JP6646596B2 - Evaluation method of unpleasant odor - Google Patents

Description

  The present specification relates to a method of evaluating an unpleasant odor using an olfactory receptor (hereinafter, also simply referred to as a receptor or an OR).

  It is said that humans recognize an odor by binding a receptor expressed in an olfactory cell to an odor component and inputting the information to the brain. It has been reported that there are 396 types of receptors in humans. In general, the receptors code-recognize the odor components, and are associated so that the plurality of odor components are recognized by the plurality of receptors. It has also been reported that each receptor recognizes a plurality of odors having similar structures, or that an odor component that activates one receptor acts as an antagonist that inhibits activation of another receptor.

  As a method for evaluating such odor components, a CRE reporter assay is known. In the CRE reporter assay, an odor component binds to OR on the cell surface, sequentially activates G protein and adenylate cyclase, and finally activates CRE binding protein (CREB), a transcription factor in the nucleus. Make use of it. The activated transcription factor further binds to a cAMP transcription region cAMP response element (CRE) on the genome to promote the expression of a downstream gene. Here, by providing a gene encoding a luminescent protein such as luciferase as such a downstream gene, the binding between the test substance and OR can be detected by a signal such as luminescence.

  The use of such a CRE reporter assay to search for an OR-specific antagonist that binds to an odor-causing substance has also been disclosed (Patent Document 1).

JP-T-2015-81820

  In the human living environment, various odors are present. Above all, the unpleasant odor caused by storing clothes and the like after washing in a dry state and the sweat odor caused by sweat after wearing are extremely unpleasant. It is. In recent years, 4-methyl-3-hexenoic acid has been identified as a freshly dried odor component and a sweat odor component, and it has been elucidated that the odor component is a metabolite of Moraxella spp. The Moraxella spp. Is an opportunistic infection of animals such as humans, but exists in living environments with a high probability. It adheres to a large amount of laundry at home and generates an unpleasant odor after drying and after wearing, even after washing.

  Since it is difficult to control the adhesion and elimination of Moraxella spp., It is similarly difficult to fundamentally eliminate this unpleasant odor component. On the other hand, it is possible to reduce such discomfort by adding fragrance.However, unpleasant odor and wearing odor are very strong, and discomfort is caused by mixing fragrance with fresh odor and wearing odor. In some cases, it increased further.

  The present specification provides a method for evaluating such an unpleasant odor, a technique for evaluating an agent for an unpleasant odor, and the like, using an olfactory receptor for a substance that smells fresh and smells worn out. The present specification also provides an inhibitor for an unpleasant odor derived from textiles such as clothing.

  The present inventors have searched for an olfactory receptor, and as a result, found an olfactory receptor that recognizes 4-methyl-3-hexenoic acid, and evaluated fresh dry odor and wearing sweat odor by using such an olfactory receptor to suppress the odorant. Or similar odor quality can be searched. According to such knowledge, the following means are provided.

[1] An evaluation method of unpleasant odor,
Contacting an evaluation object that may contain one or more test substances with one or two or more olfactory receptors selected from the group consisting of OR2AK2, OR2W1, OR51L1, and OR51V1; Assessing the responsiveness of the species or two or more olfactory receptors,
A method comprising:
[2] The method according to [1], wherein the evaluation step is a step of evaluating an unpleasant odor using the responsiveness of the one or more olfactory receptors of 4-methyl-3-hexenoic acid. .
[3] A method for searching for an agent or a candidate thereof for a receptor responding to an unpleasant odor,
Contacting one or more olfactory receptors selected from the group consisting of OR2AK2, OR2W1, OR51L1 and OR51V1 with an evaluation object that may contain one or more test substances, Assessing the responsiveness of the receptor;
Selecting the agent or a candidate thereof from the one or more test substances based on the responsiveness,
A method comprising:
[4] The search method according to [3], wherein the agent is an agonist.
[5] The search method according to [3], wherein the agent is an antagonist.
[6] The method according to [5], wherein an antagonist that suppresses the olfactory receptor responsiveness is selected to be equal to or more than phenylacetaldehyde.
[7] An unpleasant odor suppressant containing phenylacetaldehyde as an active ingredient.
[8] A method for suppressing an unpleasant odor,
A method for suppressing unpleasant odor due to 4-methyl-3-hexenoic acid using phenylacetaldehyde.
[9] A method for evaluating the odor quality of textile products,
One or two or more olfactory receptors selected from the group consisting of OR2AK2, OR2W1, OR51L1 and OR51V1 are brought into contact with a gas derived from a fiber product as an object to be evaluated, and the one or more olfactory receptors are contacted. Assessing the responsiveness of the method.
[10] A kit for evaluating the odor quality of a fiber product,
One or more olfactory receptors selected from the group consisting of OR2AK2, OR2W1, OR51L1, and OR51V1;
A kit comprising:
[11] The kit according to [10], further comprising an animal cell having the at least one olfactory receptor on a cell surface.
[12] The kit according to [10] or [11], wherein the at least one olfactory receptor is provided at a predetermined position on a solid body.

