Foot odor detection method, and method for evaluating or selecting foot odor inhibitor

Abstract

To provide a foot odor detection method, and a method for evaluating or selecting a foot odor inhibitor.SOLUTION: A foot odor detection method includes the step of measuring the levels of Corynebacterium bacteria in the resident bacteria of the skin of the subject's feet.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting foot odor and a method for evaluating or selecting foot odor control agents. [Background technology]

[0002] Body odor is generated by the metabolism of sweat, sebum, keratin waste products, etc., secreted from the living body by bacteria resident on the skin. Among body odors, foot odor is one that is strong and unpleasant. Therefore, research has been conducted on the components and bacteria that cause odor, and methods for controlling them. It has been revealed that the main component that causes foot odor is isovaleric acid (IVA) (Non-Patent Document 1), which is produced by Staphylococcus and Corynebacterium bacteria, which are normal bacteria on the foot skin, metabolizing L-leucine derived from the living body (Non-Patent Documents 2-4).

[0003] Generally, to evaluate the strength of foot odor or the odor suppression effect of a material, a method in which an odor judger performs a sensory evaluation, a method in which odor components are quantified using an analytical instrument, a method in which bacteria involved in the generation of odor are detected, etc. are used. Whichever method is used, it is desirable to be able to perform the evaluation with high accuracy and ease. Under such circumstances, a screening method for foot odor suppressants using Staphylococcus or Corynebacterium bacteria (Patent Document 1) and a method for evaluating malodors using Corynebacterium bacteria (Patent Document 2) have been reported, but there is a demand for a highly accurate index that more accurately reflects actual foot odor and the effect on foot odor.

[0004] Known bacteria detected from human foot skin include the above-mentioned Staphylococcus bacteria and Corynebacterium bacteria, as well as Micrococcus bacteria, Chitococcus bacteria, Brevibacterium bacteria, etc. (Non-Patent Document 5). Non-Patent Document 5 reports that Staphylococcus bacteria are the main cause of foot odor, while it has been shown that Micrococcus bacteria, Chitococcus bacteria, and Brevibacterium bacteria have the ability to decompose volatile fatty acids, specifically isovaleric acid, and does not suggest that bacteria of these genera can be a highly accurate indicator that more accurately reflects foot odor or the effects on foot odor. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6600473 [Patent Document 2] JP 2015-130854 A [Non-patent literature]

[0006] [Non-Patent Document 1] Br. J. Dermatol., 1990, 122, 771-776 [Non-Patent Document 2] Journal of the Japanese Society for Bacteriology 45(4), 1990, 797-800 [Non-Patent Document 3] Journal of Cosmetics and Fragrance 28(3), 2004, 177-182 [Non-Patent Document 4] Can. J. Microbiol., 2006, 52, 357-364 [Non-Patent Document 5] Flavour. Fragr. J., 2013, 28, 231-237 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides a highly accurate method for detecting foot odor or for evaluating or selecting foot odor control agents. [Means for solving the problem]

[0008] The present inventors comprehensively analyzed the bacterial flora normally present on the skin of the feet and investigated its relationship with foot odor. As a result, they unexpectedly found that, compared with previously reported bacteria causing foot odor, bacteria of the genus Chitococcus have a high correlation with the odor intensity of isovaleric acid, the main component causing foot odor, and produce large amounts of isovaleric acid, and that the use of bacteria of the genus Chitococcus makes it possible to evaluate foot odor and to evaluate or select foot odor suppressants.

[0009] That is, the present invention relates to the following 1) and 2). 1) A method for detecting foot odor, comprising a step of measuring the amount of Chitococcus bacteria in the normal bacteria present on the skin of a subject's feet. 2) A method for evaluating or selecting a foot odor suppressant, comprising the following steps (1) to (3): (1) A step of culturing bacteria of the genus Chitococcus in the presence of a test substance and L-leucine (2) A step of measuring the amount of bacteria of the genus Chitococcus or the amount of isovaleric acid in the culture obtained in (1). (3) A step of evaluating or selecting a test substance that inhibits the amount of bacteria of the genus Chitococcus or the production of isovaleric acid based on the results of the measurement in (2) as a foot odor suppressant. Effect of the Invention

[0010] According to the present invention, the foot odor of a subject can be evaluated with high accuracy. Also, according to the present invention, a substance capable of suppressing foot odor can be evaluated or selected with high accuracy. [Brief description of the drawings]

[0011] [Figure 1] 1 is a graph showing the correlation between the amount of Chitococcus bacteria and the odor intensity of isovaleric acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] All patents, non-patent publications, and other publications cited herein are hereby incorporated by reference in their entirety.

[0013] In the present invention, "foot odor" refers to the odor emanating from the feet, and also the foot odor emanating from clothing such as socks or footwear that comes into contact with the feet. Since the main causative substance of foot odor is isovaleric acid, the foot odor is preferably an isovaleric acid odor. "Suppression of foot odor" includes the reduction, elimination, or inhibition of the occurrence of foot odor, and preferably suppression of the occurrence of foot odor by inhibiting the production of isovaleric acid, and more preferably suppression of the occurrence of foot odor by inhibiting the production of isovaleric acid by Chitococcus bacteria. "Foot" refers to the part of the foot from the ankle up, including the instep (dorsal foot), sole (sole), heel, and toes, with the sole being preferred.

