Oxygen indicator packaging

Abstract

The present invention provides an oxygen detection agent package that can detect a hypoxic atmosphere, which is an anaerobic atmosphere that serves as the culture environment for anaerobic bacteria, at an early stage, and a method for detecting oxygen using the oxygen detection agent package. [Solution] An oxygen indicator package comprising an oxygen indicator that reversibly changes color depending on the presence or absence of oxygen, and a transparent packaging material without ventilation holes, wherein the oxygen indicator is contained in the packaging material and the oxygen permeability of the packaging material is 0.2 to 1.0 mL / atm·day, and an oxygen detection method comprising simultaneously sealing the oxygen indicator package and an oxygen absorber into an anaerobic bacterial culture container, wherein the oxygen indicator is colored to indicate the absence of oxygen at the time of sealing.

Description

[Technical Field]

[0001] This invention relates to packaging for oxygen indicators. [Background technology]

[0002] Conventionally, oxygen indicators utilizing variable organic dyes that reversibly change color through oxidation-reduction have been proposed and are used in conjunction with oxygen absorbers to maintain the freshness of food and the quality of medical pharmaceuticals. For example, a solid oxygen indicator comprising an organic dye such as a thiazine dye, azine dye, or oxazine dye, and a reducing agent has been disclosed (Patent Document 1). Furthermore, an oxygen indicator composition comprising a complex obtained by mixing a layered silicate exemplified by the smectite group, a cationic surfactant, an organic dye, a reducing agent, and an optional basic substance has been disclosed for the purpose of obtaining an oxygen indicator or oxygen indicator ink with improved stability (Patent Document 2).

[0003] On the other hand, the cultivation of biological samples such as tissues and cells in the fields of biology, reproduction, or biotechnology research or industry requires a gaseous environment different from that of the atmosphere. In many research fields, cell culture is performed under a low-concentration oxygen atmosphere similar to that found in living organisms. One method for creating such a low-concentration oxygen atmosphere without using special equipment is to place an oxygen absorber in a sealed container, and the aforementioned oxygen indicator is used to confirm the low-concentration oxygen state suitable for cultivation. For example, Patent Document 3 discloses a bacterial culture system that can efficiently contain oxygen absorbers, detection agents, water and other adjusting agents, and culture plates in a sealed container while keeping them separate from each other, in order to create an environment suitable for bacterial culture. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 53-120493 [Patent Document 2] Japanese Patent Publication No. 2004-045365 [Patent Document 3] Utility Model Registration No. 3034364 Gazette [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] According to the method described in Patent Document 3, cultivation and testing can be performed even in locations without special equipment. In such cases, an oxygen indicator, as described in Patent Document 1 or 2, is used to visualize the deoxygenated state and confirm the culture environment for anaerobic bacteria, etc. However, with the aforementioned indicator, it takes a long time for the system to change to an anaerobic color after deoxygenation occurs, and it takes time before the deoxygenated state can be confirmed. In particular, in anaerobic culture for clinical testing, if the culture fails due to insufficient deoxygenation, it is necessary to restart it as soon as possible, but under the above conditions, there was a problem that the decision to restart was delayed. Therefore, there was a need for a method that could detect the presence or absence of oxygen in the container in a shorter time and determine whether the inside of the container had an anaerobic atmosphere suitable for cultivation. This invention has been made in view of the above circumstances, and the object of this invention is to provide an oxygen indicator package that can detect a low-oxygen atmosphere, which is an anaerobic atmosphere that is the culture environment for anaerobic bacteria, at an early stage. [Means for solving the problem]

[0006] The inventors have discovered that an oxygen indicator package having an oxygen indicator and a transparent packaging material with a specific oxygen permeability can solve the above problems, and have completed the invention.

[0007] In other words, the present invention relates to the following [1] to

[11] . [1] An oxygen indicator package comprising an oxygen indicator that reversibly changes color depending on the oxygen concentration, and a transparent packaging material without ventilation holes, wherein the oxygen indicator is contained in the packaging material, and the oxygen permeability of the packaging material is 0.2 to 1.0 mL / atm·day. [2] The packaging material consists of a transparent film, and the oxygen permeability of the resin film is 400 to 1600 mL / m². 2 The oxygen indicator packaging described in [1] above, wherein the oxygen indicator is atm-day. [3] The oxygen indicator packaging according to [2], wherein the transparent film is made of at least one selected from the group consisting of polyolefin, cellophane, polyethylene terephthalate, polyvinyl chloride, nylon, and films laminated thereon. [4] The oxygen indicator packaging according to any one of [1] to [3], wherein the color change range of the oxygen indicator is 0.1 to 1.0% of the oxygen concentration. [5] The oxygen indicator packaging according to any one of [1] to [4], wherein the oxygen indicator comprises a supported powder, a reversible color-changing organic dye, and a reducing agent. [6] The oxygen indicator packaging according to any one of [1] to [5], wherein the color indicating the absence of oxygen of the oxygen indicator is white, and the color indicating the presence of oxygen of the oxygen indicator is blue. [7] A method for storing an oxygen indicator package, wherein the oxygen indicator package and oxygen absorber described in any one of [1] to [6] above are stored in a gas barrier container. [8] An oxygen detection method comprising simultaneously sealing an oxygen indicator package and an oxygen absorber described in any one of [1] to [6] above into an anaerobic bacterial culture vessel, wherein the oxygen indicator is colored to indicate the absence of oxygen at the time of sealing. [9] The oxygen detection method according to [8], wherein after sealing the oxygen indicator packaging, the oxygen indicator exhibits a slight color indicating the presence of oxygen.

