Method for evaluating antimicrobial activity

Abstract

To provide an antibacterial activity evaluation method capable of quantitatively evaluating antibacterial activity that vapor generated from a substance to be evaluated has.SOLUTION: An antibacterial activity evaluation method includes the following processes: (a) a process of preparing a plurality of inoculated solid culture media which is capable of proliferating bacteria to be evaluated proliferating by cell division, and in which the bacteria to be evaluated are inoculated; (b1) a process of using one of the inoculated solid culture media prepared in the process (a) as a first culture medium, and culturing the bacteria to be evaluated present in the first culture medium while allowing vapor generated from a substance to be evaluated to contact the first culture medium; (b2) a process of measuring the number of the bacteria to be evaluated present in the first culture medium after the process (b1); and (b3) a process of calculating proliferation rate of the bacteria to be evaluated in the first culture medium on the basis of the following calculation formula 1.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for evaluating antibacterial activity. Background Art

[0002] In recent years, consumers' interest in hygiene has increased, and the number of products advertised as having antibacterial and / or sterilization effects has grown. In particular, many products that realize antibacterial and / or sterilization of spaces have become common: such products diffuse gas of compounds having antibacterial and / or sterilization effects via air fresheners, diffusers, sprays, or the like, or spray a liquid containing a compound having antibacterial and / or sterilization effects and allow the gas of the compound to evaporate from the liquid. Therefore, establishing a method for evaluating the antibacterial activity of products that provide antibacterial and / or sterilization effects for spaces is an urgent issue.

[0003] Several methods for evaluating antibacterial and / or sterilization effects have been known to date. For example, Patent Document 1 discloses a method for detecting antimicrobial activity, in which a microbial culture means inoculated with a test microorganism and a test sample are placed side by side in a space enclosed by a container body provided with an opening that allows insertion and removal of the test sample container and the test microorganism culture means, the space is substantially blocked from outside air and allowed to stand for a predetermined time, and the antimicrobial activity of vapor generated from the test sample is determined based on the growth (proliferation) state of the microorganism. The document also describes a detection method that further comprises, after said determination, a step of culturing and observing the microorganism by the microbial culture means to determine the bacteriostatic and / or bactericidal activity of the test sample.

[0004] In addition, although Patent Document 2 describes an invention of antibacterial fibers, it describes a bacterial count method and a shake flask method as antibacterial activity test methods for the fibers. The bacterial count method is a method in which a bacterial solution is inoculated onto a sample (fiber) and cultured at a predetermined temperature, then the bacterial count before and after culture is measured by a conventional method. The shake flask method is a method in which a sample (fiber) and a bacterial solution are placed in an Erlenmeyer flask, shaken for 1 hour, and the bacterial count before and after shaking is measured.

[0005] Patent Document 3 describes a method for evaluating antibacterial properties of a surface to be evaluated, which comprises: a first step of inoculating an inoculum bacterial solution onto the surface to be evaluated of a ceramic product or a glass product, covering the surface with a covering film, capping it, and then storing it under constant conditions; and a second step of, after the first step, washing out bacteria adhering to the surface to be evaluated and the covering film into a measurement solution using a constant medium, and measuring the number of viable bacteria in the measurement solution. [Prior Art Document] [Patent Documents]

[0006] [Patent Document 1] Japanese Unexamined Patent Publication No. 10-108691 [Patent Document 2] Japanese Unexamined Patent Publication No. 4-156850 [Patent Document 3] Japanese Unexamined Patent Publication No. 2001-299384 [Summary of the Invention] [Problem to be Solved by the Invention]

[0007] The technology described in Patent Document 1 is useful as a method for measuring the antimicrobial activity (also called antimicrobial activity, and antimicrobial activity includes both bacteriostatic (inhibition of cell division) and bactericidal activity; the same applies hereinafter in this specification) of vapors generated from a test sample. However, in Patent Document 1, "samples in which bacterial growth (proliferation) was observed / samples in which bacterial growth was not observed" is generally judged by whether or not colonies (also called colonies, which are aggregates (cell masses) derived from single cells formed by bacteria or cultured cells in cell biology; the same applies hereinafter in this specification) appear by visual inspection, and if colonies appear, it is judged that bacterial growth was observed and uniformly judged as "(antimicrobial) activity absent". According to the inventors' studies, if the vapors generated from the test sample have relatively mild antimicrobial activity, as a result of inhibiting bacterial cell division, the number of colonies (colonies) may remain the same or hardly decrease, while the size of the colonies may decrease. Furthermore, in reality, if the vapors generated from the test sample have even milder antimicrobial activity, the number of colonies (colonies) may remain the same, and the size of the colonies may hardly change. In such cases, the technology described in Patent Document 1 cannot evaluate the antibacterial activity of gaseous samples with relatively mild antibacterial and / or disinfectant effects.

[0008] Furthermore, the technology described in Patent Document 1 allows for the determination that if bacterial growth is not observed, the sample is cultured in a steam-free environment to further check for bacterial growth, and if bacterial growth is observed, it has bacteriostatic activity; if bacterial growth is not observed, it has bactericidal activity. However, as mentioned above, the determination of "samples in which bacterial growth was observed / samples in which bacterial growth was not observed" in Patent Document 1 is based on whether colonies appear by visual inspection. Therefore, if the bacteria have grown to a degree that does not produce colonies, it will be judged as "samples in which bacterial growth was not observed," which is a problem.

[0009] The techniques described in Patent Documents 2 and 3 are methods for measuring the number of bacteria after inoculating an antimicrobial solution onto an antimicrobial sample (such as fibers, ceramics, and glass). While these methods are useful for measuring the antimicrobial activity of a solid sample surface, they cannot be directly applied to gaseous samples. Furthermore, bacteria inoculated onto a solid sample surface are cultured without nutrients for growth, so they will die even if the solid sample surface lacks antimicrobial activity. As a result, there has been a problem in quantitatively evaluating the antimicrobial activity of products used in environments where everyday bacteria proliferate.

[0010] The object of the present invention is to provide an antibacterial activity evaluation method that can quantitatively evaluate the antibacterial activity of vapors generated from a substance to be evaluated. [Means for solving the problem]

[0011] As a result of diligent research, the present inventors have discovered an antibacterial activity evaluation method that can quantitatively evaluate the antibacterial activity of vapors generated from a substance to be evaluated, and have completed the present invention. A brief overview of some of the representative inventions disclosed in this application is as follows.

[0012] [1] A method for evaluating antimicrobial activity, comprising the following steps. (a) A step of preparing multiple inoculated solid culture media on which the target bacteria, which proliferate by cell division, can grow, (b1) A step in which one of the inoculated solid media prepared in step (a) above is used as the first medium, and the target bacteria present in the first medium are cultured while vapor generated from the substance to be evaluated is brought into contact with the first medium. (b2) After step (b1), a step of measuring the number of the target bacteria present in the first culture medium, (b3) A step of calculating the growth rate of the target bacteria in the first culture medium based on the calculation formula 1 below.

[0013]

number

[0014] [2] In the antimicrobial activity evaluation method described in [1], Furthermore, a method for evaluating antimicrobial activity, including the following steps. (c1) A step in which the bacteria to be evaluated present in the second medium is cultured under the same conditions as in step (b1), except that one of the inoculated solid media prepared in step (a) above, other than the first medium, is used as the second medium, and vapors generated from the substance to be evaluated are not brought into contact with the second medium. (c2) After step (c1), a step of measuring the number of the target bacteria present in the second culture medium, (c3) A step of calculating the growth rate of the target bacteria in the second culture medium based on the calculation formula 2 below.

[0015]

number

[0016] In the antimicrobial activity evaluation method described in [3] [2], Furthermore, a method for evaluating antimicrobial activity, including the following steps. (d1) A step of calculating the antibacterial rate of the vapor generated from the substance to be evaluated against the target bacteria under the conditions of step (b1) based on the calculation formula 3 below.

[0017]

number

[0018] [4] In the antimicrobial activity evaluation method described in [1], The above step (a) is a method for evaluating antimicrobial activity, comprising the following steps. (a1) A step of preparing multiple solid culture media on which the target bacteria, which proliferate by cell division, can grow, (a2) A step of preparing the inoculated solid culture media by inoculating each of the solid culture media with the suspension of the target bacteria in the same volume using the plate coating method.

[0019] In the antimicrobial activity evaluation method described in [5] [2], The (b2) step is a method for evaluating antimicrobial activity, comprising the following steps. (b21) A step of adding liquid to the first culture medium after culturing in step (b1) above, and recovering the target bacteria present in the first culture medium as a suspension. (b22) A step of diluting the suspension obtained in step (b21) according to sequentially set dilution ratios to obtain diluted solutions with different concentrations of the target bacteria, (b23) Prepare multiple solid culture media for bacterial count measurement in which the target bacteria can grow, and solid A step of inoculating each of the culture media with the same volume of the dilution obtained in step (b22), (b24) A step of culturing each of the solid culture media for bacterial count measurement that have been inoculated with the dilution in step (b23) under the same conditions. (b25) After step (b24), a step of measuring the number of colonies in the solid culture medium for bacterial count measurement in which colonies have been formed, (b26) A step of calculating the number of target bacteria present in the first medium after culturing in step (b1) based on the following formula 4, using the dilution ratio and volume of the diluent inoculated into the solid medium for bacterial count measurement, the number of colonies formed in the solid medium for bacterial count measurement, and the volume of the liquid added to the first medium for bacterial suspension.

[0020]

number

[0021] [6] A method for evaluating antimicrobial activity, comprising the following steps. (a1) A step of preparing multiple solid culture media on which the target bacteria, which proliferate by cell division, can grow, (a2) Inoculating each of the solid culture media with the same volume of the suspension of the bacteria to be evaluated, (b1) A step in which one of the solid media inoculated with the target bacteria in step (a2) is designated as the first medium, and the target bacteria inoculated in the first medium are cultured while vapor generated from the substance to be evaluated is brought into contact with the first medium. (b2) After step (b1), a step of measuring the number of the target bacteria present in the first culture medium, (c1) A step in which the bacteria to be evaluated inoculated in the solid medium in step (a2) is designated as the second medium, and the bacteria to be evaluated inoculated in the second medium is cultured under the same conditions as in step (b1), except that vapors generated from the substance to be evaluated are not brought into contact with the second medium. (c2) After step (c1), a step of measuring the number of the target bacteria present in the second culture medium, (d2) A step of calculating the antibacterial rate of the vapor generated from the substance to be evaluated against the target bacteria under the conditions of step (b1) based on the calculation formula 5 below.

[0022]

number

[0023] In the antimicrobial activity evaluation method described in [7] [6], The aforementioned (b2) process includes the following steps (b21) to (b26): The (c2) step is a method for evaluating antimicrobial activity, comprising the following steps (c21) to (c26). (b21) A step of adding liquid to the first culture medium after culturing in step (b1) above, and recovering the target bacteria present in the first culture medium as a suspension. (b22) A step of diluting the suspension obtained in step (b21) according to sequentially set dilution ratios to obtain diluted solutions with different concentrations of the target bacteria, (b23) Prepare multiple solid culture media for measuring the bacterial count of the first culture medium in which the target bacteria to be evaluated can grow, solid A step of inoculating each of the culture media with the same volume of the dilution obtained in step (b22), (b24) A step of culturing each of the first culture medium for bacterial count measurement that has been inoculated with the dilution in step (b23) under the same conditions. (b25) After step (b24), a step of measuring the number of colonies in the first culture medium for bacterial count measurement in which colonies have been formed, (b26) A step of calculating the number of target bacteria present in the first medium after culturing in step (b1) based on the following formula 4, using the dilution ratio and volume of the diluent inoculated into the first medium for bacterial count measurement, the number of colonies formed in the first medium for bacterial count measurement, and the volume of the liquid added to the first medium for bacterial suspension.

[0024]

number

[0025] (c21) A step of adding liquid to the second culture medium after culturing in step (c1) above, and recovering the target bacteria present in the second culture medium as a suspension. (c22) A step of diluting the suspension obtained in step (c21) according to sequentially set dilution ratios to obtain diluted solutions with different concentrations of the target bacteria, (c23) Prepare multiple solid culture media for measuring the number of bacteria in the second culture medium in which the target bacteria to be evaluated can grow, and solid A step of inoculating each of the culture media with the same volume of the dilution obtained in step (c22), (c24) A step of culturing each of the solid media for measuring the bacterial count of the second medium, which has been inoculated with the dilution in step (c23), under the same conditions. (c25) After step (c24), a step of measuring the number of colonies in the second culture medium for bacterial count measurement in which colonies have been formed. (c26) A step of calculating the number of target bacteria present in the second medium after culturing in step (c1) based on the following calculation formula 4' from the dilution ratio and volume of the diluent inoculated into the second medium for bacterial count measurement, the number of colonies formed in the second medium for bacterial count measurement, and the volume of the liquid added to the second medium for bacterial suspension.

