阅读说明：本技术 细菌培养用气体浓度调节剂和使用其的细菌的培养方法 (Gas concentration regulator for bacterial culture and method for culturing bacteria using same ) 是由 山田元 于 2020-02-20 设计创作，主要内容包括：一种细菌培养用气体浓度调节剂,其包含(a)抗坏血酸类、(b)过渡金属催化剂、(c)活性炭、(d)碱金属碳酸盐或碱土金属氢氧化物和(e)水,前述抗坏血酸类和前述水浸渗于所述活性炭中,前述活性炭的平均孔径为2.0nm以上。(A gas concentration regulator for culturing bacteria, which comprises (a) an ascorbic acid compound, (b) a transition metal catalyst, (c) activated carbon, (d) an alkali metal carbonate or an alkaline earth metal hydroxide, and (e) water, wherein the activated carbon is impregnated with the ascorbic acid compound and the water, and the activated carbon has an average pore diameter of 2.0nm or more.)

1. A gas concentration regulator for bacterial culture, comprising: (a) ascorbic acids, (b) a transition metal catalyst, (c) activated carbon, (d) alkali metal carbonate or alkaline earth metal hydroxide, and (e) water, wherein the activated carbon is impregnated with the ascorbic acids and the water, and the activated carbon has an average pore diameter of 2.0nm or more.

2. The gas concentration regulator according to claim 1, wherein the activated carbon has an average pore diameter of 2.0nm or more and 5.5nm or less.

3. The gas concentration regulator according to claim 1 or 2, wherein the total pore volume of the activated carbon is 0.60cm³More than g.

4. Use of the gas concentration regulator according to any one of claims 1 to 3 for bacterial culture.

5. A method for culturing bacteria in the presence of the gas concentration regulator according to any one of claims 1 to 3.

6. A gas concentration regulator package comprising a gas concentration regulator according to any one of claims 1 to 3 packaged in a pouch-like form with a gas-permeable packaging material.

Technical Field

The present invention relates to a gas concentration regulator for culturing bacteria and a method for culturing bacteria using the same.

Background

In the culture of biological samples such as tissues and cells, which are carried out in the research field or industrial field 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 the bicarbonate buffer culture solution at pH7.4, which is the same as the normal state of blood, it is necessary to set the atmospheric carbon dioxide concentration to about 5%. In many research fields, cell culture is performed in a low-concentration oxygen atmosphere as in vivo.

As an apparatus for creating a gas atmosphere of a high-concentration carbon dioxide atmosphere and a low-concentration oxygen atmosphere, a carbon dioxide gas incubator and the like are known, and the burden of equipment cost, high-pressure gas management and the like is large. Therefore, in recent years, a method using a gas concentration regulator utilizing an oxidation reaction of ascorbic acids is widely used (see patent documents 1 and 2).

Documents of the prior art

Patent document

Patent document 1: japanese patent No. 3818324

Patent document 2: japanese patent No. 5682831

Disclosure of Invention

Problems to be solved by the invention

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

Accordingly, an object of the present invention is to provide a gas concentration regulator for culturing bacteria, which is capable of increasing the amount of carbon dioxide produced in the initial stage of oxidation reaction of ascorbic acids (1 hour after the start of the reaction).

Means for solving the problems

The inventors of the present invention conducted extensive studies and found that: the average pore diameter of the activated carbon as a carrier of the gas concentration regulator influences the amount of carbon dioxide generated in the initial stage of the oxidation reaction of ascorbic acids. The present invention has been completed based on such findings.

Namely, the present invention relates to the following.

<1> a gas concentration regulator for bacterial culture, comprising (a) an ascorbic acid compound, (b) a transition metal catalyst, (c) activated carbon, (d) an alkali metal carbonate or an alkaline earth metal hydroxide, and (e) water, wherein the activated carbon is impregnated with the ascorbic acid compound and the water, and the activated carbon has an average pore diameter of 2.0nm or more.

<2> the gas concentration regulator according to <1>, wherein the activated carbon has an average pore diameter of 2.0nm or more and 5.5nm or less.

<3>According to the above<1>Or<2>The gas concentration regulator, wherein the total pore volume of the activated carbon is 0.60cm³More than g.

