WO2016204211A1 - Procédé d'évaluation et de sélection d'un agent permettant de supprimer les odeurs associées aux composés sulfurés - Google Patents

Procédé d'évaluation et de sélection d'un agent permettant de supprimer les odeurs associées aux composés sulfurés Download PDF

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WO2016204211A1
WO2016204211A1 PCT/JP2016/067894 JP2016067894W WO2016204211A1 WO 2016204211 A1 WO2016204211 A1 WO 2016204211A1 JP 2016067894 W JP2016067894 W JP 2016067894W WO 2016204211 A1 WO2016204211 A1 WO 2016204211A1
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olfactory receptor
response
receptor polypeptide
sulfide compound
test substance
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PCT/JP2016/067894
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Japanese (ja)
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敬一 吉川
菜穂子 齋藤
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花王株式会社
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Priority claimed from JP2016116227A external-priority patent/JP6122181B2/ja
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Priority to EP16811685.3A priority Critical patent/EP3312289B1/fr
Priority to US15/737,079 priority patent/US10585087B2/en
Publication of WO2016204211A1 publication Critical patent/WO2016204211A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for evaluating or selecting an inhibitor for odor of sulfide compounds.
  • Volatile sulfur compounds generated from wastes such as drain outlets and garbage are malodorous components that make people uncomfortable even at low concentrations. These volatile sulfur compounds are produced by degradation of sulfur-containing amino acids such as cysteine and methionine contained in sewage and wastes or proteins containing them by the action of metabolic enzymes such as methionine lyase and cysteine lyase in bacteria. appear. For example, among volatile sulfur compounds that cause unpleasant odor, methyl mercaptan is produced from methionine by the action of methionine lyase, and hydrogen sulfide is produced from cysteine by the action of cysteine lyase.
  • sulfide compounds such as dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide are generated enzymatically or oxidatively from methyl mercaptan and hydrogen sulfide, which also cause bad odor.
  • Patent Document 1 discloses that a fragrance component is added as a deodorant or fragrance to a composition for cleaning, sterilizing, antifouling, and deodorizing a drain outlet or the like.
  • Patent Document 2 the action of an enzyme that generates methyl mercaptan from methionine or an enzyme that generates hydrogen sulfide from cysteine is inhibited by a specific fragrance component and is caused by a volatile sulfur compound generated from a waste or a drain outlet. It is disclosed to suppress malodors.
  • the smell is bound to the olfactory receptor on the olfactory nerve cell located in the olfactory epithelium in the upper nasal cavity, and the response of the receptor is transmitted to the central nervous system. It is recognized by. In the case of humans, it is reported that there are about 400 olfactory receptors, and the genes encoding them are about 2% 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.
  • Patent Documents 3 to 4 disclose searching for substances that suppress malodor such as hexanoic acid and skatole, using as an index the activity of olfactory receptors that specifically respond to those malodorous substances.
  • Patent Document 5 discloses searching for a substance that suppresses sweat odor, using as an index the activity of an olfactory receptor that responds to a specific carboxylic acid.
  • Patent Document 6 discloses a method for identifying an agent that modulates the function of a polypeptide by measuring the activity of the polypeptide encoding an olfactory receptor in the presence of isovaleric acid or its equivalent.
  • Patent Document 7 discloses a method of screening a library of chemical substances for compounds related to olfactory sensation by identifying a compound that specifically binds to a polypeptide encoding an olfactory receptor.
  • Patent Document 1 JP-A-2006-206882 (Patent Document 2) JP-A-2010-004971 (Patent Document 3) JP-A-2012-050411 (Patent Document 4) JP-A-2012-249614 (Patent Document 3) Literature 5) US Patent Publication No. 2013/0336910 (Patent Literature 6) International Publication No. 2006/094704 (Patent Literature 7) Japanese Translation of PCT International Publication No. 2004-504010
  • the present invention provides a method for evaluating and / or selecting a sulfide compound odor inhibitor comprising: Adding a test substance and a sulfide compound to at least one olfactory receptor polypeptide selected from the group consisting of OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with OR4S2; and Measuring the response of the olfactory receptor polypeptide to the sulfide compound;
  • a method comprising:
  • A Response to dimethyl disulfide
  • B Response to dimethyl trisulfide
  • DMDS Dimethyl disulfide
  • DMTS Dimethyl trisulfide
  • DMDS DMDS only
  • DMTS DMTS only
  • Vehicle mineral oil only
  • Error bar SE.