[0026]

number

[0027] In the antimicrobial activity evaluation method described in any one of [8] [1] to [7], The substance to be evaluated is a fragrance composition, and the method for evaluating antibacterial activity is described above. [Effects of the Invention]

[0028] According to the present invention, it is possible to provide an antibacterial activity evaluation method that can quantitatively evaluate the antibacterial activity of vapors generated from a substance to be evaluated. [Brief explanation of the drawing]

[0029] [Figure 1] Figure 1 is a flowchart showing each step of the antimicrobial activity evaluation method according to the first embodiment of the present invention. [Figure 2] Figure 2 is a flowchart showing each step of the antimicrobial activity evaluation method according to the second and third embodiments of the present invention. [Figure 3] Figure 3 is a flowchart showing each step of the antimicrobial activity evaluation method according to the fourth embodiment of the present invention. [Figure 4] Figure 4 is a flowchart showing each step of the antimicrobial activity evaluation method according to the fifth embodiment of the present invention. [Figure 5] Figure 5 is a flowchart showing each step of the antimicrobial activity evaluation method according to the sixth embodiment of the present invention. [Figure 6]Figure 6 is a side view showing a container for carrying out an antibacterial activity evaluation method according to one embodiment of the present invention. [Figure 7] Figure 7 is a schematic diagram showing the solid culture medium preparation step of an antimicrobial activity evaluation method according to one embodiment of the present invention. [Figure 8] Figure 8 is a schematic diagram showing the inoculation step of the target bacteria in the antimicrobial activity evaluation method according to one embodiment of the present invention. [Figure 9] Figure 9 is a schematic diagram showing the vapor contact culture step of the substance to be evaluated in the antibacterial activity evaluation method according to one embodiment of the present invention. [Figure 10] Figure 10 is a schematic diagram showing the vapor contact culture step of the substance to be evaluated in the antibacterial activity evaluation method according to one embodiment of the present invention. [Figure 11] Figure 11 is a schematic diagram showing the process of collecting the target bacteria in the antimicrobial activity evaluation method according to one embodiment of the present invention. [Figure 12] Figure 12 is a schematic diagram showing the process of collecting the target bacteria in the antimicrobial activity evaluation method according to one embodiment of the present invention. [Figure 13] Figure 13 is a schematic diagram showing the dilution preparation step of an antibacterial activity evaluation method according to one embodiment of the present invention. [Figure 14] Figure 14 shows images of a container cultured without contact with the fragrance composition and a container cultured while in contact with the first fragrance composition, in an example where the antibacterial activity evaluation method according to one embodiment of the present invention was applied. [Figure 15] Figure 15 shows images of containers cultured while in contact with the second fragrance composition and containers cultured while in contact with the third fragrance composition, in an example where the antibacterial activity evaluation method according to one embodiment of the present invention was applied. [Figure 16] Figure 16 shows images of containers cultured while in contact with the fourth fragrance composition and containers cultured while in contact with the fifth fragrance composition in an example where the antibacterial activity evaluation method according to one embodiment of the present invention was applied. [Figure 17]Figure 17 shows images of containers cultured while in contact with the sixth fragrance composition and containers cultured while in contact with the seventh fragrance composition in an example where the antibacterial activity evaluation method according to one embodiment of the present invention was applied. [Figure 18] Figure 18 shows images of containers cultured while in contact with the 8th fragrance composition and containers cultured while in contact with the 9th fragrance composition, in an example where the antibacterial activity evaluation method according to one embodiment of the present invention was applied. [Figure 19] Figure 19 is an explanatory diagram that allows for comparison between images of a container cultured without contact with the fragrance composition and a container cultured while in contact with the first to ninth fragrance compositions in an example where the antibacterial activity evaluation method according to one embodiment of the present invention is applied, the number of colonies formed, and the number of bacteria calculated by the antibacterial activity evaluation method according to this embodiment. [Modes for carrying out the invention]

[0030] Hereinafter, a method for evaluating antimicrobial activity according to one embodiment of the present invention (hereinafter sometimes referred to as "the antimicrobial activity evaluation method according to this embodiment") will be described in detail. In this specification, "~" means a range including the lower limit and upper limit, and unless otherwise specified, "concentration" and "%" represent "mass concentration" and "mass percentage concentration," respectively. In addition, in this specification, the prefix "the aforementioned" may not be added to previously mentioned terms and process names (for example, "the aforementioned target bacteria" may be written as "target bacteria," and "the aforementioned (a) process" may be written as "(a) process").

[0031] (First evaluation method) The antimicrobial activity evaluation method according to the first embodiment of the present invention (hereinafter sometimes referred to as the "first evaluation method") includes the following steps.

[0032] (a) A step of preparing multiple inoculated solid media on which the target bacteria, which proliferate by cell division, can grow, and on which the target bacteria have been inoculated; (b1) A step of using one of the inoculated solid media prepared in step (a) as the first medium, and culturing the target bacteria present in the first medium while bringing vapor generated from the substance to be evaluated into contact with the first medium; (b2) A step of measuring the number of target bacteria present in the first medium after step (b1); (b3) A step of calculating the growth rate of the target bacteria in the first medium based on the calculation formula 1 below.

[0033]

number

[0034] Figure 1 is a flowchart showing each step of the first evaluation method. As shown in Figure 1, in the first evaluation method, step (a) involves preparing multiple inoculated solid culture media, which are solid culture media on which the target bacteria, which proliferate by cell division, can grow, and which have been inoculated with the target bacteria. Here, in this embodiment, "bacterial count" is synonymous with the so-called total bacterial count, and is the number when counting each individual bacterial cell (the same applies hereafter). Therefore, in this embodiment, "bacterial count" and "colony count" are different concepts.

[0035] In the first evaluation method, "inoculated solid medium on which the target bacteria have been inoculated" is not particularly limited, as long as the target bacteria have been inoculated onto the solid medium by known inoculation methods, such as the pour plate method (mixing the bacteria with the solid medium (agar medium) before it solidifies and then solidifying the medium to inoculate the bacteria) or the plate coating method (dropping a bacterial solution (bacterial suspension) onto the surface of the solid medium (agar medium) after it has solidified and then spreading it with a Conlarger rod or the like). However, as will be described later, it is preferable to culture the target bacteria while allowing the vapor generated from the substance to be evaluated to come into direct contact with them in step (b1). In this case, it is preferable that the target bacteria have been inoculated onto the surface of the solid medium by the plate coating method. When the vapor generated from the substance to be evaluated is brought into contact with the surface of the inoculated solid medium (the same surface on which the bacteria have been inoculated) in step (b1), the vapor generated from the substance to be evaluated comes into direct contact with the target bacteria, so the antibacterial effect of the vapor generated from the substance to be evaluated on the target bacteria can be appropriately evaluated. Furthermore, the plate coating method has the advantage of facilitating colony observation and colony count measurement, as described later, because all colonies are formed on the surface of the solid culture medium.

[0036] Furthermore, the solid culture medium that can be used in the first evaluation method is not particularly limited, and any known solid culture medium will suffice, although details will be described later (the same applies hereinafter in the antimicrobial activity evaluation method according to this embodiment).

[0037] In the first evaluation method, and in the first evaluation method, the number of target bacteria inoculated does not need to be constant in each solid culture medium. However, as will be described later, it is preferable that the number of bacteria be constant in each solid culture medium, as this simplifies the calculation of the antimicrobial effect.

[0038] Next, in step (b1), one of the multiple inoculated solid media prepared in step (a) is selected as the first medium, and the target bacteria are cultured while vapors generated from the substance to be evaluated are brought into contact with the first medium. As a result, the target bacteria present in the first medium are cultured in a state where vapors generated from the substance to be evaluated are in contact with them. Here, in this specification, "vapors generated from the substance to be evaluated" means "vapors of volatile components contained in the substance to be evaluated," and for example, components with relatively high vapor pressure that can become gas at room temperature are also included in "vapors generated from the substance to be evaluated." Conversely, components with relatively low vapor pressure contained in the substance to be evaluated are also included in "vapors generated from the substance to be evaluated" because they evaporate together with components with high vapor pressure contained in the substance to be evaluated. In this embodiment, since the objective is to quantitatively evaluate the antibacterial activity of a product used in an environment where everyday bacteria proliferate, it is important to bring the vapors (gases) generated from the substance to be evaluated, rather than the substance to be evaluated itself (solid or liquid), such as the fragrance composition contained in the product, into contact with the target bacteria present in the solid medium.

[0039] Next, in step (b2), the number of target bacteria present in the first medium, which were cultured while being exposed to vapors generated from the substance to be evaluated in step (b1), is measured. Next, in step (b3), as shown in calculation formula 1 above, the growth rate of the target bacteria cultured in the first medium while being exposed to vapors generated from the substance to be evaluated is calculated by dividing the number of target bacteria present in the first medium after culturing in step (b1), as measured in step (b2), by the number of target bacteria present in the first medium before culturing in step (b1).

[0040] Here, the "number of target bacteria present in the first medium before culturing in step (b1)" should preferably be the number of bacteria immediately before culturing while in contact with vapors generated from the substance to be evaluated. Since it is not easy to measure the number of target bacteria immediately before culturing, in practice, after preparing the inoculated solid medium with the target bacteria in step (a) and before starting culturing in step (b1), the number of target bacteria inoculated can be used as the "number of target bacteria inoculated." For example, if the prepared inoculated solid medium is stored at a temperature at which the target bacteria cannot be cultivated, such as by refrigeration, then the "number of target bacteria present in the first medium before culturing in step (b1)" can be used.

[0041] In this embodiment, the method for measuring the number of bacteria (of the target bacteria) is not particularly limited and includes, for example, serial dilution, measurement using a bacterial count chamber, turbidity measurement, measurement by weight, and methods for measuring cellular components and biochemical activity. The serial dilution method will be described in detail in the fifth evaluation method section below. The measurement using a bacterial count chamber involves adding the liquid (sample) to which the amount of microorganisms to be measured to be measured to a bacterial count chamber (such as a hemocytometer) with a grid, and directly counting the number of cells in a certain compartment by observing it under a microscope. Turbidity measurement is a method in which the amount of transmitted light is measured using a spectrophotometer and converted to the number of bacteria present in the liquid. The measurement by weight is a method in which the weight of the bacterial cells is measured and converted to the number of bacteria. The method for measuring cellular components and biochemical activity is a method in which the amount of cellular components is measured and converted to the number of bacteria. As will be described later, the serial dilution method is the most preferable because it can accurately measure the number of viable bacteria.

[0042] Based on the above, according to the first evaluation method, the growth rate of the target bacteria is calculated when the bacteria are cultured while being exposed to vapors generated from the target substance in an environment in which the target bacteria can grow. Therefore, it is possible to quantitatively evaluate how vapors generated from the target substance suppress cell division of the target bacteria under conditions close to those of an actual living environment.

[0043] As mentioned above, in the antimicrobial activity evaluation method described in Patent Document 1, the distinction between "those in which bacterial growth (proliferation) was observed / those in which bacterial growth was not observed" is generally determined by visual inspection to see whether colonies appeared. This presented a problem in that if the number of colonies remained the same or hardly decreased while the size of the colonies decreased, or even if the number of colonies remained the same or hardly decreased while the size of the colonies remained almost unchanged, it would be judged as "those in which bacterial growth (proliferation) was observed."

[0044] In this respect, the first evaluation method measures the number of target bacteria regardless of whether colonies of the target bacteria have appeared. As a result, even if the number and size of colonies hardly change (the vapors emitted from the substance being evaluated suppress the cell division of the target bacteria to the extent that the number and size of colonies do not decrease), the degree to which the vapors emitted from the substance being evaluated suppress the cell division of the target bacteria can be accurately and quantitatively evaluated.

[0045] Furthermore, as mentioned above, the antimicrobial activity evaluation method described in Patent Document 1 has the problem that "samplings in which bacterial growth (proliferation) was observed / samplings in which bacterial growth was not observed" are generally judged by whether or not colonies appeared by visual inspection, and if the bacteria have grown to the extent that no colonies appear, it is judged as "samplings in which bacterial growth was not observed."

[0046] In this respect, the first evaluation method measures the number of target bacteria regardless of whether colonies of the target bacteria have appeared. As a result, even if the bacteria have proliferated to the extent that no colonies appear (i.e., the vapors emitted from the substance being evaluated inhibit the cell division of the target bacteria to the extent that no colonies appear), the degree to which the vapors emitted from the substance being evaluated inhibit the cell division of the target bacteria can be accurately and quantitatively evaluated.

[0047] Furthermore, as mentioned above, the antimicrobial activity evaluation methods for solid samples described in Patent Documents 2 and 3 have the problem that, because bacteria are inoculated onto the surface of the solid sample and cultured in a state where there are no nutrients for growth, the bacteria die even if there is no antimicrobial activity on the surface of the solid sample, making it difficult to quantitatively evaluate the antimicrobial activity.

[0048] In this respect, according to the first evaluation method, the target bacteria are cultured while being exposed to vapors generated from the substance under conditions where the target bacteria can proliferate. As a result, the cell division of the target bacteria is suppressed solely by the vapors generated from the substance. Consequently, it is possible to quantitatively evaluate the degree to which the vapors generated from the substance suppress the cell division of the target bacteria for products used in environments where everyday bacteria proliferate.

[0049] Furthermore, in step (b1), if vapors generated from the substance to be evaluated can be brought into contact with the first culture medium, the substance to be evaluated will permeate the first culture medium and come into contact with the bacteria to be evaluated present in the first culture medium, thereby allowing confirmation of the antibacterial effect of the substance to be evaluated against the bacteria to be evaluated. However, as mentioned above, it is preferable that the bacteria to be evaluated are inoculated onto the surface of the solid culture medium by the plate coating method, and that in step (b1), the bacteria to be evaluated are cultured while vapors generated from the substance to be evaluated are brought into contact with the surface of the first culture medium. This allows the vapors generated from the substance to be evaluated to come into direct contact with the bacteria to be evaluated, thereby allowing for appropriate evaluation of the antibacterial effect of the substance to be evaluated.

[0050] Furthermore, as a variation of the first evaluation method, two or more substances to be evaluated can be prepared and evaluated simultaneously. A variation of the first evaluation method when three substances to be evaluated are prepared includes the following steps (the same applies to the variation of the sixth evaluation method described later). Note that the following steps (b1A), (b2A), and (b3A), (b1A), (b2A), and (b3A), and (b1A), (b2A), and (b3A) are steps that correspond to steps (b1), (b2), and (b3), respectively, and letters have been added to their names for convenience to distinguish them.

[0051] (a) A step of preparing multiple solid media on which the target bacteria that grow by cell division can grow and which are inoculated with the target bacteria; (b1A) A step of designating one of the solid media prepared in step (a) as medium 1A, and culturing the target bacteria present in medium 1A while bringing vapor generated from the first target substance into contact with medium 1A; (b2A) A step of measuring the number of target bacteria present in medium 1A after step (b1A); (b3A) A step of calculating the growth rate of the target bacteria in medium 1A based on the following calculation formula 1A.

[0052]

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[0053] (b1B) A step in which one of the solid media prepared in step (a), other than medium 1A, is designated as medium 1B, and under the same conditions as in step (b1A), the target bacteria present in medium 1B are cultured, except that vapor generated from the second target substance is brought into contact with medium 1B instead of vapor generated from the first target substance; (b2B) A step in which the number of target bacteria present in medium 1B is measured after step (b1A); (b3B) A step in which the growth rate of the target bacteria in medium 1B is calculated based on the following formula 1B;

[0054]

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[0055] (b1C) A step in which one of the solid media prepared in step (a), other than medium 1A and medium 1B, is designated as medium 1C, and under the same conditions as in step (b1A), the target bacteria present in medium 1C are cultured, except that vapor generated from the third target substance is brought into contact with medium 1C instead of vapor generated from the first target substance; (b2C) A step in which the number of target bacteria present in medium 1C is measured after step (b1C); (b3C) A step in which the growth rate of the target bacteria in medium 1C is calculated based on the following calculation formula 1C.

[0056]

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[0057] Here, in a modified version of the first evaluation method, if the number of substances to be evaluated is four or more, the solid media other than the 1A, 1B, and 1C media will be designated as the 1D, 1E, etc., and the target bacteria present in each solid medium will be cultured while bringing the vapor generated from the nth substance to be evaluated (n is a natural number) into contact with the corresponding solid media, under the same conditions as in step (b1).

