Screening method for bad breath suppressing ingredients

Abstract

To provide a novel mouth odor inhibitory component and a method that can search for more effective mouth odor inhibitory components.SOLUTION: A screening method for mouth odor inhibitory components includes the following steps of: using a medium containing a test substance and a mouth odor-causing substance precursor to culture Actinomyces bacteria; and then, measuring the amounts of the mouth odor-causing substance precursor and / or the mouth odor-causing substance in the culture system to evaluate the mouth odor inhibitory effect of the test substance.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for screening for an ingredient for suppressing bad breath. [Background technology]

[0002] In recent years, awareness of bad breath has been increasing. The main cause of bad breath is substances that cause bad breath and are produced by bacteria in the oral cavity. Volatile sulfur compounds (VSCs) such as methyl mercaptan and hydrogen sulfide are known to be substances that cause bad breath, and the bacteria that produce these substances that cause bad breath have also been identified. For example, Fusobacterium nucleatum is known to be a bacterium that produces substances that cause bad breath (Patent Document 1).

[0003] Actinomyces naeslundii (hereinafter sometimes referred to as "A. naeslundii"), a normal bacterium in the oral cavity, is known to initially colonize the tooth surface and gingival sulcus (Non-Patent Document 1). However, it has not been clarified whether Actinomyces bacteria contribute to the production of substances that cause bad breath. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 09-110662 [Non-patent literature]

[0005] [Non-Patent Document 1] Palmer RJ Jr et al., J Bacteriol 2003 185:3400-3409 Summary of the Invention [Problem to be solved by the invention]

[0006] If the production mechanism of substances that cause bad breath, which are caused by bacteria normally present in the oral cavity, can be elucidated, it is expected that this will lead to the development of a screening method for ingredients that suppress bad breath, and the discovery of new ingredients that suppress bad breath (bad breath suppressants).

[0007] Therefore, an object of the present invention is to provide a screening method that utilizes the production mechanism of substances that cause bad breath, and that can search for novel or more effective bad breath suppressing components. [Means for solving the problem]

[0008] In view of the above circumstances, the present inventors conducted intensive research to solve the problems, and found that the presence of Actinomyces bacteria significantly increases the production of substances that cause bad breath. They also found that a screening method for bad breath suppressing components that utilizes this mechanism can be used to search for novel bad breath suppressing components or more effective bad breath suppressing components, and thus completed the present invention.

[0009] That is, the present invention provides a method for screening for an ingredient that suppresses bad breath, comprising the steps of culturing Actinomyces bacteria using a medium containing a test substance and a precursor of a substance that causes bad breath, and, after the step, measuring the amount of the precursor of the substance that causes bad breath and / or the substance that causes bad breath in the culture system, thereby evaluating the effect of the test substance in suppressing bad breath.

[0010] The bacterium of the genus Actinomyces is preferably Actinomyces naeslundii.

[0011] The precursor of the substance causing bad breath is preferably L-cysteine.

[0012] The step of culturing Actinomyces bacteria is preferably carried out at a pH range of 4.0 to 7.0.

[0013] The substance that causes bad breath is preferably hydrogen sulfide. Effect of the Invention

[0014] According to the screening method for bad breath suppressing components of the present invention, it is possible to search for novel bad breath suppressing components and more effective bad breath suppressing components. [Brief description of the drawings]

[0015] [Figure 1] 1 shows pH changes during H2S production by Actinomyces bacteria using L-cysteine ​​as a substrate, according to Reference Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The screening method for bad breath suppressing components of the present invention (hereinafter sometimes referred to as "the screening method of the present invention") is characterized by comprising the following steps: A step of culturing Actinomyces bacteria using a medium containing a test substance and a precursor of a substance causing bad breath (hereinafter sometimes referred to as the "culturing step"). After the above steps, a step of measuring the amount of the precursor of the bad breath causative substance and / or the bad breath causative substance in the culture system and evaluating the bad breath suppressing effect of the test substance (hereinafter sometimes referred to as the "evaluation step").

[0017] When Actinomyces bacteria (hereinafter sometimes referred to as "bad breath causative bacteria") are cultured using a medium containing precursors of bad breath causative substances, the precursors of the bad breath causative substances are metabolized by the bad breath causative bacteria to produce bad breath causative substances. Here, the presence of the test substance may suppress the production of the bad breath causative substances. In such cases, the test substance can be evaluated as having a bad breath suppressing effect. The screening method of the present invention utilizes such a mechanism.

