be 2.0 cm 3 / g or less, can be 1.8cm 3 / g or less, can be 1.5cm 3 / g or less, or 1.0cm 3 It should be noted that the total pore volume of the activated carbon in the present invention can be measured by the method described in the examples described later.

[0040] From the viewpoint of oxygen absorption performance and packing properties in a package, the content of activated carbon in the gas concentration adjuster is preferably 50 to 400 parts by mass, more preferably 75 to 300 parts by mass, based on 100 parts by mass of ascorbic acid.

[0041] (d) Alkali metal carbonate or alkaline earth metal hydroxide

[0042] In order to adjust the carbon dioxide concentration, the gas concentration regulator of the present invention comprises an alkali metal carbonate or an alkaline earth metal hydroxide. Alkali metal carbonate is used for the purpose of rapidly carrying out the oxidation reaction of ascorbic acid and controlling the reaction field in the alkaline region. On the other hand, alkaline earth metal hydroxide can be used as a carbon dioxide gas absorbent. It should be noted that when an alkali metal carbonate and an alkaline earth metal hydroxide are used in combination, it is difficult to adjust the carbon dioxide concentration, so it is not preferred to use the two in combination.

[0043] As the alkali metal carbonate, water-soluble alkali metal carbonates such as sodium carbonate, sodium hydrogen carbonate and sodium carbonate hydrate can be preferably used, and among them, sodium carbonate is particularly preferred.

[0044] As alkaline earth metal hydroxide, particularly calcium hydroxide, magnesium hydroxide or their mixture can be suitably used. When wishing to absorb a large amount of carbon dioxide in a short time, calcium hydroxide etc. can be suitably used, which has high solubility in water and a fast carbon dioxide absorption rate. And then, the generation of carbon dioxide is not constant but changes from moment to moment. For example, when forming a gas atmosphere of oxygen concentration 0%, carbon dioxide concentration 5% more rapidly, in order to absorb all oxygen in the atmosphere at once, a large amount of carbon dioxide is generated at once after the reaction of the oxidation reaction of ascorbic acid just begins, and then the generation of carbon dioxide is rapidly reduced. In this case, preferably calcium hydroxide with a fast carbon dioxide absorption rate is mixed with magnesium hydroxide with a slow carbon dioxide absorption rate.

[0045] The alkaline earth metal hydroxide is preferably in the form of a powder, and the average particle size thereof is preferably 1 to 100 μm, more preferably 2 to 50 μm.

[0046] From the viewpoint of adjusting the carbon dioxide concentration, the content of the alkali metal carbonate or alkaline earth metal hydroxide in the gas concentration adjusting agent is preferably 10 to 200 parts by mass, more preferably 15 to 150 parts by mass, and further preferably 20 to 100 parts by mass based on 100 parts by mass of ascorbic acid.

[0047] (e) Water

[0048] The gas concentration regulator of the present invention contains water necessary for the oxidation reaction of ascorbic acid.

[0049] As for water, from the viewpoint that the gas concentration regulator can be obtained in the form of a fluid solid, it is preferred to adopt a form impregnated in activated carbon. In the gas concentration regulator of the present invention, from the viewpoint of oxygen absorption performance, water is impregnated in activated carbon together with ascorbic acid. Specifically, an aqueous solution of ascorbic acid obtained by dissolving ascorbic acid in water is impregnated in activated carbon as a porous carrier. In addition, soluble components other than ascorbic acid can be dissolved in water, and insoluble components can also be dispersed.

[0050] From the viewpoint of advancing the oxidation reaction of ascorbic acid, the content of water in the gas concentration adjuster is preferably 100 to 200 parts by mass, more preferably 110 to 180 parts by mass, and further preferably 120 to 180 parts by mass based on 100 parts by mass of ascorbic acid.

[0051] (f) Other ingredients

[0052] The gas concentration adjusting agent of the present invention may contain components other than the above-mentioned components (a) to (e) as necessary within a range not impairing the effects of the present invention.

[0053] (f1) Thermoplastic resin

[0054] In order to suppress excessive heat generation accompanying the oxygen absorption reaction (oxidation reaction of ascorbic acid), the gas concentration regulator of the present invention may contain a thermoplastic resin. The type of thermoplastic resin is not particularly limited, for example, polyethylene, polypropylene, ethylene-vinyl acetate copolymer, elastomer or a mixture thereof can be used, and from the viewpoint of easy adjustment of the softening point and less influence of odor, low molecular weight polyethylene, polypropylene or a mixture thereof with a molecular weight of less than 10,000 can be preferably used.

[0055] From the viewpoint of miscibility with other components, the thermoplastic resin is preferably in the form of granules having a particle size of 1 to 500 μm, more preferably 10 to 300 μm. From the viewpoint of more effectively suppressing heat generation, the softening point of the thermoplastic resin is preferably 90 to 125°C.