[0058] In the modified version of the first evaluation method described above, in step (b1A), the target bacteria are cultured while the vapor generated from the first target substance is brought into contact with the first A medium; in step (b1B), the target bacteria are cultured under the same conditions as in step (b1A), except that the vapor generated from the second target substance is brought into contact with the first B medium instead of the vapor generated from the first target substance; and in step (b1C), the target bacteria are cultured under the same conditions as in step (b1A), except that the vapor generated from the third target substance is brought into contact with the first C medium instead of the vapor generated from the first target substance. Therefore, it is possible to perform steps (b1A), (b1B), and (b1C) simultaneously, and it is preferable to perform steps (b1A), (b1B), and (b1C) simultaneously because it is easier to standardize the culture conditions and work costs can be reduced. As described later, by preparing multiple containers in the same room (where the temperature and humidity are the same) and bringing the vapor generated from one substance to be evaluated into contact with a solid culture medium in each container (it is important to prevent the vapor generated from the substance to be evaluated from leaking out of the container), steps (b1A), (b1B), and (b1C) can be performed simultaneously, making it possible to evaluate the antimicrobial activity of multiple substances (and their vapors) in a low-cost and simple manner.

[0059] (Second evaluation method) The antimicrobial activity evaluation method according to the second embodiment of the present invention (hereinafter sometimes referred to as the "second evaluation method") includes the following steps in addition to the first evaluation method.

[0060] (c1) A step in which one of the solid media prepared in step (a), other than the first medium, is designated as the second medium, and the target bacteria present in the second medium are cultured under the same conditions as in step (b1), except that vapors generated from the substance to be evaluated are not brought into contact with the second medium; (c2) A step in which the number of target bacteria present in the second medium is measured after step (c1); (c3) A step in which the growth rate of the target bacteria in the second medium is calculated based on the following calculation formula 2.

[0061]

number

[0062] Figure 2 is a flowchart showing each step of the second evaluation method and the third evaluation method described later. As shown in Figure 2, in the second evaluation method, in step (c1), one of the solid media prepared in step (a), other than the first medium, is selected as the second medium, and the target bacteria present in the second medium are cultured under the same conditions as in step (b1), except that the vapors generated from the substance to be evaluated are not brought into contact with the second medium. Next, in step (c2), the number of target bacteria present in the second medium, which were cultured under the same conditions as in step (b1), except that the vapors generated from the substance to be evaluated are not brought into contact with the second medium, is measured. Next, in step (c3), the growth rate of the target bacteria cultured in the second medium under the same conditions as in step (b1), except that the vapors generated from the substance to be evaluated are not brought into contact with the second medium, is calculated by dividing the number of target bacteria present in the second medium after culturing in step (c1), which was measured in step (c2), by the number of target bacteria present in the second medium before culturing in step (c1).

[0063] Based on the above, the second evaluation method allows for the calculation of the growth rate of the target bacteria cultured while in contact with vapors generated from the target substance in an environment where the target bacteria can grow. It also allows for the calculation of the growth rate of the target bacteria cultured under the same conditions except that they are not in contact with vapors generated from the target substance. By comparing these growth rates, the inhibitory effect of vapors generated from the target substance on cell division of the target bacteria can be analyzed and evaluated with greater accuracy compared to the first evaluation method.

[0064] In the second evaluation method, "same conditions" as in process (b1) in process (c1) means that the temperature and time, which are general culture conditions, are the same in both process (b1) and process (c1). Although not essential, it is preferable to include the condition that humidity, atmospheric pressure, and other parameters that may be included in culture conditions are the same in "same conditions" in order to improve the accuracy of the evaluation. As in the examples described later, when process (b1) is performed in a container, it is preferable that process (c1) is also performed in the same container as process (b1), or in a container of the same shape and volume as the container used in process (b1), as "same conditions".

[0065] In step (a), multiple solid media inoculated with the target bacteria are prepared, and one of these solid media is used as the first medium in step (b1), and the other as the second medium in step (c1). Therefore, the types of media are the same. The number of target bacteria inoculated into the first medium and the number of target bacteria inoculated into the second medium do not need to match, but to improve the accuracy of the evaluation, it is preferable that the number of target bacteria inoculated into the first medium and the number of target bacteria inoculated into the second medium be of at least the same order (degree), specifically 10. 2 10 pieces or more 3 It is preferable to have fewer than one item.

[0066] As a method for matching the number of bacteria to be evaluated inoculated into the first medium and the number of bacteria to be evaluated inoculated into the second medium, an example includes, but is not limited to, a method of inoculating equal volumes of a suspension of the bacteria to be evaluated into the first medium and the second medium, as in the fourth evaluation method described below.

[0067] Furthermore, when the number of bacteria to be evaluated inoculated into the first medium is matched with the number of bacteria to be evaluated inoculated into the second medium, and the number of bacteria to be evaluated inoculated into the first medium (second medium) is 30 or more and 300 or less, the following can be adopted as a modified example of the second evaluation method. That is, in a case where the number of bacteria to be evaluated inoculated into the first medium is matched with the number of bacteria to be evaluated inoculated into the second medium, and the number of bacteria to be evaluated inoculated into the first medium (second medium) is 30 or more and 300 or less, in step (c1), the number of colonies formed by the bacteria to be evaluated inoculated into the second medium is per one medium (with an area of 50 cm 2 or more and 80 cm 2 or less), culturing is performed until the count is 30 or more and 300 or less, and said number of colonies is regarded as the number of bacteria to be evaluated inoculated into the first medium and the second medium.

[0068] In the present embodiment, since the bacteria to be evaluated proliferate by cell division, one bacterium to be evaluated proliferates and forms one colony as a result of culturing. Therefore, if culturing is performed until all bacteria can each form a colony, the number of colonies formed on the solid medium for bacterial count measurement corresponds to the number of bacteria originally inoculated. Here, the criterion for whether culturing has been completed until all bacteria can each form a colony is that the number of colonies after culturing per one medium (with an area of 50 cm 2 or more and 80 cm 2 or less) is 30 or more and 300 or less. Here, the condition that the area of the medium is 50 cm 2 or more and 80 cm 2 or less is synonymous with that when the medium is formed in a commonly used container having a substantially circular cross section (for example, the container or petri dish according to the present embodiment), the diameter of the container is 8 cm or more and 10 cm or less. Furthermore, "per one medium (area 50 cm 2More than 80cm 2 The condition that "the number of colonies after culturing per 1 cm of culture medium is between 30 and 300" is defined as "1 cm of culture medium 2 The number of colonies per square centimeter is 0.375. 2 More than 6 pieces / cm 2 This can also be rephrased as follows: Therefore, in step (c1), the second culture medium (area 50 cm²) is used. 2 More than 80cm 2 If the target bacteria inoculated in the following are cultured until they form 30 to 300 colonies, all of the bacteria should form colonies, and the number of these colonies should match the number of target bacteria inoculated in the first and second media (30 to 300).

[0069] As a result, in the modified version of the second evaluation method, the number of target bacteria inoculated into the first medium and the number of target bacteria inoculated into the second medium are made to match, and the number of target bacteria inoculated into the first medium (second medium) is set to 30 to 300, and the number of colonies formed in the second medium is equal to one medium (area 50 cm²). 2 More than 80cm 2 By culturing the colonies so that there are between 30 and 300 colonies per medium (as described below), the number of colonies can be considered as the number of target bacteria inoculated into the first and second media. Furthermore, when adopting a modified version of the second evaluation method, it is advantageous in that the number of target bacteria inoculated into the solid medium does not need to be known. In addition, in the modified version of the second evaluation method, if the first and second media are formed in transparent containers, the process of colony formation can be observed and compared with the growth rate of the target bacteria calculated in steps (b3) and (c3). That is, by setting the number of target bacteria inoculated into the first (second) medium to between 30 and 300, a maximum of only 300 colonies will be formed in the first and second media. Therefore, the growth rate of the target bacteria calculated in steps (b3) and (c3) can be predicted from the number of colonies in each medium, and it is also possible to verify whether the growth rate of the target bacteria calculated in steps (b3) and (c3) is reasonable.

[0070] In the examples described later, a modified version of the second evaluation method was applied. When cultured without contact with the fragrance composition (corresponding to the second culture medium), 235 colonies were formed. Therefore, the initial number of bacteria inoculated into this medium was considered to be 235.

[0071] Furthermore, when applying the modified version of the first evaluation method described above to the second evaluation method, one additional solid culture medium should be prepared in addition to the first A medium, first B medium, first C medium, etc. used in the modified version of the first evaluation method, and this should be used as the second culture medium in the second evaluation method. In other words, as a modified version of the second evaluation method, the first A medium, first B medium, etc., are used to culture the target bacteria while they are in contact with the first and second target substances, respectively, while the second culture medium is used to culture the target bacteria without any contact with either the first or second target substances, thus matching the culture conditions for these two culture media.

[0072] Furthermore, even if it is difficult to ensure that the number of target bacteria inoculated into the first medium (second medium) is between 30 and 300, and the modified version of the second evaluation method cannot be applied, the number of target bacteria inoculated into the first and second mediums can be measured by applying other bacterial counting methods, so there is no problem in implementing the first and second evaluation methods. In addition, when directly calculating the antimicrobial rate using the sixth evaluation method described later, it is possible to perform the evaluation even if the number of target bacteria present in the inoculated solid medium prepared in step (a) of the first and second evaluation methods (i.e., the number of bacteria present in the first and second mediums before culturing) is unknown.

[0073] (Third evaluation method) The antimicrobial activity evaluation method according to the third embodiment of the present invention (hereinafter sometimes referred to as the "third evaluation method") includes the following steps in addition to the second evaluation method.

[0074] (d1) A step to calculate the antibacterial rate of vapor generated from the substance to be evaluated against the target bacteria under the conditions of step (b1), based on the calculation formula 3 below.

[0075]

number

[0076] In other words, as shown in Figure 2, in the third evaluation method, in step (d1), as shown in calculation formula 3, the growth rate of the target bacteria cultured in the first medium while in contact with vapors generated from the target substance, calculated in step (b3), is divided by the growth rate of the target bacteria cultured in the second medium under the same conditions as in step (b1), except that vapors generated from the target substance are not in contact with the target bacteria, calculated in step (c3), to calculate the cell division inhibition rate (e.g., 0.01, 0.001, etc.). Then, by subtracting this cell division inhibition rate from 1 and multiplying by 100, the antibacterial rate (e.g., 99%, 99.9%) of vapors generated from the target substance against the target bacteria under the conditions (temperature, time, etc.) of step (b1) is calculated as a percentage.

[0077] Based on the above, the third evaluation method allows for the calculation of the antimicrobial activity of target bacteria cultured while in contact with vapors generated from the target substance in an environment where the target bacteria can proliferate. Therefore, the inhibitory effect of vapors generated from the target substance on cell division of target bacteria can be evaluated more clearly and quantitatively compared to the second evaluation method.

[0078] In the third evaluation method, expressing the antibacterial rate as a percentage makes it easier to understand, but it is not limited to this.

[0079] (Fourth evaluation method) The antimicrobial activity evaluation method according to the fourth embodiment of the present invention (hereinafter sometimes referred to as the "fourth evaluation method") limits some steps of the first evaluation method, the second evaluation method, or the third evaluation method (hereinafter referred to as the "first to third evaluation methods") as follows, wherein step (a) of the first to third evaluation methods includes the following steps.

[0080] (a1) A step of preparing multiple solid media on which the target bacteria, which proliferate by cell division, can grow; (a2) A step of inoculating each of the solid media with the same volume of the suspension of the target bacteria using the plate coating method to prepare inoculated solid media.

[0081] Figure 3 is a flowchart showing each step of the fourth evaluation method. As shown in Figure 3, in the fourth evaluation method, in step (a1), multiple solid culture media capable of growing the target bacteria are prepared. Next, in step (a2), the suspension of the target bacteria is inoculated into each of the solid culture media prepared in step (a1) by the plate spreading method in the same volume (i.e., a fixed amount each). The "plate spreading method" in step (a2), as mentioned above, refers to a method in which the bacterial solution (bacterial suspension) is dropped onto the surface of the culture medium after the solid culture medium (agar medium) has solidified, and then spread using a Conlarger rod or the like.

[0082] Based on the above, according to the fourth evaluation method, in step (a) of the first to third evaluation methods, multiple inoculated solid media can be prepared in which the number of the target bacteria to be evaluated is the same in each case. In particular, according to the fourth evaluation method, the number of bacteria inoculated into each solid medium can be easily adjusted by the volume of the suspension (amount dropped onto the solid medium). Therefore, by inoculating each solid medium with the same volume of a suspension having a constant number of bacteria per volume, the number of bacteria inoculated into each solid medium can be made the same. Note that "the number of bacteria is the same" here does not require that the number of bacteria be the same down to the single cell level, but rather means that they are at least of the same order (degree), and preferably the same with a precision of two significant figures.

[0083] Furthermore, according to the fourth evaluation method, since the target bacteria are inoculated onto the surface of the solid culture medium by the plate coating method, as mentioned above, in step (b1), the vapor generated from the target substance can be brought into direct contact with the target bacteria during cultivation, and as a result, the antibacterial effect of the vapor generated from the target substance on the target bacteria can be appropriately evaluated. In addition, according to the fourth evaluation method, since all colonies are formed on the surface of the solid culture medium, observation of colonies and measurement of the number of colonies become easier. Moreover, according to the fourth evaluation method, since the target bacteria remain on the surface of the solid culture medium (first medium) even after cultivation, in step (b21) of the fifth evaluation method described later, it becomes easy to add a liquid such as sterile water to the first medium to wash out the target bacteria and recover them as a suspension.

[0084] In the fourth evaluation method, since the target bacteria may settle in the suspension of the target bacteria over time, the suspension should be thoroughly stirred before inoculation so that the number of bacteria per unit volume remains constant (the same applies hereinafter in the antimicrobial activity evaluation method according to this embodiment). The inoculation volume of the suspension is not particularly limited, but an inoculation volume of 10 to 100 μL is preferable because it allows for easy inoculation by the plate coating method.

[0085] (5th evaluation method) A fifth embodiment of the present invention, a method for evaluating antimicrobial activity (hereinafter sometimes referred to as the "fifth evaluation method"), limits some steps of the second evaluation method as follows, wherein step (b2) of the second evaluation method includes the following steps.

[0086] (b21) A step of adding liquid to the first culture medium after culturing in step (b1) and recovering the target bacteria present in the first culture medium as a suspension; (b22) A step of diluting the suspension obtained in step (b21) according to sequentially set dilution ratios to obtain diluted solutions with different concentrations of the target bacteria; (b23) A step of preparing multiple solid culture media for measuring the number of bacteria in the first culture medium on which the target bacteria can grow, and for measuring the number of bacteria in the first culture medium solidThe steps are: inoculating each of the culture media with the same volume of the diluent obtained in step (b22); culturing each of the solid culture media for first culture media bacterial count measurement inoculated with the diluent in steps (b24) and (b23) under the same conditions; measuring the number of colonies formed on the solid culture media for first culture media bacterial count measurement after steps (b25) and (b24); and for solid culture media for first culture media bacterial count measurement in which the number of colonies measured in step (b25) is 30 or more and 300 or less, calculating the number of target bacteria present in the first culture media after culturing in step (b1) based on the dilution ratio and volume of the diluent inoculated into the solid culture media for first culture media bacterial count measurement, the number of colonies formed on the solid culture media for first culture media bacterial count measurement, and the volume of the liquid added to the first culture medium for bacterial suspension, using the following calculation formula 4.