FIG. 4 shows the receptor response of 4-methyl-3-hexenoic acid. It is a figure which shows the concentration dependence of the response to 4-methyl-3-hexenoic acid of OR2AK2, OR2W1, OR51L1, and OR51V1. It is a figure which shows suppression of OR51V1 response to 0.5 mM of 4-methyl-3-hexenoic acids. It is a figure which shows the sensory evaluation result regarding the antagonistic effect with respect to 4-methyl-3-hexenoic acid by phenylacetaldehyde.

  The present specification relates to odor evaluation and suppression using olfactory receptors, techniques for searching for odor quality, and the like. In particular, the present invention relates to a technique for evaluating and suppressing unpleasant odors using an olfactory receptor for 4-methyl-3-hexenoic acid, which is a dry odor and a sweat odor, and a technique for searching for odor. According to the evaluation method disclosed in the present specification (hereinafter, also referred to as the present evaluation method), it is possible to evaluate the receptor responsiveness of 4-methyl-3-hexenoic acid, which is a causative substance of fresh-dry odor and wearing sweat odor. . In addition, it becomes possible to search for an enhancer (agonist) and an inhibitor (antagonist) that enhance the receptor responsiveness of 4-methyl-3-hexenoic acid. In addition, analogs that cause a similar olfactory receptor response as 4-methyl-3-hexenoic acid can be searched for.

  "Odorant" refers to a substance that, when supplied to humans, causes some odor to be sensed. Generally, it is a substance that becomes at least partially gaseous at normal temperature or near body temperature.

  "Odor quality" can mean the nature of the odor. The “smell quality” can be, for example, a property expressed by a bodily, sensory, or material expression associated with a human smelling.

  The "olfactory receptor" or "receptor" may be any one of G protein-coupled receptors on olfactory cells (olfactory receptor nerves), and the origin animal species is not particularly limited. Depending on the purpose of the present evaluation method, the species of the origin animal and the type of receptor can be selected, and one or more receptors can be used as necessary. In order to evaluate the response of an odorant that can be perceived in humans, it is preferable to use a human-derived receptor.

  For example, the amino acid sequence of the human olfactory receptor and the nucleotide sequence of the DNA encoding the same are known. A base sequence containing a representative polynucleotide (cDNA) can be easily obtained from a publicly known database GenBank (http://www.ncbi.nlm.nih.gov/genbank/) or the like.

  As used herein, the term “agent” refers to an olfactory receptor that responds to a certain odorant, which enhances, suppresses, changes (modulates) or responds to the receptor responsiveness of the odorant. Refers to an agent that exerts an action such as expressing sex. In the present specification, agents exhibiting such effects are also referred to as an enhancer (agonist), a suppressor (antagonist), a modulator, a substitute, and the like, respectively. Here, to enhance or suppress the responsiveness of the receptor means to generally increase or decrease the response intensity in cooperation with an odorant. Further, changing (modulating) the responsiveness of the receptor includes increasing or decreasing the response strength of the receptor in coordination with the odorant, for example, by changing the concentration dependency or the threshold value of the receptor responsiveness. And various aspects such as acquiring reactivity to different receptors or exhibiting different reactivity only at a specific receptor among a plurality of receptors that can respond. In addition, expressing the same responsiveness means that the odorant has the receptor responsiveness, that is, that it binds to the same receptor, that multiple receptors respond in a similar pattern, and that the same response intensity is obtained. And having the same concentration dependency and response threshold.

  In this specification, a fiber product refers to a product having fibers in various forms such as woven fabric (woven fabric), knitted fabric (knitted fabric), lace, nonwoven fabric, felt, and leather. The textile product is not particularly limited, but includes, for example, clothing, towels, towels, and daily necessities that cover at least a part of the body for protecting or decorating the body of a human or the like. The textile product is also provided for washing at home or the like, although not particularly limited.

  Hereinafter, embodiments of the evaluation method and the like of the present disclosure will be described in detail.