[0056] From the viewpoint of advancing the oxidation reaction of ascorbic acid, the content of the thermoplastic resin in the gas concentration adjuster is preferably 35 to 300 parts by mass, more preferably 60 to 200 parts by mass, based on 100 parts by mass of ascorbic acid.

[0057] (f2) Formaldehyde remover

[0058] In order to mainly remove the aldehydes produced as a byproduct of the oxidation reaction of ascorbic acid, the gas concentration regulator of the present invention may further include an aldehyde scavenger. As compounds having aldehyde scavenging ability, various compounds such as amines are known, but preferably, ethylene urea, urea, arginine, lysine hydrochloride or polyallylamine having sufficient aldehyde scavenging ability, no irritating odor is observed, and high effects are exerted in a small amount are preferably mixed, and ethylene urea having high effects in a smaller amount is more preferred.

[0059] The so-called aldehyde in this specification refers to a compound having one or more formyl groups in its molecule, i.e., aldehydes. In the present invention, it typically refers to an aldehyde produced as a by-product component during oxygen absorption or bacterial culture. As the aldehyde, any aldehyde classified as aldehyde in the chemical field is included as long as it does not have an adverse effect on bacterial culture. Specifically, for example, formaldehyde, acetaldehyde, etc. are included.

[0060] From the viewpoint of removing aldehyde efficiently and inexpensively, the content of the aldehyde-removing agent in the gas concentration regulator is preferably 0.5 to 25 parts by mass, more preferably 1.0 to 10 parts by mass, and further preferably 1.0 to 5.0 parts by mass based on 100 parts by mass of ascorbic acid.

[0061] The method for producing the gas concentration regulator of the present invention is not particularly limited, and examples thereof include a method in which a transition metal catalyst, an alkali metal carbonate, an alkaline earth metal hydroxide, or the like is dissolved in an aqueous solution of ascorbic acid, and the solution is mixed and impregnated into activated carbon.

[0062] [Gas concentration regulator package]

[0063] The gas concentration adjusting agent can also be made into a gas concentration adjusting agent package by packaging the composition containing the above-mentioned components with a packaging material using a gas-permeable packaging material in whole or in part.

[0064] (Packaging Materials)

[0065] Examples of packaging materials include: a bag-shaped packaging material formed by laminating two air-permeable packaging materials; a bag-shaped packaging material formed by laminating one air-permeable packaging material and one non-air-permeable packaging material; and a bag-shaped packaging material formed by bending one air-permeable packaging material and sealing the edges together except for the bent portion.

[0066] Here, when the air-permeable packaging material and the non-air-permeable packaging material are quadrilateral, the packaging material may be: a bag-shaped bag formed by overlapping two air-permeable packaging materials and heat-sealing four sides; a bag-shaped bag formed by overlapping one air-permeable packaging material and one non-air-permeable packaging material and heat-sealing four sides; a bag-shaped bag formed by bending one air-permeable packaging material and heat-sealing three sides except the bent portion. In addition, the packaging material may be a bag-shaped bag formed by forming the air-permeable packaging material into a tube and heat-sealing both ends and the main body of the tube.

[0067] (Breathable packaging material)

[0068] As the breathable packaging material, a packaging material that is permeable to oxygen and carbon dioxide can be selected. Among them, a breathable packaging material having a breathability resistance of 600 seconds or less, more preferably 90 seconds or less based on the Gurley tester method can be suitably used. Here, the breathability resistance refers to the value measured by the method of JIS P8117 (1998). More specifically, it refers to the time required for 100 mL of air to pass through the breathable packaging material using a Gurley air permeability tester manufactured by Toyo Seiki Seisaku-sho Co., Ltd.

[0069] As the above-mentioned breathable packaging material, in addition to paper and nonwoven fabrics, breathable packaging materials that are imparted with breathability to plastic films can be used. As plastic films, for example, laminated films formed by laminating and bonding films such as polyethylene terephthalate, polyamide, polypropylene, and polycarbonate with films such as polyethylene, ionomer, polybutadiene, ethylene acrylic acid copolymer, ethylene methacrylic acid copolymer, or ethylene vinyl acetate copolymer as heat seal layers can be used. In addition, these laminates can also be used as breathable packaging materials.

[0070] As a method of imparting air permeability, various methods can be used in addition to perforation processing by cold needles or hot needles. When perforation processing is used to impart air permeability, the air permeability can be freely adjusted according to the diameter, number, material, etc. of the holes to be perforated.

[0071] The thickness of the laminated film is preferably 50 to 300 μm, particularly preferably 60 to 250 μm. In this case, compared with a case where the thickness is not within the above range, a packaging material can be obtained that maintains strength and has excellent heat-sealing properties and packaging suitability.