<4> use of the gas concentration regulator according to any one of <1> to <3> for culturing bacteria.

<5> a method for culturing bacteria, which comprises culturing bacteria in the presence of the gas concentration regulator according to any one of <1> to <3 >.

<6> a gas concentration controlling agent package, which is obtained by packaging the gas concentration controlling agent of any one of <1> to <3> in a pouch form using a gas permeable packaging material.

ADVANTAGEOUS EFFECTS OF INVENTION

The gas concentration regulator for culturing bacteria of the present invention can produce a gas atmosphere having a high carbon dioxide concentration and a low oxygen concentration in a short time because the amount of carbon dioxide generated in the oxidation reaction of ascorbic acids is large in the initial stage of the reaction (1 hour after the start of the reaction). As described later, the gas concentration is set to a predetermined value, and thus the method can be used for culturing any of anaerobic bacteria, microaerophilic bacteria, and aerobic bacteria.

Detailed Description

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

In the present specification, the term "a to B" in the description of numerical values means "a to B inclusive" (in the case of a < B) or "a to B inclusive" (in the case of a > B). In the present invention, a combination of preferred embodiments is a more preferred embodiment.

[ gas concentration regulator ]

The gas concentration regulator for bacterial culture is characterized by comprising (a) an ascorbic acid compound, (b) a transition metal catalyst, (c) activated carbon, (d) an alkali metal carbonate or an alkaline earth metal hydroxide, and (e) water, wherein the activated carbon is impregnated with the ascorbic acid compound and the water, and the activated carbon has an average pore diameter of 2.0nm or more. The gas concentration adjusting agent is preferably used in the form of a package body packed with a gas-permeable packing material with a composition comprising (a) ascorbic acids, (b) a transition metal catalyst, (c) activated carbon, (d) an alkali metal carbonate or an alkaline earth metal hydroxide, and (e) water. The gas concentration regulator of the present invention can be used for bacterial culture.

(a) Ascorbic acids

The gas concentration regulator of the present invention comprises ascorbic acids having both oxygen-absorbing ability and carbon dioxide gas-generating ability as a main agent of the oxygen-absorbing reaction.

The ascorbic acid is L-ascorbic acid and its stereoisomer and its salt and hydrate. Examples of the L-ascorbate include sodium L-ascorbate, potassium L-ascorbate, and calcium L-ascorbate. Examples of the stereoisomer of L-ascorbic acid include erythorbic acid (D-erythorbic acid). Examples of the erythorbate salt include sodium erythorbate, potassium erythorbate, and calcium erythorbate. The ascorbic acids may be used alone in 1 kind, or in combination of 2 or more kinds.

In the gas concentration regulator of the present invention, ascorbic acids are impregnated in activated carbon together with water from the viewpoint of oxygen absorption performance. Specifically, an aqueous solution of ascorbic acids obtained by dissolving ascorbic acids in water is impregnated into activated carbon as a porous carrier. In this case, the amount of the porous carrier to be used can be reduced for a high concentration of the ascorbic acid compound in the aqueous solution, and therefore the concentration of the ascorbic acid compound is preferably as close to the saturation solubility as possible. Therefore, as the ascorbic acid compound, a compound having high solubility in water is preferably selected, and specifically, sodium L-ascorbate is preferable. When sodium L-ascorbate is used, the concentration of the aqueous solution is preferably 40 to 55 mass%.

In the gas concentration regulator, the concentration of oxygen in the atmosphere is regulated by the oxidation reaction of ascorbic acids, and the concentration of carbon dioxide in the atmosphere is regulated by the generated carbon dioxide. In this oxidation reaction, carbon dioxide is theoretically generated in an equimolar amount to the oxygen molecules consumed. Due to the above principle, the carbon dioxide concentration also increases with a decrease in the oxygen concentration, but when a compound having a carbon dioxide absorbing ability, for example, an alkaline earth metal hydroxide is added to the gas concentration adjusting agent, the increase in carbon dioxide can be suppressed. Conversely, when a compound having a carbon dioxide generating ability is blended with the gas concentration adjusting agent, carbon dioxide can be generated in an amount larger than the amount of reduction in oxygen. In the gas concentration regulator of the present invention, the degree of progress of the oxidation reaction and the amount of carbon dioxide generated can be adjusted by appropriately selecting the blending component and/or the blending amount of the composition constituting the gas concentration regulator, and thus the desired oxygen concentration and carbon dioxide concentration can be adjusted in a specific atmosphere. By adjusting the oxygen concentration and the carbon dioxide concentration to desired levels, the culture environment suitable for various bacteria such as anaerobic bacteria, microaerophilic bacteria, or aerobic bacteria can be adjusted.