[10] An anaerobic atmosphere determination method comprising simultaneously sealing an oxygen indicator package and an oxygen absorber described in any one of [1] to [6] into an anaerobic bacterial culture vessel, wherein the oxygen indicator is colored to indicate the absence of oxygen at the time of sealing, and an anaerobic atmosphere is determined to exist within 4 hours.

[11] An anaerobic culture method comprising simultaneously sealing an oxygen indicator package, an oxygen absorber, and an anaerobic bacterial culture dish described in any one of [1] to [6] above into an anaerobic bacterial culture container, wherein the oxygen indicator is colored to indicate the absence of oxygen at the time of sealing. [Advantages of the Invention]

[0008] According to the present invention, an oxygen detector package capable of early detecting a low oxygen atmosphere, which is an anaerobic atmosphere, a culture environment for anaerobic bacteria, can be provided. Further, an oxygen detection method using the oxygen detector package can be provided. Since it is possible to early detect that it is an anaerobic atmosphere, the oxygen detector package of the present invention is excellent as a detector used when obtaining a culture environment for anaerobic bacteria. [Embodiments for Carrying Out the Invention]

[0009] [Oxygen Detector Package] The oxygen detector package of the present invention has an oxygen detector that reversibly changes color depending on the presence or absence of oxygen, and a transparent packaging material having no ventilation holes, and the oxygen detector is housed in the packaging material, and the oxygen transmission rate of the packaging material is 0.2 to 1.0 mL / atm·day.

[0010] [Oxygen Detector] The oxygen detector is not particularly limited as long as it indicates the presence or absence of oxygen in the container by reversibly changing color depending on the presence or absence of oxygen, but is preferably an oxygen detector containing a supported powder, a reversibly discoloring organic dye, and a reducing agent.

[0011] [Supported Powder] The supported powder can support each component such as a reversibly discoloring organic dye and a reducing agent described later, and make the oxygen detector in a homogeneous solid state. The supported powder is preferably an inorganic substance, more preferably a basic inorganic substance, still more preferably magnesium carbonate or magnesium hydroxide, and even more preferably magnesium carbonate. It is preferable that the supported powder is impregnated with each component described later as a solution, preferably an aqueous solution, and supported. The amount of the supported powder is preferably 65 to 99% by mass, more preferably 70 to 99% by mass, still more preferably 75 to 98% by mass, even more preferably 80 to 98% by mass, and even more preferably 90 to 97% by mass with respect to the total amount of the oxygen detector solid content excluding the solvent. When the amount of the reversible color-changing organic dye is within the above range, the visibility of the color change can be improved, and the oxygen detection performance of the detector can be enhanced.

[0012] (Reversible color-changing organic dye) The reversible color-changing organic dye is a redox-variable organic dye whose color changes reversibly by oxidation-reduction. The organic dye is an aromatic compound containing a long conjugated double bond system having π electrons that can move easily within the molecule. The reversible color-changing organic dye is preferably at least one selected from the group consisting of redox indicators, thiazine dyes, azine dyes, oxazine dyes, indigoid dyes, and thioindigoid dyes, and more preferably a thiazine dye. Specifically, the reversible color-changing organic dye is preferably at least one selected from the group consisting of methylene blue, new methylene blue, methylene green, barrier amine blue B, diphenylamine, ferroin, capri blue, safranine T, indigo, indigo carmine, indigo white, and indirubin, and more preferably methylene blue. The amount of the reversible color-changing organic dye is preferably 0.001 to 5% by mass, more preferably 0.001 to 1% by mass, still more preferably 0.002 to ......

[0013] (Reducing agent) The reducing agent is a compound that reduces the reversible color-changing organic dye in an environment with a low oxygen concentration. It should be noted that in the translation of , the original text seems to be incomplete. I translated it as much as possible based on the existing content. If there are any inaccuracies, please provide the complete correct text for a more accurate translation.The reducing agent is preferably a monosaccharide such as glucose, fructose, or xylose; a reducing disaccharide such as maltose; ascorbic acid and its salts; dithionic acid and its salts; cysteine ​​and its salts; more preferably a monosaccharide; even more preferably at least one selected from the group consisting of xylose and glucose; and even more preferably xylose. The amount of the reducing agent is preferably 10 to 1000 parts by mass, more preferably 20 to 500 parts by mass, even more preferably 30 to 300 parts by mass, even more preferably 50 to 200 parts by mass, and even more preferably 70 to 150 parts by mass, per 1 part by mass of the reversible color-changing organic dye. By having the amount of the reducing agent within the above range, the reversible color-changing organic dye can be appropriately reduced, and the oxygen detection performance of the detection agent can be improved.

