Gas concentration regulator for anaerobic bacteria culture and method for culturing anaerobic bacteria using the same

Abstract

A gas concentration regulator for anaerobic bacteria culture, comprising: (a) dehydroascorbic acid; (c) a transition metal catalyst; (d) activated carbon; (e) at least one selected from the group consisting of alkali metal carbonates, alkali metal hydroxides, and alkaline earth metal hydroxides; and (f) water.

Description

Technical Field

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

[0002] In the culture of biological specimens such as tissues / cells carried out in the research or industrial fields of biology, reproduction, or biotechnology, a gas environment different from the atmospheric atmosphere is required. For example, as a condition for maintaining the pH of a bicarbonate buffer system 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 culture is carried out in a low-concentration oxygen atmosphere similar to that in the living body.

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

[0004] Prior Art Documents

[0005] Patent Documents

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

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

[0008] Problems to be Solved by the Invention

[0009] In the culture of anaerobic bacteria, it is required to create a gas environment of a high-concentration carbon dioxide atmosphere. Therefore, in the oxidation reaction of ascorbic acids, it is desired to more effectively increase the amount of carbon dioxide generated.

[0010] The problem to be solved by the present invention is to provide a gas concentration regulator for culturing anaerobic bacteria that has a large amount of carbon dioxide generation.

[0011] Solutions to the Problems

[0012] The present inventors conducted intensive studies and as a result, found that: compared with the case where ascorbic acids are blended, the amount of carbon dioxide generated increases when dehydroascorbic acid is blended. Based on such an insight, the present invention has been completed.

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

[0014] <1>A gas concentration regulator for anaerobic bacteria culture, comprising: (a) dehydroascorbic acid; (c) a transition metal catalyst; (d) activated carbon; (e) at least one selected from the group consisting of alkali metal carbonates, alkali metal hydroxides, and alkaline earth metal hydroxides; and (f) water.

[0015] <2>The gas concentration regulator for anaerobic bacteria culture according to <1> above further comprises (b) ascorbic acids.

[0016] <3>In the gas concentration regulator for anaerobic bacteria culture according to <2> above, the molar ratio [(b) / (a)] of (b) ascorbic acids to (a) dehydroascorbic acid is 1.5 or less.

[0017] <4>A method for culturing anaerobic bacteria, which cultures anaerobic bacteria in the presence of the gas concentration regulator according to any one of <1> to <3> above.

[0018] <5>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 pouch with a gas-permeable packaging material.

[0019] Effects of the Invention

[0020] The gas concentration regulator for anaerobic bacteria culture of the present invention has a large amount of carbon dioxide generation and can effectively create a gas environment with a high-concentration carbon dioxide atmosphere and a low-concentration oxygen atmosphere. Detailed Embodiments

[0021] Hereinafter, an embodiment of the present invention will be described. The content of the present invention is not limited to the embodiments described below.

[0022] It should be noted that in this specification, the term "A to B" for 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.

[0023] [Gas Concentration Regulator]

[0024] The gas concentration regulator for anaerobic bacteria culture of the present invention comprises: (a) dehydroascorbic acid; (c) a transition metal catalyst; (d) activated carbon; (e) at least one selected from the group consisting of alkali metal carbonates, alkali metal hydroxides, and alkaline earth metal hydroxides; and (f) water. The gas concentration regulator may further comprise (b) ascorbic acids.

[0025] In addition, the gas concentration regulator of the present invention is preferably used as the following package, which is a package of a composition containing (a) dehydroascorbic acid, (c) transition metal catalyst, (d) activated carbon, (e) at least one selected from the group consisting of alkali metal carbonates, alkali metal hydroxides, and alkaline earth metal hydroxides, and (f) water with a gas-permeable packaging material. The composition may further contain (b) ascorbic acids.

[0026] In the gas concentration regulator of the present invention, the total of the components (a) to (f) preferably contains 50% by mass or more, more preferably 60% by mass or more, and still more preferably 70% by mass or more.

[0027] (a) Dehydroascorbic acid

[0028] The gas concentration regulator of the present invention contains dehydroascorbic acid, which has both oxygen absorption ability and carbon dioxide gas generation ability, as the main agent for the oxygen absorption reaction.