[0018] [Culture process] The culturing step of the present invention is a step of culturing Actinomyces bacteria, which are bad breath causative bacteria, using a medium containing a test substance and a precursor of a substance causing bad breath. The culturing method is not particularly limited, but examples include a method of adding the test substance and the precursor of a substance causing bad breath to a medium, and then inoculating the bacteria causing bad breath and culturing the bacteria, and a method of adding the test substance and the precursor of a substance causing bad breath to a medium inoculated with the bacteria causing bad breath and culturing the bacteria. The test substance and the precursor of a substance causing bad breath may be added to the medium at the same time or separately, and the order of addition is not particularly limited.

[0019] The Actinomyces bacteria are not particularly limited, and examples thereof include Actinomyces naeslundii, Actinomyces israelii, Actinomyces johnsonii, Actinomyces odontolyticus, Actinomyces oricola, Actinomyces viscosus, Actinomyces oris, etc. Among these, Actinomyces naeslundii is preferred in that it has a high production function of substances causing bad breath, and therefore screening accuracy can be improved.

[0020] The test substance is not particularly limited, but examples thereof include inorganic compounds, organic compounds, and animal and plant extracts.

[0021] The precursor of the substance causing bad breath is not particularly limited as long as it is a precursor of the substance causing bad breath and is decomposed (metabolized) by bacteria of the genus Actinomyces, but from the viewpoint of screening accuracy, for example, methionine, L-cysteine, homo L-cysteine, taurine, etc. are included, preferably methionine, L-cysteine, homo L-cysteine, more preferably L-cysteine. In addition, examples of the substance causing bad breath that is generated by decomposing (metabolizing) the precursor of the substance causing bad breath include volatile sulfur compounds such as hydrogen sulfide, methyl mercaptan, and dimethyl sulfide. Here, L-cysteine ​​and homo L-cysteine ​​are mainly precursors of hydrogen sulfide, and methionine is mainly precursor of methyl mercaptan.

[0022] The test substance and the precursor of the substance causing bad breath may be dissolved in a solvent as necessary. The test substance and the precursor of the substance causing bad breath may be dissolved in the same solvent, or each may be dissolved in a different solvent. Examples of the solvent include ethanol, physiological saline, phosphate buffer, and culture medium (liquid culture medium described below).

[0023] The medium is not particularly limited as long as it is a medium generally used for culturing bacteria causing bad breath, and may be either a synthetic medium or a natural medium, but is preferably a synthetic medium from the viewpoint of screening accuracy. The medium may be either a solid medium or a liquid medium, but is preferably a liquid medium from the viewpoint of screening accuracy. As the carbon source of the medium, for example, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, etc. can be used alone or in combination. As the nitrogen source of the medium, amino acids such as arginine, ammonium chloride, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean flour, casamino acid, etc. can be used alone or in combination. As the inorganic salts of the medium, inorganic salts such as sodium chloride, potassium chloride, manganese chloride, iron chloride, calcium chloride, magnesium sulfate, manganese sulfate, ferrous sulfate, zinc sulfate, copper sulfate calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, etc. can be used alone or in combination. In addition, trace nutrient sources such as biotin, thiamine, vitamins such as vitamin B6, etc. can be used as necessary. These trace nutrient sources can also be substituted with medium additives such as peptone, meat extract, yeast extract, dry yeast, corn steep liquor, etc.

[0024] The amount (content) of the test substance in the medium is not particularly limited, but is preferably 0.000001 to 10% by mass, and more preferably 0.00001 to 1% by mass. In this case, the content of the test substance refers to that in the medium immediately before the start of culture.

[0025] The concentration of the precursor of the substance causing bad breath in the medium is not particularly limited, but is preferably 0.001 to 100 mM, more preferably 0.01 to 20 mM, and particularly preferably 0.1 to 10 mM. In this case, the concentration of the precursor of the substance causing bad breath means the concentration in the medium immediately before the start of culture.

[0026] The pH of the medium in the culture step can be appropriately set depending on the type of bacteria causing bad breath and the type of medium used, and is not particularly limited, but from the viewpoint of screening accuracy, the pH is preferably 4.0 to 7.0, more preferably 5.0 to 7.0, and even more preferably 6.0 to 7.0. Note that the pH of the medium in this case refers to the pH of the medium immediately before the start of culture.