[0087]

number

[0088] Figure 4 is a flowchart showing each step of the fifth evaluation method. As shown in Figure 4, in the fifth evaluation method, in step (b21), liquid is added to the first culture medium after culturing in step (b1), and the target bacteria present in the first culture medium are recovered as a suspension. In step (b21), in order to recover the target bacteria present in the first culture medium, a liquid such as sterile water is added to the first culture medium, and the target bacteria present on the surface of the first culture medium are washed out to obtain a suspension of the target bacteria. Note that in the first evaluation method, vapors generated from the substance to be evaluated come into contact with the first culture medium, and components in the vapor may remain in the first culture medium, but these are diluted to an appropriate ratio in the next step (b22) (the dilution ratio at which the number of colonies can be measured is 10 as will be described later). 5 ~10 8 Because it is twice as much, the inventors have confirmed that the effect of the remaining substance on the culture of the target bacteria in the subsequent (b24) step is negligibly small.

[0089] Next, in step (b22), the suspension obtained in step (b21) is diluted according to progressively set dilution ratios, and diluted solutions with different concentrations of the target bacteria are obtained. Here, "progressively set dilution ratios" means dilution ratios set based on a predetermined difference or ratio, for example, (i) 10 times, 10 2 double, 10 3 double, 10 4 times..., (ii) 2 times, 2 2 double, 2 3 double, 2 4 It is preferable to set the dilution ratio as a value obtained by raising a certain number to a power, such as "times," and (i) is particularly preferable from the viewpoint that it is possible to reliably prepare one solid culture medium for bacterial count measurement in which the number of colonies is between 30 and 300. Furthermore, "concentration of the target bacteria" means the number of bacteria per unit volume in the suspension of the target bacteria (the same applies hereinafter in the antibacterial activity evaluation method according to this embodiment).

[0090] Next, in step (b23), multiple solid media for measuring the bacterial count of the first medium on which the target bacteria can grow are prepared, and the first medium bacterial count is measured solid Each culture medium is inoculated with the same volume of the dilution obtained in step (b22). Here, the "solid culture medium for bacterial count measurement" is designated as such for convenience to distinguish it from the "solid culture medium" that appears in other steps, and may be the same as or different from the "solid culture medium" prepared in step (a1) of the fourth evaluation method. Similarly, the "solid culture medium for bacterial count measurement of the first culture medium" is designated as such for convenience to distinguish it from the "solid culture medium for bacterial count measurement of the second culture medium" described later, and may be the same as or different from the "solid culture medium for bacterial count measurement of the second culture medium" prepared in step (c23) described later.

[0091] Furthermore, in step (b23), the method of inoculating the target bacteria is not particularly limited, but the volume of each diluent is usually 1 mL or more, and it is preferable to inoculate the target bacteria by the pour plate method, which facilitates inoculation at such volumes, that is, by mixing the bacteria with the solid medium (agar medium) before it solidifies and then solidifying the medium to inoculate the bacteria into the medium.Therefore, in step (b23), when inoculating the target bacteria by the pour plate method, "preparing a solid medium" means preparing a solid medium (agar medium) in powder form, granular form (before dissolution in water), or liquid form (before solidification), and then the diluent is mixed with the liquid solid medium (agar medium) (before solidification) to inoculate the target bacteria, and then the medium solidifies.On the other hand, in step (b23), when inoculating the target bacteria by the plate coating method, "preparing a solid medium" means preparing a solid medium that has solidified by the time the target bacteria are inoculated, similar to the fourth evaluation method.

[0092] Furthermore, regarding the bottom area of ​​the solid culture medium used in step (b23), in order to easily identify culture media with a colony count of 30 to 300 in steps (b26) and (c26) described later, the bottom area of ​​the solid culture medium for measuring the bacterial count of the first culture medium (solid culture medium for measuring the bacterial count of the second culture medium) is set to 50 cm². 2 More than 80cm 2 The following is preferable. This allows for visual identification and observation of colonies when 30 to 300 colonies are formed, and as a result, the number of colonies can be appropriately measured. For example, if the bottom area of ​​the solid medium for measuring bacterial count in the first medium (solid medium for measuring bacterial count in the second medium) is 50 cm² 2 Less than or 80cm 2 If it exceeds 1 cm 2 The number of colonies per square centimeter is 0.375. 2 More than 6 pieces / cm 2 The number of colonies in the culture medium identified in step (b26) should be adjusted to satisfy the following conditions.

[0093] Next, in step (b24), each of the solid media for measuring the bacterial count of the first medium, which was inoculated with each dilution in step (b23), is cultured under the same conditions. As mentioned above, in step (b24), in order to appropriately measure the number of colonies in the following step (b25), when the number of colonies in any of the solid media for measuring the bacterial count of the first medium reaches 30 to 300 (the bottom area of ​​the solid media for measuring the bacterial count of the first medium is 50 cm²), 2 Less than or 80cm 2 If it exceeds 1 cm 2 The number of colonies per square centimeter is 0.375. 2 More than 6 pieces / cm 2 The culture should be stopped when the following conditions are met.

[0094] Next, in step (b25), the number of colonies in the solid medium for bacterial count measurement of the first medium, on which colonies have formed after step (b24), is measured, and solid medium for bacterial count measurement of the first medium with a colony count of 30 to 300 is identified. If a solid medium for bacterial count measurement with a colony count of 30 to 300 is not obtained, the process can be repeated from step (b22) with a different dilution ratio.

[0095] Next, in step (b26), the number of target bacteria present in the first medium after culturing in step (b1) is calculated based on the above formula 4, using the dilution ratio and volume of the dilution solution inoculated into the first medium for bacterial count measurement, which has a colony count of 30 or more and 300 or less identified in step (b25), the volume of the liquid added to the first medium for bacterial suspension, and the number of colonies formed on the first medium for bacterial count measurement.

[0096] Here, we will explain why, in calculation formula 4, we used the "volume of liquid added to the first medium for bacterial suspension" rather than the "volume of suspension recovered from the first medium." As mentioned above, in step (b21), in order to recover the target bacteria present in the first medium, a liquid such as sterile water is added to the first medium, and the target bacteria present on the surface of the first medium are washed out to obtain a suspension of the target bacteria. At this time, it is not easy to recover 100% of the obtained suspension, but if the suspension of bacteria is uniform (i.e., if the number of bacteria per volume of suspension is constant), the following equation can be transformed.

[0097]

number

[0098] In calculation formula 4, terms (1) to (4) are added for explanatory purposes. (1) × (2) × (3) gives the number of bacteria present in the suspension recovered from the first medium. If the number of bacteria per unit volume of suspension is constant, multiplying the number of bacteria present in the suspension recovered from the first medium by (4) gives the number of bacteria present in the liquid added to the first medium for bacterial suspension, which corresponds to the number of target bacteria present in the first medium after culturing in step (b1). As a result, the numerator of (2) and the denominator of (4) cancel each other out, and in calculation formula 4, the "volume of the suspension recovered from the first medium" becomes unnecessary, and it is sufficient to know the "volume of the liquid added to the first medium for bacterial suspension". In other words, as long as the bacterial suspension is performed reliably, it is not necessary to recover 100% of the suspension, and the calculation is not affected by the volume of the recovered suspension, making it easier to implement.

[0099] Furthermore, in the fifth evaluation method, the number of bacteria is measured by culturing the target bacteria, so the number calculated by steps (b21) to (b26) is the number of viable cells (live cells) of the target bacteria present in the first medium after culturing in step (b1), which has been recovered as a suspension. Generally, the antibacterial effect of the substance being evaluated (vapor) includes both bacteriostatic effect (inhibition of cell division) and bactericidal effect. If, by other methods, the sum of viable and dead bacteria is obtained as the number of target bacteria recovered as a suspension, and that number does not change from the initial number, it is impossible to distinguish whether cell division was simply inhibited by the bacteriostatic effect or whether the bacteria were killed. On the other hand, when calculating the number of viable cells as in the fifth evaluation method, if the target bacteria are killed by the vapor generated from the substance being evaluated, the value will be lower than the initial number. Therefore, in the fifth evaluation method, the bactericidal effect of the substance being evaluated can also be appropriately evaluated.

[0100] Note that the number of bacteria (viable cells) of the target bacteria calculated by steps (b21) to (b26) of the fifth evaluation method may be expressed in terms of colony forming units (CFU), but in this specification it will be expressed in terms of "individuals".

[0101] Here, we will explain step (b26) of the fifth evaluation method in detail based on a specific example. Table 1 below shows the relationship between the volume of the suspension recovered in step (b21), the volume of each diluent inoculated in step (b23), the dilution ratio of each diluent obtained in step (b22) (dilution ratios set in stages), the number of colonies formed on each solid medium for measuring bacterial count in the first medium measured in step (b25), and the number of bacteria of the target bacteria calculated in step (b26) when the fifth evaluation method is applied to a certain target bacterium.

[0102] [Table 1]

[0103] As shown in Table 1, colonies were formed at a dilution ratio of 10 7These are solid media for measuring the bacterial count of the first medium, inoculated with a dilution smaller than 1:1, and these correspond to the "solid media for measuring the bacterial count of the first medium on which colonies have formed" in step (b24). In step (b25), the number of colonies in the "solid media for measuring the bacterial count of the first medium on which colonies have formed" is measured, and as a result, in step (b26), the dilution ratio that satisfies the condition of "having 30 or more colonies and 300 or less" from among the "solid media for measuring the bacterial count of the first medium on which colonies have formed" is 10 6 A solid medium for measuring the bacterial count of the first medium is used in the case of a 2x ratio. Subsequently, by substituting the "dilution ratio of the diluent inoculated into the solid medium for measuring the bacterial count of the first medium," the "volume of the diluent inoculated into the solid medium for measuring the bacterial count of the first medium," the "volume of the liquid added to the first medium for bacterial suspension," and the "number of colonies formed on the solid medium for measuring the bacterial count of the first medium" into formula 4, the number of target bacteria present in the first medium after culturing in step (b1), which was recovered as a suspension, is calculated as follows: 2.2 × 10⁻⁶ 9 It can be calculated as one unit.

[0104]

number

[0105] Furthermore, this bacterial count measurement method is based on the premise that one target bacterium grows and forms one colony after being cultured in the first medium for bacterial count measurement. Therefore, the target bacterium needs to grow through cell division. In other words, reviewing steps (b21) to (b26) based on the above results, the first medium after culturing in step (b1) contains 2.2 × 10⁶ target bacteria. 9 In the (b21) step, 10 mL of sterile water is added to the first medium for bacterial suspension, and when the target bacteria are uniformly suspended in this sterile water, the number of target bacteria per 10 mL of sterile water added to the first medium is 2.2 × 10 9 This is one sample, of which 6 mL contains the target bacteria (1.34 × 10⁶). 9 The individual particles were recovered as a suspension. This suspension was diluted with each of the above dilution ratios, but the dilution ratio was 10 6For a double dilution, 1.34 × 10 per 1 mL of dilution. 9 ÷6÷10 6 This means there were 223 bacteria to be evaluated. This dilution ratio is 10 6 When a 1:1 dilution is inoculated onto the solid medium for measuring bacterial count in the first culture medium and cultured, one bacterium will multiply by cell division in its vicinity until it reaches approximately 100,000 or more cells, at which point it will form a colony. Once all the bacteria have been cultured to the point where they can each form a colony, the number of colonies formed on the solid medium for measuring bacterial count in the first culture medium will correspond to the original number of bacteria inoculated. In other words, in step (b24), this dilution ratio is 10 6 When the diluted solution is inoculated into the solid medium for measuring the bacterial count of the first medium and cultured, 223 colonies are formed. Step (b25) measures that the number of colonies on this solid medium for measuring the bacterial count of the first medium is 223, and as a result, step (b26) calculates that the target bacteria present in the first medium after culture in step (b1), which was recovered as a suspension, is 2.2 × 10⁻⁶ according to formula 4 above. 9 The number of bacteria can be calculated as follows. Here, for the sake of explanation, the number of target bacteria in the suspension recovered from the first culture medium is also calculated. However, as mentioned above, the volume of the suspension recovered from the first culture medium is canceled out and is not needed for the calculation. Therefore, if the volume of the liquid (sterile water) added to the first culture medium for bacterial suspension is known, the number of target bacteria present in the first culture medium can be directly determined from formula 4.

[0106] Note that the dilution ratio is 10 7 In the case of double the amount, the number of colonies was 20, so it does not meet the condition of "the number of colonies is between 30 and 300" and is therefore not accepted. This is because the dilution ratio is 10 7 If the number doubles, it's possible that the target bacteria have proliferated to the point where no colonies are forming, and in this case, it won't be reflected in the colony count.

[0107] Also, the dilution ratio is 10 5 In the case of a 2x dilution, the number of colonies was 2000 or more, so it did not meet the condition of "the number of colonies being between 30 and 300," and was therefore not adopted. This is because the dilution ratio was 10 5In the case of double the number, we know that the number of colonies is more than 2000, but it is difficult to count them accurately because there are too many colonies.

[0108] Note that although not shown in Table 1, the dilution ratio is 10 4 If the number of colonies is less than twice, then it is 10 5 Needless to say, counting the colonies becomes even more difficult than when there are twice as many.

[0109] Furthermore, the fifth evaluation method can naturally be applied not only to process (b2) but also to process (c2). In other words, a modified version of the fifth evaluation method is to limit some of the processes of the third evaluation method, so that process (c2) of the third evaluation method includes the following processes.

[0110] (c21) A step of adding liquid to the second medium after culturing in step (c1) and recovering the target bacteria present in the second medium as a suspension; (c22) A step of diluting the suspension obtained in step (c21) according to sequentially set dilution ratios to obtain diluted solutions with different concentrations of the target bacteria; (c23) A step of preparing multiple solid media for measuring the bacterial count of the second medium in which the target bacteria can grow, and for measuring the bacterial count of the second medium solid The process involves inoculating each of the culture media with the same volume of the diluent obtained in step (c22), culturing each of the second culture mediums for bacterial count measurement inoculated with the diluent in steps (c24) and (c23) under the same conditions, measuring the number of colonies formed on the second culture medium for bacterial count measurement after steps (c25) and (c24), and for the second culture medium for bacterial count measurement in which the number of colonies measured in step (c25) is 30 or more and 300 or less, calculating the number of target bacteria present in the second culture medium after culturing in step (c1) based on the following calculation formula 4' from the dilution ratio and volume of the diluent inoculated into the second culture medium for bacterial count measurement, the volume of the liquid added to the second culture medium for bacterial suspension, and the number of colonies formed on the second culture medium for bacterial count measurement.