[0029] Dehydroascorbic acid is a compound obtained by oxidizing ascorbic acid and generates diketogulonic acid by hydrolysis. In addition, by adjusting a suitable catalyst and reaction environment, the decarbonation reaction and oxidation reaction are repeated to absorb oxygen while generating carbon dioxide. Dehydroascorbic acid can also be generated by oxidizing ascorbic acids.

[0030] In the gas concentration regulator of the present invention, from the viewpoint of oxygen absorption performance, dehydroascorbic acid is preferably impregnated in activated carbon together with water. Specifically, an aqueous solution of dehydroascorbic acid in which dehydroascorbic acid is dissolved in water is preferably impregnated in activated carbon as a porous carrier.

[0031] In the gas concentration regulator, the oxygen in the atmosphere is absorbed by the oxidation reaction of dehydroascorbic acid to adjust its concentration, and the carbon dioxide generated is used to adjust the carbon dioxide concentration in the atmosphere. It should be noted that in this oxidation reaction, theoretically, carbon dioxide equimolar or more than the consumed oxygen molecules is generated. Due to the above principle, when the oxygen concentration decreases, the carbon dioxide concentration increases accordingly.

[0032] (b) Ascorbic acids

[0033] The gas concentration regulator of the present invention may contain ascorbic acids. Ascorbic acid is cheaper and easier to obtain than dehydroascorbic acid and has both oxygen absorption ability and carbon dioxide gas generation ability as the main agent for the oxygen absorption reaction.

[0034] Ascorbic acids refer to L-ascorbic acid, its stereoisomers, salts and hydrates. As L-ascorbate salts, sodium L-ascorbate, potassium L-ascorbate, calcium L-ascorbate, etc. can be cited. As stereoisomers of L-ascorbic acid, isoascorbic acid (D-isoascorbic acid), etc. can be cited. As isoascorbate salts, sodium isoascorbate, potassium isoascorbate, calcium isoascorbate, etc. can be cited. Ascorbic acids can be a single kind or two or more kinds can be used in combination.

[0035] In the gas concentration regulator of the present invention, from the viewpoint of oxygen absorption performance, it is preferable that ascorbic acids are impregnated in activated carbon together with water. Specifically, it is preferable to impregnate an aqueous solution of ascorbic acids obtained by dissolving ascorbic acids in water into activated carbon as a porous carrier. At this time, a higher concentration of the aqueous solution of ascorbic acids can reduce the amount of the porous carrier used, so the concentration of ascorbic acids is preferably set to a concentration as close as possible to the saturation solubility. Therefore, as ascorbic acids, it is preferable to select compounds with high solubility in water. From the viewpoints of solubility in water and ease of acquisition, ascorbic acid or sodium ascorbate is preferable as ascorbic acids. When using ascorbic acid or sodium ascorbate, it is suitable to set the concentration of the aqueous solution to 40 to 55% by mass.

[0036] When the gas concentration regulator of the present invention contains (b) ascorbic acids, the molar ratio [(b) / (a)] of (b) ascorbic acids to (a) dehydroascorbic acid is preferably 1.5 or less, more preferably 1.2 or less, further preferably 1.0 or less, further preferably 0.7 or less, further preferably 0.5 or less, and further preferably 0.1 or less. When culturing anaerobic bacteria, it is used by being put into an airtight container with gas barrier properties, and the above composition is sufficient at the time of its input.

[0037] (c) Transition metal catalyst

[0038] The gas concentration regulator of the present invention contains a transition metal catalyst that promotes the oxidation reaction of dehydroascorbic acid and ascorbic acids.

[0039] The transition metal catalyst is a catalyst having metal compounds such as salts and oxides of transition metals. As transition metals, iron, manganese, zinc, copper, and cobalt are suitable. As salts of transition metals, they include halides and inorganic acid salts of transition metals, for example, chlorides and sulfates of transition metals. As representative examples, anhydrous salts or hydrates of ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, manganese chloride, zinc sulfate, copper sulfate, copper chloride, cobalt sulfate, etc. can be cited, and among them, ferrous sulfate heptahydrate with good solubility in water and good compatibility is preferred.

