JP6104058B2 - Search method for mold odor inhibitor - Google Patents

Description

  The present invention relates to a method for searching for a mold odor inhibitor.

Moldy odor caused by molds, such as food, drinking water, air conditioner (air conditioner) filters, etc., gives a sense of discomfort to humans even if it is a weak odor.
The mold odor is felt by the presence of substances produced by mold growth, and 2-methylisoborneol and diosmine are known as typical causative substances (Non-patent Documents 1 and 2). Since this substance also causes the musty odor of tap water, a standard value of 0.00001 mg / L or less is set in the Japanese Waterworks Law.

  As countermeasures against the mold odor generated, a technique for bacteriostatic using an antibacterial component against mold that causes mold odor, a technique for masking with a fragrance having a stronger odor than mold odor, etc. have been proposed. The technology does not deodorize the generated mold odor component, and the mold odor cannot be eliminated fundamentally and is not immediately effective. In addition, discomfort may occur due to the smell of the fragrance.

By the way, in mammals such as humans, the smell is bound to the olfactory receptor on the olfactory nerve cell existing in the olfactory epithelium in the upper nasal cavity, and the response of the receptor is transmitted to the central nervous system. Is recognized. In humans, it is reported that there are 396 olfactory receptors, and the genes encoding them are about 3% of all human genes.
In general, olfactory receptors and odor molecules are associated with each other in a plurality of combinations. That is, each olfactory receptor receives a plurality of odor molecules with similar structures with different affinities, while each odorant receptor is received by a plurality of olfactory receptors. Furthermore, it has been reported that an odor molecule that activates one olfactory receptor acts as an antagonist that inhibits activation of another olfactory receptor. The combination of these multiple olfactory receptor responses has led to the recognition of individual odors.

  Therefore, even when the same odor molecule is present, if another odor molecule is present at the same time, the receptor response is inhibited by the other odor molecule, and the finally recognized odor may be completely different. This mechanism is called olfactory receptor antagonism. Unlike the deodorization method by adding another odor such as perfume or fragrance, the odor modulation by this receptor antagonism can specifically lose the recognition of malodors such as mold odor, Moreover, since there is no discomfort due to the smell of the fragrance, it is a preferred deodorizing means. The present inventor has found that malodors such as hexanoic acid and skatole can be suppressed by using the receptor antagonism (Patent Documents 1 and 2).

  For olfactory receptor antagonism, identify an olfactory receptor corresponding to the target musty odor causative substance, and search for and identify a substance that exhibits an effective olfactory receptor antagonistic action against the musty odor causing substance. It must be, but such a search is not easy. Conventionally, odor evaluation has been performed by a sensory test by an expert. However, the sensory test has problems that it is necessary to train an expert who can evaluate the odor and that throughput is low.

JP 2012-50411 A JP 2012-249614 A

Kenji Kimura, Science of Food No.288 Page.27-37 (2002) Nobuo Hamada, Junji Masuda, Shoji Fukuyama, Life Sanitation Vol.43 No.4 Page.135-143 (1999)

  The present invention provides a method for efficiently searching for a musty odor inhibitor using an olfactory receptor response as an index.

  The present inventor succeeded in newly identifying an olfactory receptor that responds to a causative odor causative substance, and suppresses the mold odor by masking with the olfactory receptor antagonism by using the response of the olfactory receptor as an index. It has been found that it is possible to evaluate and select a new mold odor inhibitor.

That is, this invention relates to the search method of the mold odor inhibitor including the following processes.
Adding a test substance and a musty odor causing substance to an olfactory receptor selected from OR11A1 and OR2M3;
Measuring the response of the olfactory receptor to the musty odor causing substance;
Identifying a test substance that suppresses the response of the olfactory receptor based on the measured response;
A step of selecting the identified test substance as a mold odor inhibitor.

  According to the present invention, there is no problem such as low immediate effect and discomfort based on the odor of the fragrance, which has occurred in the conventional odor eliminating method using the odor eliminating agent and the fragrance, and the mold odor is specific. Therefore, it is possible to efficiently search for a mold odor inhibitor that can be deodorized easily.