(Method of evaluating unpleasant odor)
This evaluation method is a method for evaluating an unpleasant odor. In the present specification, the unpleasant odor includes a fresh dry odor such as a rag smell generated when the textile is stored after washing or after washing, and a sweat odor or a sweaty odor generated after wearing or using the textile. Can be.

  This evaluation method involves contacting an evaluation object that may contain one or more test substances with one or more olfactory receptors selected from the group consisting of OR2AK2, OR2W1, OR51L1, and OR51V1. And evaluating the responsiveness of the one or more olfactory receptors. All of these receptors have been identified by the present inventors as having responsiveness to 4-methyl-3-hexenoic acid.

  All of these four types of receptors can be said to be responsive olfactory receptors for substances that smell fresh and dry sweat. Therefore, by using one or two or more of these receptors, it is possible to evaluate the presence or absence, the degree, and the like of such an unpleasant odor. Although any of these receptors may be used, OR51V1 is preferably used from the viewpoint of responsiveness to 4-methyl-3-hexenoic acid. More preferably, only OR51V1 is used. In order to perform various evaluations more reliably, OR2AK2 is used in addition to OR51V1, OR51V1, OR2AK2, and OR2W1 are more preferably used, and OR51V1, OR2AK2, OR2W1, and OR51L1 are more preferably used.

  This evaluation method can use the receptor responsiveness of 4-methyl-3-hexenoic acid as an unpleasant smell substance. For example, the degree of unpleasant odor to be evaluated can be evaluated based on the responsiveness of the receptor due to 4-methyl-3-hexenoic acid. Further, for example, based on the responsiveness of the receptor due to 4-methyl-3-hexenoic acid, the effect of the test substance in the evaluation target on the responsiveness can be evaluated.

  In the present evaluation method, the receptor responsiveness of another odor substance can be used as the odor substance. For example, it may be 5-methyl-2-hexenoic acid or 5-methyl-4-hexenoic acid, which is an isomer of 4-methyl-3-hexenoic acid. Furthermore, 2-methylbutyric acid, 4-methylpentanoic acid, 4-methyl-3-pentenoic acid, 4-methylhexanoic acid, isobutyric acid, and isovaleric acid (the above are smell substances for wearing sweat odor). You may.

  Receptors used in this evaluation method include, in addition to the receptor itself present on the olfactory nerve cells, the same polypeptide as the receptor or a polypeptide functionally equivalent to the receptor, such as genetic engineering. What is produced in A polypeptide having a function equivalent to that of a receptor can be referred to as a polypeptide that, when expressed on a cell membrane, binds to and activates an intended odorant, similarly to a receptor. As used herein, the receptor is preferably a human receptor.

  The amino acid sequences of the various human receptors described above are already known, and are proteins comprising amino acids represented by the following SEQ ID NOs. Each of the above-mentioned receptors is a receptor expressed in human olfactory cells, and a base sequence containing a representative polynucleotide (cDNA) can be easily obtained from a known database.

  Such a receptor is, in addition to the polypeptide consisting of the above amino acid sequences, 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, of the amino acid sequence. As described above, more preferably, a polypeptide consisting of an amino acid sequence having 99% or more identity, which can respond to each odorant, can be used.

  Such polypeptides can be obtained by using a mutant of a receptor obtained by using a known method to obtain a pattern of responsiveness to one or more of the above-mentioned odorants having responsiveness already confirmed. It can be evaluated whether or not it can be used in the present evaluation method.

  In the present specification, the identity or similarity of a base sequence or an amino acid sequence is determined by comparing sequences, as is known in the art, for two or more proteins or two or more proteins. The relationship between nucleotides. In the art, "identity" refers to the sequence between protein or polynucleotide sequences, as determined by the alignment between the protein or polynucleotide sequences, or, in some cases, the alignment between a series of such sequences. Means the degree of sequence invariance. Similarity also refers to the correlation between protein or polynucleotide sequences, as determined by the alignment between the protein or polynucleotide sequences, or, in some cases, the alignment between a series of partial sequences. Means the degree of More specifically, it is determined by sequence identity and conservation (substitutions that maintain physicochemical properties in a particular amino acid or sequence in a sequence). The similarity is referred to as “Similarity” in a BLAST sequence homology search result described later. The method of determining identity and similarity is preferably the one designed to provide the longest alignment between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, a BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)). The conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.

  The receptor may be used in any form as long as the receptor function required in the present disclosure is not lost. For example, the receptor may be a tissue or cell that naturally expresses the receptor, such as an olfactory receptor or an olfactory cell isolated from a living body, or a culture thereof, as well as a gene that expresses the olfactory receptor. It can be used in the form of a recombinantly engineered cell or culture thereof, or in the form of an artificial lipid bilayer with OR. All of these forms are included in the receptor used in the present invention. Preferably, the receptor is expressed in cells for each receptor. Those skilled in the art can appropriately prepare these various forms of receptors based on known techniques.