[0014] (Other ingredients) The oxygen indicator may also contain other components such as cyclodextrins, inclusion agents such as layered silicates, non-color-changing colorants, and basic substances. Furthermore, a solvent may be used to impregnate and support the supported powder with components such as reversibly color-changing organic dyes.

[0015] Inclusion agents such as cyclodextrins and layered silicates can encapsulate reversible color-changing organic dyes, improving lightfastness, storage stability, and color change responsiveness. The amount of the inclusion agent is preferably 1 to 100 parts by mass, more preferably 3 to 50 parts by mass, even more preferably 5 to 30 parts by mass, even more preferably 7 to 25 parts by mass, and even more preferably 10 to 20 parts by mass, per 1 part by mass of the reversibly color-changing organic dye. Including the above amount of inclusion agent can improve light resistance, storage stability, and color change responsiveness.

[0016] Non-coloring colorants, when combined with reversible color-changing organic dyes, can make color changes more noticeable depending on the environment. Preferred non-coloring colorants are immutable organic dyes, more preferably red food additives such as acid red or phloxine B, and even more preferably phloxine B. The amount of the non-coloring colorant is preferably 0.1 to 3.0 parts by mass, more preferably 0.1 to 2.5 parts by mass, even more preferably 0.2 to 2.0 parts by mass, even more preferably 0.3 to 1.5 parts by mass, and even more preferably 0.5 to 1.0 parts by mass, per 1 part by mass of the reversibly color-changing organic dye. Including the above amount of non-coloring colorant can improve the visibility of the color change and enhance the oxygen detection performance of the detection agent.

[0017] Basic substances can be used to enhance the reducing activity of reducing agents. The basic substance is preferably at least one selected from the group consisting of hydroxides and carbonates. Examples of hydroxides include sodium hydroxide, potassium hydroxide, and calcium hydroxide, while examples of carbonates include sodium carbonate, potassium carbonate, calcium carbonate, and sodium bicarbonate.

[0018] The solvent is used to dissolve each component into a solution and impregnate the supported powder. Aqueous solvents are preferred as the solvent, and water is more preferred. Water alone may be used as the solvent, or a water-soluble organic solvent such as alcohol may be used. The solvent may also be partially or completely removed by drying after impregnation. The amount of solvent used can be adjusted as appropriate depending on the amount of each component, the solubility of each component, the amount of supported powder, the liquid absorption performance of the supported powder, etc., but preferably it is 5 to 80 parts by mass, more preferably 8 to 70 parts by mass, even more preferably 10 to 60 parts by mass, even more preferably 15 to 50 parts by mass, and even more preferably 20 to 40 parts by mass per 100 parts by mass of supported powder. A homogeneous detection agent can be obtained by using the above amount of solvent.

[0019] (Characteristics and manufacturing method of oxygen indicator) As described above, the oxygen indicator preferably contains a reversible color-changing organic dye, and the color change should be recognizable by the eye. Preferably, the color change should be either white and blue, or pink and bluish-purple, for oxygen-free and oxygen-rich states, with the combination of white and blue being more preferable. In other words, it is more preferable that the color indicating the absence of oxygen in the oxygen indicator is white, and the color indicating the presence of oxygen is blue. The combination of white and blue in the color change improves visual recognition and shortens the time required to determine an anaerobic atmosphere, which is an effect of the present invention. Furthermore, the color change range of the oxygen indicator is preferably 0.1 to 1.0% of the oxygen concentration. By having the color change range within this range, the presence of an anaerobic atmosphere can be determined more accurately.

[0020] As long as the oxygen indicator has the above-described configuration, there are no restrictions on its manufacturing method, but it is preferable to manufacture it in the following manner. First, a reversible color-changing organic dye, a reducing agent, and other optional components are dissolved in a solvent to obtain a solution. Next, the solution is impregnated into a supported powder, and this is compressed into tablets to obtain an oxygen indicator in tablet form. The tablet form improves the visibility of the color, making it easier to distinguish subtle color changes. It also makes it easier to put into packaging materials. If the oxygen indicator does not contain a supported powder, it is preferable to impregnate the solution into thread, paper, cloth, etc., to form a film, sheet, or thread.