[0111]

number

[0112] In the modified version of the fifth evaluation method, the number of bacteria is measured by culturing the target bacteria. Therefore, steps (c21) to (c26) calculate the number of viable bacteria among the target bacteria present in the second medium after culturing in step (c1), which is recovered as a suspension. Consequently, when calculating the number of viable bacteria of the target bacteria present in the first medium after culturing in step (b1), which is recovered as a suspension, using the fifth evaluation method, these can be appropriately compared and evaluated using the same criteria by calculating the number of viable bacteria of the target bacteria present in the second medium after culturing in step (c1) using the modified version of the fifth evaluation method.

[0113] (6th evaluation method) The antimicrobial activity evaluation method according to the sixth embodiment of the present invention (hereinafter sometimes referred to as the "sixth evaluation method") includes the following steps.

[0114] (a1) A step of preparing multiple solid media on which the target bacteria to be evaluated, which proliferate by cell division, can grow; (a2) A step of inoculating each of the solid media with the same volume of the suspension of the target bacteria to be evaluated; (b1) A step of designating one of the solid media inoculated with the target bacteria to be evaluated in step (a2) as the first medium, and culturing the target bacteria inoculated in the first medium while bringing the vapor generated from the substance to be evaluated into contact with the first medium; (b2) A step of measuring the number of target bacteria present in the first medium after step (b1); (c1) (a 2) A step in which one of the solid media inoculated with the target bacteria in the first step is designated as the second medium, and the target bacteria inoculated in the second medium are cultured under the same conditions as in step (b1), except that vapors generated from the substance to be evaluated are not brought into contact with the second medium; (c2) After step (c1), the number of target bacteria present in the second medium is measured; (d2) Based on the calculation formula 5 below, the antibacterial rate of vapors generated from the substance to be evaluated against the target bacteria under the conditions of step (b1) is calculated.

[0115]

number

[0116] Figure 5 is a flowchart showing each step of the sixth evaluation method. As shown in Figure 5, the sixth evaluation method is almost the same as a single method combining the first evaluation method (see Figure 1), the second evaluation method (see Figure 2), the third evaluation method (see Figure 2), and the fourth evaluation method (see Figure 3). However, the sixth evaluation method differs in that it does not require that the number of target bacteria present in the inoculated solid medium prepared in step (a) of the first and second evaluation methods (i.e., the number of bacteria present in the first and second media before culturing) be known, and that the concentration of the suspension of target bacteria inoculated in step (a2) of the fourth evaluation method is not known.

[0117] The following explains why the number of bacteria present in the first and second culture media before culturing, and the concentration of the suspension (number of bacteria per unit volume), may be unknown. As explained in the section "3. Evaluation Method," the antimicrobial effect of the vapor generated from the substance to be evaluated against the target bacteria under the conditions of step (b1) is given by the calculation formula 3, which is repeated below.

[0118]

number

[0119] Here, the growth rate in the first culture medium in calculation formula 3 is given by calculation formula 1, which is reproduced below, as explained in the section on "First Evaluation Method".

[0120]

number

[0121] Furthermore, the "growth rate in the second medium" in calculation formula 3 is given by calculation formula 2, which is reproduced below, as explained in the section on "Second Evaluation Method".

[0122]

number

[0123] Here, as explained in the section "4. Evaluation Method," in step (a2), since the same volume of the suspension of the target bacteria (which has a constant number of bacteria per unit volume) is inoculated into each solid culture medium, the "number of bacteria present in the first culture medium before culturing" and the "number of bacteria present in the second culture medium before culturing" are the same. Therefore, substituting formulas 1 and 2 into formula 3 and rearranging, we get the following formula 5.

[0124]

number

[0125] Based on the above, in the sixth evaluation method, although it is necessary to inoculate each solid culture medium with the suspension of the target bacteria in the same volume in step (a2), it is not necessary for the number of bacteria present in the first and second culture media before culturing to be known, and it is acceptable (not a problem) if the concentration (number of bacteria per unit volume) of the suspension of the target bacteria inoculated into the first and second culture media is unknown. Therefore, it is advantageous in that it is simpler and is expected to reduce the number of steps compared to a single method that combines the first, second, third, and fourth evaluation methods. Furthermore, as shown in Figure 5, in the sixth evaluation method, there is no step (b3) in the second evaluation method and no step (c3) in the third evaluation method (see Figure 2), and the antimicrobial rate can be directly determined from the number of bacteria present in the first culture medium and the second culture medium after culturing. This is advantageous in that it is simpler and is expected to reduce the number of steps compared to the third evaluation method.

[0126] Furthermore, by combining the sixth evaluation method, the fifth evaluation method, and a modified version of the fifth evaluation method, the sixth evaluation method can calculate the number of viable target bacteria present in the first medium after culturing in step (b1) recovered as a suspension, and the number of viable target bacteria present in the second medium after culturing in step (c1) recovered as a suspension. Therefore, the bactericidal effect of the target substance can be appropriately evaluated in the antimicrobial rate calculated by the sixth evaluation method (see the section on the fifth evaluation method for details).

[0127] (Bacteria to be evaluated) In the antimicrobial activity evaluation method according to this embodiment, suitable (evaluable) target organisms for evaluation include, as mentioned above, bacteria that proliferate by cell division or certain yeasts. Examples include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Propionibacterium acnes, Corynebacterium, Bacillus species, Klebsiella pneumoniae, Enterococcus species, Candida species (yeast), Saccharomyces species (yeast), and Malassezia species (common skin yeast, skin disease bacteria). On the other hand, multicellular organisms such as fungi do not satisfy the condition that a single bacterium, when cultured, proliferates and forms a single colony, and are therefore unsuitable as target organisms for evaluation according to this embodiment.

[0128] (Solid culture medium) In the antimicrobial activity evaluation method according to this embodiment, the usable solid culture medium is not particularly limited as long as it contains nutrients such as a carbon source and a nitrogen source and is capable of promoting the growth of the target bacteria. For example, if the target bacteria are Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus enterococcus, etc., examples of suitable media include SCDLP medium, blood agar medium, chocolate medium, BRB lactose agar medium, heart infusion medium, brain heart infusion medium, SCD medium, ordinary agar medium, and standard agar medium. If the target bacteria are Candida, Saccharomyces, or Malassezia, examples of suitable media include YM medium, Sabouraud agar medium, potato dextrose agar medium (PDA medium), SD medium, and YPD medium.

[0129] The solid culture medium according to this embodiment may further contain a selective agent that suppresses bacteria other than the target bacteria. For example, if the target bacteria are multidrug-resistant bacteria such as multidrug-resistant Pseudomonas aeruginosa or multidrug-resistant Acinetobacter, MacConkey agar, DHL agar, NAC agar, etc. can be used. On the other hand, if the target bacteria are vancomycin-resistant enterococci, bile-esculin-azido agar, KF streptococcus agar, etc. can be used. Furthermore, if the target bacteria are vancomycin-resistant Staphylococcus aureus or vancomycin-moderately resistant Staphylococcus aureus, mannitol salt medium, egg yolk-added mannitol salt medium, egg yolk salt medium, Staphylococcus No. 110 medium, Baird-Parker agar, etc. can be used.

[0130] Furthermore, commonly used solidifying agents such as agar and agarose can be used in the solid culture medium. The inclusion of enzyme substrate in the solid culture medium does not affect the present invention.

[0131] (container) This section describes containers that can be used in various culture steps in the antibacterial activity evaluation method according to this embodiment. In the first evaluation method shown in Figure 1, the second evaluation method shown in Figure 2, and the sixth evaluation method shown in Figure 5, any container capable of containing (storing) the culture medium and the substance to be evaluated is acceptable for culturing while bringing the vapor generated from the substance to be evaluated into contact with the culture medium. Specific examples include a container capable of containing a petri dish with the culture medium formed on it and the substance to be evaluated, or a cylindrical transparent container capable of forming a culture medium and having a predetermined height (see the examples described later).

[0132] Furthermore, in the (b1) vapor contact culture process of the substance to be evaluated according to this embodiment, it is not necessary for the space to be formed as a container, as long as it is a space that can accommodate the petri dish containing the culture medium and the substance to be evaluated; it can be carried out in a room or other similar location. In addition, the vapor generated from the substance to be evaluated can be allowed to evaporate naturally and come into contact with the surface of the culture medium, or it can be forcibly brought into contact with the surface of the culture medium using, for example, an aeration device.

[0133] As shown in the examples described later, in order to reduce implementation costs and / or to evaluate multiple target substances simultaneously, the bottom is transparent and the bottom area is 50 cm². 2 More than 80cm 2 It is preferable to use a cylindrical container with the following dimensions (diameter 8 cm to 10 cm) and height 10 cm to 20 cm: a transparent bottom and a base area of ​​50 cm². 2 More than 80cm 2 By using a container with a diameter of 8 cm to 10 cm, a solid culture medium (agar medium) can be formed at the bottom of the container, allowing the colonies formed on the medium to be observed from outside the container and the number of colonies to be measured. Furthermore, the bottom surface area of ​​the container should be 50 cm². 2 More than 80cm 2 By setting the container to the following dimensions (diameter 8 cm to 10 cm), a solid culture medium (agar medium) can be formed at the bottom of the container, similar to a typical petri dish (diameter 8 cm to 10 cm). When 30 to 300 colonies are formed, the colonies can be distinguished and observed, allowing for accurate measurement of the colony count. Furthermore, by setting the container height to 10 cm to 20 cm, as shown in Figure 9 of the examples described later, the container can be inverted, and the substance to be evaluated (filter paper impregnated with the substance) can be placed at the bottom of the container (corresponding to the top of the container before inversion) for cultivation. This ensures that the solid culture medium formed at the top of the container (corresponding to the bottom of the container before inversion) and the substance to be evaluated are at a predetermined distance from each other, allowing the vapor generated from the substance to be evaluated to appropriately contact the surface of the solid culture medium while culturing the target bacteria. In addition, to prevent saturation of the container with vapor generated from the substance to be evaluated, the container may have ventilation holes in a part of it, or the bottom of the container may be slightly elevated to allow for ventilation.

[0134] The container according to this embodiment can be made of plastic or other resin, glass, aluminum, or stainless steel, but it is preferable that the material is transparent so that the inside can be observed, and that it is possible to perform commonly used sterilization procedures to make the inside sterile. As a result, from the viewpoint of operability, impact resistance, chemical resistance, etc., the container according to this embodiment is preferably a glass, substantially cylindrical container as shown in Figure 6, which will be used in the later-described embodiment.

[0135] (Substances to be evaluated) In the antimicrobial activity evaluation method according to this embodiment, the substance to be evaluated is not particularly limited as long as it contains volatile components, but it is preferable that the substance to be evaluated is a fragrance composition. As mentioned above, in recent years, consumer interest in hygiene has increased, and the number of products claiming antimicrobial and / or disinfectant properties has increased. In particular, many products have emerged that disinfect and / or disinfect a space by diffusing a gas of a compound having antimicrobial and / or disinfectant properties using air fresheners, diffusers, or sprays, or by spraying a liquid containing a compound having antimicrobial and / or disinfectant properties and allowing the gas of the compound to evaporate from the liquid. Many of these products contain fragrance compositions. Fragrance compositions use volatile fragrance compounds as active ingredients, and if the fragrance compound has antimicrobial and / or disinfectant properties, there is no need to add antimicrobial and / or disinfectant agents separately. For these reasons, establishing a method for evaluating the antimicrobial activity of fragrance compositions is an urgent issue, and the antimicrobial activity evaluation method according to this embodiment can solve this problem.

[0136] Furthermore, since products that claim to have antibacterial and / or disinfectant properties in a space are likely to come into contact with the human body, there is an increasing demand for products that prioritize safety (toxicity, irritation, sensitization, etc.) and have relatively mild antibacterial and / or disinfectant effects. In this respect, the antibacterial effect of fragrance compositions is milder than that of other antibacterial agents, and the antibacterial activity evaluation method according to this embodiment is suitable when the substance to be evaluated is a fragrance composition, as it can appropriately evaluate the antibacterial effect regardless of the strength of the antibacterial effect.

[0137] In this embodiment, "fragrance composition having antibacterial activity" means (i) a fragrance composition having antibacterial activity containing a fragrance compound having antibacterial activity as an active ingredient, or (ii) a fragrance composition having antibacterial activity containing a volatile compound (not a fragrance compound) having antibacterial activity and other fragrance compounds as active ingredients, which can be added to various articles.

[0138] Here, "containing as an active ingredient" a fragrance compound having antibacterial activity means containing it in an amount sufficient to exert the desired antibacterial effect. Therefore, in this embodiment, the fragrance composition having antibacterial activity may contain only the fragrance compound having antibacterial activity, but it may also contain other fragrance components or other additives such as solvents, as long as the desired fragrance is not impaired. The form of the fragrance composition having antibacterial activity according to this embodiment is not particularly limited, and water-soluble fragrance compositions, oil-soluble fragrance compositions, and emulsified fragrance compositions are examples.

[0139] The concentration of the antibacterial fragrance compound in the fragrance composition according to this embodiment (the total concentration if multiple antibacterial fragrance compounds are included; the same applies hereinafter) can be arbitrarily determined depending on the target of the fragrance composition. The concentration of the antibacterial fragrance compound relative to the total mass of the fragrance composition according to this embodiment can be in the range of 0.1 ppt to 100%, preferably 0.001 ppm to 50%, and more preferably 0.1 ppm to 10%. More specifically, the concentration of the fragrance compound having antibacterial activity can be set within any combination of the following ranges, but is not limited to these limits: a lower limit of 0.1 ppt, 10 ppt, 100 ppt, 1 ppb, 10 ppb, 100 ppb, 1 ppm, 10 ppm, 1000 ppm, 1%, 10%, or 50%, and an upper limit of 100%, 50%, 10%, 1%, 1000 ppm, 100 ppm, 10 ppm, 1 ppm, 100 ppb, 10 ppb, 1 ppb, 100 ppt, 10 ppt, or 1 ppt.

[0140] In this embodiment, the fragrance compounds having antibacterial activity are not particularly limited, but examples of fragrance compounds having antibacterial activity include citral, cinnamaldehyde, thymol, octanal, perillaldehyde, and geraniol. Examples of essential oils having antibacterial activity include tea tree oil, lavender, thyme, cinnamon bark oil, peppermint oil, palmarose oil, lemongrass oil, and yuzu oil.