[0014] In the present invention, "detecting" foot odor means clarifying the presence or absence of foot odor or the intensity of foot odor, and can also be expressed in other terms such as testing, measuring, judging, or evaluation assistance.

[0015] As shown in the Examples below, the indigenous bacterial flora of the skin of the feet was comprehensively analyzed to investigate the relationship with foot odor. The amount of Chitococcus bacteria showed a significantly high positive correlation with the odor intensity of isovaleric acid, which is the main component of foot odor (Table 5). Surprisingly, this correlation was higher than that of Staphylococcus bacteria and Corynebacterium bacteria, which produce isovaleric acid and are known to be the cause of foot odor (Table 5). In addition, Chitococcus schroeteri was isolated from the skin of the feet of a subject who had a strong odor intensity of isovaleric acid. This bacterium had a higher ability to produce isovaleric acid than Staphylococcus capitis, which is an isovaleric acid-producing bacterium (Table 6). In addition to Chitococcus schroeteri, Chitococcus aerolatus was also isolated from the same subject. Furthermore, the applicant has discovered that Sanguinea oleifera extract has the effect of suppressing the production of isovaleric acid by normal skin bacteria and can be used to suppress foot odor, and has previously filed a patent application (Patent Application No. 2022-062780). When Chitococcus bacteria were cultured in the presence of Sanguinea oleifera extract, the production of isovaleric acid by the Chitococcus bacteria was suppressed. Therefore, the amount of Chitococcus bacteria can be a highly accurate indicator for detecting foot odor in a subject. Furthermore, the amount of Chitococcus bacteria or the amount of isovaleric acid produced by Chitococcus bacteria can be a highly accurate indicator for evaluating or selecting foot odor suppressants.

[0016] In one aspect, the present invention provides a method for detecting foot odor, comprising a step of measuring the amount of Chitococcus bacteria in the normal bacteria on the foot skin of a subject. Here, the foot skin indigenous bacteria refers to bacteria that are indigenous to the skin of human feet (preferably the soles).

[0017] Examples of bacteria belonging to the genus Kytococcus include Kytococcus schroeteri, Kytococcus aerolatus, Kytococcus sedentarius, etc. In the foot odor detection method of the present invention, the bacteria belonging to the genus Kytococcus are preferably bacteria belonging to the genus Kytococcus including at least one species selected from the group consisting of Kytococcus schroeteri and Kytococcus aerolatus, more preferably at least one species selected from the group consisting of Kytococcus schroeteri and Kytococcus aerolatus, and even more preferably Kytococcus schroeteri.

[0018] In the foot odor detection method of the present invention, the method for measuring the amount of bacteria of the genus Chitococcus in the bacteria normally present on the skin of the foot is not particularly limited. For example, such a method includes a method of analyzing the genus or species of bacteria present in the bacteria normally present on the skin of the foot based on the base sequence of the 16S ribosomal RNA (rRNA) gene contained in the foot flora genomic DNA, and measuring the amount of bacteria of the genus Chitococcus in combination with the copy number of the 16S rRNA gene contained in the genomic DNA. The method will be described below.

[0019] 1) Collecting bacteria from the skin of the feet Bacteria on the skin of the foot can be collected using a sterile urethane swab (Culture Swab EZ, BD Japan). Specifically, the sterile urethane swab can be soaked in saline and rubbed back and forth against the skin of the foot (e.g., 4.5 cm x 4.5 cm) several times. The sample can be used for 16S rRNA gene sequencing immediately after collection, but can also be frozen and stored at -80°C in the swab form.

[0020] 2) Extraction of genomic DNA from bacterial samples For the bacterial samples collected on the foot skin in 1), nucleic acids are liberated using known methods such as the lytic enzyme method (BMC Microbiol., 2004, 4:16) or the bead method (Science, 2008, 320, 1647-1651), and then genomic DNA is extracted using known methods for isolating and extracting DNA, such as commonly used methods such as the phenol-chloroform method (Mol. Biol., 1986, 191, 615-624) or the guanidine method (Science, 2005, 308:1635-1638).

[0021] 3) Measurement of total bacterial count 2) The copy number of the 16S rRNA gene contained in the bacterial genomic DNA extracted is measured. That is, the bacterial genomic DNA is used as a template to amplify all or a part of the region of the 16S rRNA gene by PCR, and the copy number of the 16S rRNA gene contained in the bacterial genomic DNA is calculated using a calibration curve created using DNA with a known copy number of the 16S rRNA gene as a template. In this case, it is preferable that the PCR primer is set to a region that is universally conserved among bacterial genera or species. Examples of such primers include, but are not limited to, those shown in Table 1, and forward and reverse can be used in appropriate combination so that the desired region is amplified. Note that, although the copy number of the 16S rRNA gene possessed by each bacterial species differs, it has been confirmed that there is a correlation between the 16S rRNA gene copy number measured by PCR and the bacterial amount (bacterial number) measured by colony counting, and the value of the copy number of the 16S rRNA gene contained in the bacterial genomic DNA can be regarded as the total bacterial amount.