[0040] From the viewpoint of promoting the oxidation reaction of dehydroascorbic acid and ascorbic acids, the content of the transition metal catalyst in the gas concentration regulator is preferably 1 to 30 parts by mass, more preferably 5 to 25 parts by mass, and still more preferably 10 to 20 parts by mass, relative to a total of 100 parts by mass of dehydroascorbic acid and ascorbic acids.

[0041] (d) Activated carbon

[0042] The gas concentration regulator of the present invention contains activated carbon. Activated carbon has the function of being a carrier for impregnating an aqueous solution of dehydroascorbic acid and an aqueous solution of ascorbic acids, and due to its large specific surface area, it has a large contact area with air and has the function of promoting the progress of the oxidation reaction.

[0043] As the activated carbon, for example, activated carbon manufactured by various production methods such as using sawdust, coal, coconut shells, etc. as raw materials and activating with steam or activating with a drug such as zinc chloride can be used. In addition, for the activated carbon, in order to load an aqueous solution of dehydroascorbic acid, an aqueous solution of ascorbic acids, etc. onto the activated carbon and fill it in granules in a sachet, granular activated carbon is preferably used. From the viewpoints of oxygen absorption performance and fillability (fluidity) in the package, the particle size of the granular activated carbon is preferably 0.1 to 2 mm, and more preferably 0.5 to 1 mm.

[0044] From the viewpoints of oxygen absorption performance and fillability in the package, the content of the activated carbon in the gas concentration regulator is preferably 50 to 400 parts by mass, and more preferably 75 to 300 parts by mass, relative to a total of 100 parts by mass of dehydroascorbic acid and ascorbic acids.

[0045] (e) Alkali metal carbonates, alkali metal hydroxides, and alkaline earth metal hydroxides

[0046] The gas concentration regulator of the present invention contains at least one selected from the group consisting of alkali metal carbonates, alkali metal hydroxides, and alkaline earth metal hydroxides. At least one selected from the group consisting of alkali metal carbonates, alkali metal hydroxides, and alkaline earth metal hydroxides is used to rapidly progress the oxidation reaction of dehydroascorbic acid and ascorbic acids and to control the reaction field in an alkaline region.

[0047] As the alkali metal carbonate, water-soluble alkali metal carbonates such as sodium carbonate, sodium bicarbonate, and sodium carbonate hydrate can be suitably used, and sodium carbonate is particularly preferred.

[0048] As the alkali metal hydroxide, potassium hydroxide and sodium hydroxide can be mentioned, and sodium hydroxide is preferred.

[0049] As the alkaline earth metal hydroxide, calcium hydroxide and magnesium hydroxide can be mentioned.

[0050] Among alkali metal carbonates, alkali metal hydroxides, and alkaline earth metal hydroxides, from the viewpoint of solubility in water when forming a salt with ascorbic acid, the gas concentration regulator of the present invention preferably contains an alkali metal carbonate and / or an alkali metal hydroxide.

[0051] From the viewpoint of neutralizing hydrolysis products, the content of the alkali metal hydroxide in the gas concentration regulator is preferably equimolar to the total molar amount of dehydroascorbic acid and ascorbic acids.

[0052] From the viewpoint of regulating the carbon dioxide concentration, the content of the alkali metal carbonate in the gas concentration regulator is preferably 10 to 200 parts by mass, more preferably 50 to 200 parts by mass, and further preferably 100 to 150 parts by mass with respect to a total of 100 parts by mass of dehydroascorbic acid and ascorbic acids.

[0053] (f) Water

[0054] The gas concentration regulator of the present invention contains water required for the oxidation reaction of dehydroascorbic acid and ascorbic acids.

[0055] Regarding water, from the viewpoint that the gas concentration regulator can be obtained in the form of a flowable solid, it is preferably in a form impregnated in activated carbon. In the gas concentration regulator of the present invention, from the viewpoint of oxygen absorption performance, it is preferable that water is impregnated in activated carbon together with dehydroascorbic acid and ascorbic acids. Specifically, it is preferable to impregnate an aqueous solution of dehydroascorbic acid and an aqueous solution of ascorbic acids in which dehydroascorbic acid or ascorbic acids are dissolved in water into activated carbon as a porous carrier. In addition, soluble components other than dehydroascorbic acid and ascorbic acids may be dissolved in water, and insoluble components may be dispersed.