Response of olfactory receptors to diosmin. The horizontal axis represents individual olfactory receptors, and the vertical axis represents response intensity. Response of olfactory receptor to 2-methylisoborneol. The horizontal axis represents individual olfactory receptors, and the vertical axis represents response intensity. Response of olfactory receptors to various concentrations of diosmin. Error bar = ± SE. ●: OR11A1, ○: mock (expressing a pME18S vector not incorporating a receptor, a cell not expressing a receptor) Olfactory receptor response to various concentrations of 2-methylisoborneol. Error bar = ± SE. ●: OR2M3, ○: mock (expressing cells that do not express the receptor, pME18S vector not incorporating the receptor)

  In this specification, the term “masking” relating to odor refers to all means for making the target odor unrecognized or weakening recognition. “Masking” may include chemical means, physical means, biological means, and sensory means. For example, as masking, any means for removing odor molecules that cause the target odor from the environment (for example, adsorption and chemical decomposition of odor molecules), to prevent the target odor from being released into the environment. (For example, containment), a method of adding another odor such as a fragrance or a fragrance to make it difficult to recognize the target odor, and the like.

  In the present specification, “masking by olfactory receptor antagonism” is one form of the above-mentioned “masking” in a broad sense, and by applying the target odor molecule and other odor molecules together, It is a means to change the odor recognized by an individual by inhibiting the receptor response to the target odor molecule by the molecule. Masking by olfactory receptor antagonism is distinguished from means for canceling the target odor by another strong odor, such as a fragrance, even if the means uses other odor molecules. An example of masking by olfactory receptor antagonism is the case of using a substance that inhibits the response of the olfactory receptor, such as an antagonist (antagonist). If a substance that inhibits the response is applied to the receptor of the odor molecule that causes a specific odor, the response of the receptor to the odor molecule is suppressed, so that the odor finally perceived by the individual is changed. Can do.

  The method for searching for a mold odor inhibitor according to the present invention includes: a) adding a test substance and a musty odor causing substance to any one of olfactory receptors selected from OR11A1 and OR2M3; b) response of the olfactory receptor C) a step of identifying a test substance that suppresses the response of the olfactory receptor based on the measured response, and d) a step of selecting the identified test substance as a mold odor inhibitor ,including.

In the method of the present invention, the test substance and the mold odor-causing substance are added to the olfactory receptor that responds to the mold odor.
The olfactory receptor used in the method of the present invention is selected from OR11A1 and OR2M3.
OR11A1 is an olfactory receptor expressed in human olfactory cells, and is registered in GenBank as GI: 277754165. OR11A1 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, encoded by a gene having the gene sequence represented by SEQ ID NO: 1. As shown in Examples below, OR11A1 is diosmine (IUPAC name: (4S, 4aS, 8aR) -4,8a-dimethyl-1,2,3,4,5,6,7,8-octahydronaphthalene-4a -Concentration-dependent response to (all).
Also, from an amino acid sequence having a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more with respect to the amino acid sequence of OR11A1. Thus, polypeptides having responsiveness to diosmin are also included in the olfactory receptor used in the method of the present invention.

OR2M3 is an olfactory receptor expressed in human olfactory cells, and is registered in GenBank as GI: 52317199. OR2M3 is a protein consisting of an amino acid sequence represented by SEQ ID NO: 4 encoded by a gene having a gene sequence represented by SEQ ID NO: 3. As shown in Examples below, OR2M3 responds to 2-methylisoborneol in a concentration-dependent manner.
In addition, from the amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more with respect to the amino acid sequence of OR2M3. Thus, polypeptides having responsiveness to 2-methylisoborneol are also included in the olfactory receptor used in the method of the present invention.

  In the method of the present invention, any one of the olfactory receptors may be used alone, or a combination of both may be used, but a substance that more reliably suppresses the musty odor is evaluated or selected. In such a case, it is preferable to use a combination of OR11A1 and OR2M3.

  Since OR11A1 responds to the odor of diosmin and OR2M3 responds to the odor of 2-methylisoborneol (FIGS. 1 and 2), substances that suppress the response of these receptors are masked by olfactory receptor antagonism. Changes can be made in the perception of diosmin odor or 2-methylisoborneol odor in the center, and as a result, these odors can be suppressed. Such diosmin and 2-methylisoborneol are odor molecules produced by mold (actinomycetes) and cyanobacteria, and are representative causative substances of mold odor (Non-patent Documents 1 and 2). Therefore, the substance that suppresses the response of OR11A1 and / or OR2M3 can suppress the mold odor by masking based on olfactory receptor antagonism.

As the fungus odor causative substance used in the present invention, it is preferable to use diosmin when OR11A1 is used as an olfactory receptor and 2-methylisoborneol when OR2M3 is used as an olfactory receptor.
Moreover, examples of the mold odor suppressed by the mold odor suppressant searched for by the method of the present invention include a scent of diosmin and a scent of 2-methylisoborneol.