[0022] [Table 1]

[0023] 4) Sequencing of the 16S rRNA gene in genomic DNA The sequence of the 16S rRNA gene contained in the bacterial genomic DNA extracted in 2) is determined. That is, the sequence of the 16S rRNA gene characteristic of each bacterial genus or species is determined, and the flora structure of the indigenous bacteria on the skin of the foot is analyzed based on the sequence data. For this reason, it is necessary to select the region of the 16S rRNA gene to be sequenced so as to reflect the flora structure, but in order to perform the analysis quickly and eliminate sequence reading errors, it is desirable to determine and compare the sequence of a short region as long as it reflects the characteristics of the sequence of each bacterial genus or species. The region of the 16S rRNA gene to be sequenced is amplified by PCR, and in this case, the primers are preferably set to a region that is universally conserved among bacterial genera or species, such as, but not limited to, those shown in Table 1.

[0024] After purifying the amplified PCR product, sequencing is performed. Any known method can be used for sequencing, but for example, the next-generation ultra-high-speed sequencing device, such as MiSeq (Illumina), can be used to rapidly sequence the product. It is known that the 16S rRNA gene of bacteria contains regions (V1 to V9) whose nucleotide sequences are not conserved among bacterial species and are highly variable. Therefore, it is desirable to determine the nucleotide sequence of at least one of such regions, for example, the region including V1 and V2, or the region including V3 and V4. The region including V1 and V2 can be amplified, for example, by a primer set of SEQ ID NOs: 9 and 10.

[0025] The obtained sequence data can be analyzed using analysis software such as Qiime (Quantitative Insights Into Microbial Ecology) for the obtained nucleotide sequence data group, and each sequence can be identified according to the system of The Ribosomal Database Project (Lan, Y et al., Using the RDP classifier to predict taxonomic novelty and reduce the search space for finding novel organisms. PLoS One 2012. 7:e32491.). In addition, homology searches using the BLAST algorithm against gene sequence databases such as the NCBI nucleatide database, NCBI 16S microbial rRNA database, Greengenes database, and SILVA are also possible (J. Mol. Biol., 1990, 215(3):403-410).

[0026] 5) Calculation of the amount of Chitococcus bacteria Next, the amount of bacteria of the genus Chitococcus is calculated from the total amount of bacteria measured in 3) and the abundance ratio of the bacteria obtained in 4).

[0027] Alternatively, a method for measuring the amount of Chitococcus bacteria in the indigenous bacteria on the skin of the foot can be used to measure the amount of Chitococcus bacteria in the indigenous bacteria on the skin of the foot based on a base sequence characteristic of Chitococcus bacteria contained in the genomic DNA of the foot flora, preferably the base sequence of the 16S rRNA gene of Chitococcus bacteria. The method will be described below.

[0028] The amount of bacteria of the genus Chitococcus is measured based on the base sequence characteristic of bacteria of the genus Chitococcus contained in the bacterial genomic DNA extracted in 2) from the bacterial sample on the skin of the foot collected in 1) above. That is, the base sequence characteristic of bacteria of the genus Chitococcus is amplified by PCR using the bacterial genomic DNA as a template, and the amount of bacteria of the genus Chitococcus contained in the bacterial genomic DNA is measured using a calibration curve created using the genomic DNA of bacteria of the genus Chitococcus with a known amount of bacteria as a template. The PCR primer in this case is a primer that specifically recognizes and amplifies the base sequence characteristic of bacteria of the genus Chitococcus.

[0029] In this manner, the amount of Chitococcus bacteria in the normal bacteria on the skin of the subject's feet is measured, and the subject's foot odor is detected based on the measurement results.

[0030] In one embodiment, the foot odor of a subject can be detected by comparing the amount of bacteria of the genus Chitococcus in the normal bacteria on the skin of the subject's foot with a reference value. For example, the amount of bacteria of the genus Chitococcus in a group that does not have foot odor, specifically isovaleric acid odor, is obtained in advance as standard data, and the reference value can be appropriately determined based on statistical values ​​such as the average value and standard deviation of the amount of bacteria based on the data. Examples of groups that do not have foot odor include a group of people who receive a score of 0 or a group of people who receive a score of 1 or 0 when an odor judgement expert uses a tube to eliminate odors from other than the sole of the foot and smells the foot odor from a position about 5 cm away from the sole of the foot on a 6-point scale: score 5: very strong foot odor, score 4: strong foot odor, score 3: foot odor that can be easily detected, score 2: foot odor is detectable but weak odor (recognition threshold), score 1: very slight odor (detection threshold, cannot tell what odor it is), and score 0: no odor. In one example, when a group of people who do not have foot odor has a score of 1 or 0, the reference value for the amount of bacteria of the genus Chitococcus is 2.68 x 10 in terms of the amount of 16S rRNA genes per sole area. 4 copies / cm 2 (LOG 104.4275). However, in this embodiment, the reference value is not limited to the above value, and an appropriate value that can reflect the actual presence or absence of foot odor or the intensity of foot odor can be set appropriately.