  As the receptor, a cell that naturally expresses the receptor, such as an olfactory cell, or a recombinant cell that is genetically engineered to express the receptor, or a culture thereof is preferably used. The recombinant cell can be prepared by transforming a cell with a vector into which a gene encoding a receptor has been incorporated. As described above, the cell into which the gene encoding the receptor is introduced by genetic modification is the same as that for cultured animal cells that are contacted with the test substance, and for the same domain of any of the class, order, family, genus, and species. It preferably belongs to Preferably, they belong to the same domain in the order of species, genus, family, order and class.

  In addition, it is preferable that a gene encoding the receptor transport protein 1S (RTP1s) is also introduced into cells that express the receptor. RTP1s has a function of assisting the cell membrane expression of the receptor. Examples of RTP1s that can be used for producing the above-mentioned recombinant cell include human RTP1s. Note that an RTP1s mutant gene may be used instead of the RTP1s gene.

(Evaluation target that may contain test substance)
In this evaluation method, an evaluation target which may contain one or more test substances is brought into contact with a receptor. It is sufficient that the evaluation target includes, or may include, at least one test substance. When the object to be evaluated is brought into contact with the receptor, a gas, typically a biocompatible gas, for example, air can be used as the medium.

  The test substance is not particularly limited in its substance state, and may be a gas, a liquid, or a solid, as long as it causes the receptor to respond when contacted with the receptor. The test substance can typically be in a gaseous state.

  The test substance is not particularly limited. In addition to naturally occurring substances, they may be chemically or biologically synthesized substances. The test substance may be a single substance or a combination of two or more substances.

  The test substance may be an odor substance having a fresh dry odor and a sweat odor, or may be an analog (substitute agent) having the same action as the odor substance or a candidate thereof. In addition, as the test substance, an inhibitor or a candidate thereof having an inhibitory effect of reducing the responsiveness of the receptor due to a substance smelling freshly dried odor or wearing sweat odor, an enhancer or a candidate thereof for enhancing the responsiveness of the receptor, etc. No. Such an enhancer, an inhibitor, or a test substance that is a candidate for them is applied in combination with a substance that smells dry or smells worn. For example, a combination of an odor substance and a candidate for an inhibitor and a odor substance and a candidate for an enhancer can be mentioned. The test substance is, for example, a gas derived from an individual textile product (eg, a gas obtained from a textile product after washing) or a culture derived from Moraxella bacteria, as a source of fresh odor or wearing odor. It may be one or more substances of known or unknown structure that may be contained in the gas. In this case, a gas generated from such a fiber product can be evaluated.

  In addition, the gas derived from textile products can be obtained by various methods. For example, a gas obtained by storing a fiber product under certain conditions (for example, a closed space where microorganisms can propagate but in a closed space, etc.), and a gas obtained by culturing microorganisms extracted from a fiber product (headspace gas) Can be obtained as

  In order to evaluate the receptor responsiveness of the evaluation target that may contain such a test substance, a step of bringing the evaluation target into contact with the receptor is performed. As the evaluation target, for example, the test substance is used at a constant concentration, at a different concentration, or in a state in which two or more are combined.

  The step of contacting the evaluation target with the receptor can be performed by culturing cells retaining the receptor in the presence of the evaluation target. As the culture conditions, the culture conditions for the receptor-expressing cells can be appropriately selected. For example, as described above, a serum-free medium containing an appropriate antibiotic can be used as the medium. The culturing time needs to be long enough to allow the response evaluation by the receptor. For example, it can be about one hour or more and several hours or less.

  Evaluation using a receptor can be carried out in the same manner as in the past. For example, a form in which the response of a test substance is evaluated by using activation of a CRE-binding protein via a receptor can be used. The protocol for the mode is not particularly limited. For example, utilizing the fact that the activated CRE-binding protein binds to CRE and promotes expression of a downstream gene, a reporter gene is introduced as the downstream gene, and the expression level of the reporter gene is measured. Is also good. The reporter gene can be appropriately selected by those skilled in the art, such as a gene encoding a known photoprotein. The expression level of the reporter gene is appropriately determined depending on the type of the reporter gene used.

  Receptor-mediated activation pathways for CRE binding proteins are well known to those of skill in the art, as described above. By bringing the receptor into contact with various odor molecules and binding them, G protein and adenylate cyclase (AC) are sequentially activated in the cell, and cAMP is increased. Along with this, protein kinase A (PKA) is phosphorylated and activates the downstream signaling system. On the other hand, PKA translocates into the nucleus and activates the transcription factor CRE binding protein (CREB).