The test substance used in the method of the present invention is not particularly limited as long as it is a substance desired to be used as a mold odor inhibitor. The test substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, etc., and may be a compound, a composition or a mixture. Good.
In the method of the present invention, the test substance and the musty odor-causing substance may be added simultaneously or in any order.

  In the method of the present invention, the olfactory receptor can be used in any form as long as the function of the receptor is not lost. For example, an olfactory receptor is a tissue or cell that naturally expresses an olfactory receptor, such as an olfactory receptor or olfactory cell isolated from a living body, or a culture thereof; a membrane of an olfactory cell carrying the olfactory receptor Used in the form of a recombinant cell or culture thereof genetically engineered to express the olfactory receptor; a membrane of the recombinant cell; and an artificial lipid bilayer membrane having the olfactory receptor Can be done. All of these forms fall within the scope of the olfactory receptor used in the present invention.

  In a preferred embodiment, cells that naturally express olfactory receptors such as olfactory cells, recombinant cells genetically engineered to express olfactory receptors, or cultures thereof are used. The recombinant cell can be produced by transforming a cell using a vector incorporating a gene encoding an olfactory receptor.

Suitably, in order to promote cell membrane expression of the olfactory receptor, RTP1S is introduced together with the receptor, preferably RTP1S and RTP2 are introduced together with the receptor. An example of RTP1S that can be used for the production of the recombinant cell is human RTP1S. Human RTP1S is registered with GenBank as GI: 50234917. Human RTP1S is a protein consisting of an amino acid sequence represented by SEQ ID NO: 6 encoded by a gene having a gene sequence represented by SEQ ID NO: 5. Human RTP2 is registered with GenBank as GI: 258547120. Human RTP2 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 10, encoded by a gene having the gene sequence represented by SEQ ID NO: 9.
Further, instead of human RTP1S, the amino acid sequence of human RTP1S (SEQ ID NO: 6) is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and still more preferably 98%. A polypeptide that has an amino acid sequence having a sequence identity of at least% and promotes the expression of the olfactory receptor membrane in the same manner as human RTP1S may be used. For example, the RTP1S mutant used in the examples of the present specification is a protein consisting of the amino acid sequence represented by SEQ ID NO: 8 and encoded by the gene having the gene sequence represented by SEQ ID NO: 7. It is a protein that has 78.9% sequence identity with the amino acid sequence shown in Fig. 6 and has the function of promoting expression in the membrane of the olfactory receptor and can be used for the production of the above-mentioned recombinant cells. . Alternatively, mouse RTP1S (Saito H., Chi Q., Zhuang H., Matsunami H., Mainland JD Sci Signal., 2009, 2: ra9) is also 89% identical to the amino acid sequence shown in SEQ ID NO: 6. It is a protein that has the ability to promote the expression of the olfactory receptor membrane in the membrane and can be used to produce the above recombinant cells.

  In this specification, the sequence identity of a base sequence and an amino acid sequence is calculated by the Lippman-Pearson method (Lipman-Pearson method; Science, 227, 1435, (1985)). Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (Ver. 5.1.1; software development), the unit size to compare (ktup) should be set to 2. Is calculated by

  According to the method of the present invention, following the addition of the test substance and the mold odor causing substance, the response of the olfactory receptor to the mold odor causing substance is measured. The measurement may be performed by any method known in the art as a method for measuring the response of the olfactory receptor, such as a calcium imaging method. For example, when the olfactory receptor is activated by an odor molecule, it is known to increase the amount of intracellular cAMP by activating adenylate cyclase coupled with intracellular Gαs (Mombaerts P Nat Neurosci. 5. 263-278). Therefore, the response of the olfactory receptor can be measured by using the amount of intracellular cAMP after addition of the odor molecule as an index. Examples of the method for measuring the amount of cAMP include an ELISA method and a reporter gene assay method.

  Next, based on the measured response of the olfactory receptor, the inhibitory effect of the test substance on the response of the receptor is evaluated, and the test substance that suppresses the response is identified. The inhibitory effect can be evaluated, for example, by comparing the response of the receptor to the mold odor-causing substance measured when different concentrations of the test substance are added. More specific examples include mold odors between the higher concentration test substance added group and the lower test substance added group; between the test substance added group and the non-added group; or before and after the test substance addition. Compare the response of the receptor to the causative agent. When the response of the olfactory receptor is suppressed by addition of the test substance or by addition of a higher concentration of the test substance, the test substance can be identified as a substance that suppresses the response of the olfactory receptor.