[0056] From the viewpoint of carrying out the oxidation reaction of dehydroascorbic acid and ascorbic acids, the content of water in the gas concentration regulator is preferably 30 to 500 parts by mass, more preferably 50 to 300 parts by mass, and further preferably 80 to 200 parts by mass with respect to a total of 100 parts by mass of dehydroascorbic acid and ascorbic acids.

[0057] (g) Other components

[0058] The gas concentration regulator of the present invention may contain components other than the above components (a) to (f) as needed within a range that does not impair the effects of the present invention.

[0059] (g1) Thermoplastic resin

[0060] In order to suppress excessive heat generation accompanying the oxygen absorption reaction (oxidation reaction of dehydroascorbic acid and ascorbic acids), the gas concentration regulator of the present invention may contain a thermoplastic resin. The type of the thermoplastic resin is not particularly limited. For example, polyethylene, polypropylene, ethylene-vinyl acetate copolymer, elastomer, or a mixture thereof can be used. From the viewpoints of easy adjustment of the softening point and less influence of odor, low molecular weight polyethylene, polypropylene, or a mixture thereof having a molecular weight of 10,000 or less is preferably used.

[0061] From the viewpoint of miscibility with other components, the thermoplastic resin is preferably a granular material having a particle size of 1 to 500 μm, more preferably 10 to 300 μm. In addition, from the viewpoint of more effectively suppressing heat generation, the softening point of the thermoplastic resin is preferably 90 to 125°C.

[0062] From the viewpoint of carrying out the oxidation reaction of dehydroascorbic acid and ascorbic acids, the content of the thermoplastic resin in the gas concentration regulator is preferably 100 to 1000 parts by mass, more preferably 300 to 500 parts by mass, relative to a total of 100 parts by mass of dehydroascorbic acid and ascorbic acids.

[0063] (g2) Aldehyde scavenger

[0064] In order to mainly remove aldehydes by-produced during the oxidation reaction of dehydroascorbic acid and ascorbic acids, the gas concentration regulator of the present invention may further contain an aldehyde scavenger. As compounds having aldehyde scavenging ability, various compounds such as amines are well-known, but ethylenethiourea, urea, arginine, lysine hydrochloride, or polyallylamine having sufficient aldehyde scavenging ability, not found to produce irritating odor, and exhibiting high effects in a small amount are preferably blended, and ethylenethiourea having high effects in an even smaller amount is more preferably used.

[0065] The aldehyde referred to in this specification means a compound having one or more formyl groups in its molecule, that is, an aldehyde. 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 an aldehyde in the chemical field may be included as long as it does not have an adverse effect on bacterial culture. Specifically, for example, it includes formaldehyde, acetaldehyde, etc.

[0066] From the viewpoints of effectively and inexpensively removing aldehydes, the content of the aldehyde scavenger in the gas concentration regulator is preferably 0.5 to 25 parts by mass, more preferably 1 to 10 parts by mass, and further preferably 1 to 5 parts by mass, relative to a total of 100 parts by mass of dehydroascorbic acid and ascorbic acids.

[0067] (g3) Covering material

[0068] The gas concentration regulator of the present invention may also have a covering material on the outside of the granulated product of the composition containing the above components (a) to (f). As the covering material, from the viewpoint of adsorbing trace odor components generated by the oxygen absorption reaction, porous particles such as activated carbon, zeolite, and silicate can be mentioned. In addition, from the viewpoint of improving the fluidity of the gas concentration regulator and easily filling and packaging the gas concentration regulator in a packaging material, talc, magnesium stearate, calcium stearate, etc. can be mentioned. These auxiliaries can be used alone, or two or more kinds can be used in combination as needed. In addition, these auxiliaries can also be easily obtained as commercially available products.