(b) Transition metal catalyst

The gas concentration regulator of the present invention contains a transition metal catalyst that promotes the oxidation reaction of ascorbic acids.

The transition metal catalyst is a catalyst having a metal compound such as a salt or an oxide of a transition metal. As transition metals, iron, manganese, zinc, copper, cobalt are suitable. As the salt of the transition metal, a halide and an inorganic acid salt of the transition metal are included, and for example, a chloride and a sulfate of the transition metal. Typical examples thereof include anhydrous or hydrous salts of ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, manganese chloride, zinc sulfate, copper chloride, and cobalt sulfate, and among them, ferrous sulfate heptahydrate having good solubility in water and good compounding properties is preferable.

The content of the transition metal catalyst in the gas concentration regulator is preferably 5 to 25 parts by mass, more preferably 10 to 20 parts by mass, based on 100 parts by mass of the ascorbic acid compound, from the viewpoint of accelerating the oxidation reaction of the ascorbic acid compound.

(c) Activated carbon

The gas concentration regulator of the present invention comprises activated carbon. The activated carbon functions as a carrier impregnated with an aqueous solution of ascorbic acids, and has a large specific surface area, so that the activated carbon has a large contact area with air and a function of accelerating the progress of an oxidation reaction.

As the activated carbon, for example, activated carbon produced by various production methods such as steam activation using sawdust, coal, coconut shell, or the like as a raw material, and drug activation using zinc chloride or the like can be used. In addition, granular activated carbon is preferably used in order to load an aqueous solution of an ascorbic acid compound or the like on activated carbon and fill the activated carbon in a small bag in granular form. The particle diameter of the granular activated carbon is preferably 0.1 to 2mm, more preferably 0.5 to 1mm, from the viewpoint of oxygen absorption performance and filling property (fluidity) in a package.

In the present invention, the average pore diameter of the activated carbon is 2.0nm or more. By setting the average pore diameter of the activated carbon to 2.0nm or more, the oxidation reaction of ascorbic acids in the gas concentration regulator for bacterial culture of the present invention generates a large amount of carbon dioxide in the initial stage of the reaction (1 hour after the start of the reaction), and a gas atmosphere of a high-concentration carbon dioxide atmosphere and a low-concentration oxygen atmosphere can be created in a short time. The average pore diameter of the activated carbon is preferably 2.2nm or more, and more preferably 2.5nm or more, from the viewpoint of the amount of carbon dioxide generated in the initial stage of the oxidation reaction of ascorbic acids (1 hour after the start of the reaction). The upper limit of the average pore diameter of the activated carbon may be 5.5nm or less, 5.0nm or less, 4.5nm or less, or 4.0nm or less. The average pore diameter of the activated carbon in the present invention can be measured by the method described in the examples described later.

In the present invention, the total pore volume of the activated carbon is preferably 0.60cm from the viewpoint of the amount of carbon dioxide generated in the initial stage of the reaction (1 hour after the start of the reaction) in the oxidation reaction of ascorbic acids³A value of at least g, more preferably 0.65cm³A concentration of 0.68cm or more³More than g. The upper limit of the total pore volume of the activated carbon may be 2.0cm³Less than g, 1.8cm³Less than g, may be 1.5cm³Less than g, and may be 1.0cm³The ratio of the carbon atoms to the carbon atoms is less than g. The total pore volume of the activated carbon in the present invention can be measured by the method described in the examples described below.

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, based on 100 parts by mass of the ascorbic acid compound, from the viewpoint of oxygen absorption performance and filling property in a package.