  For example, if the receptor response in the test substance addition group is suppressed to 80% or less, preferably 50% or less, compared to the control group, the test substance can be selected as a mold odor inhibitor. When both OR11A1 and OR2M3 are used as olfactory receptors, it is sufficient that the response of any one of the receptors used is suppressed, but it is preferable that the responses of both receptors are suppressed.

  The test substance identified in the above procedure changes the recognition of the mold odor in the center by masking based on olfactory receptor antagonism by suppressing the response of the olfactory receptor to the musty odor used in the above procedure. It is a substance that can be generated and as a result can prevent the individual from recognizing the mold odor. Therefore, the test substance identified by the above procedure is selected as a musty odor inhibitor for the musty odor used in the above procedure.

  The mold odor suppressor selected by the method of the present invention can be used to suppress the mold odor by olfactory masking based on suppression of the response of the olfactory receptor to the mold odor, and also suppress the mold odor Can be used for the manufacture of a compound or composition. In addition to the mold odor suppressor, the compound or composition for suppressing the mold odor is a component having another deodorizing effect, or any component used in the deodorant or deodorant, for example, a fragrance, a powder component , Liquid fats and oils, solid fats and oils, waxes, hydrocarbons, plant extracts, Chinese herbal ingredients, higher alcohols, lower alcohols, esters, long chain fatty acids, surfactants (nonionic surfactants, anionic surfactants, cations Surfactants, amphoteric surfactants, etc.), sterols, polyhydric alcohols, humectants, water-soluble polymer compounds, thickeners, film agents, bactericides, preservatives, UV absorbers, retention agents, cooling sensations Agent, warming agent, stimulant, sequestering agent, sugar, amino acids, organic amines, synthetic resin emulsion, pH adjuster, antioxidant, antioxidant assistant, oil, powder, capsules, chelating agent , Inorganic salt, organic salt pigment Thickeners, bactericides, antiseptics, fungicides, colorants, antifoaming agents, extenders, modulators, organic acids, polymers, polymer dispersants, enzymes, enzyme stabilizers, etc., depending on the purpose You may contain.

  As other components having a deodorizing effect that can be contained in the above-mentioned mold odor suppressing compound or composition, any known deodorant having a chemical or physical deodorizing effect can be used. Deodorant active ingredient extracted from each part of leaf, petiole, fruit, stem, root, bark, etc. (eg, green tea extract); lactic acid, gluconic acid, succinic acid, glutanic acid, adipic acid, malic acid, tartaric acid , Maleic acid, fumaric acid, itaconic acid, citric acid, benzoic acid, salicylic acid and other organic acids, various amino acids and their salts, glyoxal, oxidizing agents, flavonoids, catechins, polyphenols; porous activated carbon, zeolite, etc. Substances; inclusion agents such as cyclodextrins; photocatalysts; various masking agents and the like.

With respect to the above-described embodiment, the following aspects are further disclosed in the present invention.
<1> A method for searching for a mold odor inhibitor including the following steps:
Adding a test substance and a musty odor causing substance to an olfactory receptor selected from OR11A1 and OR2M3;
Measuring the response of the olfactory receptor to the musty odor causing substance;
Identifying a test substance that suppresses the response of the olfactory receptor based on the measured response;
A step of selecting the identified test substance as a mold odor inhibitor.
<2> The method according to <1>, wherein the mold odor is a scent of diosmin or 2-methylisoborneol.
<3> When OR11A1 is selected as the olfactory receptor, the mold odor causing substance is diosmin, and when OR2M3 is selected as the olfactory receptor, the mold odor causing substance is 2-methylisoborneol. Method.
<4> The olfactory receptor is an olfactory receptor expressed on a cell that naturally expresses the olfactory receptor or on a recombinant cell genetically engineered to express the olfactory receptor. <1 The method of>-<3>.
<5> The method according to <1> to <4>, further comprising a step of measuring a response of an olfactory receptor to which no test substance is added.
<6> If the response of the olfactory receptor to which the test substance is added is suppressed to 80% or less with respect to the response of the olfactory receptor to which the test substance is not added, the test substance is selected as a mold odor inhibitor The method of <5>.
<7> The method according to <1> to <6>, wherein the step of measuring the response of the receptor is performed by a reporter gene assay.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Example 1 Identification of Olfactory Receptor Responding to Mold Odor 1) Cloning of Human Olfactory Receptor Gene Human olfactory receptor is based on sequence information registered in GenBank, and human genomic cDNA female (G1521: Promega) is used as a template. The PCR method was used for cloning. Each gene amplified by the PCR method is incorporated into the pENTR vector (Invitrogen) according to the manual, and the NotI and AscI created downstream of the Flag-Rho tag sequence on the pME18S vector using the NotI and AscI sites present on the pENTR vector. Recomposed to the site.