[0031] For example, if the amount of bacteria of the genus Chitococcus in the skin bacteria normally present on the foot of a subject is greater than a reference value, the subject can be evaluated as having foot odor, and if not, the subject can be evaluated as not having foot odor. In this case, the more the amount of bacteria of the genus Chitococcus in the skin bacteria normally present on the foot of a subject is greater than the reference value, the stronger the foot odor of the subject can be evaluated. For example, if the amount of bacteria of the genus Chitococcus in the skin bacteria normally present on the foot of a subject is statistically significantly higher than the reference value, the subject can be evaluated as having foot odor, and if not, the subject can be evaluated as not having foot odor. In this case, the more the amount of bacteria of the genus Chitococcus in the skin bacteria normally present on the foot of a subject is higher than the reference value, the stronger the foot odor of the subject can be evaluated. For example, if the amount of bacteria of the genus Chitococcus in the normal skin bacteria of the subject's foot is preferably 110% or more, more preferably 150% or more, and even more preferably 200% or more relative to the reference value, the subject can be evaluated as having foot odor, and if not, the subject can be evaluated as not having foot odor.In this case, the more the amount of bacteria of the genus Chitococcus in the normal skin bacteria of the subject's foot is greater than the reference value, the stronger the foot odor of the subject can be evaluated.

[0032] In another embodiment, the amount of bacteria of the genus Chitococcus in the normal bacteria on the skin of the foot of a subject is measured over time (for example, monthly), and compared with the measurement result of the previous measurement, thereby detecting a change in the foot odor of the subject.For example, if the amount of bacteria of the genus Chitococcus in the normal bacteria on the skin of the foot of a subject is decreased compared to the previous measurement, the foot odor of the subject can be detected as having improved, if there is no change, the foot odor of the subject can be detected as having not changed, and if there is an increase, the foot odor of the subject can be detected as having worsened.

[0033] In yet another embodiment, the comparison with the reference value and the comparison over time can be combined.In this embodiment, each measurement result of the measurement over time of the amount of bacteria of the genus Chitococcus in the skin bacteria of the subject's foot is compared with the reference value by the above-mentioned procedure, and the measurement results from the second time onwards are also compared with the previous measurement results by the above-mentioned procedure.In this embodiment, the comparison result with the reference value and the comparison result with the previous measurement results are comprehensively examined, so that the foot odor of the subject can be detected in more detail.

[0034] The foot odor detection method of the present invention may further include a step of measuring the bacterial count of Staphylococcus bacteria and / or Corynebacterium bacteria, which are known foot odor-causing bacteria among the normal bacteria on the skin of the subject's feet. The means for measuring the bacterial count and the manner of detecting foot odor based on the measurement results are the same as in the case of Chitococcus bacteria. In this case, the foot odor of the subject can be detected in more detail by comprehensively examining the results of the detection of the subject's foot odor using the bacterial count of Chitococcus bacteria as an indicator and the results of the detection of the subject's foot odor using the bacterial count of Staphylococcus bacteria and / or Corynebacterium bacteria as an indicator.

[0035] The foot odor detection kit is a kit for detecting the foot odor of a subject according to the foot odor detection method of the present invention. The kit of the present invention contains a reagent for measuring the bacterial quantity of Chitococcus bacteria. Examples of the reagent for measuring the bacterial quantity of Chitococcus bacteria include a reagent for nucleic acid amplification containing a primer that specifically recognizes and amplifies a base sequence characteristic of Chitococcus bacteria. In addition to the above, the kit can also contain a standard sample for creating a calibration curve, a PCR reagent, a labeling reagent, a buffer solution, instruments and controls required for the test, a tool for collecting bacteria from the sole of the subject's foot (e.g., a sterile urethane swab for collecting bacteria, etc.), a reagent for storing the collected bacterial specimen, a storage container, a reagent for extracting genomic DNA from the collected bacterial specimen, etc.

[0036] As described above, the amount of Chitococcus bacteria among the normal bacteria on the skin of the feet correlates well with foot odor, and Chitococcus bacteria have a high ability to produce isovaleric acid. Therefore, a substance that reduces the amount of Chitococcus bacteria or the amount of isovaleric acid produced can suppress foot odor.

[0037] Therefore, in another aspect, the present invention provides a method for evaluating or selecting a foot odor suppressing agent, the method comprising the following steps (1) to (3). (1) A step of culturing bacteria of the genus Chitococcus in the presence of a test substance and L-leucine (2) A step of measuring the amount of bacteria of the genus Chitococcus or the amount of isovaleric acid in the culture obtained in (1). (3) A step of evaluating or selecting a test substance that inhibits the amount of bacteria of the genus Chitococcus or the production of isovaleric acid based on the results of the measurement in (2) as a foot odor suppressant.

[0038] In the method for evaluating or selecting a foot odor suppressant of the present invention, the Chitococcus bacteria may be the same as those described in the foot odor detection method of the present invention, and preferably includes at least one species selected from the group consisting of Chitococcus schroeteri and Chitococcus aerolatus, more preferably at least one species selected from the group consisting of Chitococcus schroeteri and Chitococcus aerolatus, and even more preferably Chitococcus schroeteri. These can be obtained from public microorganism depositories. For example, Chitococcus schroeteri and Chitococcus aerolatus can be obtained from the RIKEN BioResource Research Center (RIKEN BRC) as, for example, JCM12136 and JCM17971, respectively. Alternatively, those isolated from human foot skin may be used.