[0027] The culture time can be appropriately set depending on the type of bad breath causative bacteria and the type of culture medium used, and is not particularly limited. From the viewpoint of ensuring the time required for the bad breath causative bacteria to decompose the precursors of substances causing bad breath, the culture time is preferably 0.5 to 72 hours, and more preferably 3 to 48 hours.

[0028] The culture temperature can be appropriately set depending on the type of bacteria causing bad breath and the type of culture medium used, and is not particularly limited. From the viewpoint of enabling the bacteria causing bad breath to grow well, the culture temperature is preferably 20 to 50°C, more preferably 30 to 45°C, and even more preferably 35 to 40°C.

[0029] [Evaluation process] The evaluation step of the present invention is a step of measuring the amount of the precursor of the bad breath causative substance and / or the bad breath causative substance in the culture system after the culture step, and evaluating the bad breath suppression effect of the test substance. That is, it means any one of the steps of measuring the amount of the precursor of the bad breath causative substance contained in the culture system after the culture step and evaluating the bad breath suppression effect of the test substance, measuring the amount of the bad breath causative substance contained in the culture system after the culture step and evaluating the bad breath suppression effect of the test substance, and measuring the amount of both the precursor of the bad breath causative substance and the bad breath causative substance contained in the culture system after the culture step and evaluating the bad breath suppression effect of the test substance.

[0030] "In the culture system" refers collectively to the solid phase, liquid phase, and gas phase in the culture utensil after the culture step. In this step, for example, when the precursor of the substance causing bad breath is contained in the liquid phase (e.g., liquid medium), the amount of the precursor of the substance causing bad breath contained in the liquid phase may be measured to evaluate the effect of the test substance in suppressing bad breath. In addition, for example, when the substance causing bad breath is present in the gas phase, the amount of the substance causing bad breath contained in the gas phase may be measured to evaluate the effect of the test substance in suppressing bad breath.

[0031] The evaluation process may be divided into a process of measuring the amount of precursors of halitosis-causing substances and / or halitosis-causing substances in the culture system, as explained in the evaluation methods [3] and [4] below, and a process of evaluating the halitosis-suppressing effect of the test substance from the measured amount of precursors of halitosis-causing substances and / or halitosis-causing substances.

[0032] When the bacteria causing bad breath are cultured in the presence of precursors of substances causing bad breath, the precursors are decomposed (metabolized) by the bacteria causing bad breath, and the substances causing bad breath are produced. Here, the presence of a test substance in the culture medium can change the amount of the precursors of the substances causing bad breath and the substances causing bad breath. For example, if the metabolic pathway of the bacteria causing bad breath to produce the substances causing bad breath is inhibited by the presence of the test substance, and the ability to produce the substances causing bad breath is reduced, the decomposition of the precursors of the substances causing bad breath is suppressed. In addition, if the bacteria causing bad breath are killed because the test substance has a bactericidal effect, the amount of the substances causing bad breath produced is reduced, and the decomposition of the precursors of the substances causing bad breath is also suppressed. Furthermore, it is also conceivable that the test substance acts directly on the precursors of the substances causing bad breath, which prevents the bacteria causing bad breath from decomposing the precursors of the substances causing bad breath, and as a result, the decomposition of the precursors of the substances causing bad breath is suppressed. Although the behavior of the precursors of bad breath causative substances has been described using examples only in which the decomposition of the precursors of bad breath causative substances is inhibited, the evaluation process of the present invention can also evaluate cases in which the presence of the test substance promotes the decomposition of the precursors of bad breath causative substances. As described above, the evaluation process of the present invention can be said to be a process in which the behavior of the precursors of bad breath causative substances and the substances that cause bad breath are comprehensively taken into consideration to evaluate the bad breath suppression effect of the test substance.

[0033] Methods for measuring the amount of halitosis causative substance precursors or halitosis causative substances in a culture system include, for example, a method using an analytical instrument and a method for judging "odor" by a sensory test. Here, the method using an analytical instrument is effective in that absolute evaluation is possible, resulting in high screening accuracy, and that evaluation can be performed on many test substances at once, since only a small amount of halitosis causative substance precursors is required, compared to the method using an analytical instrument. On the other hand, the method using a sensory test is effective in that evaluation can be performed easily without complicated operations, compared to the method using an analytical instrument.