(d) Alkali metal carbonate or alkaline earth metal hydroxide

The gas concentration adjusting agent of the present invention contains an alkali metal carbonate or an alkaline earth metal hydroxide for adjusting the carbon dioxide concentration. Alkali metal carbonates are used for the purpose of rapidly proceeding oxidation reactions of ascorbic acids and controlling the reaction field in the alkaline region. On the other hand, an alkaline earth metal hydroxide may be used as the carbon dioxide gas absorbent. When an alkali metal carbonate and an alkaline earth metal hydroxide are used in combination, it is difficult to adjust the carbon dioxide concentration, and therefore it is not preferable to use both in combination.

As the alkali metal carbonate, a water-soluble alkali metal carbonate such as sodium carbonate, sodium hydrogen carbonate, or sodium carbonate hydrate can be suitably used, and among them, sodium carbonate is particularly preferable.

As the alkaline earth metal hydroxide, calcium hydroxide, magnesium hydroxide or a mixture thereof can be suitably used in particular. When it is desired to absorb a large amount of carbon dioxide in a short time, an alkaline earth metal hydroxide having high solubility in water and a high carbon dioxide absorption rate, such as calcium hydroxide, can be suitably used. Further, the amount of carbon dioxide generated is not constant but varies from moment to moment. For example, when a gas atmosphere having an oxygen concentration of 0% and a carbon dioxide concentration of 5% is formed more rapidly, a large amount of carbon dioxide is generated at a time immediately after the start of the oxidation reaction of ascorbic acids in order to absorb all oxygen in the atmosphere at a time, and then the amount of generated carbon dioxide rapidly decreases. In this case, it is preferable to mix calcium hydroxide having a high carbon dioxide absorption rate with magnesium hydroxide having a low carbon dioxide absorption rate.

The alkaline earth metal hydroxide is preferably a powder, and the average particle diameter thereof is preferably 1 to 100 μm, more preferably 2 to 50 μm.

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 adjuster is preferably 10 to 200 parts by mass, more preferably 15 to 150 parts by mass, and still more preferably 20 to 100 parts by mass, based on 100 parts by mass of the ascorbic acid compound.

(e) Water (W)

The gas concentration regulator of the present invention contains water necessary for the oxidation reaction of ascorbic acids to proceed.

The water is preferably impregnated into the activated carbon from the viewpoint that the gas concentration adjuster can be obtained as a solid having fluidity. In the gas concentration regulator of the present invention, from the viewpoint of oxygen absorption performance, activated carbon is impregnated with water together with ascorbic acids. Specifically, an aqueous solution of ascorbic acids obtained by dissolving ascorbic acids in water is impregnated into activated carbon as a porous carrier. In addition, soluble components other than ascorbic acid may be dissolved in water, or insoluble components may be dispersed.

From the viewpoint of allowing the oxidation reaction of ascorbic acids to proceed, the content of water in the gas concentration regulator is preferably 100 to 200 parts by mass, more preferably 110 to 180 parts by mass, and still more preferably 120 to 180 parts by mass, based on 100 parts by mass of ascorbic acids.

(f) Other ingredients

The gas concentration regulator of the present invention may contain components other than the above-described components (a) to (e) as necessary within a range not impairing the effects of the present invention.

(f1) Thermoplastic resin

The gas concentration regulator of the present invention may contain a thermoplastic resin in order to suppress excessive heat generation accompanying the progress of the oxygen-absorbing reaction (oxidation reaction of ascorbic acid compounds). The kind of the thermoplastic resin is not particularly limited, and for example, polyethylene, polypropylene, an ethylene-vinyl acetate copolymer, an 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 having a molecular weight of 10000 or less can be suitably used.

The thermoplastic resin is preferably a particulate material having a particle diameter of 1 to 500. mu.m, more preferably 10 to 300. mu.m, from the viewpoint of miscibility with other components. From the viewpoint of more effectively suppressing heat generation, the softening point of the thermoplastic resin is preferably 90 to 125 ℃.

The content of the thermoplastic resin in the gas concentration regulator is preferably 35 to 300 parts by mass, more preferably 60 to 200 parts by mass, based on 100 parts by mass of the ascorbic acid compound, from the viewpoint of allowing the oxidation reaction of the ascorbic acid compound to proceed.