[0039] The concentration and amount of the Chitococcus bacteria used, and the concentration and amount of the test substance used may be appropriately set based on the growth phase of the Chitococcus bacteria, the morphology, chemical properties, cytotoxicity, etc. of the test substance. The concentration of L-leucine is not particularly limited, but the final concentration during the culture in step (1) is preferably 0.5 to 100 mM, and more preferably 10 to 20 mM.

[0040] The culture conditions for the bacteria of the genus Chitococcus are not particularly limited as long as they are conditions under which the bacteria of the genus Chitococcus can grow well. For example, the culture conditions can be 30 to 45°C, preferably 35 to 40°C, under aerobic conditions, preferably with shaking. The culture period can be appropriately set in consideration of the form, chemical properties, cytotoxicity, etc. of the test substance. Usually, the culture period is, for example, 12 to 60 hours, preferably 24 to 48 hours. The culture medium can be a medium that is usually used for culturing bacteria of the genus Chitococcus. The culture medium can be trypticase soy medium, blood agar medium, soybean casein digest agar medium, Zobell medium, etc. The container used for the culture is not particularly limited as long as it does not affect the growth of the bacteria of the genus Chitococcus, and ordinary glass bottles, glass tubes, centrifuge tubes, plastic containers, etc. can be used.

[0041] Next, the amount of bacteria of the genus Chitococcus or the amount of isovaleric acid produced in the obtained culture is measured. The amount of bacteria of the genus Chitococcus can be measured in the same manner as the amount of bacteria of the genus Chitococcus in the foot odor detection method of the present invention. Alternatively, since the bacteria present in the culture are essentially only bacteria of the genus Chitococcus, a conventional method for measuring the amount of bacteria can be used, for example, a method for measuring the number of bacteria by smearing the culture on a plate, measuring absorbance, measuring calorimetry, measuring ATP, measuring impedance, or measuring the number of bacteria by real-time PCR. The amount of isovaleric acid can be measured according to a method known in the art, for example, using LC / MS, GC / MS, etc., under the conditions described in detail in the Examples below. The amount of isovaleric acid can also be determined by a sensory evaluation based on an odor evaluation shown in the Examples below.

[0042] Furthermore, based on the measured results, a test substance that inhibits the production of isovaleric acid is evaluated or selected as a foot odor suppressant. Such evaluation or selection is performed, for example, by comparing a test group in which bacteria of the genus Chitococcus are cultured in the presence of a test substance with a control group in which bacteria of the genus Chitococcus are cultured in the absence of the test substance or in the presence of a control substance (hereinafter collectively referred to as control). Alternatively, evaluation is performed by comparing the measurement results between various concentrations of the test substance.

[0043] For example, when the amount of bacteria of the genus Chitococcus or the amount of isovaleric acid in the test group is smaller than that in the control group, the test substance can be evaluated or selected as a foot odor suppressant. In this case, it can be judged whether the amount of bacteria of the genus Chitococcus or the amount of isovaleric acid in the test group is statistically significantly smaller than that in the control group. Alternatively, it can be judged whether the amount of bacteria of the genus Chitococcus or the amount of isovaleric acid in the test group is a certain amount or less, for example, 90% or less, preferably 70% or less, more preferably 50% or less, when the amount of bacteria of the genus Chitococcus or the amount of isovaleric acid in the control group is 100%. Alternatively, it can be judged whether the score in the test group is preferably 0.5 points or more lower, more preferably 1 point or more lower than that in the control group, when the amount of isovaleric acid is scored based on the odor intensity score of the sensory evaluation shown in the Examples below.

[0044] The foot odor suppressant thus obtained can be used by being incorporated into cosmetics, quasi-drugs, medicines, etc., for the purpose of suppressing foot odor. EXAMPLES

[0045] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0046] Example 1 Analysis of foot odor and indigenous bacteria on the soles of the feet (1) Study participants The study was conducted on 26 men in their 20s to 50s. The subjects had stopped using foot sprays, foot creams, etc. for about two months prior to the measurement.

[0047] (2) Odor evaluation The evaluation was carried out by four or more testers who were experienced in odor sensory evaluation. For each of the left and right soles of the test participants' feet, a tube made by cutting a drinking cup (Shimojima Package Plaza) to a height of 4.5 cm from the mouth was placed about 5 cm away from the skin on the sole of the foot on the toe side, and the odor was sniffed from above and scored and evaluated according to the odor intensity and odor quality classification criteria below. Note that the components in parentheses for each classification in the odor quality classification indicate the representative components that exhibit the odor of that classification. The intensity of each odor quality in the odor quality classification is also scored according to the odor intensity criteria below.