[0021] <Packaging material> The packaging material of the oxygen indicator packaging of the present invention is a packaging material that does not have ventilation holes, is transparent, and has an oxygen permeability of 0.2 to 1.0 mL / atm·day. The packaging material of the oxygen indicator packaging of the present invention is transparent. Because it is transparent, the color of the oxygen indicator, which reversibly changes color depending on the presence or absence of oxygen, can be visually observed, and the color of the oxygen indicator allows for recognition of an anaerobic state where oxygen is absent. The packaging material used in the oxygen indicator packaging of the present invention is a packaging material that does not have ventilation holes. By not having ventilation holes, it is possible to control the amount of oxygen permeability of the packaging material within an appropriate range. In this invention, "ventilation hole" refers to a vent with a cross-sectional area of ​​0.005 mm² at its narrowest point. 2 This refers to the through-holes described above. Specific examples of ventilation holes include holes made by cutting a portion of the packaging film, holes obtained by puncturing with a needle, and holes created by inserting thread-like or tubular materials between layers of film to allow for ventilation. The aforementioned packaging material has an oxygen permeability of 0.2 to 1.0 mL / atm·day. In this invention, the oxygen permeability of the packaging material refers to the daily oxygen permeability between the space containing the oxygen indicator (inside the packaging) and the space surrounding the oxygen indicator packaging (outside the packaging) of the packaging material used for one oxygen indicator packaging. More specifically, it can be calculated using the following formula. Oxygen permeability of packaging material = Oxygen permeability of transparent film (mL / m²) 2 (atm·day) × effective area (m²) 2 ) Here, the effective area refers to the area of ​​the transparent film that directly separates the space containing the oxygen indicator (inside the packaging) from the space surrounding the oxygen indicator packaging (outside the packaging).

[0022] The oxygen permeability of the packaging material is 0.2 to 1.0 mL / atm·day, preferably 0.3 to 1.0 mL / atm·day, more preferably 0.4 to 1.0 mL / atm·day, even more preferably 0.4 to 0.8 mL / atm·day, and even more preferably 0.4 to 0.6 mL / atm·day. By having the oxygen permeability of the packaging material within this range, it is possible to obtain a package that can detect a low-oxygen atmosphere at an early stage.

[0023] (Transparent film) The aforementioned packaging material is transparent, and there are no restrictions on its material as long as its oxygen permeability is 0.2 to 1.0 mL / atm·day, but it is preferably made of a transparent film. The transparency of the transparent film does not need to be completely light-transmitting; it only needs to be transparent enough that the color change of the detection agent inside the packaging material is clearly visible. However, higher transparency is preferable because it improves visibility.

[0024] The transparent film is preferably a film made up of at least one selected from the group consisting of polyolefin, cellophane, polyethylene terephthalate, polyvinyl chloride, nylon, and films laminated therefrom. Examples of material combinations that constitute a laminated film, which is a film formed by laminating the aforementioned materials, include nylon and polyethylene, nylon and polypropylene, polypropylene and polyethylene, polypropylene coated with polyvinylidene chloride and polyethylene, polypropylene and polyvinyl chloride, stretched polypropylene and unstretched polypropylene, nylon and polyethylene coated with polyvinylidene chloride, polyethylene terephthalate and polyethylene, polyethylene terephthalate and polypropylene, cellophane and polyethylene, cellophane and polypropylene, and the like. Among these, the transparent film is more preferably a film comprising at least one selected from the group consisting of polyolefin and films laminated with various polyolefins, still more preferably a film comprising at least one selected from the group consisting of polyethylene, polypropylene, and films laminated with various polyethylene and polypropylene, yet still more preferably a film comprising at least one selected from the group consisting of polypropylene and films laminated with various polypropylene, yet still more preferably a film comprising at least one selected from the group consisting of biaxially stretched polypropylene, unstretched polypropylene, and films laminated with the above polypropylene, yet still more preferably a film laminated with one or more selected from the group consisting of biaxially stretched polypropylene and unstretched polypropylene, yet still more preferably a film laminated with biaxially stretched polypropylene and unstretched polypropylene By the transparent film being composed of the above films, it has excellent visibility, can appropriately adjust the oxygen permeation amount of the packaging material, and thus can obtain a package capable of detecting a low oxygen atmosphere at an early stage.

[0025] The oxygen permeability of the transparent film is preferably 400 to 1600 mL / m 2 ·atm·day, more preferably 500 to 1500 mL / m 2 ·atm·day, more preferably 600 to 1200 mL / m 2 ·atm·day, still more preferably 700 to 1000 mL / m 2 ·atm·day, yet still more preferably 800 to 900 mL / m 2 ·atm·day. By the oxygen permeability of the transparent film being within the above range, the oxygen permeation amount of the packaging material can be easily and appropriately adjusted, and thus a package capable of detecting a low oxygen atmosphere at an early stage can be obtained.

[0026] <Form and Manufacturing Method of Oxygen Detector Package> The oxygen indicator packaging of the present invention has an oxygen permeability of 0.2 to 1.0 mL / atm·day and is transparent enough to allow visual observation of the color change of the oxygen indicator inside. The form is not limited. Examples of forms include bags and boxes, with bags being preferred. In the case of a bag-shaped oxygen indicator packaging, it is preferable to have a transparent film, which is a preferred packaging material, with its edges bonded together so that the oxygen indicator can be sealed inside.

[0027] There are various methods for manufacturing such oxygen indicator packaging, and for example, the following methods can be used. (1) A method of folding a single transparent film, sealing two ends other than the folded portion, inserting an oxygen indicator, and then sealing the opening to form a bag-shaped oxygen indicator package. (2) A method of overlapping two transparent films, sealing three edges, inserting the oxygen indicator, and then sealing the opening to form a bag-shaped oxygen indicator package. (3) A method of sealing a single transparent film to form a tube, then sealing one of the openings, inserting the oxygen indicator, and then sealing the remaining opening to form a bag-shaped oxygen indicator package.