[0034] Examples of methods using analytical equipment include analytical methods using chromatography such as gas chromatography-mass spectrometry, gas chromatography-hydrogen flame ionization detection, gas chromatography-flame photometric detection (using a flame photometric detector as a detector), gas chromatography-electron capture detection, high performance liquid chromatography-ultraviolet-visible spectroscopy, and high performance liquid chromatography-mass spectrometry, as well as detection methods using semiconductor sensors. The content of volatile sulfur compounds as substances causing bad breath in a culture system can be determined, for example, by gas chromatography-flame photometric detection. In analytical methods using chromatography, the content of the precursors of the substances causing bad breath in a culture system can be calculated from the area of ​​the peaks corresponding to the precursors of the substances causing bad breath.

[0035] In the evaluation step of the present invention, the method for evaluating the effect of a test substance on suppressing bad breath may be, for example, the following evaluation methods [1] to [4]. [1] Methods for evaluating the amount of precursors and / or substances that cause bad breath directly [2] A method of evaluation based on a comparison of the measured amount of precursors of substances causing bad breath and / or substances causing bad breath with the amount of precursors of substances causing bad breath used. [3] A method for evaluating the amount of precursors and / or substances causing bad breath obtained by using two or more test substances. [4] A method of evaluation in which the measured amount of precursors of substances causing bad breath and / or substances causing bad breath is compared with the measured amount of precursors of substances causing bad breath and / or substances causing bad breath obtained in a similar manner except that the test substance is not used.

[0036] Evaluation method [1] is carried out at the same time as measuring the amount of precursors of bad breath causative substances and / or bad breath causative substances in the culture system. For example, if it is confirmed that all of the precursors of bad breath causative substances have disappeared as a result of the measurement, it can be evaluated that the test substance does not have an effect of suppressing bad breath. Also, if it is confirmed that no substances causing bad breath are produced as a result of the measurement, it can be evaluated that the test substance has a good effect of suppressing bad breath. Therefore, this evaluation method does not require the complicated operations of evaluation methods [2] to [4] and can be carried out easily. On the other hand, this evaluation method is qualitative, and although it is possible to judge whether or not the test substance has an effect of suppressing bad breath, it is preferable to carry out evaluation methods [2] to [4] when quantitatively judging the effect of suppressing bad breath of the test substance.

[0037] The evaluation method [2] is [2-1] a method for evaluating the halitosis suppressing effect of a test substance by comparing the measured amount of the precursor of the halitosis causing substance with the amount of the precursor of the halitosis causing substance used, or [2-2] a method for evaluating the halitosis suppressing effect of a test substance by comparing the measured amount of the halitosis causing substance with the amount of the precursor of the halitosis causing substance used. Here, "amount of the precursor of the halitosis causing substance used" means the amount of the precursor of the halitosis causing substance contained in the medium before the start of the culture in the culture step. The above [2-1] is exemplified by a method for measuring the amount of the precursor of the halitosis causing substance in the medium after the culture step is completed, and measuring the amount of the precursor of the halitosis causing substance contained in the medium before the start of the culture, and evaluating the halitosis suppressing effect of the test substance from the change in these amounts. The above [2-2] is exemplified by a method for evaluating the halitosis suppressing effect of a test substance by calculating the amount of the undecomposed precursor of the halitosis causing substance from the measured amount of the precursor of the halitosis causing substance in the culture system after the culture step is completed, and comparing this with the amount of the precursor of the halitosis causing substance used. This evaluation method is easy to implement in that it does not require the preparation of a comparison target, as in evaluation methods [3] and [4]. However, since this evaluation method does not take into account influences other than those of the test substance and the presence of the bacteria that cause bad breath (for example, the self-decomposition of the precursors of substances that cause bad breath due to heat), it is preferable to use evaluation methods [3] and [4] when it is necessary to judge the bad breath suppression effect of the test substance more precisely.

[0038] The evaluation process in the evaluation method [2] is specifically as follows: A step of measuring the amount of the precursor of the bad breath causative substance and / or the bad breath causative substance in the culture system after the culturing step, and measuring the amount of the precursor of the bad breath causative substance used, and evaluating the bad breath suppressing effect of the test substance from the change in these amounts.