[0069] (Method for manufacturing gas concentration regulator)

[0070] The method for manufacturing the gas concentration regulator of the present invention is not particularly limited. For example, the following methods can be mentioned: a method of preparing an aqueous solution of dehydroascorbic acid in which dehydroascorbic acid, a transition metal catalyst, an alkali metal hydroxide, etc. are dissolved, and mixing this solution with activated carbon and an alkali metal carbonate and impregnating it into the activated carbon. In addition, the following method can also be mentioned: preparing an aqueous solution of ascorbic acid in which ascorbic acid, a transition metal catalyst, an alkali metal hydroxide, etc. are dissolved, and on the other hand, preparing an aqueous solution of dehydroascorbic acid in which dehydroascorbic acid, a transition metal catalyst, an alkali metal hydroxide, etc. are dissolved, and mixing these solutions with activated carbon and an alkali metal carbonate and impregnating it into the activated carbon.

[0071] [Gas concentration regulator package]

[0072] For the gas concentration regulator, by packaging the composition containing the above components with a packaging material that uses all or part of a breathable packaging material, a gas concentration regulator package can also be produced.

[0073] (Packaging material)

[0074] As the packaging material, the following can be mentioned: a packaging material formed by laminating two breathable packaging materials to form a bag shape; a packaging material formed by laminating one breathable packaging material and one non-breathable packaging material to form a bag shape; a packaging material formed by bending one breathable packaging material and sealing the edges to each other except at the bent part to form a bag shape.

[0075] Here, when the breathable packaging material and the non-breathable packaging material are quadrilateral, the packaging material can be mentioned: a bag formed by overlapping two breathable packaging materials and heat-sealing the four sides; a bag formed by overlapping one breathable packaging material and one non-breathable packaging material and heat-sealing the four sides; a bag formed by bending one breathable packaging material and heat-sealing three sides except at the bent part. In addition, the packaging material can also be a bag formed by making a breathable packaging material into a tubular shape and heat-sealing both ends and the main body part of the tubular body.

[0076] Ventilation packaging material

[0077] As the ventilation packaging material, a packaging material that can permeate oxygen and carbon dioxide can be selected. Among them, a ventilation packaging material with an air permeability resistance of 600 seconds or less, more preferably 90 seconds or less, based on the Gurley type test machine method can be suitably used. Here, the air permeability 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 permeate through the ventilation packaging material using a Gurley type air permeability tester manufactured by Toyo Seiki Seisakusho Co., Ltd.

[0078] As the above-mentioned ventilation packaging material, in addition to paper and non-woven fabric, a ventilation packaging material obtained by imparting ventilation to a plastic film can also be used. As the plastic film, for example, a laminated film obtained by laminating and bonding a film such as polyethylene terephthalate, polyamide, polypropylene, polycarbonate, etc. and a film such as polyethylene, ionomer, polybutadiene, ethylene / acrylic copolymer, ethylene / methacrylic acid copolymer or ethylene / vinyl acetate copolymer as a heat-sealing layer can be used. In addition, these laminates can also be used as ventilation packaging materials.

[0079] As a method for imparting ventilation, in addition to the perforation process based on cold needles and hot needles, various methods can be adopted. When imparting ventilation by perforation, the ventilation can be freely adjusted according to the diameter, number, material, etc. of the perforated holes.

[0080] In addition, the thickness of the laminated film is preferably 50 to 300 μm, particularly preferably 60 to 250 μm. At this time, compared with the case where the thickness is not within the above range, a packaging material with excellent strength retention, heat-sealability, and packaging adaptability can be produced.

[0081] In order to maintain the function of the above-mentioned gas concentration regulator package for a long time, it is preferable to store it in a gas-barrier container or bag before use and take it out from the gas-barrier container or bag for use when in use. In addition, when the gas concentration regulator package is used for the purpose of culturing bacteria, it is preferable to sterilize the package in advance using γ-rays or the like.

[0082] [Cultivation method of anaerobic bacteria]

[0083] The cultivation method of anaerobic bacteria of the present invention is a method for cultivating anaerobic bacteria in the presence of a gas concentration regulator. Specifically, it can be implemented by the following method: After setting the gas concentration regulator (preferably a gas concentration regulator package) together with a culture container containing anaerobic bacteria and a culture medium in a gas-barrier sealed container, sealing it, and leaving the sealed container standing at a temperature suitable for bacterial culture.