[0048] <Odor Intensity> Score 5: Very strong foot odor Score 4: Strong foot odor Score 3: Easily detectable foot odor Score 2: Foot odor is detectable but weak (cognitive threshold) Score 1: Very slight odor (detection threshold: no idea what the odor is) Score 0: Odorless

[0049] <Odor quality classification> E1: Light sourness (acetic acid / propionic acid) E2: Stuffy odor (diacetyl acetoin) E3: Heavy sourness (isovaleric acid) E4: Body odors other than those mentioned above (aldehydes, lactones, amines, apocrine odor components, etc.) H: Fragrances and other odors other than body odor are strong and cannot be rated

[0050] (3) Analysis of skin resident bacteria (3-1) Collection of skin resident bacteria Microorganisms were collected from the skin using a swab for collecting microorganisms (BD BBL Culture Swab EZ (BD Japan)) according to the following procedure. 1) Collection site (4.5cm x 4.5cm (approx. 20cm) 2 Marking the ). 2) Soak the cotton ball at the tip of the swab in saline (Otsuka Pharmaceutical). 3) Scrub the collection site four times with the swab. 4) After collection, cut off the tip of the swab and place only the cotton ball portion into a 1.5 mL microtube (Eppendorf). 5) Repeat steps 2) to 4) for three swabs on the same area. 6) Store the microtube temporarily on ice and then at -80°C.

[0051] (3-2) Extraction of genomic DNA from bacteria Genomic DNA was extracted from the collected skin resident bacteria in the following manner. First, the frozen samples were thawed. 600 μL of DNA Isolation Buffer [Tris-HCl pH 8.0 (NIPPON GENE) (final concentration 10 mM), EDTA (NIPPON GENE) (final concentration 1 mM), NaCl (Invitrogen) (final concentration 100 mM), Triton X-100 (SIGMA) (final concentration 2% (v / v)), SDS (NIPPON GENE) (final concentration 1% (w / v))] was added to the thawed 1.5 mL microtube (Eppendorf), and the mixture was stirred at maximum speed for 1 minute using a high-speed shaker (Cute Mixer, Tokyo Rikakikai). After stirring, 450 μL of the suspension was collected and added to a 15 mL conical tube (Falcon) containing 1,200 mg of zirconia beads (ZircoPrep Mini, Nippon Genetics Co., Ltd.) and 1 mL of phenol / chloroform / isoamyl alcohol aqueous solution (25:24:1) (NIPPON GENE). The above procedure was performed on two cotton swabs to prepare a total of 900 μL of bacterial suspension. The 15 mL Falcon tube containing 900 μL of bacterial suspension was stirred at maximum speed for 5 minutes using a high-speed shaker (Cute Mixer, Tokyo Rikakikai), and then centrifuged at 4°C and 9,000 rpm for 10 minutes. 700 μL of the aqueous phase was mixed with MaXtract High Density (Qiagen) containing 1 mL of phenol / chloroform / isoamyl alcohol aqueous solution (25:24:1) (NIPPON GENE). After stirring at maximum speed for 5 minutes using a high-speed shaker (Cute Mixer, Tokyo Rikakikai), the mixture was centrifuged at 20°C and 12,000 rpm for 5 minutes, and 600 μL of the aqueous phase was collected in a 1.5 mL microtube (Eppendorf), mixed with 4 μL of a coprecipitant (Ethachinmate, Fujifilm Wako Pure Chemical Co., Ltd.) and 60 μL of 3M sodium acetate pH 5.2 (NIPPON GENE). In addition, 600 μL of isopropanol (Fujifilm Wako Pure Chemical Co., Ltd.) was mixed, and the mixture was incubated at 4°C for 10 minutes after inversion. After centrifugation at 14,000 rpm and 4°C for 10 minutes, the supernatant was removed, and 600 μL of 70% (v / v) ethanol (Fujifilm Wako Pure Chemical Co., Ltd.) was mixed and washed by tapping.After centrifuging again at 14,000 rpm at 4° C. for 10 minutes, the supernatant was removed and the pellet was dried naturally. Finally, the DNA was dissolved in 200 μL of TE Buffer pH 8.0 (NIPPON GENE) to obtain a DNA solution.

[0052] (3-3) Measurement of bacterial load by real-time PCR Real-time PCR was performed using a QuantStudio3 real-time PCR system (Thermo Fisher Scientific) with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). The primers used for real-time PCR are shown in Table 2. Reactions using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) were performed in 20 μL of reaction solution (2×SYBR Green master mix, 0.4 μM each forward primer and reverse primer, 9.2 μL sample solution), and were performed at 50°C for 2 minutes and 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The copy number of the calibration curve plasmid (pUC118-S.epi27F / 338R) was calculated based on the average molecular weight per bp of 660 and the Avogadro's number of 6.022×10 23 It was calculated as: The values ​​measured above indicate the copy number of the rRNA gene. Although the number of rRNA genes possessed differs depending on the bacterial species, a previous study found a correlation between the number of rRNA genes measured by real-time PCR and the number of bacteria measured by colony counting (results omitted), so in this analysis, the value of the rRNA gene copy number is considered to be the amount of bacteria measured by real-time PCR.