[0141] Furthermore, as described above, the fragrance composition having antibacterial activity according to this embodiment may contain any other compound or component in addition to the fragrance compound having antibacterial activity. Other optional compounds or components include various fragrance compounds or fragrance compositions, water-soluble polymer compounds (gum arabic, karaya gum, tragacanth gum, sodium alginate, xanthan gum, cellulose derivatives, cross-linked polyacrylic acid, polydimethylmethylene piperidium chloride, etc.), oily components (oils and fats, waxes, higher alcohols (cetyl alcohol (cetanol), 2-hexyldecanol, stearyl alcohol, isostearyl alcohol, cetostearyl alcohol, oleyl alcohol, arachidodecanol, behenyl alcohol, 2-octyldodecanol, lauryl alcohol, myristyl alcohol, decyltetradecanol, lanolin alcohol, etc.), hydrocarbons (mineral oil, polyethylene, microcrystalline wax, petrolatum), higher fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, isostearic acid, 12-hydroxystearic acid, oleic acid, lanolin fatty acid, etc.), alkyl glyceryl ethers (batyl alcohol, chymyl alcohol, ceraki (Such as alcohols, isostearyl glyceryl ether, etc.), esters (diisopropyl adipate, isononyl isononanoate, octyldodecyl ricinoleate, cholesteryl / lanosteryl unsaturated fatty acids with 10-30 carbon atoms, cetyl lactate, ethylene glycol di-2-ethylhexanoate, pentaerythritol fatty acid ester, dipentaerythritol fatty acid ester, diisostearyl malate, dioctyl succinate, cetyl 2-ethylhexanoate), silicones), surfactants (ionic surfactants and nonionic surfactants are examples, and ionic surfactants include cationic surfactants (lauryltrimethylammonium chloride, cetyltrimethylammonium chloride, stearyltrimethylammonium chloride, distearyldimethylammonium chloride, behenyltrimethylammonium chloride, cetyltrimethylammonium bromide, stearyltrimethylammonium bromide, lanolin fatty acid aminopropylethyldimethylammonium ethyl sulfate, stearyltrimethylammonium saccharin,Cetyltrimethylammonium saccharin, behenyltrimethylammonium methyl sulfate, etc., anionic surfactants (alkyl ether sulfates, alkyl sulfates, alkenyl ether sulfates, alkenyl sulfates, olefin sulfonates, alkanesulfonates, saturated or unsaturated fatty acid salts, alkyl or alkenyl ether carboxylates, α-sulfone fatty acid salts, N-acyl amino acid type surfactants such as sodium cocoyl glutamate, phosphate mono or diester type surfactants, sulfosuccinate esters, etc.), and amphoteric surfactants (cocobetaine, lauramidopropyl betaine, cocamidopropyl betaine, sodium lauroamphoacetate, sodium cocoamphoacetate, lauryl betaine, etc.), sugars, preservatives (amounts of sugars). Examples of substances that can be used include sodium benzoate, methylparaben, phenoxyethanol, etc., chelating agents, stabilizers (phenacetin, 8-hydroxyquinoline, acetanilide, sodium pyrophosphate, barbituric acid, uric acid, tannic acid, etc.), pH adjusters (citric acid, tartaric acid, lactic acid, malic acid, succinic acid, fumaric acid, maleic acid, pyrophosphate, gluconic acid, glucuronic acid, benzoic acid, 2-amino-2-methyl-1,3-propanediol, basic amino acids, etc.), plant extracts, vitamins, UV absorbers, oil-soluble dyes, functional substances, fish extracts, livestock extracts, plant extracts, yeast extracts, animal and plant proteins, hydrolyzed animal and plant proteins, starch, dextrin, sugars, amino acids, nucleic acids, organic acids, solvents, etc. Examples of various fragrance compounds or compositions include synthetic fragrance compounds (synthetic fragrances), natural essential oils, and natural fragrances, as described in "Japan Patent Office Gazette, Collection of Well-Known and Conventional Technologies (Fragrances), Part II: Food Fragrances, published January 14, 2000," "Japan Patent Office Gazette, Collection of Well-Known and Conventional Technologies (Fragrances), Part III: Cosmetic Fragrances, published June 15, 2001," "Survey on the Actual Use of Food Fragrance Compounds in Japan" (FY2000 Ministry of Health and Welfare Science Research Report, Japan Fragrance Manufacturers Association, published March 2001), and "Synthetic Fragrances: Chemistry and Product Knowledge" (Revised and Enlarged Edition published December 20, 2016, edited by the Synthetic Fragrance Editorial Committee, Chemical Daily Co., Ltd.).

[0142] Specific examples of synthetic fragrance compounds include hydrocarbon compounds such as monoterpenes like α-pinene, β-pinene, γ-terpinene, myrcene, camphene, and limonene; sesquiterpenes such as valencene, cedrene, caryophyllene, and longifolene; and 1,3,5-undecatriene.

[0143] Specific examples of synthetic fragrance compounds include alcohol compounds such as saturated alkanols (butanol, pentanol, 3-octanol, hexanol), unsaturated alcohols (Z)-3-hexen-1-ol, prenol, 2,6-nonadienol), terpene alcohols (linalool, geraniol, citronellol, tetrahydromyrcenolic acid, farnesol, nerolidol, cedrol, α-terpineol, terpinen-4-ol, borneol), and aromatic alcohols (benzyl alcohol, phenylethyl alcohol, cinnamyl alcohol).

[0144] Specific examples of synthetic fragrance compounds include aldehyde compounds such as saturated aldehydes like acetaldehyde, hexanal, octanal, decanal, and hydroxycitronellal; unsaturated aldehydes like (E)-2-hexenal and 2,4-octadienal; terpene aldehydes like citronellal, citral, myrthenal, and perillaldehyde; and aromatic aldehydes like benzaldehyde, cinnamyl aldehyde, vanillin, ethyl vanillin, heliotropin, and p-tolylaldehyde.

[0145] Specific examples of synthetic fragrance compounds include ketone compounds such as saturated and unsaturated ketones like 2-heptanone, 2-undecanone, 1-octen-3-one, acetoin, and 6-methyl-5-hepten-2-one (methylheptenone); diketones and hydroxyketones such as diacetyl, 2,3-pentanedione, maltol, ethylmaltol, cyclotene, and 2,5-dimethyl-4-hydroxy-3(2H)-furanone; terpene ketones such as carvone, menthone, and nootkatone; ketones derived from terpene decomposition products such as α-ionone, β-ionone, and β-damascenone; and aromatic ketones such as raspberry ketone.

[0146] Specific examples of synthetic fragrance compounds include furan or ether compounds such as furfuryl alcohol, furfural, rose oxide, linalool oxide, mentfuran, theaspiran, estragole, eugenol, and 1,8-cineole.

[0147] Specific examples of synthetic fragrance compounds include aliphatic esters such as ethyl acetate, isoamyl acetate, octyl acetate, ethyl butyrate, ethyl isobutyrate, isoamyl butyrate, ethyl 2-methylbutyrate, ethyl isovalerate, 2-methylbutyl isobutyrate, ethyl hexanoate, allyl hexanoate, ethyl heptanoate, ethyl caprylate, isoamyl isovalerate, and ethyl nonanoate; terpene alcohol esters such as linalyl acetate, geranyl acetate, lavandulyl acetate, terpenyl acetate, terpinyl acetate, and neryl acetate; and aromatic esters such as benzyl acetate, methyl salicylate, methyl cinnamate, cinnamyl propionate, ethyl benzoate, cinnamyl isovalerate, and ethyl 3-methyl-2-phenylglycidate.

[0148] Specific examples of synthetic fragrance compounds include lactone compounds such as saturated lactones like γ-decalactone, γ-dodecalactone, δ-decalactone, and δ-dodecalactone, and unsaturated lactones like 7-decene-4-olide and 2-decene-5-olide.

[0149] Specific examples of synthetic fragrance compounds include acidic compounds such as saturated and unsaturated fatty acids like acetic acid, butyric acid, isovaleric acid, caproic acid, octanoic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid.

[0150] Specific examples of synthetic fragrance compounds include nitrogen-containing compounds such as indole, skatole, pyridine, alkyl-substituted pyrazines, methyl anthranilate, and trimethylpyrazine.

[0151] Specific examples of synthetic fragrance compounds include sulfur-containing compounds such as methanethiol, dimethyl sulfide, dimethyl disulfide, allyl isothiocyanate, 3-methyl-2-buten-1-thiol, 3-methyl-2-butanethiol, 3-methyl-1-butanethiol, 2-methyl-1-butanethiol, 3-mercaptohexanol, 4-mercapto-4-methyl-2-pentanone, 3-mercaptohexyl acetate, p-menta-8-thiol-3-one, and furfuryl mercaptan.

[0152] Examples of natural essential oils include sweet orange, bitter orange, petitgrain, lemon, bergamot, mandarin, neroli, peppermint, spearmint, lavender, chamomile, rosemary, eucalyptus, sage, basil, rose, hyacinth, lilac, geranium, jasmine, ylang-ylang, anise, clove, ginger, nutmeg, cardamom, cedar, cypress, vetiver, patchouli, and labdanum.

[0153] Examples of various animal and plant extracts, such as fish extracts, livestock extracts, plant extracts, or yeast extracts, include extracts of herbs or spices, extracts of coffee, green tea, black tea, or oolong tea, and milk or milk products and various enzyme hydrolysates such as lipases and / or proteases thereof.

[0154] Furthermore, the antibacterial fragrance composition according to this embodiment can be prepared by adding an antibacterial fragrance compound to a suitable solvent or dispersion medium by known methods. That is, the form of the antibacterial fragrance composition according to this embodiment may include a solution in which the antibacterial fragrance compound or other components are dissolved in a water-soluble or oil-soluble solvent, an emulsified preparation, and the like.

[0155] Examples of water-soluble solvents include ethanol, methanol, acetone, tetrahydrofuran, acetonitrile, 2-propanol, methyl ethyl ketone, glycerin, propylene glycol, dipropylene glycol, and 1,3-butylene glycol.

[0156] Examples of oil-soluble solvents include vegetable oils and fats, animal oils and fats, refined oils and fats (for example, processed oils such as medium-chain triglycerides and short-chain triglycerides such as triacetin and tributyline), various essential oils, triethyl citrate, liquid paraffin, limonene, isopropyl myristate, and methyl dihydroabiethate (Harcoline).

[0157] Furthermore, to obtain an emulsified formulation, the fragrance sustained-release compound can be emulsified with a water-soluble solvent and an emulsifier. The method for emulsifying the fragrance sustained-release compound is not particularly limited, and an emulsified solution with excellent stability can be obtained by using emulsifiers of various types that have been conventionally used for emulsifying fragrance compositions, such as fatty acid monoglycerides, fatty acid diglycerides, fatty acid triglycerides, propylene glycol fatty acid esters, sucrose fatty acid esters, polyglycerin fatty acid esters, lecithin, modified starch, sorbitan fatty acid esters, quillaja extract, gum arabic, tragacanth gum, guar gum, karaya gum, xanthan gum, pectin, alginic acid and its salts, carrageenan, gelatin, casein quillaja saponin, and sodium caseinate, and emulsifying them using a homomixer, colloid mill, rotary disc homogenizer, high-pressure homogenizer, etc. Furthermore, in order to stabilize the emulsification, the water-soluble solvent may be used in addition to water by adding one or more of the following polyhydric alcohols: glycerin, propylene glycol, sorbitol, maltitol, sucrose, glucose, trehalose, sugar solution, reduced starch syrup, etc.

[0158] The antimicrobial fragrance composition according to this embodiment may further contain, if necessary, components commonly used in fragrance compositions. These components may include, for example, solvents such as water and ethanol, or fragrance fixatives such as ethylene glycol, propylene glycol, dipropylene glycol, glycerin, hexyl glycol, benzyl benzoate, triethyl citrate, diethyl phthalate, Harcolin, medium-chain triglyceride, and medium-chain diglyceride.

[0159] <Use on various items> The antibacterial fragrance composition according to this embodiment can be used by adding it to various articles or fragrance compositions used therein. Specifically, the antibacterial fragrance composition according to this embodiment may be added to various articles by itself, or it may be added to various articles in combination with one or more water-soluble fragrances, emulsified fragrance compositions, any fragrance compound, or natural essential oils (for example, fragrance compounds and / or fragrance compositions described in the aforementioned "Japan Patent Office Gazette, Collection of Well-Known and Conventional Technologies (Fragrances) Part II Food Fragrances," "Japan Patent Office Gazette, Collection of Well-Known and Conventional Technologies (Fragrances) Part III Cosmetic Fragrances," "Survey on the Actual Use of Food Fragrance Compounds in Japan," and "Synthetic Fragrances: Chemistry and Product Knowledge").

[0160] The articles to which the antibacterial fragrance composition according to this embodiment can be added are not particularly limited, but specific examples include consumer goods such as food and beverages, cosmetics, and pharmaceuticals and hygiene products. The antibacterial fragrance composition according to this embodiment is preferably used by adding it to cosmetics.

[0161] The foods and beverages to which the antibacterial fragrance composition according to this embodiment can be added are not particularly limited, but examples include food-based beverages such as fruit juices, vegetable drinks, sports drinks, honey drinks, soy milk, vitamin supplements, mineral supplements, nutritional drinks, nourishing drinks, lactic acid bacteria drinks, and dairy drinks; beverages such as green tea, black tea, oolong tea, herbal tea, and coffee drinks; alcoholic beverages such as chuhai, cocktail drinks, sparkling wine, fruit wine, and medicinal wine; and confectionery such as caramel, candy, and tablets.

[0162] Furthermore, the cosmetics or pharmaceutical / hygiene products to which the antibacterial fragrance composition according to this embodiment can be added are not particularly limited, but examples include: soap, body wash, bath additives, detergents, shampoos, conditioners, hair treatments, hair styling products, lotions, basic cosmetics, facial cleansers, finishing cosmetics, topical skin preparations, hygiene products, antiperspirants, deodorants, bath additives, mouthwashes, toothpaste, perfumes, colognes, eau de toilettes, etc., which are products for keeping a person's appearance clean or beautiful; household products and environmental hygiene products that maintain the function or cleanliness of various items in a home or household, such as laundry detergents, fabric softeners, starches, household cleaners, bath cleaners, dish soaps, bleaches, mold removers, floor waxes, fragrances, deodorizers, repellents, incense, etc.; and items such as inks, stationery, toys, and other daily necessities.

[0163] When applying the antibacterial fragrance composition according to this embodiment to various articles such as food and beverages and cosmetics, the concentration of the antibacterial fragrance compound in the various articles can be arbitrarily determined according to the fragrance of the article and the desired degree of antibacterial effect.

[0164] When the antibacterial fragrance composition according to this embodiment is applied to cosmetics or pharmaceuticals and hygiene products, an example of the concentration of the antibacterial fragrance compound contained in the antibacterial fragrance composition according to this embodiment is within the range of 1 ppm to 10%, preferably 10 ppm to 1%, relative to the total mass of the cosmetics or pharmaceuticals and hygiene products. More specifically, the lower limit can be any of 1 ppm, 10 ppm, 100 ppm, 0.1%, or 1%, and the upper limit can be any of 10%, 1%, 0.1%, 100 ppm, or 10 ppm, and the concentration can be within any combination of these lower and upper limits, but is not limited to these. [Examples]

[0165] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples.