[0028] Examples of sealing methods include a method of heat sealing by melting a transparent film with heat, and a method of bonding by applying an adhesive, but the heat sealing method is preferred.

[0029] [Storage method for oxygen indicator packaging] The present invention relates to a method for storing oxygen indicator packaging, which involves storing the oxygen indicator packaging and an oxygen absorber in a gas barrier container. The oxygen indicator packaging described above is preferably stored in an oxygen-free environment. Storing it in an oxygen-free environment prevents deterioration due to color changes, and because it has already adopted the oxygen-free color when used in anaerobic bacterial culture, it can be used as is. One method of storing it in an oxygen-free environment is to seal it in a gas-barrier bag with an oxygen absorber (for example, Mitsubishi Gas Chemical Co., Ltd., product name: AGELESS® ZP-100). When sealed with an oxygen absorber, the color change to the oxygen-free state is usually completed within one day. Gas barrier containers are not particularly limited as long as they are airtight and substantially gas barrier properties, but they are preferably made of non-permeable materials from the viewpoint of blocking external ventilation. Furthermore, transparent materials are more preferable because the color of the oxygen indicator can be easily confirmed. Specifically, these include multilayer sheets and films with laminated structures such as polyethylene terephthalate / aluminum vapor deposition / polyethylene, stretched polypropylene / polyvinyl alcohol / polyethylene, and polyvinylidene chloride coated stretched nylon / polyethylene, as well as nylon-based co-extruded multilayer sheets and films with oxygen permeability of 0.05 to 20 mL / m². 2 A container made of a laminate with a 24hr·atm (25℃, 50%RH) rating can be easily used. In addition to the above, metal cans, glass bottles, plastic containers, etc., can also be used as gas barrier containers.

[0030] [Oxygen detection method] The present invention relates to an oxygen detection method in which an oxygen indicator package and an oxygen absorber are simultaneously sealed in an anaerobic bacterial culture container, wherein the oxygen indicator changes color to indicate the absence of oxygen at the time of sealing. This detection method allows for the early detection of a hypoxic atmosphere, which is an anaerobic environment that serves as the culture environment for anaerobic bacteria.

[0031] <Oxygen absorber> The oxygen absorber is not limited as long as it absorbs oxygen in the anaerobic bacterial culture vessel and creates an oxygen-free anaerobic atmosphere, but the following oxygen absorbers are preferred.

[0032] The aforementioned oxygen absorber is preferably a composition that absorbs oxygen and generates carbon dioxide, packaged in a breathable packaging material. The carbon dioxide generating oxygen absorber can be composed of any combination of an oxygen absorbing composition, a carbon dioxide generating composition, or an oxygen absorbing / carbon dioxide generating composition, but it is preferable to be composed of a single-component type of oxygen absorbing / carbon dioxide generating composition.

[0033] As the oxygen scavenging agent composition used, for example, known carbon dioxide generating oxygen scavenging agents can be used. Examples of this type of oxygen scavenging agent include oxygen scavenging compositions in which reducing organic substances are the main component of the oxygen absorption reaction, and oxygen scavenging agents that combine iron as the main component with a carbon dioxide generating agent such as a bicarbonate. Among these, carbon dioxide generating oxygen scavenging agents mainly composed of ascorbic acid, such as ascorbic acid or erythorbic acid and their salts, are particularly preferred due to their ease of adjustment of oxygen absorption amount, oxygen absorption rate, carbon dioxide generation rate, and carbon dioxide generation amount, as well as ease of adjustment of oxygen concentration and carbon dioxide concentration based on these factors, ease of handling, and ease of manufacture.

[0034] The oxygen absorber is used by packaging the oxygen absorber composition in a breathable packaging material. The packaging material for the oxygen absorber can be any material that is breathable and allows good permeability of oxygen and carbon dioxide. Suitable packaging materials include nonwoven fabric made of synthetic fibers, synthetic paper, microporous film, or paper, and composite packaging materials that further incorporate open polyolefin, warif, etc., as reinforcing materials.

[0035] <Anaerobic bacteria culture vessel> The anaerobic bacterial culture container is not particularly limited as long as it can accommodate the required amount of the oxygen indicator packaging, the oxygen absorber, and the anaerobic bacterial culture dishes described later, but the following type of container is preferred.

[0036] The material of the anaerobic bacterial culture container is preferably one that has excellent gas barrier properties and good transparency. This allows for a rapid anaerobic atmosphere to be created inside the container, and the color change of the oxygen indicator packaging can be visually observed. The aforementioned material is preferably polyester resin, polyacrylic resin, polycarbonate resin, vinyl chloride, polyethylene, polypropylene, polystyrene, or laminates thereof, and more preferably polycarbonate resin. There are no particular restrictions on the shape of the anaerobic bacterial culture container, but bag-shaped and box-shaped containers are preferred, with box-shaped containers being more preferred.