[0053] [Table 2]

[0054] (3-4) Bacterial flora analysis using next-generation sequencers MiSeq (Illumina) was used for the analysis. The analysis region was amplified using the primers for amplifying the V1V2 region of 16S rRNA shown in Table 3. The reaction was performed in 25 μL of reaction solution (genomic DNA 2.5 μL, 1 μM each forward primer and reverse primer 10 μL, 2 × KAPA HiFi HotStart ReadyMix 12.5 μL) and reacted at 95 ° C for 3 minutes, followed by 25 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds, followed by 5 minutes of reaction at 72 ° C. The genetic information obtained was analyzed using Qiime (Quantitative Insights Into Microbial Ecology) (http: / / qiime.org / ) to identify the bacterial species. The data obtained was analyzed using Rx64 3.5.3 (https: / / cran.r-project.org / ), Easy R Ver.1.40 (EZR) (https: / / www.jichi.ac.jp / saitama-sct / SaitamaHP.files / download.html), and Excel (Microsoft Office version 2018). The amount of each type of bacteria was calculated from the total amount of bacteria obtained by real-time PCR and the composition rate obtained from the results of bacterial flora analysis using a next-generation sequencer.

[0055] [Table 3]

[0056] (4) Isolation and identification of indigenous skin bacteria from the soles of the feet The isolation and identification of the skin commensal bacteria were performed by the following method. First, the cryopreserved samples were thawed. 0.5 mL of 10% glycerol aqueous solution was added to the thawed 1.5 mL microtube (Eppendorf) and suspended, and then seeded on egg yolk-added mannitol salt agar medium (BBL) or tryptone soya agar medium supplemented with 5 g / L Tween (registered trademark) 80 and 500 mg / L fosfomycin (SCD agar medium) (Nissui), and cultured under aerobic conditions at 37 °C. After culturing, single colonies were picked up, suspended in 5 mL of LP diluent "Daigo" (Fujifilm wako), and then seeded again on the same medium (egg yolk-added mannitol medium or SCD agar medium). After culturing, single colonies were picked up, and species identification was performed based on the sequence information of the 16S rRNA gene.

[0057] (5) Measurement of isovaleric acid (IVA) production After culturing at 37 °C for 24 hours on SCD agar medium, the cells were scraped off with 10 platinum loops (Kenis 2 mm Φ) and suspended in 2 mL of physiological saline. 100 μL of this suspension was added to 2 mL of 5 / 5 medium (0.2% L-leucine, 0.05% Beef extract (Difco), 0.5% Hypopeptone S (Nissui), 0.75% sodium chloride), and cultured with shaking under aerobic conditions at 37 °C for 24 hours. 20 μL of deuterium-labeled isovaleric acid (IVA-d7, 100 μM) (manufactured by CDN isotopes) was added as an internal standard substance to 40 μL of the test solution obtained by filtering the above shaking culture solution through a 0.20 μm disk membrane filter (ADVANTEC). Subsequently, 20 μL of 3-nitrophenyl hydrazine (3-NPH, manufactured by Tokyo Chemical Industry) solution (200 mM) and 20 μL of EDC (manufactured by Tokyo Chemical Industry) solution (containing 6% pyridine, 120 mM) were added, and the solution diluted 10-fold with 10% acetonitrile was used as the measurement sample. The measurement sample was subjected to LC / MS under the following conditions to quantify the isovaleric acid production.

[0058] <LC / MS conditions> HPLC system: SHIMADZU Nexera Column: CAPCELL CORE (2.7 μm, 2.1 × 50 mm) Mobile phase: A) 0.1% HCOOH aq B) CH3CN Flow rate: 0.5mL / min Injection volume: 10μL

[0059] [Table 4]

[0060] (6) Results (6-1) Correlation analysis between the amount of various bacteria and the odor intensity of isovaleric acid The number of bacterial species detected by the bacterial flora analysis was 275 at the genus level. Among them, genera that were present in 5% or more of the total of 5200% when the presence rate of the bacterial species contained in each sample (52 samples in total from the left and right feet of 26 subjects) was added up in all samples (52 samples in total from the left and right feet of 26 subjects) and were detected on the foot skin in Non-Patent Document 5, Corynebacterium bacteria, Micrococcus bacteria, Kytococcus bacteria, Staphylococcus bacteria, and Brevibacterium bacteria, were analyzed for their association with isovaleric acid odor intensity. The isovaleric acid odor intensity is a value obtained by scoring E3 in the odor quality classification according to the odor intensity standard. As a result, Table 5 shows the bacterial genera in which a significant difference (p<0.05 Spearman's rank correlation coefficient) was confirmed. Among these, the bacterial genus with the highest correlation coefficient was Chitococcus (r=0.756, p<0.05), followed by Staphylococcus (r=0.740, p<0.05) and Corynebacterium (r=0.642, p<0.05). This demonstrated that Chitococcus bacteria are superior markers for determining foot odor than Corynebacterium and Staphylococcus bacteria (Table 5). The correlation between the amount of Chitococcus bacteria and the intensity of the isovaleric acid odor is shown in Figure 1. If an individual is judged to have foot odor when their isovaleric acid odor intensity score is 2 or higher, which is the recognition threshold, the amount of Chitococcus bacteria at that time is 2.68 × 10 16S rRNA gene amount per sole area. 4 copies / cm 2 (LOG 10 4.4275).

