JPS6143995B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an oxygen scavenger for easily creating a gas atmosphere with a constant carbon dioxide concentration for culturing anaerobic bacteria. Since anaerobic bacteria do not have a cytochrome enzyme system, they cannot utilize molecular oxygen and cannot grow using normal culture methods. In other words, since it grows only in anoxic conditions,
It was necessary to maintain the bacterial growth medium completely in an oxygen-free state, requiring various measures and special equipment and devices. Moreover, the operation of these tools and devices required considerable skill. Many methods are known for removing molecular oxygen, that is, free oxygen, for culturing such anaerobic bacteria, as described below. (a) Multilayer method: This is the simplest method, and involves layering petrolatum, liquid paraffin, agar, etc. on the surface of the culture medium to prevent air from entering. (b) High-rise solid medium culture method: Mixed culture on glucose agar, ordinary agar, etc. that have been boiled and rapidly cooled, or puncture culture. This method takes advantage of the fact that oxygen does not penetrate deep into the culture medium. (c) Air displacement method: Place the culture medium inoculated with bacteria in a thick-walled glass tank such as an iron tank or a desiccator.
The air in the tank is removed using a vacuum pump and grown either as is or by replacing it with hydrogen or nitrogen gas.
Hosoya's anaerobic incubator is relatively commonly used in this method. Although this method is reliable as a physical method, it requires various equipment such as a vacuum pump, gas cylinder, and pressure gauge, and requires complicated operations. (d) Chemical oxygen absorption method: This method uses a chemical reaction to absorb and remove oxygen, keeping the inside of the sealed system anaerobic. Depending on the chemicals used, there are the Schuton-Sak method and Rosenthal's chromium sulfate method. Shutonsak method: A method in which anaerobic bacteria grow by absorbing a mixture of pyrogalic acid and sodium carbonate. Rosenthal's chromium sulfuric acid method: A method of generating hydrogen and absorbing oxygen through the reaction of metallic chromium and sulfuric acid. Cr＋H ₂ SO ₄ →CrSO ₄ +H ₂ 4CrSO ₄ +2H ₂ SO ₄ +O ₂ →2Cr(SO ₄ )3+2H ₂ O (e) Method of adding reducing agent etc. to the medium: Adding a reducing substance to the medium It creates an anaerobic state. Such media include liver slice liver broth,
Examples include Hosoya cysteine bouillon. (f) Biological method: A method of culturing anaerobic bacteria in a container by reducing molecular oxygen using the respiration of aerobic bacteria. Bacterium moss containing Bacillus marcescens or Bacillus subtilis that has been extensively cultured in advance is required. Each of the above methods has its advantages depending on its purpose, but each requires complicated operations to achieve an anaerobic state. Furthermore, in recent years, it has been thought that carbon dioxide gas is necessary for culturing anaerobic bacteria, and culture in an oxygen-free carbon dioxide atmosphere is becoming common. The purpose of the present invention is to provide an oxygen scavenger that can easily create an anaerobic atmosphere with a constant carbon dioxide concentration that meets the above-mentioned requirements, and which has been used only in large-scale hospitals or medical research institutions equipped with conventional bacteriological laboratories. This makes culturing of anaerobic bacteria, which was previously possible, now possible at public health centers, general clinics, and hospitals. Another object of the present invention is to provide an oxygen scavenger that can be used in the method for culturing anaerobic bacteria as disclosed in Japanese Patent Application No. 147246/1989 filed by the present inventors. Through a chemical reaction, the oxygen scavenger of the present invention absorbs and removes molecular oxygen in a sealed system within 6 hours, and at the same time generates carbon dioxide gas to achieve an atmosphere with a carbon dioxide concentration suitable for the type of anaerobic bacteria. and can be maintained in that state. Specifically, the oxygen scavenger of the present invention comprises ascorbic acid or a salt thereof, two or more alkaline compounds, and a metal salt. The present invention will be explained in detail below. The main agent preferably has a particle size of 50 to 80 mesh, and among them, sodium L-ascorbate has excellent reactivity. The two or more alkaline compounds are used to maintain the pH within the reaction system of the reaction shown below from neutral to weakly alkaline, and to adjust the concentration of carbon dioxide gas generated while uniformly adding moisture. This alkaline compound contains at least an alkali carbonate and an alkali hydrogen carbonate containing water of crystallization. Specifically, an alkali carbonate with water of crystallization is
Na ₂ CO ₃・10H ₂ O, BeCO ₃・4H ₂ O, MgCO ₃・
Examples include 3H ₂ O, KNaCO ₃ .6H ₂ O, and examples of alkali hydrogen carbonate include KHCO ₃ .NaHCO ₃ . The metal salt is used to speed up the oxygen absorption rate of the main agent, and iron compounds or copper compounds are suitable.
Among them, ferrous sulfate heptahydrate, which has a high solubility, is suitable. The reaction of the oxygen scavenger of the present invention will be explained below using sodium L-ascorbate as the main agent. Furthermore, the oxygen scavenger of the present invention contains activated carbon in order to speed up the above reaction. Specific formulation examples of the present invention are shown below. Γ Combination example Sodium L-ascorbate 6 parts by weight Sodium carbonate, decahydrate 8 to 12 parts by weight Sodium hydrogen carbonate 6 to 12 parts by weight Ferrous sulfate, heptahydrate 1 part by weight Activated carbon 5 parts by weight Select within the above composition ratio By doing so, the oxygen concentration in the sealed system can be reduced to below the detection limit by gas chromatograph within 6 hours, and a carbon dioxide concentration of about 30 to 50% can be obtained. Furthermore, the final shape of the composition is not limited, and may be in the form of powder granules or doublets. For this purpose, it is possible to add a small amount of crystalline cellulose talc, gum arabic, dextrin, starch, sodium phosphate, etc. as a shape-imparting agent. Since the present invention has the composition and blending ratio as described above, water can be uniformly distributed in the composition, and the reactions of oxygen absorption and carbon dioxide gas generation can be made constant. If free water is added externally during mixing, filling, or use, the reaction will occur, but the water may not be distributed uniformly or
The reaction does not proceed uniformly, which is not preferable for obtaining the desired gas atmosphere. In addition, if the alkaline compound does not contain at least alkali carbonate or alkali hydrogen carbonate, and if it is alkali carbonate alone, it will not be possible to maintain the carbon dioxide concentration above 30%, while if it is only alkali hydrogen carbonate, the oxygen absorption rate will be low. slow and unable to achieve the desired gas concentration. Next, a method of using the oxygen scavenger of the present invention will be explained. The amount of oxygen scavenger used varies depending on the amount of air in the sealed system and the allowable time required for oxygen absorption and removal, and generally an air permeability of 5,000 to 10,000 seconds/100 c.c. (JIS P8117 Gurley air permeability) is required. Packaging and use with packaging materials that have The actual method of using the oxygen scavenger of the present invention is to use a jar inoculated with anaerobic bacteria to be cultured, a Kolben culture test tube, a jar containing various test specimens, or a jar with low gas permeability such as oxygen, carbon dioxide, and water vapor. It is only necessary to place it in a flexible synthetic resin bag, and it is possible to obtain an anaerobic state extremely easily. Further, the oxygen scavenger of the present invention hardly generates heat or absorbs heat when it starts reacting with the atmosphere, and the reaction starts over a wide temperature range. Therefore, it can be used for culturing not only medium- and high-temperature bacteria, but also low-temperature bacteria. Furthermore, since it does not emit or absorb moisture during reaction with the atmosphere, it does not cause excessive drying of the culture medium in a sealed container. It does not cause dew condensation. Examples of the present invention will be described below. <Example 1> Using an oxygen absorber having the composition shown in Table 1 below, the oxygen concentration and carbon dioxide concentration in a sealed system were measured.