  In addition, as a method of measuring the increased amount of cAMP, an ELISA method can be used. Further, for example, the response of the receptor may be performed by any method known in the art as a method for measuring the response of the receptor, for example, a calcium imaging method or the like.

  Based on the responsiveness of the receptor to the evaluation target thus obtained, the effect of the test substance that can be included in the evaluation target on the receptor, that is, whether the test substance acts on the receptor, the degree, The response threshold, concentration dependence, and response pattern for multiple receptors are identified.

  For example, when the test substance is a certain odor substance, based on the responsiveness of the receptor to the odor substance, the action of the odor substance on the receptor, that is, the odor substance acts on the receptor Whether or not, its degree, its response threshold, concentration dependence, and its response pattern to multiple receptors are identified.

  Further, for example, one test substance is used as a smell of fresh odor such as 4-methyl-3-hexenoic acid or a sweat odor, and another test substance is used as a receptor for 4-methyl-3-hexenoic acid. When an agent for responsiveness or its candidate is used, it can be identified whether the agent enhances or suppresses the effect of the odorant on the receptor.

  Further, for example, when the test substance is a substance that may have the same odor as fresh odor or sweat odor when worn, the odor substance is directed to a receptor for an odor substance such as 4-methyl-3-hexenoic acid. It can be evaluated whether it shows the same responsiveness as responsiveness.

  Depending on the mode of evaluation, a comparison object or control may be required. In that case, for example, for a control substance or a combination thereof, responsiveness can be obtained using similar receptors under similar conditions, and the results can be used as a control evaluation. The evaluation target can also be evaluated by comparing the control evaluation thus obtained with the evaluation result of the responsiveness of the evaluation target. In addition, as a control, the evaluation result of the responsiveness of a test substance or a control substance to cultured animal cells that do not express the receptor can also be used.

  Based on the above, according to the present evaluation method, evaluation of unpleasant odor such as dry odor or worn sweat odor of one or more possible test substances, that is, human having one or more test substances Can be evaluated for the intensity of the unpleasant odor perceived as a dry odor or a sweat odor.

  In addition, the present evaluation method can be implemented in various modes depending on the mode of the evaluation target. That is, the present invention can also be implemented as a method for evaluating the effect of one or more test substances that can be included in the evaluation target on a receptor that responds to a dry odor or an unpleasant odor. For example, in one embodiment, as one test substance, 4-methyl-3-hexenoic acid, which is a smell of fresh dry odor and sweat odor, and a suppressor candidate as another test substance are received. By evaluating the responsiveness when brought into contact with the body and the responsiveness under the same conditions except that only 4-methyl-3-hexenoic acid is used, the candidate inhibitor as the test substance is identified as 4- It can be evaluated whether or not to suppress receptor responsiveness due to methyl-3-hexenoic acid. More specifically, when the inhibitor candidate is present in the presence of 4-methyl-3-hexenoic acid and the receptor responsiveness is reduced, the substance is a receptor for 4-methyl-3-hexenoic acid. It can be selected as a responsiveness inhibitor candidate. According to such an embodiment, a candidate for an enhancer that enhances the receptor responsiveness of 4-methyl-3-hexenoic acid can also be selected.

  Further, for example, as another aspect, there is a possibility that one test substance may exhibit similar or similar receptor responsiveness to 4-methyl-3-hexenoic acid, which is a substance smelling dry odor and wearing odor. By evaluating the responsiveness of a certain candidate to contact with a receptor and the responsiveness under the same conditions except that 4-methyl-3-hexenoic acid is used, the candidate substance becomes methyl hexanoate. It can be evaluated whether or not it shows similar or similar receptor responsiveness.

  From the above, the present evaluation method can also be carried out as a method for searching for an agent for a receptor that responds to a dry odor or a sweat odor, that is, an inhibitor, an enhancer, a modulator, and a substitute.

(Method of searching for an agent or a candidate thereof for a receptor responding to an unpleasant odor)
The search method disclosed in the present specification provides an evaluation object that may contain one or more test substances, and one or more olfactory senses selected from the group consisting of OR2AK2, OR2W1, OR51L1, and OR51V1 A step of evaluating the responsiveness of the olfactory receptor by contacting with a receptor, and a step of selecting the agent or a candidate thereof from the one or more test substances based on the responsiveness, Can be prepared. As described above, an agent or a candidate thereof is selected by using an unpleasant odor substance as at least one test substance and at least one agent or a candidate thereof as at least one other test substance. can do.