2) Preparation of pME18S-RTP1S and pME18S-RTP2 vectors The RTP1S mutant gene (SEQ ID NO: 7) encoding the RTP1S mutant (SEQ ID NO: 8) was incorporated into the EcoRI and XhoI sites of the pME18S vector. Similarly, the human RTP2 gene (SEQ ID NO: 9) encoding human RTP2 (SEQ ID NO: 10) was incorporated into the EcoRI and XhoI sites of the pME18S vector.

3) Preparation of olfactory receptor-expressing cells HEK293 cells each expressing 374 types of human olfactory receptor were prepared. A reaction solution having the composition shown in Table 1 was prepared and allowed to stand in a clean bench for 15 minutes, and then added to each well of a 96-well plate (BD). Next, 100 μl of HEK293 cells (3 × 10 ⁵ cells / cm ² ) were seeded in each well and cultured for 24 hours in an incubator maintained at 37 ° C. and 5% CO ₂ .

4) Luciferase assay The olfactory receptor expressed in HEK293 cells increases the amount of intracellular cAMP by coupling with intracellular Gαs and activating adenylate cyclase. For measurement of odor response, a luciferase reporter gene assay was used in which the increase in intracellular cAMP level was monitored as a luminescence value derived from the firefly luciferase gene (fluc2P-CRE-hygro). In addition, a renilla luciferase gene fused to the downstream of the CMV promoter (hRluc-CMV) was introduced at the same time, and used as an internal standard for correcting errors in gene transfer efficiency and cell number.
75 μl of a solution containing diosmin (Wako Pure Chemical Industries, Ltd.) or 2-methylisoborneol (Wako Pure Chemical Industries, Ltd.) prepared with CD293 medium (Invitrogen) was removed from the culture prepared in 3) above. Added. The cells were cultured for 4 hours in a CO ₂ incubator to fully express the luciferase gene in the cells. The activity of luciferase was measured using a Dual-Glo ^™ luciferase assay system (Promega) according to the operation manual of the product. A value obtained by dividing the luminescence value derived from firefly luciferase induced by stimulation with diosmine or 2-methylisoborneol by the luminescence value in cells not subjected to stimulation was calculated as a fold increase and used as an index of response intensity.

5) Results As a result of measuring responses to diosmin (300 μM) for 374 types of olfactory receptors, only the olfactory receptor OR11A1 showed a response to diosmin (FIG. 1A). The diosmin response of OR11A1 was concentration dependent (FIG. 2A). From this, it became clear that OR11A1 shows a response to diosmin. OR11A1 is a novel diosmin receptor that has not previously been found to respond to diosmin.
Similarly, as a result of measuring responses to 2-methylisoborneol (1 mM) for 374 types of olfactory receptors, only the olfactory receptor OR2M3 showed a response to 2-methylisoborneol (FIG. 1B). The 2-methylisoborneol response of OR2M3 was concentration dependent (FIG. 2B). This revealed that OR2M3 showed a response to 2-methylisoborneol. OR2M3 is a novel 2-methylisoborneol receptor that has not previously been found to respond to 2-methylisoborneol.

Claims (6)

Method for searching for mold odor suppressor comprising the following steps:
Adding a test substance and a musty odor causing substance to an olfactory receptor selected from OR11A1 and OR2M3;
Measuring the response of the olfactory receptor to the musty odor causing substance;
Identifying a test substance that suppresses the response of the olfactory receptor based on the measured response;
A step of selecting the identified test substance as a mold odor inhibitor.   The method according to claim 1, wherein the mold odor is a odor of diosmin or 2-methylisoborneol.   The olfactory receptor is an olfactory receptor expressed on a cell that naturally expresses the olfactory receptor or on a recombinant cell that has been genetically engineered to express the olfactory receptor. The method described.   The method according to any one of claims 1 to 3, further comprising a step of measuring a response of an olfactory receptor without adding a test substance.   If the response of the olfactory receptor to which the test substance is added is suppressed to 80% or less with respect to the response of the olfactory receptor to which the test substance is not added, the test substance is selected as a mold odor inhibitor, Item 5. The method according to Item 4.   6. The method according to any one of claims 1 to 5, wherein the step of measuring the receptor response is performed by a reporter gene assay.