(f2) Aldehyde removing agent

The gas concentration adjusting agent of the present invention may further contain an aldehyde removing agent in order to mainly remove aldehydes by-produced as oxidation of ascorbic acids proceeds. Various compounds such as amines are known as the compound having an aldehyde-removing ability, but ethylene urea, arginine, lysine hydrochloride, or polyallylamine which has a sufficient aldehyde-removing ability, does not cause any irritating odor, and exhibits a high effect in a small amount is preferably blended, and ethylene urea having a high effect in a smaller amount is more preferable.

The term "aldehyde" as used herein refers to a compound having 1 or more formyl groups in the molecule, i.e., an aldehyde. In the present invention, the aldehyde is typically produced as a byproduct during oxygen inhalation or bacterial culture. The aldehyde includes any aldehyde classified into aldehydes in the chemical field as long as it does not adversely affect the culture of bacteria. Specifically, examples thereof include formaldehyde, acetaldehyde and the like.

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 still more preferably 1.0 to 5.0 parts by mass, based on 100 parts by mass of the ascorbic acid compound, from the viewpoint of efficiently and inexpensively removing an aldehyde.

The method for producing the gas concentration regulator of the present invention is not particularly limited, and examples thereof include the following methods: the method includes dissolving a transition metal catalyst, an alkali metal carbonate or an alkaline earth metal hydroxide, or the like in an aqueous solution of ascorbic acid, mixing the solution, and impregnating the activated carbon with the mixed solution.

[ gas concentration regulator Package ]

The gas concentration controlling agent can be packaged in a gas concentration controlling agent package by packaging a composition containing the above components in a packaging material using a gas permeable packaging material for all or a part thereof.

(packaging Material)

Examples of the packaging material include: laminating 2 sheets of air-permeable packaging material to form a bag-shaped packaging material; laminating 1 sheet of air-permeable packaging material and 1 sheet of non-air-permeable packaging material to form a bag-shaped packaging material; the 1-sheet air-permeable packaging material was folded and the edges were sealed with each other except for the folded portion to produce a bag-like packaging material.

Here, when the air-permeable packing material and the air-impermeable packing material are quadrangular, the packing material includes: overlapping 2 sheets of air-permeable packaging material, and heat-sealing 4 edges to obtain a bag-shaped product; overlapping 1 sheet of air-permeable packaging material and 1 sheet of non-air-permeable packaging material, and heat-sealing 4 edges to obtain a bag-shaped product; bending 1 sheet of air-permeable packaging material, and heat-sealing 3 sides except the bending part to obtain a bag-like product. The packaging material may be a gas-permeable packaging material formed into a tubular shape, and both ends of the tubular body and the main body portion may be heat-sealed to form a bag-like shape.

(air-permeable packaging Material)

As the gas-permeable packaging material, a packaging material that is permeable to oxygen and carbon dioxide may be selected. Among them, an air-permeable packaging material having an air permeability resistance of 600 seconds or less, more preferably 90 seconds or less, by the Gurley type testing method can be suitably used. Here, the air permeability resistance means a value measured by the method of JIS P8117 (1998). More specifically, it means the time required for 100mL of air to pass through the air-permeable packaging material using a Gurley air permeability measuring apparatus manufactured by Toyo Seiki Seisaku-Sho.

As the air-permeable packing material, an air-permeable packing material in which air permeability is imparted to a plastic film may be used in addition to paper and nonwoven fabric. As the plastic film, for example, a laminated film obtained by laminating and bonding a film such as polyethylene terephthalate, polyamide, polypropylene, polycarbonate, or the like and a film such as polyethylene, ionomer, polybutadiene, ethylene acrylic acid copolymer, ethylene methacrylic acid copolymer, ethylene vinyl acetate copolymer, or the like as a heat seal layer can be used. In addition, these laminates can also be used as breathable packaging materials.

As a method for imparting air permeability, various methods can be employed in addition to the perforation processing by cold needles and hot needles. When the air permeability is provided by the punching process, the air permeability can be freely adjusted according to the diameter, number, material, and the like of the punched holes.

The thickness of the laminated film is preferably 50 to 300 μm, and particularly preferably 60 to 250 μm. In this case, a packaging material having holding strength, heat sealability, and packaging suitability can be obtained as compared with a case where the thickness is not within the above range.