[0072] In order to maintain the function of the gas concentration adjusting agent package for a long time, it is preferred that the package is stored in a gas-tight container or bag before use and taken out of the gas-tight container or bag when used. In addition, when the gas concentration adjusting agent package is used for bacterial culture, it is preferred that the package be sterilized in advance using gamma rays or the like.

[0073] [Bacterial culture method]

[0074] The bacterial culture method of the present invention is a method for culturing bacteria in the presence of a gas concentration regulator. Specifically, it can be implemented in the following manner: after the gas concentration regulator (preferably a gas concentration regulator package) is placed in a gas-tight closed container together with a culture container containing bacteria and a culture medium, it is sealed, and the closed container is left to stand at a temperature suitable for bacterial culture, thereby implementing. At this time, for the purpose of measuring the amount of aldehyde produced in the gas-tight closed container, regulating the humidity in the container, etc., an open container containing distilled water can also be provided in the closed container. As an open container, in addition to the culture container, a beaker, a flask, etc. can be exemplified, preferably a container of the same type as the culture container containing bacteria and a culture medium.

[0075] The oxygen concentration in the sealed container is preferably 6 to 14% by volume when culturing microaerobic bacteria, and is preferably 1% by volume or less when culturing anaerobic bacteria.

[0076] The carbon dioxide concentration in the sealed container is preferably 1 to 10% by volume, more preferably 2 to 10% by volume, and further preferably 2 to 9% by volume when culturing microaerobic bacteria. When culturing anaerobic bacteria, it is preferably 10% by volume or more, more preferably 12% by volume or more, and further preferably 14% by volume or more.

[0077] Suitable oxygen and carbon dioxide concentrations vary depending on the bacteria, but it is important to reach the desired concentration in a short time. When cultivating microaerobic bacteria, the oxygen concentration of the oxidation reaction of ascorbic acid in the initial stage of the reaction (after 1 hour from the start of the reaction) is preferably 2 to 18% by volume, more preferably 3 to 17% by volume, and further preferably 4 to 16% by volume. The carbon dioxide concentration of the oxidation reaction of ascorbic acid in the initial stage of the reaction (after 1 hour from the start of the reaction) is preferably 1 to 10% by volume, more preferably 2 to 10% by volume, and further preferably 2 to 9% by volume. In addition, when cultivating anaerobic bacteria, the oxygen concentration of the oxidation reaction of ascorbic acid in the initial stage of the reaction (after 1 hour from the start of the reaction) is preferably 2% by volume or less, more preferably 1% by volume or less. The carbon dioxide concentration of the oxidation reaction of ascorbic acid in the initial stage of the reaction (after 1 hour from the start of the reaction) is preferably 10% by volume or more, more preferably 12% by volume or more, and further preferably 14% by volume or more.

[0078] The culture temperature is preferably 20 to 45°C, particularly preferably 25 to 40°C.

[0079] In the bacterial culture method of the present invention, the atmosphere in the gas-tight container before the culture is implemented does not need to be specially controlled, for example, it can also be air. Fill the gas-tight container with air, and when the culture method of the present invention is implemented using a gas concentration regulator that can produce carbon dioxide of the same volume as the absorbed oxygen, if the carbon dioxide concentration is set to 2-10% by volume, the oxygen concentration is about 11-19% by volume, and if the carbon dioxide concentration is set to 3-8% by volume, the oxygen concentration is about 13-18% by volume. However, in the culture method using the gas concentration regulator of the present invention, the oxygen concentration is not particularly limited. In addition, as a suitable bacterial culture condition, the aldehyde concentration dissolved in the culture medium is preferably less than 2 mg / L, more preferably less than 1.5 mg / L, and further preferably less than 1.0 mg / L.

[0080] The bacterium used in the culture method of the present invention is not particularly limited. The culture method of the bacterium of the present invention can also be used for the culture of any bacterium of anaerobic bacteria, microaerobic bacteria and aerobic bacteria. There is no particular restriction on the culture medium used in the culture method of the present invention. Since the commonly used culture medium can be directly used, the culture medium suitable for the cultured bacteria can be freely selected. In addition, the culture container is not particularly limited as long as the air permeability with the outside of the container is ensured, and the volume, shape, material, etc. can all be adopted as any one suitable for culture. The culture container with a lid portion can be suitably used, but the air permeability with the outside of the container must also be ensured at this time.

[0081] The gas-barrier sealed container used in the bacterial culture method is a container that blocks the flow of gas inside and outside the container to maintain the oxygen and carbon dioxide concentrations formed by the gas concentration regulator for a long time. Containers made of glass, metal, plastics such as polycarbonate, etc. are often used, but gas-barrier films and laminates thereof can also be used.

[0082] According to the culture method of the present invention, it is possible to perform microscopic observation and transportation of bacteria in a suitable gas atmosphere without using a gas tank and a gas regulator.