  • Wastes such as drain outlets and garbage generate various volatile sulfur compounds due to bacterial decay and cause bad odor.
  • One of the main causative substances of the malodor is sulfide compounds such as dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide. Therefore, control of the odor of these sulfide compounds is required.
  • the present inventor succeeded in newly identifying an olfactory receptor that specifically responds to a sulfide compound that is a causative substance of malodor from wastes such as drains and garbage.
  • the present inventor evaluates and / or evaluates substances that can suppress the odor of sulfide compounds by olfactory receptor antagonism by using the response of the olfactory receptor or a polypeptide having the same function as an index. It was found that selection is possible.
  • “inhibition of odor by olfactory receptor antagonism” means that by applying both the target odor molecule and another molecule, the other molecule inhibits the receptor response to the target odor molecule. As a result, it is a means for suppressing the odor recognized by the individual. Suppression of odor by olfactory receptor antagonism is distinguished from means for concealing the target odor by the fragrance of a fragrance, such as deodorization by a fragrance, even if the means uses other molecules.
  • An example of odor suppression by olfactory receptor antagonism is the case of using a substance that inhibits the response of olfactory receptors such as an antagonist (antagonist). If a substance that inhibits the response is applied to the receptor of the odor molecule that brings about a specific odor, the response of the receptor to the odor molecule is suppressed, so the odor finally perceived by the individual is suppressed. Can do.
  • olfactory receptor polypeptide refers to an olfactory receptor or a polypeptide having a function equivalent thereto, and a polypeptide having a function equivalent to an olfactory receptor is similar to an olfactory receptor.
  • a function that increases the amount of intracellular cAMP by activating adenylate cyclase in combination with intracellular G ⁇ s, which can be expressed on the cell membrane and activated by binding of odor molecules (Nat. Neurosci., 2004, 5: 263-278).
  • nucleotide sequence and amino acid sequence identity is calculated by the Lippman-Pearson method (Lipman-Pearson method; Science, 1985, 227: 1435-41). Specifically, using the homology analysis (Search homology) program of genetic information software Genetyx-Win (Ver. 5.1.1; software development), perform analysis with unit size to compare (ktup) as 2. Is calculated by
  • “at least 80% identity” with respect to nucleotide and amino acid sequences refers to 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably It means 98% or more, preferably 99% or more identity.
  • the “sulfide compound” refers to a compound represented by the following formula (I).
  • R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms.
  • Examples of the linear or branched alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, and Hexyl is mentioned.
  • Examples of the straight-chain or branched alkenyl group having 1 to 6 carbon atoms include vinyl, propenyl, allyl, butenyl, and methylbutenyl.
  • R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms, and the linear or branched chain having 1 to 4 carbon atoms
  • alkyl or alkenyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, and butenyl.
  • R 1 and R 2 are the same or different and are methyl, ethyl, propyl or isopropyl, and more preferably R 1 and R 2 are methyl.
  • n represents an integer of 1 to 5, preferably an integer of 1 to 3, and more preferably 2 or 3.
  • the “sulfide compound” is a volatile substance having a structure represented by the formula (I). More preferable examples of the “sulfide compound” include dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), allyl methyl sulfide, trimethyl sulfide, and the like, and more preferable examples include DMDS. And DMTS.
  • the “sulphide compound odor” suppressed by the present invention is an odor generated by the “sulfide compound” described above, and is preferably an odor generated by DMDS or DMTS.
  • the “sulphide compound odor” suppressed by the present invention may be a malodor emitted from spoiled garbage, sewage, or a drain.
  • OR4S2 As shown in FIGS. 1 and 2, the present inventor has identified OR4S2 as an olfactory receptor that specifically responds to a sulfide compound among many olfactory receptors. OR4S2 responds to various sulfide compounds in a concentration-dependent manner. Therefore, a substance that suppresses the response of OR4S2 or a polypeptide having a similar function causes a change in the recognition of the odor of the sulfide compound based on olfactory receptor antagonism, and as a result, the odor of the sulfide compound is changed. It can be selectively suppressed.
  • the present invention provides a method for evaluating and / or selecting a sulfide compound odor inhibitor.