[0037] The volume and shape of the anaerobic bacteria culture vessel can be appropriately selected according to the size of the anaerobic bacteria culture dish and the number of anaerobic bacteria culture dishes to be placed in one anaerobic bacteria culture vessel. As a commercially available product, Aneropack® (manufactured by Mitsubishi Gas Chemical Co., Ltd.), which is an anaerobic bacteria culture set including anaerobic bacteria culture vessels with an internal volume of 0.4 L to 7.0 L, can be suitably used. The oxygen absorber should preferably be selected according to the internal volume of the anaerobic bacteria culture vessel, and from the viewpoint of culturing anaerobic bacteria, an oxygen absorber that can create an anechoic state in about 1 to 2 hours is preferred, and an oxygen absorber that can create an anechoic state in about 1 hour is more preferred.

[0038] <Detection Method> The oxygen detection method of the present invention first involves simultaneously sealing an oxygen indicator package, which has a color indicating the absence of oxygen, and an oxygen absorber into an anaerobic bacterial culture container. Then, the anaerobic bacterial culture container is sealed. Here, oxygen indicator packets, which are colored to indicate the absence of oxygen, are quickly placed into the anaerobic bacterial culture container, which has been stored in an oxygen-free environment beforehand, and then sealed. Gradually, the oxygen concentration inside the container decreases, creating an oxygen-free environment (anaerobic atmosphere). In the oxygen detection method of the present invention, preferably, after the oxygen indicator package is sealed, the oxygen indicator exhibits a slight color indicating the presence of oxygen. That is, after the oxygen indicator package is sealed, the oxygen indicator contained in the oxygen indicator package temporarily exhibits a slight color indicating the presence of oxygen. From this, it can be seen that the initial oxygen concentration in the container is high. Then, the color gradually changes to one that indicates the absence of oxygen, revealing that the inside of the container is an anaerobic environment. In the oxygen detection method of the present invention, even if the initial oxygen concentration in the container is high, the oxygen indicator only briefly exhibits a color that indicates the presence of oxygen. Therefore, the time it takes for the color to change back to one that indicates the absence of oxygen is extremely short, making it possible to determine that an anaerobic atmosphere is present in a short time.

[0039] On the other hand, if there is a defect in the container or oxygen absorber and an anaerobic atmosphere is not created (for example, if the oxygen concentration is 1% or higher), the oxygen indicator will gradually change from an appearance that slightly shows a color indicating the presence of oxygen to a color that clearly indicates the presence of oxygen, so it is possible to determine in a short time that an anaerobic atmosphere could not be created. Because it is possible to determine early that an anaerobic atmosphere could not be created in this way, the oxygen detection method of the present invention is particularly suitable for applications where it is necessary to repeat the process quickly, such as anaerobic culture in clinical tests.

[0040] [Method for determining anaerobic atmosphere] The present invention provides a method for determining an anaerobic atmosphere, which involves simultaneously sealing the oxygen indicator package and the oxygen absorber into an anaerobic bacterial culture container, wherein the oxygen indicator is colored to indicate the absence of oxygen at the time of sealing, and the presence of an anaerobic atmosphere is determined within 4 hours. As described above, by using the oxygen detection agent packaging, it is possible to determine in a shorter time that the environment for culturing anaerobic bacteria has become a low-oxygen anaerobic atmosphere. The oxygen indicator using the aforementioned reversible color-changing organic dye changes color very slowly from an aerobic state to an anaerobic state. Therefore, using conventional oxygen indicators required at least 5 hours to determine the presence of an anaerobic atmosphere. According to the anaerobic atmosphere determination method of the present invention, it is possible to determine that an anaerobic atmosphere exists within 4 hours. The time required to determine that an anaerobic atmosphere exists is preferably within 4 hours, more preferably within 3 hours, even more preferably within 2 hours, and even more preferably within 1 hour. A shorter time required to determine that an anaerobic atmosphere exists is preferable.

[0041] The anaerobic atmosphere determination method of the present invention is preferably carried out using the oxygen detection agent package, oxygen absorber, and anaerobic bacterial culture container described in the oxygen detection method, and in the same manner as described in the oxygen detection method.

[0042] [Anaerobic Bacteria Culture Method] The present invention relates to an anaerobic bacterial culture method, which involves simultaneously sealing the oxygen indicator package, the oxygen absorber, and the anaerobic bacterial culture dish into an anaerobic bacterial culture container, wherein the oxygen indicator changes color to indicate the absence of oxygen at the time of sealing. The present invention's method for culturing anaerobic bacteria is preferably carried out using the oxygen detection agent package, oxygen absorber, and anaerobic bacteria culture container described in the oxygen detection method, and in the same manner as described in the oxygen detection method. Furthermore, the anaerobic bacterial culture method of the present invention is preferably carried out using the oxygen detection agent package, oxygen absorber, and anaerobic bacterial culture container described in the anaerobic atmosphere determination method, and in the same manner as described in the anaerobic atmosphere determination method. Therefore, it is possible to determine whether an anaerobic atmosphere is present in a short time, and it is also possible to determine early if an anaerobic atmosphere could not be created. Thus, the anaerobic bacterial culture method of the present invention is particularly suitable for applications where it is necessary to repeat the process quickly, such as anaerobic culture in clinical tests.