[0084] There is no particular limitation on the culture medium used in the culture method of the present invention, and it can be directly applied by ordinary users. Therefore, the culture medium suitable for the bacteria to be cultured can be freely selected. Suitable for the bacteria culture conditions, the concentration of aldehyde dissolved in the culture medium is preferably 2 mg / L or less, more preferably 1.5 mg / L or less, and further preferably 1.0 mg / L or less.

[0085] In addition, the culture temperature is preferably 20 to 45 °C, and particularly preferably 25 to 40 °C.

[0086] There is no particular limitation on the culture container as long as it ensures air permeability with the outside of the container, and any volume, shape, material, etc. suitable for culture can be adopted. A culture container with a lid can be suitably used, but in this case, air permeability with the outside of the container must also be ensured.

[0087] The culture method of the bacteria of the present invention can be used for the culture of anaerobic bacteria. In the culture method of the present invention, the atmosphere in the airtight container with gas barrier property before the culture is carried out is not particularly limited, and it can also be air, but from the viewpoint of culturing anaerobic bacteria, a nitrogen atmosphere is preferred.

[0088] The airtight container with gas barrier property used in the bacteria culture method is one that hinders the flow of gas inside and outside it and maintains the oxygen and carbon dioxide concentrations formed by the gas concentration regulator input for a long time. Containers made of plastics such as glass, metal, polycarbonate, etc. are often used, but airtight films and their laminates can also be used.

[0089] At this time, for the purpose of measuring the production amount of aldehyde generated in the airtight container with gas barrier property and adjusting the humidity in the container, etc., an open container containing distilled water can also be provided in the airtight container. As the open container, in addition to the culture container, beakers, flasks, etc. can also be exemplified, and a container of the same type as the culture container containing bacteria and culture medium is preferred.

[0090] From the viewpoint of culturing anaerobic bacteria, the oxygen concentration in the airtight container is preferably 0.1% by volume or less within 24 hours. In addition, the carbon dioxide concentration in the airtight container is preferably 11.5% by volume or more, more preferably 12% by volume or more, further preferably 13% by volume or more, and further preferably 15% by volume or more.

[0091] In bacterial culture, it becomes particularly important to achieve the desired oxygen concentration and carbon dioxide concentration within a short period of time. From the perspective of culturing anaerobic bacteria, the oxygen concentration at the start of the oxidation reaction of dehydroascorbic acid and ascorbic acid (3 hours after the start of the reaction) is preferably 0.50% by volume or less, more preferably 0.30% by volume or less, and further preferably 0.15% by volume or less. The carbon dioxide concentration at the start of the oxidation reaction of dehydroascorbic acid and ascorbic acid (3 hours after the start of the reaction) is preferably 11.5% by volume or more, more preferably 12% by volume or more, further preferably 13% by volume or more, and further preferably 15% by volume or more.

[0092] According to the culturing method of the present invention, microscopic observation and transportation of bacteria in a suitable gas atmosphere can be carried out without using a gas cylinder and a gas regulator.

[0093] Examples

[0094] Hereinafter, the present embodiment will be described in detail using examples and comparative examples. However, the present embodiment can be appropriately changed as long as the effects of the present invention are exhibited. It should be noted that "parts" in the examples and comparative examples refer to parts by mass unless otherwise specified.

[0095] Production Example 1

[0096] (Preparation of ascorbic acid aqueous solution)

[0097] Under a nitrogen atmosphere, 30.6 g of L-ascorbic acid, 46.2 g of a 15% by mass aqueous sodium hydroxide solution, and 4.8 g of ferrous sulfate heptahydrate were mixed to prepare an ascorbic acid aqueous solution with a concentration of 2.13 mmol / g.

[0098] Production Example 2

[0099] (Preparation of dehydroascorbic acid aqueous solution)

[0100] Under a nitrogen atmosphere, 4.5 g of dehydroascorbic acid, 6.9 g of a 15% by mass aqueous sodium hydroxide solution, and 0.7 g of ferrous sulfate heptahydrate were mixed to prepare a dehydroascorbic acid aqueous solution with a concentration of 2.13 mmol / g.