  • the method comprises adding a test substance and a sulfide compound to at least one olfactory receptor polypeptide selected from the group consisting of OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with OR4S2; and Measuring the response of the olfactory receptor polypeptide to the sulfide compound.
  • a test substance that suppresses the response of the olfactory receptor polypeptide is identified based on the measured response.
  • the identified test substance is selected as an inhibitor of sulfide compound odor.
  • the method of the present invention may be a method performed in vitro or ex vivo.
  • a test substance and the sulfide compound are added to the olfactory receptor polypeptide having responsiveness to the sulfide compound.
  • 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 sulfide compound odor suppressor.
  • the test substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, etc., or may be a compound, a composition or a mixture. Good.
  • the olfactory receptor polypeptide used in the method of the present invention is at least one selected from the group consisting of OR4S2 and polypeptides having at least 80% identity in amino acid sequence with OR4S2.
  • OR4S2 is an olfactory receptor expressed in human olfactory cells.
  • OR4S2 is registered in GenBank as GI: 116517324.
  • OR4S2 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, encoded by the gene having the nucleotide sequence represented by SEQ ID NO: 1.
  • the polypeptide having at least 80% identity in the amino acid sequence with OR4S2 is at least 80%, for example, 80% or more, preferably 85% or more, more preferably 90% or more, further with the amino acid sequence represented by SEQ ID NO: 2.
  • Preferred is a polypeptide having an amino acid sequence having an identity of 95% or more, more preferably 98% or more, and still more preferably 99% or more, and responsiveness to the sulfide compound.
  • the olfactory receptor polypeptide used in the method of the present invention may be at least one selected from the olfactory receptor polypeptides described above, but may be a combination of any two or more.
  • OR4S2 is used.
  • the olfactory receptor polypeptide can be used in any form as long as it does not lose responsiveness to the sulfide compound.
  • the olfactory receptor polypeptide is a tissue or cell that naturally expresses the olfactory receptor polypeptide, such as an olfactory receptor or olfactory cell isolated from a living body, or a culture thereof; the olfactory receptor A membrane of an olfactory cell carrying a polypeptide; a recombinant cell or culture thereof genetically engineered to express the olfactory receptor polypeptide; a membrane of the recombinant cell having the olfactory receptor polypeptide; It can be used in the form of an artificial lipid bilayer membrane having the olfactory receptor polypeptide. All of these forms fall within the scope of the olfactory receptor polypeptide used in the present invention.
  • the olfactory receptor polypeptide is a cell that naturally expresses the olfactory receptor polypeptide, such as a mammalian olfactory cell, or genetically engineered to express the olfactory receptor polypeptide. It can be a recombinant cell, or a culture thereof. Preferred examples include recombinant human cells that have been genetically engineered to express the olfactory receptor polypeptide. The recombinant cell can be produced by transforming the cell using a vector incorporating a gene encoding the olfactory receptor polypeptide.
  • a gene encoding RTP (receptor-transporting protein) is introduced into the cell together with the gene encoding the olfactory receptor polypeptide.
  • a gene encoding RTP1S is introduced into a cell together with a gene encoding the olfactory receptor polypeptide.
  • RTP1S includes human RTP1S.
  • Human RTP1S is a protein consisting of an amino acid sequence represented by SEQ ID NO: 4 encoded by a gene having a nucleotide sequence represented by SEQ ID NO: 3 registered in GenBank as GI: 50234917.
  • the response of the olfactory receptor polypeptide to the sulfide compound 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, for example, measurement of intracellular cAMP amount.
  • the olfactory receptor is activated by an odor molecule, it is known to increase the amount of intracellular cAMP by activating adenylate cyclase in combination with intracellular G ⁇ s (Nat. Neurosci., 2004, 5: 263-278).
  • the response of the olfactory receptor polypeptide 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.
  • Other methods for measuring the response of the olfactory receptor polypeptide include calcium imaging.
  • Yet another method includes measurement by electrophysiological techniques.
  • a cell such as Xenopus oocyte
  • the olfactory receptor polypeptide is co-expressed with other ion channels
  • the activity of the ion channel on the cell is measured by a patch clamp method
  • the response of the olfactory receptor polypeptide is measured by measuring with a two-electrode membrane potential fixation method or the like.