[0039] Evaluation method [3] is a method for evaluating the halitosis suppressing effect of a test substance by comparing the measured amounts of halitosis-causing substance precursors and / or halitosis-causing substances obtained from two or more test substances. In other words, a culture process is carried out for the test substance of interest and a test substance other than the test substance (sometimes referred to as "other test substances"), the amount of halitosis-causing substance precursors and / or halitosis-causing substances in each culture system is measured, and these are compared to evaluate the halitosis suppressing effect of the test substance. This evaluation method is excellent in that it is possible to precisely evaluate the halitosis suppressing effect of the test substance. In particular, it is excellent in that it is possible to precisely compare the halitosis suppressing effect of two or more test substances.

[0040] The screening method of the present invention in the case where the evaluation step in the evaluation method [3] is adopted is specifically as follows. A step of culturing bacteria causing bad breath using a medium containing a test substance and a precursor of a substance causing bad breath (referred to as "culturing step 1"). A step of measuring the amount of the precursor of the substance causing bad breath and / or the substance causing bad breath in the culture system after the culture step 1. A step of culturing the bacteria causing bad breath in the presence of the precursors of the substances causing bad breath and other test substances (referred to as "cultivation step 2"). A step of measuring the amount of the precursor of the substance causing bad breath and / or the substance causing bad breath in the culture system after the culturing step 2. A step of evaluating the halitosis suppressing effect of the test substance by comparing the measured amount of the halitosis causative substance precursor and / or the halitosis causative substance when the test substance is used with the measured amount of the halitosis causative substance precursor and / or the halitosis causative substance when other test substances are used.

[0041] Evaluation method [4] is a method for evaluating the halitosis suppressing effect of a test substance by comparing the measured amount of the precursors of halitosis causing substances and / or the substances themselves with the measured amount of the precursors of halitosis causing substances and / or the substances themselves obtained in the same manner except that the test substance is not used. In other words, a culture step is carried out for the target test substance, and a culture step is carried out in the same manner except that the test substance is not used, the amount of the precursors of halitosis causing substances and / or the substances themselves in each culture system is measured, and the halitosis suppressing effect of the test substance is evaluated by comparing these amounts. This evaluation method is excellent in that it is possible to quantitatively and precisely evaluate the halitosis suppressing effect of a test substance.

[0042] The screening method of the present invention in the case where the evaluation step in the evaluation method [4] is adopted is specifically as follows. A step of culturing bacteria causing bad breath using a medium containing a precursor of a substance causing bad breath and a test substance (referred to as "culturing step A"). After the culture step A, a step of measuring the amount of the precursor of the bad breath causative substance and / or the bad breath causative substance in the culture system (referred to as "measurement step A"). A step of culturing bacteria causing bad breath using a medium containing a precursor of a substance causing bad breath (referred to as "culturing step B"). After the culture step B, a step of measuring the amount of the precursor of the bad breath causative substance and / or the bad breath causative substance in the culture system (referred to as "measurement step B"). A step of evaluating the bad breath suppressing effect of the test substance by comparing the measured amount of the bad breath causative substance precursor and / or the bad breath causative substance in the measurement step A with the measured amount of the bad breath causative substance precursor and / or the bad breath causative substance in the measurement step B.

[0043] [Viable bacteria count measurement process] The screening method of the present invention may further include a viable cell count measurement step after the culturing step. The viable cell count measurement step is a step of measuring the viable cell count of the bacteria causing bad breath in the culture medium, and may be performed either before or after the evaluation step, or may be performed simultaneously with the evaluation step.

[0044] When evaluating the effect of a test substance on suppressing (inhibiting) the production of bad breath causative substances by bad breath causative bacteria (bad breath suppression effect), the effect is classified into an effect of suppressing the production of bad breath causative substances by sterilizing (destroying) the bad breath causative bacteria (sometimes called a "bactericidal effect"), and an effect of suppressing the production of bad breath causative substances by inhibiting the metabolism of bad breath causative bacteria (sometimes called a "metabolic inhibition effect"). If the screening method of the present invention includes a viable cell count measurement step, it is possible to determine whether the bad breath causative bacteria are alive or dead, making it possible to evaluate each of the bactericidal effect and metabolic inhibition effect of the test substance. In other words, if there is a bad breath suppression effect and the bad breath causative bacteria are killed, it can be determined that the test substance has a bactericidal effect, and if there is a bad breath suppression effect and the bad breath causative bacteria are alive, it can be determined that the test substance has a metabolic inhibition effect.