[0083] Example

[0084] Hereinafter, the present embodiment will be described in detail using Examples and Comparative Examples, but the present embodiment can be modified as appropriate as long as the effects of the present invention are exhibited. It should be noted that "parts" in Examples and Comparative Examples refer to parts by mass unless otherwise specified.

[0085] The activated carbon used in Examples and Comparative Examples is shown in Table 1. The average pore diameter and total pore volume of the activated carbon were measured by the following method.

[0086] (Average pore size and total pore volume of activated carbon)

[0087] In the determination of the average pore size and total pore volume of activated carbon, "BELSORP-max" manufactured by Microtrac BEL was used, and about 0.1 g of the sample was subjected to vacuum degassing at 130°C under pretreatment conditions, and the nitrogen adsorption isotherm in liquid nitrogen (77K) was measured. The total pore volume and average pore size were calculated using the BET multipoint method using the accompanying software.

[0088] [Table 1]

[0089] Table 1

[0090]

[0091] Example 1

[0092] 6 g of ferrous sulfate heptahydrate was dissolved in 100 g of an aqueous solution of sodium L-ascorbate (concentration 45% by mass), the aqueous solution was impregnated into 60 g of activated carbon (C1), and then 70 g of a low molecular weight polyethylene and 20 g of sodium carbonate decahydrate were added and mixed to obtain a gas concentration regulator (1).

[0093] Separately, a small air-permeable bag of Japanese paper measuring 90 mm in length and 55 mm in width, which was laminated with a porous polyethylene film, was filled with 5 g of the gas concentration adjuster (1) to obtain a gas concentration adjuster package (1).

[0094] Embodiments 2 to 4

[0095] In Example 1, except that the activated carbon (C1) was changed to activated carbons (C2) to (C4), respectively, gas concentration adjusters (2) to (4) and gas concentration adjuster packages (2) to (4) were obtained in the same manner as in Example 1.

[0096] Comparative Example 1

[0097] In Example 1, a gas concentration adjusting agent (5) and a gas concentration adjusting agent package (5) were obtained in the same manner as in Example 1 except that the activated carbon (C1) was changed to the activated carbon (C5).

[0098] [evaluate]

[0099] The oxygen concentration and carbon dioxide gas concentration of the gas concentration adjusting agent packages (1) to (5) obtained in the examples and comparative examples were measured by the following method.

[0100] (Oxygen concentration and carbon dioxide gas concentration)

[0101] The gas concentration regulator package, two petri dishes for aldehyde measurement (60 mm in diameter, 5 mL of distilled water added), and 2.5 L of air were sealed in a bag (400×220 mm) of nylon film coated with polyvinylidene chloride, and the bag was kept warm in a constant temperature bath at 35°C to measure the temporal changes in the gas components in the bag. The temporal changes in the oxygen and carbon dioxide gas concentrations were simultaneously measured using an oxygen / carbon dioxide gas analyzer "CheckMate 3" (manufactured by MOCON Europe).

[0102] [Table 2]

[0103] Table 2

[0104]

[0105] As can be seen from Table 2, in Comparative Example 1 using activated carbon (C5) having an average pore size of less than 2.0 nm, the oxidation reaction of ascorbic acid has a low carbon dioxide concentration 1 to 2 hours after the reaction starts, and the amount of carbon dioxide generated in the initial stage of the reaction is small. In contrast, in the embodiment using activated carbon having an average pore size of 2.0 nm or more, the oxidation reaction of ascorbic acid has a high carbon dioxide generation concentration 1 hour after the reaction starts, and a gas environment of a high-concentration carbon dioxide atmosphere and a low-concentration oxygen atmosphere can be produced in a short time.

Claims

1. A gas concentration regulator for bacterial culture, comprising: (a) ascorbic acid, (b) a transition metal catalyst, (c) activated carbon, (d) an alkali metal carbonate or an alkaline earth metal hydroxide, and (e) water, wherein the ascorbic acid and the water are impregnated in the activated carbon, the average pore size of the activated carbon is greater than or equal to 2.0 nm and less than or equal to 5.5 nm, and the total pore volume of the activated carbon is 0.60 cm 3 / g or above, In the measurement of the average pore size and total pore volume of activated carbon, "BELSORP-max" manufactured by Microtrac BEL was used. 0.1 g of the sample was degassed under vacuum at 130°C under pretreatment conditions, and the nitrogen adsorption isotherm in liquid nitrogen at 77K was measured. The total pore volume and average pore size were calculated using the BET multi-point method using the accompanying software.

2. Use of the gas concentration regulator according to claim 1 in bacterial culture. 3 . A method for culturing bacteria, comprising culturing bacteria in the presence of the gas concentration regulator according to claim 1 .

4. A gas concentration adjusting agent package, which is obtained by packaging the gas concentration adjusting agent according to claim 1 into a small bag shape using an air-permeable packaging material.

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