  • the test substance that suppresses the response can be identified.
  • the effect of the test substance can be evaluated by comparing the response of the olfactory receptor polypeptide to which the test substance is added to the sulfide compound to the response to the sulfide compound in the control group.
  • the olfactory receptor polypeptide to which the test substance of different concentration was added the olfactory receptor polypeptide to which the test substance was not added, the olfactory receptor polypeptide to which the control substance was added, and the test substance were added.
  • the olfactory receptor polypeptide before the treatment, cells not expressing the olfactory receptor polypeptide, and the like can be mentioned.
  • the effect of the test substance on the response of the olfactory receptor polypeptide is between the test substance addition group of the higher concentration and the test substance addition group of the lower concentration, the test substance addition group and the non-addition group.
  • the test substance is a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound Can be identified as
  • the test substance When the response in the test substance addition group is suppressed as compared with the control group, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound.
  • the response of the olfactory receptor polypeptide in the test substance addition group measured by the above procedure is preferably 60% or less, more preferably 50% or less, and even more preferably 25% or less compared to the control group. If suppressed, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound.
  • the test substance is added to the sulfide compound. It can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to.
  • the test substance identified by the above procedure is a substance that can suppress the recognition of the odor of the sulfide compound by the individual by suppressing the response of the olfactory receptor to the sulfide compound. Therefore, the test substance identified by the above procedure can be selected as an inhibitor of sulfide compound odor.
  • the substance selected as the odor suppressor for sulfide compounds by the method of the present invention can suppress the odor of sulfide compounds by suppressing the response of olfactory receptors to sulfide compounds.
  • the substance selected by the method of the present invention may be an active ingredient of a sulfide compound odor inhibitor.
  • the substance selected by the method of this invention can be contained in the compound or composition for suppressing the smell of a sulfide compound as an active ingredient for suppressing the smell of a sulfide compound.
  • the substance selected by the method of the present invention can be used for the manufacture of a sulfide compound odor suppressor or for the manufacture of a compound or composition for suppressing the odor of a sulfide compound. .
  • the sulfide compound is represented by the following formula (I): R 1- [S] n -R 2 (I) (In the formula, R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5)
  • the method of [1] which is a compound represented by these.
  • R 1 and R 2 are Preferably, they are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms, More preferably, it is the same or different and represents methyl, ethyl, propyl or isopropyl, More preferably, R 1 and R 2 represent methyl, [2] The method according to the above.
  • n is preferably an integer of 1 to 3, more preferably 2 or 3.
  • OR4S2 is preferably a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
  • the polypeptide having at least 80% identity in amino acid sequence with OR4S2 is preferably 80% or more, more preferably 85% or more, and still more preferably, with the amino acid sequence represented by SEQ ID NO: 2. 90% or more, still more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more of an amino acid sequence, and a polypeptide having responsiveness to the sulfide compound.
  • the method according to any one of [1] to [7].
  • the method further comprises measuring a response to the sulfide compound in a control group, and the control group comprises: The olfactory receptor polypeptide to which the test substance has not been added; The olfactory receptor polypeptide to which a control substance is added; The olfactory receptor polypeptide before addition of the test substance; or a cell in which the olfactory receptor polypeptide is not expressed, Is that way.
  • test substance is added to the test substance. Identifying as a substance that suppresses the response of the olfactory receptor polypeptide to sulfide compounds; Further comprising a method.
  • the response of the olfactory receptor polypeptide to which the test substance is added to the sulfide compound is preferably 60% or less, more preferably 50% or less, and even more preferably 25% with respect to the response to the sulfide compound in the control group. Identifying the test substance as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound, if suppressed to: Further comprising a method.
  • At least one olfactory receptor polypeptide selected from the group consisting of the OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with the OR4S2 expresses the olfactory receptor polypeptide
  • the recombinant cell is Preferably, a cell into which a gene encoding the olfactory receptor polypeptide and a gene encoding RTP1S are introduced, More preferably, it is a cell into which a gene encoding the olfactory receptor polypeptide and a gene encoding human RTP1S are introduced.
  • At least one olfactory receptor polypeptide selected from the group consisting of the OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with the OR4S2, the above [14] or [15] The method according to any one of [1] to [13], wherein the recombinant cell or culture thereof is used.