In order to maintain the function of the gas concentration regulator package for a long period of time, it is preferable that: before use, the container or bag is stored in a gas-tight container or bag, and when used, the container or bag is taken out from the gas-tight container or bag for use. When the gas concentration regulator package is used for culturing bacteria, it is preferable to sterilize the package with gamma rays or the like.

[ method of culturing bacteria ]

The method for culturing bacteria of the present invention is a method for culturing bacteria in the presence of a gas concentration regulator. Specifically, it can be implemented as follows: the method is carried out by placing a gas concentration adjusting agent (preferably a gas concentration adjusting agent package) in a gas-tight sealed container together with a culture container containing bacteria and a culture medium, sealing the sealed container, and allowing the sealed container to stand at a temperature suitable for culturing the bacteria. In this case, for the purpose of measuring the amount of aldehyde produced in the gas-tight sealed container, adjusting the humidity in the container, and the like, an open-type container containing distilled water may be provided in the sealed container. The open type vessel may be exemplified by a beaker, a flask, etc., in addition to a culture vessel, and is preferably the same kind of vessel as the culture vessel containing the bacteria and the culture medium.

The oxygen concentration in the closed container is preferably 6 to 14% by volume when microaerophilic bacteria are cultured, and preferably 1% by volume or less when anaerobic bacteria are cultured.

The carbon dioxide concentration in the closed container is preferably 1 to 10% by volume, more preferably 2 to 10% by volume, and still more preferably 2 to 9% by volume when the microaerophilic bacteria are cultured. When anaerobic bacteria are cultured, the culture medium is preferably 10% by volume or more, more preferably 12% by volume or more, and still more preferably 14% by volume or more.

The appropriate oxygen and carbon dioxide concentrations vary by bacteria, but it is important to achieve the desired concentrations in a short time. In the case of culturing the microaerophilic bacterium, the oxygen concentration of the oxidation reaction of ascorbic acid in the initial stage of the reaction (1 hour after the start of the reaction) is preferably 2 to 18% by volume, more preferably 3 to 17% by volume, and still more preferably 4 to 16% by volume. The carbon dioxide concentration in the oxidation reaction of ascorbic acids at the initial stage of the reaction (1 hour after the start of the reaction) is preferably 1 to 10% by volume, more preferably 2 to 10% by volume, and still more preferably 2 to 9% by volume. In addition, in the case of culturing anaerobic bacteria, the oxygen concentration of the oxidation reaction of ascorbic acids at the initial stage of the reaction (1 hour after the start of the reaction) is preferably 2% by volume or less, more preferably 1% by volume or less. The carbon dioxide concentration in the oxidation reaction of ascorbic acids at the initial stage of the reaction (1 hour after the start of the reaction) is preferably 10% by volume or more, more preferably 12% by volume or more, and still more preferably 14% by volume or more.

The culture temperature is preferably 20 to 45 ℃ and particularly preferably 25 to 40 ℃.

In the method for culturing bacteria of the present invention, the atmosphere in the gas-tight vessel before the culture is carried out does not need to be particularly controlled, and may be, for example, air. When the culture method of the present invention is carried out using a gas concentration adjusting agent capable of generating carbon dioxide in a volume equal to that of the absorbed oxygen by filling air into a gas-tight container, the oxygen concentration is about 11 to 19% by volume when the carbon dioxide concentration is 2 to 10% by volume, and the oxygen concentration is about 13 to 18% by volume when the carbon dioxide concentration is 3 to 8% by volume. However, in the culture method using the gas concentration regulator of the present invention, the oxygen concentration is not particularly limited. The concentration of aldehyde dissolved in the medium is preferably 2mg/L or less, more preferably 1.5mg/L or less, and still more preferably 1.0mg/L or less as the culture conditions of the bacteria.

The bacterium used in the culture method of the present invention is not particularly limited. The method of culturing bacteria of the present invention can also be used for culturing any of anaerobic bacteria, microaerophilic bacteria, and aerobic bacteria. The medium used in the culture method of the present invention is not particularly limited, and a medium usually used can be used as it is, and therefore a medium suitable for the bacteria to be cultured can be freely selected. The culture container is not particularly limited as long as it has gas permeability to the outside of the container, and any container suitable for culture can be used, such as volume, shape, and material. A culture container having a lid portion can be suitably used, but in this case, it is also necessary to ensure air permeability to the outside of the container.