  In the present search method, in addition to the receptor and the test substance, the step of evaluating responsiveness can be carried out in the same manner as the above-described present evaluation method. In addition, the selection step can also be appropriately performed based on the obtained evaluation result of the responsiveness in the above-described embodiment or the like. In evaluating an agent, when a plurality of receptors recognize one odorant, it is preferable to use a plurality of receptors, and more preferably to use all the receptors having a responsiveness.

  The search method also uses a gaseous substance obtained from a textile product after washing or a textile product after sweating and an agent or a candidate thereof, using a receptor that responds to a dry odor or a sweat odor to wear. By evaluating the receptor responsiveness, it is possible to identify the effect of the candidate agent on the receptor responsiveness, and to select the agent or the candidate corresponding to the odor generated under specific conditions. Become. In this case, it is preferable to perform the same receptor responsiveness evaluation using only the gas derived from the fiber product as the evaluation target and to compare.

  In the present search method, the agent to be searched can be an enhancer (agonist) that enhances receptor responsiveness. By searching for and finding agonists, removal of such agonists is an indicator of the suppression of dry odor or sweat odor. The remover of the agonist or the adsorbent of the agonist becomes a certain dry odor or sweat odor suppressant. In the present search method, the agent to be searched can be an inhibitor (antagonist) that suppresses receptor responsiveness. By searching for and finding an antagonist, it can be used as a malodor inhibitor for freshly dried odor or sweat odor.

  In the present search method, for example, an antagonist that suppresses the olfactory receptor responsiveness to be equal to or more than phenylacetaldehyde can be selected. Phenylacetaldehyde is an antagonist discovered by the present inventors that can suppress the responsiveness of OR51V1 by 4-methyl-3-hexenoic acid. Therefore, by using phenylacetaldehyde as an indicator of antagonism or as a lead compound, a better antagonist can be searched for.

(Method of evaluating the odor quality of textile products)
The method for evaluating the odor quality of a textile product disclosed in the present specification is a method for evaluating a gas derived from a textile product obtained from a textile product after washing or a textile product after perspiration or after use, by using OR2AK2, OR2W1, The method may include a step of contacting one or more olfactory receptors selected from the group consisting of OR51L1 and OR51V1 to evaluate the responsiveness of the one or more olfactory receptors. According to this evaluation method, various types of detergents, softeners, fragrances for clothes, bleaches, sizing agents, fragrances for the body, deodorants for the body, etc. which can be used in conjunction with washing, wearing or use of textile products By evaluating the textile products treated with the agent and the gas obtained from the textile products after further wearing the agents, the effect of suppressing various types of laundry-related agents, such as fresh odor and wearing sweat odor, is evaluated. be able to. In addition, it is possible to evaluate what kind of washing conditions, drying conditions, and wearing conditions contribute to the generation and suppression of freshly dried odor, worn sweat odor, and the like.

(Kit for evaluating the odor quality of textile products)
The kit for evaluating the odor quality of a textile product disclosed herein can include one or more olfactory receptors selected from the group consisting of OR2AK2, OR2W1, OR51L1, and OR51V1. . According to this kit, various evaluations regarding unpleasant odors such as fresh odors and sweat odors derived from textiles can be easily performed.

  The form of the receptor included in the kit is not particularly limited, and may be any of the various embodiments described above. For example, a kit for a transformation vector for expressing the receptor on the cell surface, a kit containing cells expressing the receptor on the cell surface, cells containing the receptor expressing the receptor on the cell surface are not contained. A kit including an immobilized solid phase body is exemplified. Specifically, animal cells having individual receptors on the cell surface can be used. Further, it may be a solid phase body that contains cells expressing the receptor on the cell surface. Typically, a plate provided with a plurality of, for example, 48 or 96 wells is exemplified. Also, an array in which such cells are immobilized can be mentioned. The array can include, for example, cell immobilization regions in a matrix. Such a solid phase body can be prepared by appropriately applying a known cell immobilization technique.

  The expression vector that can be provided by the kit can be appropriately constructed by those skilled in the art based on the nucleotide sequence of the DNA encoding the receptor to be used.