[0166] [Example 1] Evaluation of antibacterial activity of fragrance composition (1) The following describes specific examples of evaluating the antibacterial activity of fragrance compositions by implementing the antibacterial activity evaluation method according to this embodiment. In Example 1, Escherichia coli was used as the target bacterium for evaluation, and in Example 2, described later, Staphylococcus aureus was used.

[0167] <Container> First, the container used in the antimicrobial activity evaluation method according to this embodiment will be described. Figure 6 is a side view of the container 10 for carrying out the antimicrobial activity evaluation method according to this embodiment. As shown in Figure 6, the container 10 is made of transparent glass, is roughly cylindrical (8 cm in diameter, 18 cm in length, and 900 mL in volume), and has an opening 11 at one end in the longitudinal direction. Multiple containers 10 were prepared for use in the process described later (hereinafter, when it is necessary to distinguish between containers, they will be referred to as containers 10a, 10b, 10c, etc. for convenience).

[0168] <Culture> Next, the results of evaluating the antibacterial activity of the first to ninth fragrance compositions using the antibacterial activity evaluation method according to this embodiment will be explained with illustrations of each step. Figures 7 to 13 are schematic diagrams showing each step of the antibacterial activity evaluation method according to this embodiment. Figures 14 to 18 are images of containers cultured without contact with the fragrance compositions and containers cultured with contact with the first to ninth fragrance compositions in this embodiment. Figure 19 is an explanatory diagram that allows comparison between images of containers cultured without contact with the fragrance compositions and containers cultured with contact with the first to ninth fragrance compositions in this embodiment, the number of colonies formed, and the number of bacteria calculated by the antibacterial activity evaluation method according to this embodiment.

[0169] As shown in FIG. 7, first, a solid medium (XM-G agar medium) 20 was formed on the bottom of a container 10 (this corresponds to step (a1): preparing a solid medium of the fourth evaluation method shown in FIG. 3, or step (a1): preparing a solid medium of the sixth evaluation method shown in FIG. 5). Here, the XM-G agar medium is a medium used for detecting Escherichia coli and coliform bacteria in food and environmental materials, and is listed in the Food Sanitation Inspection Guidelines 2004 and Ministry of the Environment Notification No. 62 (2021). More specifically, the XM-G agar medium is a gram-negative bacterial selective medium supplemented with two chromogenic enzyme substrates (X-GLUC, MAGENTA-GAL), which allows identification of Escherichia coli by blue color development and coliform bacteria by red color development. The composition of the XM-G agar medium is as follows.

[0170] <Composition of XM-G medium: in 39.3 g (per 1 L of medium)> Peptone: 10.0 g Sodium pyruvate: 1.0 g L-tryptophan: 1.0 g D-sorbitol: 1.0 g Sodium chloride: 5.0 g Sodium dihydrogen phosphate: 2.2 g Disodium hydrogen phosphate: 2.7 g Potassium nitrate: 1.0 g Sodium lauryl sulfate: 0.2 g 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC): 0.1 g 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside (MAGENTA-GAL): 0.1 g Agar: 15.0 g (pH 7.0±0.2)

[0171] 39.3 g of granular medium was dissolved by heating in 1 L of purified water, autoclaved at 121°C for 15 minutes, then 20 mL of the resulting solution was added into the container 10 and solidified, whereby the solid medium 20 was formed on the bottom of the container 10.

[0172] Additionally, this step was also performed for other containers 10, whereby a plurality of containers 10 each having the solid medium 20 formed therein were prepared.

[0173] Next, as shown in Figure 8, 100 μL of a suspension of E. coli 30 was applied to the surface of the solid culture medium 20 formed at the bottom of the container 10 by a planar coating method so that the number of bacteria per culture medium (container) was between 30 and 300 (corresponding to (a2) the inoculation step of the target bacteria in the fourth evaluation method shown in Figure 3, or (a2) the inoculation step of the target bacteria in the sixth evaluation method shown in Figure 5). This process was also performed on another container 10 in which solid culture medium 20 was formed, so that multiple containers 10 containing solid culture medium 20 inoculated with E. coli 30 were prepared (corresponding to (a) the preparation step of solid culture medium inoculated with the target bacteria in the first evaluation method shown in Figure 1).

[0174] Next, as shown in Figure 9, one of the prepared containers 10 (hereinafter referred to as "container 10a") was inverted so that its opening 11 faced downwards. Then, a filter paper 40 (hereinafter referred to as "filter paper 40a") impregnated with 200 μL of the first fragrance composition was placed near the opening 11, and cultivation was started in this state. At this time, the first fragrance composition evaporated from the filter paper 40a, and the vapor of the first fragrance composition came into contact with the surface of the solid culture medium 20 (hereinafter referred to as "solid culture medium 20a"), and E. coli 30 (hereinafter referred to as "E. coli 30a") was cultured at 35°C for 1 day (24 hours) (this corresponds to the (b1) vapor contact culture step of the first evaluation method shown in Figure 1, the (b1) vapor contact culture step of the second evaluation method shown in Figure 2, or the (b1) vapor contact culture step of the sixth evaluation method shown in Figure 5, and the solid culture medium 20a in this container 10a corresponds to the first culture medium). In this embodiment, in addition to the first fragrance composition, second to ninth fragrance compositions were prepared, and the same steps performed on the first fragrance composition were also performed on the second to ninth fragrance compositions. Specifically, second to ninth fragrance compositions were prepared, and filter papers 40b to 40i impregnated with each of the second to ninth fragrance compositions were prepared. While each of the second to ninth fragrance compositions was brought into contact with the surface of the solid culture media 20b to 20i in containers 10b to 10i, E. coli 30b to 30i present in the solid culture media 20b to 20i were cultured under the same culture conditions as in container 10a.

[0175] In addition, a separate container 10 (hereinafter referred to as "container 10x") was prepared from containers 10a to 10i, and as shown in Figure 9, the culture of E. coli 30 (hereinafter referred to as "E. coli 30x") present in solid medium 20 (hereinafter referred to as "solid medium 20x") was started under the same conditions as containers 10a to 10i (35°C for 1 day (24 hours)) except that filter paper 40x without the fragrance composition was placed on it (this corresponds to the (c1) non-contact vapor culture step of the third evaluation method shown in Figure 2, or the (c1) non-contact vapor culture step of the sixth evaluation method shown in Figure 5, and the solid medium 20x in this container 10x corresponds to the second medium).

[0176] As shown in Figure 10, since container 10 is transparent, the solid culture medium 20 can be observed from the other end in the longitudinal direction (from the direction indicated by the white arrow in Figure 10), and the colonies formed by E. coli 30 could be visually confirmed. Figures 14 to 18 show images actually observed in container 10x (no fragrance composition) and containers 10a to 10i (first to ninth fragrance compositions). Figure 19 shows the measurement results of the number of colonies in container 10x (no fragrance composition) and containers 10a to 10i (first to ninth fragrance compositions) (other values ​​in Figure 19 will be described later).

[0177] <Calculating the number of viable bacteria present in each culture medium after incubation> Next, as shown in Figure 11, the cultured container 10 was placed upright with the opening 11 facing upwards, and a total of 10 mL of sterile water 50 was poured into the container 10 through the opening 11 to suspend the bacteria. The E. coli 30 present on the surface of the solid culture medium 20 was washed with the sterile water 50 to obtain a suspension 60, and then, as shown in Figure 12, 8 mL of the suspension 60 was recovered from the container 10 (corresponding to the (b21) bacterial recovery step of the fifth evaluation method shown in Figure 4).

[0178] Next, as shown in Figure 13, the suspension 60 was diluted stepwise by a dilution ratio of 10 times to prepare dilutions 61, 62, 63, etc. (this corresponds to the dilution preparation step (b22) of the fifth evaluation method shown in Figure 4).

[0179] Next, solid culture media for bacterial count measurement were prepared for inoculation with diluents 61, 62, 63, etc. XM-G agar was used as the solid culture medium for bacterial count measurement. 39.3 g of granular culture medium was dissolved in 1 L of purified water and autoclaved at 121°C for 15 minutes. This was divided into 15 mL portions, and 1 mL each of diluents 61, 62, 63, etc. was mixed into each. These portions were then placed in containers 101, 102, 103, etc., and solidified using the pour plate method (corresponding to the diluent inoculation step (b23) in the fifth evaluation method shown in Figure 4). In this example, each container 101, 102, 103, etc. had a base area of ​​55 cm². 2 The procedure was performed in a petri dish, but other containers capable of forming a culture medium, such as the aforementioned container 10 (however, with a base area of ​​50 cm²), are also available. 2 More than 80cm 2 The following is preferable: )

[0180] Next, as shown in Figure 13, the solid culture media in each container 101, 102, 103, etc., inoculated with dilutions 61, 62, 63, etc., were cultured under the same conditions (corresponding to the culture step (b24) of the fifth evaluation method shown in Figure 4).

[0181] Next, as shown in Figure 13, the colonies 70 formed on the solid culture media of each container 101, 102, 103, etc., inoculated with dilutions 61, 62, 63, etc., were visually observed and the number of colonies was measured. Figure 13 is a schematic diagram and does not accurately reflect the number of colonies, but as a concrete example, the number of colonies in container 101 was over 2000, the number of colonies in container 102 was 190, and the number of colonies in container 103 was 20. As a result, (base area 55 cm²) 2 Container 102 was identified that had between 30 and 300 colonies per container (corresponding to the colony count measurement step (b25) of the fifth evaluation method shown in Figure 4).

[0182] Next, in container 102 where the number of identified colonies is 30 or more and 300 or less, the dilution ratio of the diluent inoculated into the solid culture medium in this container (for example, 10 7Based on the above calculation formula 4 (specifically, calculation formula 4a), the number of E. coli bacteria is calculated as 1.9 × 10¹⁶ times the volume (1 mL), the volume of sterile water added to the solid medium 20a in container 10 for bacterial suspension (10 mL), and the number of colonies formed on the solid medium (specifically, 190). 10 This was calculated as the number of individuals (corresponding to step (b26) of the fifth evaluation method shown in Figure 4, which is the step for calculating the number of bacteria to be evaluated).

[0183]

number

[0184] The number of viable E. coli 30a cells present in the solid culture medium 20a of container 10a, which was cultured while in contact with the vapor of the first fragrance composition, is 1.9 × 10⁶, as shown in Figure 19. 10 It can also be expressed as CFU. Furthermore, considering the volume of sterile water (10 mL) added to the solid medium 20a in container 10 for bacterial suspension, 1.9 × 10 9 It can also be expressed as CFU / mL.

[0185] In the same manner as described above, the number of viable E. coli 30b to 30i cells present in solid media 20b to 20i of containers 10b to 10i, which were cultured while being exposed to the vapors of the second to ninth fragrance compositions, was calculated. Similarly, the number of viable E. coli 30x cells present in solid media 20x of container 10x, which was cultured without being exposed to the vapors of the first to ninth fragrance compositions, was calculated (corresponding to steps (c21) to (c26), which are variations of the fifth evaluation method described above). Figure 19 shows the calculation results of the number of viable cells in container 10x (no fragrance composition) and containers 10a to 10i (first to ninth fragrance compositions) (the growth rate in Figure 19 will be explained in the next step).

[0186] <Calculation of growth rate for each culture medium> Next, based on the above formula 1, the growth rates in the solid media 20a to 20i of containers 10a to 10i were calculated. Here, the case of container 10a will be explained in detail. Applying the above formula 1 to container 10a results in the following formula 1a.

[0187]

number

[0188] Here, the number of E. coli bacteria inoculated into solid medium 20x (without fragrance composition) in container 10x is the same as the number of E. coli bacteria inoculated into solid mediums 20a to 20i (first to ninth fragrance compositions) in containers 10a to 10i (because the same suspension was inoculated into solid medium 20x and solid mediums 20a to 20i in the same volume). Furthermore, as shown in Figure 19, the number of colonies formed in solid medium 20x (without fragrance composition) was 235, satisfying the condition of being between 30 and 300. As a result, this number of colonies was considered to be the number of E. coli bacteria (initial bacterial count) inoculated into solid medium 20x and solid mediums 20a to 20i (corresponding to a modified version of the second evaluation method described above).

[0189] Based on the above, substituting the number of colonies formed on solid medium 20x (used as a substitute for the number of bacteria present in solid medium 20a before culturing) and the number of bacteria present in solid medium 20a after culturing (calculated in the previous step) into formula 1a, the growth rate in solid medium 20a of container 10a is 8.1 × 10⁻⁶. 7 The following calculation was obtained. The growth rate in solid media 20b to 20i of containers 10b to 10i can also be calculated similarly based on the above calculation formula 1, so the details are omitted (corresponding to (b3) the calculation step for the growth rate of the target bacteria in the first evaluation method shown in Figure 1). Similarly, the growth rate in solid media 20x of container 10x can also be calculated similarly based on the above calculation formula 2, so the details are omitted (corresponding to (c3) the calculation step for the growth rate of the target bacteria in the second evaluation method shown in Figure 2).

[0190] Figure 19 shows the calculated growth rates for container 10x (without fragrance composition) and containers 10a to 10i (1st to 9th fragrance compositions).

[0191] As shown in Figure 19, by visual observation of colonies and measurement of the number of colonies, as in the conventional method, it can be qualitatively evaluated that the second fragrance composition (container 10b, number of colonies: 0), the third fragrance composition (container 10c, number of colonies: 133), the fourth fragrance composition (container 10d, number of colonies: 0), the sixth fragrance composition (container 10f, number of colonies: 177), the seventh fragrance composition (container 10g, number of colonies: 0), and the ninth fragrance composition (container 10i, number of colonies: 207) have antibacterial effects, as they have fewer colonies than the number of colonies (235) of the container without fragrance composition (container 10x).

[0192] However, for the first fragrance composition (container 10a, colony count: 275), the fifth fragrance composition (container 10e, colony count: 242), and the eighth fragrance composition (container 10h, colony count: 243), which had colony counts similar to or even higher than the no-fragrance composition (container 10x) (235), it is not possible to evaluate whether they have an antibacterial effect. Furthermore, for the second fragrance composition (container 10b, colony count: 0), the fourth fragrance composition (container 10d, colony count: 0), and the seventh fragrance composition (container 10g, colony count: 0), which had zero colonies, it is necessary to uniformly conclude that "no bacterial growth was observed."