[0061] [Table 5]

[0062] (6-2) Measurement of isovaleric acid production by indigenous skin bacteria strains isolated from the soles of the feet In order to confirm the isovaleric acid production ability of the main bacterial species actually present on the skin of the feet, we attempted to isolate skin-resident bacteria from samples collected from two subjects with strong foot odor. 150 strains, 75 each, were isolated from the two subjects, and the production of isovaleric acid during culture was confirmed by sensory evaluation, and 20 strains were selected. The amount of isovaleric acid was measured for the selected 20 strains using the method of Example 1 (5). As a result, the isolated strains 5P-28-2 (698 mg / L) and 5P-32-2 (544 mg / L) had a higher isovaleric acid production ability than the production amount (414 mg / L) of Staphylococcus capitis, which is considered to be an isovaleric acid producing bacterium in the above-mentioned Non-Patent Document 4 (Table 6). As a result, an isovaleric acid producing strain was isolated.

[0063] [Table 6]

[0064] (6-3) Identification of bacterial species isolated from foot skin Six strains were selected from the isovaleric acid-producing strains, and the species were identified based on the 16S rRNA gene sequence. As a result, they were identified as two strains of Staphylococcus hominis, two strains of Kytococcus schroeteri, one strain of Staphylococcus cohnii, and one strain of Micrococcus luteus. Among them, both 5P-28-2 and 5P-32-2, which were high isovaleric acid-producing strains, were Kytococcus schroeteri (Table 7).

[0065] [Table 7]

[0066] Therefore, we tried to isolate bacteria again from the swab sample of subject No. 5, from which Kytococcus schroeteri was isolated, using SCD agar medium supplemented with 5g / L Tween 80 and 500mg / L fosfomycin. We identified the species of the eight strains based on the 16S rRNA gene sequence, and isolated seven Kytococcus schroeteri strains and one Kytococcus aerolatus strain.

[0067] Example 2 Inhibition of isovaleric acid production from Chitococcus bacteria by Sanguisorba officinalis extract The present applicant discovered that Sanguisorba officinalis extract has the effect of suppressing the production of isovaleric acid by skin-resident bacteria and can be used to suppress foot odor, and has previously filed a patent application (Patent Application No. 2022-062780). Therefore, the applicant used Sanguisorba officinalis extract to confirm its effect on the production of isovaleric acid by Chitococcus bacteria.

[0068] (1) Method Sanguisorba officinalis extract was prepared from the extract ALN (solid concentration: 1.3 w / v%, extraction solvent: 50% ethanol, Maruzen Pharmaceutical Co., Ltd.). Chitococcus schröetheri (5p-28-2 strain) was cultured on SCD agar medium at 37°C for 24 hours, after which 10 loopfuls (Kennis 2mm diameter) of the cells were scraped off and suspended in 2mL of physiological saline (Otsuka Pharmaceutical). 100μL of this suspension was added to 2mL of 5 / 5 medium (final concentration: 0.2% L-leucine, 0.05% Beef extract (Difco), 0.5% Hypolypeptone S (Nissui), 0.75% salt), and 24μL of the test sample (final concentration: 0.25v / v% sanguisorba officinalis extract) or solvent control (solvent component of the test sample) was added, and the mixture was shake-cultured at 37°C for 24 hours under aerobic conditions. Thereafter, the amount of isovaleric acid produced in the culture medium was quantified by LC / MS under the conditions of Example 1 (5). The production rate of isovaleric acid (IVA) was calculated by the following formula.

[0069]

number

[0070] (2) Results When Chitococcus schroeteri was cultured in 5 / 5 medium, the addition of 0.25v / v% of Sanguisorba officinalis extract increased the isovaleric acid production rate to 45.2% compared to the solvent control, demonstrating that it is possible to use Chitococcus bacteria to discover foot odor inhibitors.

Claims

1. A method for detecting foot odor, comprising a step of measuring the amount of bacteria of the genus Kytococcus among the normal bacteria present on the skin of a subject's feet.

2. The method according to claim 1, further comprising a step of comparing the amount of Chitococcus bacteria in the normal bacteria on the skin of the subject's feet with a reference value to determine the presence or absence of foot odor or the intensity of foot odor.

3. The amount of Chitococcus bacteria in the normal skin bacteria of the subjects' feet was 2.68 x 10 16S rRNA gene amount per sole area. ４ Copies / cm ２ The method of claim 1, further comprising determining that the subject has foot odor if the test result is greater than or equal to 100 mg / mL.

4. A method for evaluating or selecting a foot odor suppressant, comprising the following steps (1) to (3): (1) Culturing Chitococcus bacteria in the presence of a test substance and L-leucine (2) A step of measuring the amount of bacteria of the genus Chitococcus or the amount of isovaleric acid in the culture obtained in (1). (3) A step of evaluating or selecting a test substance that suppresses the amount of bacteria of the genus Chitococcus or the production of isovaleric acid as a foot odor suppressant based on the results of the measurement in (2).

5. 5. The method according to claim 4, wherein a test substance that suppresses the production of isovaleric acid in the presence of the test substance to 90% or less compared to a control is evaluated or selected as a foot odor suppressant.

6. The method according to any one of claims 1 to 5, wherein the foot odor is an isovaleric acid odor.

7. The method according to any one of claims 1 to 5, wherein the bacteria of the genus Chitococcus include at least one species selected from the group consisting of Chitococcus shroeteri and Chitococcus aerolatus.

8. A foot odor detection kit for use in the method according to any one of claims 1 to 3, which contains a reagent for measuring the amount of bacteria of the genus Chitococcus.

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