(Unpleasant odor inhibitor and method)
The unpleasant odor suppressor disclosed in the present specification can contain phenylacetaldehyde as an active ingredient. Further, the method for suppressing an unpleasant odor disclosed in the present specification can suppress unpleasant odors such as a dry odor and a sweat odor caused by 4-methyl-3-hexenoic acid using phenylacetaldehyde. Phenylacetaldehyde has been found by the present inventors to suppress the receptor responsiveness of 4-methyl-3-hexenoic acid. Phenylacetaldehyde itself has a odor like honey like sweet, rose scent, fresh, grass scent. It is contained in foods and plants such as buckwheat and chocolate, and is used as a raw material for preparing fragrances and flavors. According to phenylacetaldehyde, receptor responsiveness to 4-methyl-3-hexenoic acid can be reduced in a concentration-dependent manner.

  According to the present inhibitor and the present method, phenylacetaldehyde is present in the vicinity of an olfactory receptor or in an environment where an unpleasant odor is likely to occur or in an environment in which an unpleasant odor is generated. Thereby, an unpleasant odor can be reduced. Thus, for example, it can be provided as a softener, detergent, clothing fragrance, bleach, sizing agent, body fragrance, or body deodorant containing phenylacetaldehyde.

  The content of phenylacetaldehyde in the present inhibitor is not particularly limited, and is appropriately set according to the use form, the generation level of unpleasant odor, and the like. For example, in the case of a spray agent directly supplied to a textile product after washing, wearing or in use by spraying or the like, the concentration can be about 0.01 mM to 100 mM.

  Hereinafter, the present invention will be described specifically with reference to examples, but these do not limit the present invention.

(Evaluation of response of all human olfactory receptors to 4-methyl-3-hexenoic acid)
The human olfactory receptor was cloned by PCR based on the registered sequence information. Each gene amplified by the PCR method was incorporated into Flexi Vector (Promega) according to a conventional method, and pF5K CMV-neo Flexi Vector was prepared using the SgfI and PmeI sites.

After culturing the HEK293 cells in a DME medium containing 10% fetal bovine serum (FBS) -penicillin / streptomycin, a reaction solution having the composition shown in Table 1 was prepared, and left for 30 minutes in a clean bench. (L-lysine-coated white plate). Next, HEK293 cells were seeded in each well at a rate of 3.0 × 10 ⁵ / cm ² and cultured in a CO ₂ incubator for 24 hours.

  The CRE response was measured using a luciferase reporter gene assay that monitors the increase in the amount of intracellular cAMP as a luminescence value derived from the firefly luciferase gene. Furthermore, a gene fused with the Renilla luciferase gene downstream of the CMV promoter was simultaneously transfected and used as an internal standard for correcting errors in transfection efficiency and cell number.

After culturing for 24 hours, the medium was removed, target 4-methyl-3-hexenoic acid (1 mM) prepared in a serum-free medium was added, and left in a CO ₂ incubator for 4 hours. Luciferase activity was measured according to the protocol attached to Dual-Glo Luciferase assay system (Promega). The value obtained by dividing the luminescence value derived from firefly luciferase induced by the odor substance stimulation by the luminescence value of the control group (non-odor treatment group) was used as an index of response intensity as Fold induction of luciferase. FIG. 1 shows the results of evaluating the responsiveness to 4-methyl-3-hexenoic acid.

  As shown in FIG. 1, four olfactory receptors specifically responding to 4-methyl-3-hexenoic acid could be newly identified.

(Evaluation of responsiveness depending on the concentration of 4-methyl-3-hexenoic acid)
Cells in which each receptor (OR), OR2AK2, OR2W1, OR51L1, and OR51V1, were expressed together with RTP1s were produced in the same procedure as in Example 1, cultured for 24 hours, and the medium was removed. A serum-free medium containing each concentration of hexenoic acid (10, 30, 100, 300, 1000 μM) was added, and the mixture was allowed to stand in a CO ₂ incubator for 4 hours, followed by 4-methyl-3-hexene as in Example 1. The response to acid concentration was examined. The results are shown in FIG.

  As shown in FIG. 2, it was confirmed that the responsiveness of each of these receptors increased depending on the concentration of the odorant that responded. Also, the receptor was responsive to 4-methyl-3-hexenoic acid over a broad concentration range, generally from 10 μM to 1000 μM. Among them, it was found that 51V1 exhibited high response and concentration dependency. From these results, the receptor found by the present inventors that responds to 4-methyl-3-hexenoic acid responds to 4-methyl-3-hexenoic acid in a concentration-dependent manner, and qualitatively and quantitatively determines odorants. It has been found that a comprehensive evaluation is also possible.