[Table] Each oxygen scavenger with the above composition was packaged in a breathable packaging material, sealed in a gas barrier plastic bag (vinylidene chloride coated polypropylene/polyethylene) with an air capacity of 600 ml, and kept at 35 to 37°C for 6 hours. The subsequent gas concentration was measured. The results are shown in Table 2.

[Table] As is clear from Table 2, the oxygen scavenger A-2 of the present invention provided a predetermined gas concentration, but the others did not provide a predetermined gas concentration. <Example 2> Using the oxygen absorber of Example 1, the oxygen absorber sealed in a breathable bag 2 as shown in FIG.
intermedius (hereinafter referred to as A bacteria), B.
mecaninogenicus SS・mecanigenicus (hereinafter referred to as B
A sheath 1 inoculated with bacteria (hereinafter referred to as bacteria) and streptococcus (hereinafter referred to as C bacteria) and absorbent cotton containing an anaerobic indicator Fildesumaculantsis indicator were sealed in the gas barrier plastic bag 4 used in Example 1. The cells were cultured at 37°C for 48 hours, and the state of bacterial growth was observed from the outside. At the same time, Ageless Z-200 (manufactured by Mitsubishi Gas Chemical Co., Ltd.) was used as an oxygen scavenger below the oxygen scavenger in Example 1.
A similar experiment was conducted using The results are shown in Table 3.

【table】

[Table] 〓 ±: Only partially bred〓

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing an example of use of the present invention.

Claims (1)

[Claims] 1 Ascorbic acid or its salt, consisting of at least a carbonate containing water of crystallization and an alkaline compound containing an alkali bicarbonate, and a metal compound, 6
An oxygen absorber for anaerobic bacterial culture that reduces the oxygen gas concentration in a sealed system to 0.1% or less and the carbon dioxide concentration to 30-50% within hours.