[0101] Example 1

[0102] Under a nitrogen atmosphere, 10 g of activated carbon, 15 g of polyethylene powder, and 4.7 g of sodium carbonate were measured and mixed. To this powder, 10.1 g of the ascorbic acid aqueous solution and 10.1 g of the dehydroascorbic acid aqueous solution were added and mixed to produce a deoxidizer composition. 6.9 g of this deoxidizer composition was filled into an aluminum bag and sealed.

[0103] An aluminum bag containing the above deoxidizer was sealed in a nylon bag together with 750 mL of air. The aluminum bag was opened inside the nylon bag, and the deoxidation experiment was started. The oxygen concentration and carbon dioxide concentration after 3 hours were measured by gas chromatography. As a result, the oxygen concentration was 0.20% by volume and the carbon dioxide concentration was 13.6% by volume. In addition, the oxygen concentration and carbon dioxide concentration after 24 hours were measured by gas chromatography. As a result, the oxygen concentration was 0.00% by volume and the carbon dioxide concentration was 13.7% by volume.

[0104] Example 2

[0105] Under a nitrogen atmosphere, 10 g of activated carbon, 15 g of polyethylene powder, and 4.7 g of sodium carbonate were measured and mixed. 20.3 g of an aqueous solution of dehydroascorbic acid was added to the powder and mixed to produce a deoxidizer composition. 6.9 g of the deoxidizer composition was filled into an aluminum bag and sealed.

[0106] An aluminum bag containing the above deoxidizer was sealed in a nylon bag together with 750 mL of air. The aluminum bag was opened inside the nylon bag, and the deoxidation experiment was started. The oxygen concentration and carbon dioxide concentration after 3 hours were measured by gas chromatography. As a result, the oxygen concentration was 0.10% by volume and the carbon dioxide concentration was 18.5% by volume. In addition, the oxygen concentration and carbon dioxide concentration after 24 hours were measured by gas chromatography. As a result, the oxygen concentration was 0.00% by volume and the carbon dioxide concentration was 18.6% by volume.

[0107] Comparative Example 1

[0108] Under a nitrogen atmosphere, 10 g of activated carbon, 15 g of polyethylene powder, and 4.7 g of sodium carbonate were measured and mixed. 20.3 g of an aqueous solution of ascorbic acid was added to the powder and mixed to produce a deoxidizer composition. 6.9 g of the deoxidizer composition was filled into an aluminum bag and sealed.

[0109] An aluminum bag containing the above deoxidizer was sealed in a nylon bag together with 750 mL of air. The aluminum bag was opened inside the nylon bag, and the deoxidation experiment was started. The oxygen concentration and carbon dioxide concentration after 3 hours were measured by gas chromatography. As a result, the oxygen concentration was 0.55% by volume and the carbon dioxide concentration was 10.7% by volume. In addition, the oxygen concentration and carbon dioxide concentration after 24 hours were measured by gas chromatography. As a result, the oxygen concentration was 0.00% by volume and the carbon dioxide concentration was 11.0% by volume.

Claims

1. A gas concentration regulator for anaerobic bacteria culture, comprising: (a) dehydroascorbic acid; (c) anhydrous or hydrated ferrous sulfate; (d) activated carbon; (e) alkali metal carbonate and alkali metal hydroxide, wherein the alkali metal carbonate is sodium carbonate, sodium bicarbonate or sodium carbonate hydrate, and the alkali metal hydroxide is sodium hydroxide or potassium hydroxide; and (f) water.

2. The gas concentration regulator for anaerobic bacteria culture according to claim 1, further comprising (b) ascorbic acids.

3. The gas concentration regulator for anaerobic bacteria culture according to claim 2, wherein, (b) the molar ratio of ascorbic acids to (a) dehydroascorbic acid, i.e., (b) / (a), is 1.5 or less.

4. A method for culturing anaerobic bacteria, which cultures anaerobic bacteria in the presence of the gas concentration regulator according to any one of claims 1 to 3.

5. A gas concentration regulator package, which is formed by packaging the gas concentration regulator according to any one of claims 1 to 3 into a pouch shape with a gas-permeable packaging material.