(Response suppression of OR51V1 by phenylacetaldehyde)
Cells in which each receptor (OR), OR2AK2, OR2W1, OR51L1, and OR51V1, were expressed together with RTP1s were produced in the same procedure as in Example 1, cultured for 24 hours, and the medium was removed. hexenoic acid (0.5 mM) and phenyl acetaldehyde and acetophenone (respectively, 0mM, 0.1mM, 0.3mM, 0.5mM , 1mM, 3mM and 5 mM) was added to serum-free medium containing, 4 hours CO ₂ incubator And the response was examined in the same manner as in Example 1. The results are shown in FIG.

  As shown in FIG. 3, it was found that phenylacetaldehyde specifically suppressed the response of OR51V1 to 4-methyl-3-hexenoic acid. From the above, it was found that phenylacetaldehyde was effective as an unpleasant odor suppressant due to the antagonistic action of 4-methyl-3-hexenoic acid.

(Sensory evaluation of unpleasant odor control ability by phenylacetaldehyde)
20 μl of 4-methyl-3-hexenoic acid diluted 10,000 times in mass ratio with polyethylene glycol was dropped on a cotton ball put in a glass bottle, and 20 μl of a solution in which phenylacetaldehyde was diluted 200 times in mass ratio was dropped. The glass bottle was sealed, and 4-methyl-3-hexenoic acid and phenylacetaldehyde were sufficiently evaporated. As a control, a sample without addition of phenylacetaldehyde was also prepared.

  The gas volatilized from these bottles was subjected to sensory evaluation by seven panelists skilled in odor evaluation. In the evaluation, the degree of unpleasant odor was evaluated on a scale of 0 to 5.0. The average of the evaluation results of the seven persons is shown in FIG.

  As shown in FIG. 4, the unpleasant intensity of the sample without addition exceeded 3, but the intensity of the sample with addition of phenylacetaldehyde was less than 0.5. From the above, the inhibitory effect of phenylacetaldehyde on 4-methyl-3-hexenoic acid could be confirmed by sensory evaluation by humans.

Claims (7)

A method for searching for an inhibitor for an unpleasant odor derived from 4-methyl-3-hexenoic acid of a textile product,
An evaluation target containing 4-methyl-3-hexenoic acid and one or more test substances, and the following olfactory receptor peptides;
(A) OR2AK2, an olfactory receptor peptide having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and showing olfactory receptor responsiveness equivalent to OR2AK2 (b) OR2W1, sequence An olfactory receptor peptide having an amino acid sequence having 90% or more identity with the amino acid sequence represented by No. 2 and exhibiting olfactory receptor responsiveness equivalent to OR2W1;
(C) OR51L1, an olfactory receptor peptide having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 3 and showing olfactory receptor responsiveness equivalent to OR51L1, and (d) OR51V1 Selected from the group consisting of: an olfactory receptor peptide having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and exhibiting olfactory receptor responsiveness equivalent to that of OR51V1; Contacting one or two or more olfactory receptor peptides containing a receptor peptide and expressed on the cell surface with an olfactory receptor peptide, and evaluating the responsiveness of the olfactory receptor peptide;
Selecting the inhibitor from the one or more test substances based on the responsiveness,
A method comprising: The search method according to claim 1 , wherein the one or more olfactory receptor peptides further include the olfactory receptor peptide (a). The search method according to claim 1 or 2 , wherein the inhibitor that suppresses the responsiveness of the olfactory receptor peptide (d) is selected to be equal to or more than phenylacetaldehyde.     An inhibitor of an unpleasant odor derived from 4-methyl-3-hexenoic acid, comprising phenylacetaldehyde as an active ingredient. A method for suppressing unpleasant odor,
A method for suppressing unpleasant odor derived from 4-methyl-3-hexenoic acid using phenylacetaldehyde. A kit for searching for an unpleasant odor inhibitor derived from 4-methyl-3-hexenoic acid of a textile product,
The following olfactory receptor peptides:
(A) OR2AK2, an olfactory receptor peptide having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and showing olfactory receptor responsiveness equivalent to OR2AK2 (b) OR2W1, sequence An olfactory receptor peptide having an amino acid sequence having 90% or more identity with the amino acid sequence represented by No. 2 and exhibiting olfactory receptor responsiveness equivalent to OR2W1;
(C) OR51L1, an olfactory receptor peptide having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 3 and exhibiting olfactory receptor responsiveness equivalent to OR51L1, and (d) OR51V1 Selected from the group consisting of: an olfactory receptor peptide having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and exhibiting olfactory receptor responsiveness equivalent to that of OR51V1; One or more olfactory receptor peptides, including a receptor peptide, expressed on the cell surface,
A kit comprising: The kit according to claim 6 , wherein one or more cells expressing the one or more olfactory receptor peptides on the cell surface are provided at predetermined positions on a solid phase body.