[0045] The method for measuring the viable cell count is not particularly limited, but examples include a colony counting method in which a portion of the culture medium is applied to an agar medium and the number of colonies formed after culturing for a certain period of time is counted, a method in which the number of bacteria in the culture medium is measured by observation under a microscope, and a bioluminescence method in which the amount of ATP contained in the viable bacteria is measured to calculate the number of bacteria.

[0046] [Culture equipment] In the screening method for halitosis suppressing components of the present invention, the use of a specific culture device makes it possible to more efficiently evaluate the halitosis suppressing effect of a test substance.

[0047] Since most of the bacteria that normally inhabit the oral cavity are anaerobic, they must be cultured under anaerobic conditions. The general method of culturing anaerobic bacteria involves placing a culture vessel containing a medium in an anaerobic incubator and replacing the culture vessel with an anaerobic condition. Therefore, the culture vessel must at least be an open system.

[0048] Here, as in the present invention, when culturing bacteria normally present in the oral cavity (especially bacteria causing bad breath) in the presence of precursors of substances causing bad breath and then measuring the substances causing bad breath in the culture system, the substances causing bad breath (i.e. "smell") are often gaseous at room temperature, and if the culture vessel is an open system, the smell will escape from the culture vessel, resulting in a problem that the measurement of the generated substances causing bad breath is inaccurate and the reproducibility is low. For this reason, a method has been adopted in which the lid of the culture vessel is loosened to a certain extent to culture the culture vessel so that the culture vessel can be replaced in an anaerobic state, so that floating bacteria do not enter the vessel, and so that the "smell" does not escape from the culture vessel as much as possible, but even with this method, the accuracy and reproducibility of the measurement are not satisfactory.

[0049] Therefore, the inventors have discovered a culture device (sometimes referred to as the "culture device of the present invention") that can replace the atmosphere inside a container with an anaerobic state when culturing anaerobic bacteria, prevents floating bacteria from entering the container, and prevents gases produced by the anaerobic bacteria (e.g., substances that cause bad breath) from escaping the container.

[0050] The culture instrument of the present invention is a culture instrument comprising a culture vessel and a plug having an aeration tube, the culture vessel having a liquid inlet, and the plug attached to the liquid inlet. The aeration tube is arranged so that gas outside the culture vessel can be exchanged with gas inside the culture vessel. The aeration tube preferably has a rubber tube joined to its tip located outside the culture vessel. The liquid inlet of the culture vessel is sealed by the plug, and the joint between the sealing part and the aeration tube and the rubber tube is preferably covered with aluminum foil, paraffin, or the like to prevent airborne bacteria from entering the vessel.

[0051] The volume of the culture vessel varies depending on the type of anaerobic bacteria to be cultured, but is preferably 0.001 to 10 L, more preferably 0.01 to 1 L, and particularly preferably 0.1 to 0.3 L. The shape of the culture vessel is not particularly limited, but examples thereof include round-bottom flasks, eggplant flasks, Erlenmeyer flasks, flat-bottom flasks, and Sakaguchi flasks. The material of the culture vessel is not particularly limited, but examples thereof include glass and plastic. The length of the vent tube is not particularly limited, but is preferably 2 to 40 cm, more preferably 3 to 30 cm, and particularly preferably 5 to 15 cm. The inner diameter of the vent tube is not particularly limited, but is preferably 0.1 to 2 cm, more preferably 0.2 to 1 cm, and particularly preferably 0.25 to 0.5 cm. In addition, the length of the rubber tube when a rubber tube is provided at the tip of the vent tube outside the vessel is not particularly limited, but is preferably 2 to 40 cm, more preferably 3 to 30 cm, and particularly preferably 5 to 15 cm. The inner diameter of the rubber tube is not particularly limited, but is, for example, preferably 0.1 to 2 cm, more preferably 0.2 to 1 cm, and particularly preferably 0.25 to 0.5 cm. EXAMPLES

[0052] The present invention will be described in more detail below based on experimental examples, but the present invention is not limited to these.