  • the measurement of the response of the olfactory receptor polypeptide is measurement of intracellular cAMP amount by ELISA or reporter gene assay, measurement by calcium imaging, or electrophysiological measurement.
  • Example 1 Identification of Olfactory Receptor Responsive to Sulfide Compound (1) Cloning of Human Olfactory Receptor Gene Human olfactory receptor is a human genomic DNA female (G1521: Promega) based on sequence information registered in GenBank. Cloning was performed by PCR using a template. Each gene amplified by the PCR method is incorporated into a pENTR vector (Invitrogen) according to the manual, and NotI and AscI prepared 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.
  • pENTR vector Invitrogen
  • HEK293 cells expressing any one of 428 human olfactory receptors were prepared.
  • a reaction solution having the composition shown in Table 2 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).
  • HEK293 cells (3 ⁇ 10 5 cells / cm 2 ) were seeded in 90 ⁇ L each well, and cultured for 24 hours in an incubator maintained at 37 ° C. and 5% CO 2 .
  • cells (Mock) under conditions that do not express olfactory receptors were also prepared and used in the experiment.
  • Luciferase assay The olfactory receptor expressed in HEK293 cells is coupled with intracellular G ⁇ s to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP.
  • a luciferase reporter gene assay was used to monitor the increase in intracellular cAMP level as a luminescence value derived from the firefly luciferase gene (fluc2P-CRE-hygro).
  • a renilla luciferase gene fused with the CMV promoter downstream was simultaneously introduced, and used as an internal standard for correcting errors in gene transfer efficiency and cell number.
  • the medium was removed from the culture prepared in (3) above, and 75 ⁇ L of a solution containing a sulfide compound prepared with DMEM (Nacalai) was added.
  • the sulfide compound was dimethyl disulfide (DMDS) or dimethyl trisulfide (DMTS) 100 ⁇ M.
  • the cells were cultured for 3 hours in a CO 2 incubator to fully express the luciferase gene in the cells.
  • the luciferase activity was measured using a Dual-Glo TM luciferase assay system (Promega) according to the operation manual of the product.
  • a value fLuc / hRluc obtained by dividing the luminescence value derived from firefly luciferase by the luminescence value derived from Renilla luciferase was calculated.
  • a value obtained by dividing fLuc / hRluc derived from firefly luciferase induced by sulfide compound stimulation by fLuc / hRluc in cells not subjected to sulfide compound stimulation was calculated as a fold increase and used as an index of response intensity.
  • OR4S2 exhibits a specific response to both DMDS and DMTS (FIG. 1). ).
  • the response of OR4S2 to DMDS and DMTS was concentration dependent (FIG. 2).
  • OR4S2 also responded to dimethyl sulfide (DMS) at concentrations of 1 mM and 3 mM (FIG. 3).
  • DMS dimethyl sulfide
  • OR4S2 did not respond to 3 mM methyl mercaptan, which is also a volatile sulfur compound (FIG. 4). Therefore, OR4S2 is a sulfide compound receptor having responsiveness to various sulfide compounds.
  • OR4S2 is a novel sulfide compound receptor that has not been found to respond to sulfide compounds.
  • Example 2 Search for Sulfur Compound Odor Suppressor Based on Olfactory Receptor Response (1) Luciferase Assay HEK293 expressing OR4S2 (SEQ ID NO: 2) in the same procedure as in Examples 1 (1) to (3) Cells were made. According to the procedure of Example 1 (4), the response (fLuc / hRluc value) of the olfactory receptor to DMDS in the presence and absence of the test substance was measured by luciferase reporter gene assay.
  • fLuc / hRluc value (X ′) for DMTS single stimulation divided by fLuc / hRluc value (Y) of cells not subjected to DMTS stimulation (X ′ / Y) is DMTS response value (Fold). (increase).
  • the fLuc / hRluc value (Z ′) induced by co-stimulation of DMTS and the test substance was similarly compared with the fLuc / hRluc value (Y ) To calculate (Z ′ / Y) and set it as “Fold increment”.
  • the addition concentration of DMTS to the culture was 300 ⁇ M, and the addition concentration of the test substance was 100 ⁇ M.
  • Example 3 Inhibitory ability of sulfide compound odor by OR4S2 antagonist The inhibitory effect of sulfide compound odor by OR4S2 antagonist cis-4-heptenal identified in Example 2 and 1,4-cineole was confirmed by a sensory test. .