[0193] In contrast, as shown in Figure 19, focusing on the growth rate of each container calculated by the antibacterial activity evaluation method according to this embodiment, the growth rate of container without fragrance composition (container 10x) is 9.4 × 10 7Compared with ), the growth rate of each of the first to ninth perfume compositions is lower, so it can be quantitatively evaluated that all of the first to ninth perfume compositions have an antibacterial effect. In particular, as described above, the number of colonies of the first perfume composition (container 10a, number of colonies: 275), the fifth perfume composition (container 10e, number of colonies: 242), and the eighth perfume composition (container 10h, number of colonies: 243) was larger than that of the case without perfume composition (container 10x, number of colonies: 235). However, regarding the size of the colonies, it seemed that they could be the same, larger, or smaller, and it was ambiguous whether they had an antibacterial effect or not. On the other hand, when judged by the growth rate, it was found that all of them have a lower growth rate than the case without a perfume composition, and it was shown that the antibacterial activity evaluation method according to the present embodiment can accurately evaluate the antibacterial effect.

[0194] Further, as shown in FIG. 19, regarding the second perfume composition (container 10b, number of colonies: 0), the fourth perfume composition (container 10d, number of colonies: 0), and the seventh perfume composition (container 10g, number of colonies: 0) which had 0 colonies, it is obvious that they have an antibacterial effect because no colonies appeared, but there was no way to compare the strength of the antibacterial effect. On the other hand, when judged by the growth rate, it could be determined that the antibacterial effect of these perfume compositions showed completely different trends. That is, the second perfume composition (container 10b, growth rate: 4.3×10 4 ) and the seventh perfume composition (container 10g, growth rate: 3.4×10 5 ) have a growth rate larger than 1, and bacteria proliferated to an extent that no colonies appeared, and it was found that the vapor of the perfume composition inhibited the cell division of Escherichia coli to an extent that no colonies appeared. On the other hand, the fourth perfume composition (container 10d, growth rate: 2.1×10 -1 ) has a growth rate smaller than 1, and it was found that the perfume composition partially sterilized Escherichia coli.

[0195] Therefore, according to the antibacterial activity evaluation method of the present embodiment, even when no colonies appear, the degree of inhibition of E. coli cell division by the fragrance composition can be correctly and quantitatively evaluated. In addition, the situation where the vapor of the fragrance composition inhibits E. coli cell division to such an extent that no colonies appear can only be evaluated by performing measurement in an environment where E. coli can grow, and it has been shown that the antibacterial activity evaluation method according to the present embodiment is useful, for example, for evaluating the antibacterial effect of products used in environments where bacteria grow in daily life.

[0196] From the above results, the strength of the antibacterial effect against E. coli of the first to ninth fragrance compositions can also be correctly determined by eliminating ambiguous factors such as the number of colonies and the size of colonies, and the first to ninth fragrance compositions can be ranked in the order from the strongest to the weakest antibacterial effect as 4th > 2nd > 7th > 3rd > 9th > 6th > 1st > 5th > 8th.

[0197] As described above, according to the antibacterial activity evaluation method of the present embodiment, the growth rate of E. coli cultured while bringing the vapor of various fragrance compositions into contact in an environment where E. coli can grow is calculated, and thus it was shown that how the vapor of various fragrance compositions inhibits E. coli cell division can be quantitatively evaluated under conditions close to actual living environments.

[0198] Note that, in the antibacterial activity evaluation method according to the present embodiment, observation of colonies in each container and measurement of the number of colonies are not required. By setting the number of bacteria per container (medium) to 30 or more and 300 or less, and culturing such that the number of colonies in each container is about 30 or more and 300 or less, conventional qualitative evaluation can also be performed at the same time, and comparison and examination of antibacterial activity based on different evaluation methods can also be performed.

[0199] <Calculation of antibacterial rate of fragrance composition against Escherichia coli> Next, the antibacterial rates of the first to ninth fragrance compositions against Escherichia coli were calculated. Here, the case of the first fragrance composition will be described in detail. When the above formula 3 is applied to the first fragrance composition, the following formula 3a is obtained.

[0200]

number

[0201] Thus, the antibacterial effect of the first fragrance composition against E. coli was calculated to be 14%.

[0202] Furthermore, if the antibacterial rate is to be determined directly from the number of bacteria without calculating the growth rate, applying the above formula 5 to the first fragrance composition results in the following formula 5a.

[0203]

number

[0204] Thus, the antibacterial effect of the first fragrance composition against E. coli was calculated to be 14%. The antibacterial effects of the second to ninth fragrance compositions against E. coli can be similarly calculated based on the above calculation formulas 3 or 5, so the details are omitted (corresponding to the antibacterial effect calculation step (d1) of the third evaluation method shown in Figure 2 or the antibacterial effect calculation step (d2) of the sixth evaluation method shown in Figure 5). Table 2 shows the calculation results of the antibacterial effects of the first to ninth fragrance compositions against E. coli calculated above (Table 2 also shows the calculation results of the antibacterial effects of the second to ninth fragrance compositions against Staphylococcus aureus, which will be calculated in Example 2 described later, to facilitate comparison with E. coli).

[0205] [Table 2]

[0206] As shown in Table 2, the antibacterial activity evaluation method according to this embodiment allows for the calculation of the antibacterial rate of E. coli cultured while being exposed to vapors of various fragrance compositions in an environment where E. coli can grow. This demonstrates that the inhibitory effect of vapors of various fragrance compositions on E. coli cell division can be evaluated quantitatively and more clearly than the aforementioned growth rate.

[0207] Further, when directly determining the antibacterial rate from the number of bacteria present in each culture medium after culture by calculation formula 5a, it was simpler than calculation formula 3a and a reduction in the number of steps could be achieved.

[0208] [Example 2] Evaluation of antibacterial activity of perfume composition (2) In Example 2, the antibacterial activity evaluation method according to the present embodiment was applied in the same manner as in Example 1, except that Staphylococcus aureus was used instead of Escherichia coli as the bacteria to be evaluated, the medium was a standard agar medium, and the culture conditions were 35°C for 2 days (48 hours). Here, the standard agar medium is a medium used for measuring the number of viable bacteria in food and water, and it is published in the Ministerial Ordinance on Component Specifications etc. of Milk and Milk Products, Food Sanitation Inspection Guidelines (2018), Water Supply Test Methods (2011), ISO 4833-1:2013, and complies with the composition of Plate Count Agar used in the viable cell count method of the American Public Health Association. More specifically, the standard agar medium contains a well-balanced nitrogen source (peptone), carbon source (glucose), vitamin source and mineral source (yeast extract), and thus has the ability to support the growth (proliferation) of a wide range of bacteria. In addition, since the medium is colorless, it is easy to confirm the presence or absence of colonies, so it is suitable for measuring the number of viable bacteria.

[0209] <In 23.5 g of standard agar medium (for 1 L)> Peptone from casein: 5.0 g Yeast extract: 2.5 g Glucose: 1.0 g Agar: 15.0 g (pH 7.1±0.1)

[0210] In Example 2, when forming the solid medium 20 in the container 10, 23.5 g of granular medium was dissolved by heating in 1 L of purified water, after autoclaving at 121°C for 15 minutes, 20 mL of the liquid medium maintained at about 50°C was added into the container 10, and the solid medium 20 was formed at the bottom of the container 10 by solidifying the liquid medium.

[0211] Table 2 shows the calculation results of the antibacterial rates against Staphylococcus aureus for the 2nd to 9th perfume compositions calculated in the same manner as in Example 1.

[0212] Based on the above, it has been shown that, according to the antibacterial activity evaluation method of this embodiment, regardless of the type of bacteria, as long as it is a single-celled organism, the antibacterial rate of the target bacteria cultured while being exposed to the vapors of various fragrance compositions in an environment in which the target bacteria can grow can be calculated, thus enabling quantitative evaluation of the inhibitory effect of the vapors of various fragrance compositions on the cell division of the target bacteria. [Explanation of Symbols]

[0213] 10, 10a~10i, 10x: Container (glass bottle) 20, 20a~20i, 20x: Solid culture medium 30,30a~30i,30x: Escherichia coli 40, 40a~40i, 40x: filter paper 50: Sterile water 60: Suspension 61, 62, 63: Diluent 70: Colony 101, 102, 103: Container (Petri dish)

Claims

1. A method for evaluating antimicrobial activity, including the following steps. (a) A step of preparing multiple inoculated solid culture media on which the target bacteria, which proliferate by cell division, can grow, (b1) A step in which one of the inoculated solid media prepared in step (a) is used as the first medium, and the target bacteria present in the first medium are cultured while the vapor generated from the substance to be evaluated is brought into contact with the first medium. (b2) After step (b1), a step of measuring the number of the target bacteria present in the first culture medium, (b3) A step of calculating the growth rate of the target bacteria in the first culture medium based on the following formula 1, [Math 1] (c1) A step in which the bacteria to be evaluated present in the second medium is cultured under the same conditions as in step (b1), except that one of the inoculated solid media prepared in step (a) above, other than the first medium, is used as the second medium, and vapors generated from the substance to be evaluated are not brought into contact with the second medium. (c2) After step (c1), a step of measuring the number of the target bacteria present in the second culture medium, (c3) A step of calculating the growth rate of the target bacteria in the second culture medium based on the calculation formula 2 below, [Math 2] (e1) A step after step (b1) and before step (b2) of observing the colonies of the target bacteria in the first culture medium and measuring the number of colonies, (e2) A step after step (c1) and before step (c2) of observing the colonies of the target bacteria in the second culture medium and measuring the number of colonies.

2. In the antimicrobial activity evaluation method according to claim 1, Furthermore, a method for evaluating antimicrobial activity, including the following steps. (d1) A step of calculating the antibacterial rate of the vapor generated from the substance to be evaluated against the target bacteria under the conditions of step (b1) based on the calculation formula 3 below. [Math 3]

3. In the antimicrobial activity evaluation method according to claim 1, The above step (a) is a method for evaluating antimicrobial activity, comprising the following steps. (a1) A step of preparing multiple solid culture media on which the target bacteria to be evaluated, which proliferate by cell division, can grow. (a2) A step of preparing the inoculated solid culture media by inoculating each of the solid culture media with the suspension of the target bacteria in the same volume using the plate coating method.

4. In the antimicrobial activity evaluation method according to claim 1, The (b2) step is a method for evaluating antimicrobial activity, comprising the following steps. (b21) A step of adding liquid to the first culture medium after culturing in step (b1) and recovering the target bacteria present in the first culture medium as a suspension. (b22) A step of diluting the suspension obtained in step (b21) according to sequentially set dilution ratios to obtain diluted solutions with different concentrations of the target bacteria, (b23) A step of preparing multiple solid culture media for bacterial count measurement in which the target bacteria can grow, and inoculating each of the solid culture media for bacterial count measurement with the same volume of the dilution obtained in step (b22), (b24) A step of culturing each of the solid culture media for bacterial count measurement that have been inoculated with the dilution in step (b23) under the same conditions. (b25) A step after step (b24) of measuring the number of colonies in the solid culture medium for bacterial count measurement in which colonies have been formed. (b26) A step of calculating the number of target bacteria present in the first medium after culturing in step (b1) based on the following calculation formula 4, using the dilution ratio and volume of the diluent inoculated into the solid medium for bacterial count measurement, the number of colonies formed in the solid medium for bacterial count measurement, and the volume of the liquid added to the first medium for bacterial suspension. [Math 4]

5. A method for evaluating antimicrobial activity, including the following steps. (a1) A step of preparing multiple solid culture media on which the target bacteria to be evaluated, which proliferate by cell division, can grow. (a2) Inoculating each of the solid culture media with the same volume of the suspension of the bacteria to be evaluated, (b1) A step in which one of the solid media inoculated with the target bacteria in step (a2) is designated as the first medium, and the target bacteria inoculated in the first medium are cultured while vapor generated from the substance to be evaluated is brought into contact with the first medium. (b2) After step (b1), a step of measuring the number of the target bacteria present in the first culture medium, (c1) A step in which the bacteria to be evaluated inoculated in the solid medium in step (a2) is designated as the second medium, and the bacteria to be evaluated inoculated in the second medium is cultured under the same conditions as in step (b1), except that vapors generated from the substance to be evaluated are not brought into contact with the second medium. (c2) After step (c1), a step of measuring the number of the target bacteria present in the second culture medium, (d2) A step of calculating the antibacterial rate of the vapor generated from the substance to be evaluated against the target bacteria under the conditions of step (b1) based on the calculation formula 5 below. [Math 5] (e1) A step after step (b1) and before step (b2) of observing the colonies of the target bacteria in the first culture medium and measuring the number of colonies, (e2) A step after step (c1) and before step (c2) of observing the colonies of the target bacteria in the second culture medium and measuring the number of colonies.

6. In the antimicrobial activity evaluation method described in claim 5, The (b2) step includes the following steps (b21) to (b26): The (c2) step is a method for evaluating antimicrobial activity, comprising the following steps (c21) to (c26). (b21) A step of adding liquid to the first culture medium after culturing in step (b1) and recovering the target bacteria present in the first culture medium as a suspension. (b22) A step of diluting the suspension obtained in step (b21) according to sequentially set dilution ratios to obtain diluted solutions with different concentrations of the target bacteria, (b23) A step of preparing multiple solid media for measuring the bacterial count of the first culture medium in which the target bacteria to be evaluated can grow, and inoculating each of the first solid media for measuring the bacterial count of the first culture medium with the same volume of the dilution obtained in step (b22), (b24) A step of culturing each of the first culture medium for bacterial count measurement that has been inoculated with the dilution in step (b23) under the same conditions. (b25) After step (b24), a step of measuring the number of colonies in the first culture medium for bacterial count measurement in which colonies have been formed. (b26) A step of calculating the number of target bacteria present in the first medium after culturing in step (b1) based on the following calculation formula 4, using the dilution ratio and volume of the diluent inoculated into the first medium for bacterial count measurement, the number of colonies formed in the first medium for bacterial count measurement, and the volume of the liquid added to the first medium for bacterial suspension. [Math 6] (c21) A step of adding liquid to the second culture medium after culturing in step (c1) above, and recovering the target bacteria present in the second culture medium as a suspension. (c22) A step of diluting the suspension obtained in step (c21) according to sequentially set dilution ratios to obtain diluted solutions with different concentrations of the target bacteria, (c23) A step of preparing multiple solid media for measuring the bacterial count of the second medium on which the target bacteria to be evaluated can grow, and inoculating each of the solid media for measuring the bacterial count of the second medium with the same volume of the dilution obtained in step (c22), (c24) A step of culturing each of the solid media for measuring the bacterial count of the second medium, which has been inoculated with the dilution in step (c23), under the same conditions. (c25) After step (c24), a step of measuring the number of colonies in the second culture medium for bacterial count measurement in which colonies have been formed. (c26) A step of calculating the number of target bacteria present in the second medium after culturing in step (c1) based on the following calculation formula 4' from the dilution ratio and volume of the diluent inoculated into the second medium for bacterial count measurement, the number of colonies formed in the second medium for bacterial count measurement, and the volume of the liquid added to the second medium for bacterial suspension. [Number 7]

7. In the antimicrobial activity evaluation method according to any one of claims 1 to 6, The substance to be evaluated is a fragrance composition, and the method for evaluating antibacterial activity is described above.

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