[0053] [Bacterial pre-culture] (Preculture of Actinomyces bacteria) Preculture was performed using A. naeslundii ATCC 19039. The specific method is as follows. A colony of A. naeslundii ATCC19039 was added to 20 mL of TSB medium (trypticase soy medium supplemented with 1 mg / mL yeast extract, 5 μg / mL hemin, and 1 μg / mL menadione) and cultured anaerobically at 37°C for 48 hours. 0.1 mL of this bacterial solution was added to 20 mL of TSB medium and cultured anaerobically at 37°C for 22 hours.

[0054] Actinomyces israelii, Actinomyces johnsonii, Actinomyces odontolyticus, Actinomyces oricola, and Actinomyces viscosus were precultured in the same manner as Actinomyces naeslundii.

[0055] [Bacterial pre-culture] (Preculture of Fusobacterium bacteria) Preculture was performed using F. nucleatum ATCC 25586. The specific method is as follows. A colony of F. nucleatum was added to 15 mL of medium containing 1 g / mL trypticase peptone, 1 mg / mL yeast extract, 1 g / mL Biosate peptone, 1.92 g / mL brain heart infusion, 5 μg / mL hemin, 1 μg / mL menadione, 0.2 mM dipotassium hydrogen phosphate, 0.3 mM potassium dihydrogen phosphate, 4.8 mM sodium bicarbonate, 72 μM calcium chloride, 1.4 mM sodium chloride, and 66 μM magnesium sulfate, and anaerobically cultured for 23 hours at 37° C. 1 mL of this bacterial solution was added to 20 mL of the medium, and anaerobically cultured for 20 hours at 37° C.

[0056] [Preparation of mCDM] A medium (Modified Chemically Defined Medium, mCDM) having the composition shown in Table 1 was prepared. The specific method is as follows. Dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, sodium chloride, and manganese chloride tetrahydrate were dissolved in pure water (MilliQ water) and sterilized by autoclave. The other reagents were dissolved in pure water (MilliQ water), sterilized by filtration with a 0.2 μm membrane filter, and then mixed to prepare the solution.

[0057] [Table 1]

[0058] [GC-FPD analysis conditions] The analysis conditions for GC-FPD in the Reference Examples and Examples are as follows. Apparatus:GC-14B Column:ZO-1H 3.1m×3.2mm id (Shinwa Chemical Industrial Ltd.) Column Temp.: 70℃ Injection mode:Splitless (180℃) Carrier Gas: Nitrogen (constant flow rate 50mL / min) Transfer temperature: 150℃ Detector Temp.: 180℃

[0059] [Culture equipment] The culture equipment used in the Reference Examples and Examples was a 200 mL glass Erlenmeyer flask fitted with a silicone stopper having a stainless steel vent tube (length: 10 cm, inner diameter: 0.3 mm) with a rubber tube (length: 10 cm, inner diameter: 0.3 mm) at the end located outside the culture vessel, and then covered with aluminum foil from the sealed part of the flask to the joint between the vent tube and the rubber tube.

[0060] [Reference example 1-1] The culture medium was prepared by adding L-cysteine ​​to mCDM to a final concentration of 5 mM and adjusting the pH to 6.5. The A. naeslundii culture was washed with PBS and then measured for OD. 600 The culture medium was adjusted to a value of 1.0. 10 mL of the bacterial solution resuspended in this culture medium was mixed with 30 mL of mCDM in an Erlenmeyer flask and cultured in an anaerobic jar at 37°C for 15 hours. After culture, 10 mL of headspace gas in the flask was collected, and 1 mL was subjected to GC-FPD analysis.

[0061] [Reference example 1-2] The same procedure was performed except that the A. naeslundii bacterial liquid was replaced with a bacterial liquid of Fusobacterium nucleatum subsp. nucleatum ATCC 25586. After cultivation, 10 mL of headspace gas was collected from the flask, and 1 mL was subjected to GC-FPD analysis.

[0062] [Reference example 1-3] The same procedure was carried out except that the A. naeslundii bacterial liquid was not used. After the culture, 10 mL of headspace gas in the flask was collected, and 1 mL of the gas was subjected to GC-FPD analysis.

[0063] The amount of H2S generated in Reference Example 1-1 was 345.7±33.58 ppm. The amount of H2S generated in Reference Example 1-2 was 28.91±1.6 ppm. The amount of H2S generated in Reference Examples 1-3 was 0.16±0.01 ppm.