[0043] The anaerobic bacterial culture dish used in the anaerobic bacterial culture method of the present invention can be any so-called sterile petri dish, such as a round petri dish, a square petri dish, a microtest plate, or a multiwell plate.

[0044] Although embodiments of the present invention have been described above, the present invention is not limited to the embodiments described above, and includes all aspects included in the concept and claims of the present invention, and can be modified in various ways within the scope of the present invention. [Examples]

[0045] [Manufacturing of oxygen indicator packaging] Example 1 (Oxygen indicator 1 that is white in the absence of oxygen (anaerobic conditions) and blue in the presence of oxygen (aerobic conditions)) 15 mL of an aqueous solution containing 0.45 g of cyclodextrin mixture (product name: Celldex TB-50, Nippon Shokuhin Kako Co., Ltd.), 0.03 g of methylene blue, and 2.5 g of xylose was mixed with 50 g of magnesium carbonate and impregnated. After that, 3 g was taken out and formed into a tablet to obtain oxygen indicator 1.

[0046] (Manufacturing of oxygen indicator packaging 1) The obtained oxygen indicator 1 is placed in a bag made of a laminated film of biaxially oriented polypropylene and unoriented polypropylene (size 22mm x 35mm, thickness 45μm, oxygen permeability 860mL / m²). 2 The oxygen indicator was sealed in an ATM (day) container, heat-sealed, and then packaged to obtain an oxygen indicator package. The oxygen indicator packaging was placed in a transparent gas barrier container together with a commercially available oxygen absorber (product name: Ageless ZP, manufactured by Mitsubishi Gas Chemical Co., Ltd.), sealed, and stored for 24 hours to obtain an oxygen indicator packaging 1 in which the oxygen indicator was white. The oxygen permeability of the packaging material (a laminated film bag) of the oxygen indicator packaging was 0.52 mL / atm·day. The effective area of ​​the packaging material was 600 mm². 2 That was the case.

[0047] Example 2 (Oxygen indicator 2, which is white in the absence of oxygen (anaerobic conditions) and blue in the presence of oxygen (aerobic conditions)) After mixing 20.50g of Mg(OH), 0.03g of methylene blue, 2.5g of glucose, and 2g of water, the mixture was formed into tablets to obtain oxygen indicator 2.

[0048] (Manufacturing of oxygen indicator packaging 2) Using the obtained oxygen indicator 2, an oxygen indicator package 2 in which the oxygen indicator is white was obtained in the same manner as in Example 1. The oxygen permeability of the packaging material (a laminated film bag) of the oxygen indicator packaging was 0.52 mL / atm·day. The effective area of ​​the packaging material was 600 mm². 2 That was the case.

[0049] Example 3 (Oxygen indicator 3: Pink in anaerobic conditions and bluish-purple in aerobic conditions) 15 mL of an aqueous solution containing 0.45 g of cyclodextrin mixture (product name: Celldex TB-50, Nippon Shokuhin Kako Co., Ltd.), 0.03 g of methylene blue, 0.02 g of the non-color-changing organic dye phloxine, and 2.5 g of xylose was mixed with 50 g of magnesium carbonate and impregnated. After that, 3 g was taken out and formed into a tablet to obtain oxygen indicator 3.

[0050] (Manufacturing of oxygen indicator packaging 3) Using the obtained oxygen indicator 3, an oxygen indicator package 3 in which the oxygen indicator is pink was obtained in the same manner as in Example 1. The oxygen permeability of the packaging material (a laminated film bag) of the oxygen indicator packaging was 0.52 mL / atm·day. The effective area of ​​the packaging material was 600 mm². 2 That was the case.

[0051] Comparative Example 1 (Manufacturing of oxygen indicator packaging 4) A laminated film (33mm x 42mm, 45μm thick) made of biaxially oriented polypropylene film and unoriented polypropylene was heat-sealed on three sides with a width of 10mm, sandwiching a thread between the two films to obtain a bag made of a laminated film of biaxially oriented polypropylene and unoriented polypropylene. The oxygen indicator 3 obtained in Example 3 was sealed in the laminated film bag with a thread (made of polyester with a thickness of 0.15mm) sandwiched inside, and the bag was heat-sealed to obtain an oxygen indicator packaging body having the portion where the thread was sandwiched as a ventilation hole. The oxygen indicator package was placed in a transparent gas barrier container together with a commercially available oxygen absorber (product name: Ageless ZP, manufactured by Mitsubishi Gas Chemical Co., Ltd.), sealed, and stored for 24 hours to obtain an oxygen indicator package 4 in which the oxygen indicator was pink.