The gas-tight sealed container used in the method of culturing bacteria is one that prevents the flow of gas between the inside and outside of the container and maintains the concentration of oxygen and carbon dioxide generated by the gas concentration regulator introduced for a long period of time. Containers made of glass, metal, plastic such as polycarbonate, and the like are often used, but gas barrier films and laminates thereof may also be used.

According to the culture method of the present invention, microscopic observation and transportation of bacteria under a suitable gas atmosphere can be performed without using a gas tank and a gas regulator.

Examples

Hereinafter, this embodiment will be described in detail using examples and comparative examples, but this embodiment may be appropriately modified as long as the effects of the present invention are exhibited. In the examples and comparative examples, "part" means part by mass unless otherwise specified.

The activated carbons used in the examples and comparative examples are shown in table 1. The average pore diameter and the total pore volume of the activated carbon were measured by the following methods.

(average pore diameter and Total pore volume of activated carbon)

In the measurement of the average pore diameter and the total pore volume of the activated carbon, about 0.1g of a sample was degassed under a vacuum condition at 130 ℃ under a pretreatment condition using "BELSORP-max" manufactured by Microtrac BEL company, and the nitrogen adsorption isotherm in liquid nitrogen (77K) was measured. The total pore volume and the average pore diameter were determined by the BET multipoint method using the attached software.

[ Table 1]

TABLE 1

Example 1

After 6g of ferrous sulfate heptahydrate was dissolved in 100g of an aqueous solution (concentration: 45 mass%) of sodium L-ascorbate, 60g of activated carbon (C1) was impregnated with the aqueous solution, and 70g of low-molecular-weight polyethylene and 20g of sodium carbonate decahydrate were mixed together to obtain a gas concentration adjusting agent (1).

Further, a gas concentration controlling agent package (1) was obtained by filling 5g of the gas concentration controlling agent (1) into a gas permeable pouch of 90mm in the longitudinal direction × 55mm in the transverse direction of Japanese paper laminated with a perforated polyethylene film.

Examples 2 to 4

Gas concentration regulators (2) to (4) and gas concentration regulator packages (2) to (4) were obtained in the same manner as in example 1 except that the activated carbon (C1) was changed to activated carbons (C2) to (C4) in example 1.

Comparative example 1

A gas concentration adjuster (5) and a gas concentration adjuster package (5) were obtained in the same manner as in example 1, except that in example 1, activated carbon (C1) was changed to activated carbon (C5).

[ evaluation ]

The oxygen gas concentration and the carbon dioxide gas concentration of the gas concentration regulator packages (1) to (5) obtained in examples and comparative examples were measured by the following methods. The results are shown in Table 2.

(oxygen concentration and carbon dioxide gas concentration)

A package of a gas concentration regulator, 2 dishes for aldehyde measurement (diameter: 60mm, 5mL of distilled water added thereto), and 2.5L of air were sealed in a bag (400X 220mm) of a nylon film coated with polyvinylidene chloride, and the bag was kept at 35 ℃ in a thermostatic bath, and the change with time of the gas component in the bag was measured. The changes with time in the concentrations of oxygen and carbon dioxide gases were simultaneously measured using an oxygen/carbon dioxide gas analyzer "CheckMate 3" (manufactured by MOCON Europe Co., Ltd.).

[ Table 2]

TABLE 2

As is clear from Table 2, in comparative example 1 using activated carbon (C5) having an average pore diameter of less than 2.0nm, the concentration of carbon dioxide in the oxidation reaction of ascorbic acids was low 1 to 2 hours after the start of the reaction, and the amount of carbon dioxide generated in the initial stage of the reaction was small. On the other hand, in the example using the activated carbon having an average pore diameter of 2.0nm or more, the oxidation reaction of ascorbic acids was high in the carbon dioxide generation concentration 1 hour after the start of the reaction, and the gas atmosphere of the high-concentration carbon dioxide atmosphere and the low-concentration oxygen atmosphere could be prepared in a short time.