[0064] [Reference example 2] L-cysteine ​​was added to mCDM to a final concentration of 0, 0.1, 0.3, or 0.5 mM, and the pH was adjusted to 6.5 to prepare the culture medium. The A. naeslundii culture was washed with PBS, and the OD 600 The culture medium was adjusted so that the ratio of the bacterial culture to the total bacterial count was 1.0. 10 mL of the bacterial culture resuspended in the culture medium was mixed with 30 mL of mCDM in an Erlenmeyer flask and cultured in an anaerobic jar at 37°C for 15 hours. After culture, 10 mL of headspace gas was collected from the flask, and 1 mL of the gas was subjected to GC-FPD analysis. The results are shown in Table 2.

[0065] [Table 2]

[0066] As can be seen from Table 2, production of H2S was confirmed in the culture medium to which L-cysteine ​​was added.

[0067] [Reference example 3] L-cysteine ​​was added to mCDM to a final concentration of 5 mM, and the pH was adjusted to 3.5, 5.5, 6.5, or 7.5 to prepare culture solutions. The prepared A. naeslundii bacterial solution was washed with PBS, and the OD 600 The ratio was adjusted to 1.0. 10 mL of the bacterial solution resuspended in this culture medium and 30 mL of mCDM were mixed in an Erlenmeyer flask and cultured in an anaerobic jar at 37°C for 15 hours. After culture, 10 mL of headspace gas in the flask was collected, and 1 mL was subjected to GC-FPD analysis.

[0068] The same procedure was carried out except that the A. naeslundii bacterial solution was replaced with a solution of Actinomyces israelii, Actinomyces johnsonii, Actinomyces odontolyticus, Actinomyces oricola, or Actinomyces viscosus. Also, the same procedure was carried out except that the A. naeslundii bacterial solution was not used.

[0069] Based on the above experiments, the pH change during H2S production using L-cysteine ​​as a substrate was analyzed, and the results are shown in Figure 1. As is clear from Figure 1, it was revealed that Actinomyces bacteria have the ability to produce H2S. It was also revealed that A. naeslundii has the highest H2S production ability among Actinomyces bacteria, and that the H2S production ability of A. naeslundii is particularly remarkable in an environment of pH 6.5.

[0070] [Experimental Example 1] A. naeslundii was suspended in mCDM to which L-cysteine ​​was added as a substrate to a final concentration of 0.5 mM, and cetylpyridinium chloride (CPC) was added as a test substance to a final concentration of 0.001% by mass, and cultured under an environment of pH 6.5. After culture, 10 mL of headspace gas in the flask was collected, and 1 mL was subjected to GC-FPD analysis for GC-FPD analysis. As a result, the measured value of H2S was 0.13 ppm. In addition, the H2S production rate was measured using a blank without the addition of cetylpyridinium chloride. As a result, the measured value of H2S was 170 ppm.

[0071] As described above, it was revealed that the amount of H2S production was suppressed by adding CPC. This confirmed that the present invention has a screening function.

Claims

1. A method for screening for an ingredient for suppressing bad breath, comprising the steps of: A step of culturing Actinomyces naeslundii, Actinomyces johnsonii, or Actinomyces viscosus as a bacterium of the genus Actinomyces using a medium containing a test substance and a precursor of a substance causing bad breath, wherein the precursor of the substance causing bad breath is at least one selected from the group consisting of methionine, L-cysteine, and homo-L-cysteine. After the above steps, a step of measuring the amount of a precursor of a substance causing bad breath and / or a substance causing bad breath in the culture system to evaluate the bad breath suppressing effect of the test substance, in which the precursor of a substance causing bad breath is at least one selected from the group consisting of methionine, L-cysteine, and homo-L-cysteine, and the substance causing bad breath is a volatile sulfur compound.

2. 2. The method for screening an ingredient for suppressing bad breath according to claim 1, wherein the bacterium of the genus Actinomyces is Actinomyces naeslundii.

3. 3. The method for screening an ingredient for suppressing bad breath according to claim 1 or 2, wherein the precursor of a substance causing bad breath is L-cysteine.

4. The method for screening an oral odor suppressing component according to any one of claims 1 to 3, wherein the step of culturing the Actinomyces bacterium is carried out at a pH in the range of 4.0 to 7.

0.

5. 5. The method for screening an ingredient for suppressing bad breath according to claim 1, wherein the substance that causes bad breath is hydrogen sulfide.

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