[0052] [Evaluation of anaerobic atmosphere detection capabilities] Two 2.5L jar containers (standard 2.5L rectangular jar for Aneropack, manufactured by Mitsubishi Gas Chemical Company, Inc.) were prepared for each example and each comparative example. Of the two containers, the gasket on the lid of one of the containers was shifted to make it insufficiently airtight, and this was designated as a poorly airtight container. A small oxygen concentration meter was placed in each of the aforementioned jar containers. Next, the oxygen indicator packets obtained from the transparent gas barrier containers in the examples and comparative examples were opened and removed, and placed in the jar containers. Subsequently, an oxygen absorber (Anelopack Kenki, manufactured by Mitsubishi Gas Chemical Co., Ltd.) was placed in the jar containers, and the lids were securely closed. Of the two containers mentioned above, the one with sufficient airtightness, excluding the poorly sealed container, was designated as the anaerobic atmosphere container. Furthermore, the entire process from opening the transparent gas barrier container, removing the oxygen indicator packet, and placing it in the jar container before closing the lid was completed within 2 minutes.

[0053] The oxygen concentration in each container was measured 30 minutes, 1 hour, and then every hour thereafter, starting from the moment the lids of the jar containers were closed, and the color of the oxygen indicator was observed visually. The point at which the color of the oxygen indicator sealed in the anaerobic atmosphere container changed was defined as the point at which the anaerobic atmosphere in the anaerobic atmosphere container was confirmed, and this was defined as the anaerobic atmosphere determination time. A shorter anaerobic atmosphere determination time is preferable because it allows for earlier detection of the low-oxygen atmosphere, which is the culture environment for anaerobic bacteria. The results are shown in Table 1. In the evaluation of Examples 1 and 2, the color of the oxygen indicator sealed in the anaerobic atmosphere container changed slightly one hour after the jar container lid was closed (it changed from "slightly blue" to "almost white"). However, the point at which the color change was clearly recognizable, two hours later, was considered the point at which the anaerobic atmosphere in the anaerobic atmosphere container could be confirmed.

[0054] [Table 1]

[0055] As shown in Table 1, the oxygen indicator packaging of the example can detect a low-oxygen atmosphere more than two hours earlier than the oxygen indicator packaging of the comparative example. Furthermore, since the oxygen indicator packaging of the example shows a change to an aerobic color even when applied to a poorly sealed container, it can detect that the container is not sealed earlier than the oxygen indicator packaging of the comparative example. Therefore, the oxygen indicator packaging of the present invention can detect a hypoxic atmosphere, which is an anaerobic atmosphere that is the culture environment for anaerobic bacteria, at an early stage, and is particularly suitable as an oxygen indicator packaging used in anaerobic cultures in clinical tests.

Claims

1. It comprises an oxygen indicator that reversibly changes color depending on the presence or absence of oxygen, and a transparent packaging material that does not have ventilation holes. The oxygen indicator is contained in the packaging material. An oxygen indicator packaging having an oxygen permeability of 0.2 to 1.0 mL / atm·day.

2. The packaging material consists of a transparent film, and the oxygen permeability of the transparent film is 400 to 1600 mL / m². ２ The oxygen indicator packaging according to claim 1, wherein it is an ATM Day.

3. The oxygen indicator packaging according to claim 2, wherein the transparent film is made of at least one selected from the group consisting of polyolefin, cellophane, polyethylene terephthalate, polyvinyl chloride, nylon, and films laminated thereon.

4. The oxygen indicator packaging according to any one of claims 1 to 3, wherein the color change range of the oxygen indicator is 0.1 to 1.0% of the oxygen concentration.

5. The oxygen indicator packaging according to any one of claims 1 to 4, wherein the oxygen indicator comprises a supported powder, a reversibly color-changing organic dye, and a reducing agent.

6. The oxygen indicator packaging according to any one of claims 1 to 5, wherein the color indicating the absence of oxygen in the oxygen indicator is white, and the color indicating the presence of oxygen in the oxygen indicator is blue.

7. A method for storing an oxygen indicator package, comprising storing the oxygen indicator package and oxygen absorber described in any one of claims 1 to 6 in a gas barrier container.

8. An oxygen detection method comprising simultaneously sealing an oxygen indicator packaging and an oxygen absorber according to any one of claims 1 to 6 into an anaerobic bacterial culture container, wherein the oxygen indicator is colored to indicate the absence of oxygen at the time of sealing.

9. The oxygen detection method according to claim 8, wherein after sealing the oxygen indicator packaging, the oxygen indicator exhibits a slight color indicating the presence of oxygen.

10. An anaerobic atmosphere determination method comprising simultaneously sealing an oxygen indicator package and an oxygen absorber according to any one of claims 1 to 6 into an anaerobic bacterial culture container, wherein the oxygen indicator is colored to indicate the absence of oxygen at the time of sealing, and it is determined that the atmosphere is anaerobic within 4 hours.

11. An anaerobic culture method comprising simultaneously sealing an oxygen indicator packaging, an oxygen absorber, and an anaerobic bacteria culture dish according to any one of claims 1 to 6 into an anaerobic bacteria culture container, wherein the oxygen indicator is colored to indicate the absence of oxygen at the time of sealing.

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