  • DMDS cis-4-heptenal, or 1,4-cineole
  • a 0.1% (v / v) solution of mineral oil was prepared.
  • DMTS a 0.01% (v / v) solution of mineral oil was prepared.
  • the glass bottle containing the cotton ball was covered and allowed to stand at 37 ° C. for 1 hour, and then used as a test sample for a sensory test.
  • a glass bottle containing only a cotton ball containing a solution of DMDS or DMTS as a reference sample and a glass bottle containing only a cotton ball containing mineral oil (Vehicle) as a target sample were prepared.
  • the sensory test was performed in a single blind manner by 10 evaluators for DMDS and 11 evaluators for DMTS. The test was started from 14:00 after 1 and a half hours had passed since the meal. In order to prevent the spread of odors, the test was basically performed near the draft. In order to eliminate the effect of adaptation to the odor of sulfide compounds, the perceived intensity of DMDS or DMTS odor was appropriately confirmed during the test, and a break was taken if necessary.
  • the evaluators were divided into two groups, one group in the order cis-4-heptenal and then 1,4-cineole, the other group in the order 1,4-cineole and then cis-4-heptenal in DMDS or The inhibitory effect of DMTS odor was evaluated. The test sample was replaced with a new one after evaluation by three evaluators.
  • each evaluator has the following five levels of criteria: “DMDS (or DMTS) odor is 1: unknown, 2: perceptible, 3: easy to understand, 4: feel strong, 5: endurable “I feel so strong” that the DMDS (or DMTS) odor intensity of DMDS (or DMTS) alone is 3, and the DMDS (or DMTS) odor intensity of each test sample in increments of 1.0 to 0.5.
  • the evaluation was made in nine stages up to zero. The average value of the evaluation results by each evaluator was obtained.
  • the results of the sensory test are shown in FIG.
  • the OR4S2 antagonists cis-4-heptenal and 1,4-cineole both suppressed the odor intensity of DMDS and DMTS. From the above results, it is clear that the odor of sulfide compounds such as DMDS and DMTS is suppressed by the OR4S2 antagonist, and therefore the odor inhibitor of sulfide compounds can be efficiently searched based on the response of OR4S2. It was done.

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Abstract

La présente invention permet d'identifier des substances qui suppriment les odeurs associées aux composés sulfurés. L'invention concerne un procédé d'évaluation et/ou de sélection d'un agent permettant de supprimer les odeurs associées aux composés sulfurés, le procédé comprenant les étapes consistant à ajouter une substance d'essai et un composé sulfuré au récepteur OR4S2 et à au moins un type de polypeptide des récepteurs olfactifs choisi au sein d'un groupe constitué de polypeptides présentant au moins 80 % d'identité avec OR4S2 au niveau d'une séquence d'acides aminés ; et à mesurer la réponse du polypeptide des récepteurs olfactifs au composé sulfuré.
PCT/JP2016/067894 2015-06-17 2016-06-16 Procédé d'évaluation et de sélection d'un agent permettant de supprimer les odeurs associées aux composés sulfurés WO2016204211A1 (fr)

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US15/737,079 US10585087B2 (en) 2015-06-17 2016-06-16 Method for evaluating and selecting agent for suppressing odors of sulfide compounds

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JP2016116227A JP6122181B2 (ja) 2015-06-17 2016-06-10 スルフィド化合物の臭いの抑制剤の評価又は選択方法
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WO2019131789A1 (fr) * 2017-12-27 2019-07-04 高砂香料工業株式会社 Procédé de criblage de substances qui suppriment l'odeur corporelle caractéristique des personnes âgées
JPWO2019131789A1 (ja) * 2017-12-27 2020-12-17 高砂香料工業株式会社 加齢臭抑制素材のスクリーニング方法
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WO2021149813A1 (fr) 2020-01-22 2021-07-29 Kao Corporation Composition de fragrance, distributeur de parfum et procédé de réduction des mauvaises odeurs
WO2023074766A1 (fr) * 2021-10-28 2023-05-04 味の素株式会社 Procédé de criblage d'une substance qui supprime l'odeur d'acide aminé et procédé de suppression d'odeur d'acide aminé

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