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    • 专利摘要:
    It is desired to identify a substance which inhibits an odor caused by 2,5-dimethyl-4-hydroxy-3 (2H) -furanone. The present invention provides a method of finding an inhibitor of an odor caused by 2,5-dimethyl-4-hydroxy-3 (2H) -furanone, comprising: adding a test substance and from 2,5-dimethyl-4-hydroxy-3 (2H) -furanone to an OR5K1 odor receptor or to a polypeptide having at least 80% amino acid sequence identity therewith; measuring the response of the olfactory receptor or the polypeptide to 2,5-dimethyl-4-hydroxy-3 (2H) -furanone; and identifying a test substance inhibiting the response of the odor receptor or polypeptide, based on the measured response.
    公开号:BE1022332B1
    申请号:E2014/5024
    申请日:2014-10-22
    公开日:2016-03-24
    发明作者:Aya Namba；Naoko Saito；Michiaki Inoue；Tsuyoshi Toyabe
    申请人:Kao Corporation；
    IPC主号:
    专利说明:

    METHOD FOR SEARCHING FOR AN INHIBITOR OF ODOR CAUSED BY THE
    furaneol
    Description [Field of the Invention] [0001]
    The present invention relates to a method of investigating an odor inhibitor caused by furanole.
    [Background of the invention]
    A large number of malodorous molecules that differ in their polarity or molecular weight are observed in our living environments. So far, various deodorization processes have been developed to deodorize these various malodorous molecules. In general, such deodorization processes are generally classified into biological processes, chemical processes, physical processes and sensory processes. Among the malodorous molecules, the strongly polar short-chain fatty acids or the amines can be deodorized by the chemical process, that is to say by a neutralization reaction. Sulfur compounds, such as a thiol, can be deodorized by the physical process, i.e. by adsorption treatment. However, there still remains a large number of smelly molecules that can not be processed by conventional deodorization processes. In addition, the problem of the deodorization process based on the adsorption treatment is that it tends to result in reissue of a bad odor. In addition, these conventional methods can even mask odors other than the bad smell of interest. Therefore, there is a demand for a deodorizing process capable of overcoming these problems.
    [0003]
    Another known method involves deodorizing a bad odor by greatly increasing the perception of a different odor with a fragrance. In this process, on the other hand, the smell of the fragrance can cause discomfort. In addition, it is necessary to search for an odoriferous substance which exhibits an effective deodorant effect on the malodorous substance of interest in order to mask the bad smell by a different smell, such as that of a perfume, a fragrance, or the like. So far, sensory tests have been conducted by experts to evaluate odors. Such sensory tests, on the other hand, are associated with certain problems such as the need to train experts able to evaluate odors and low yields. Therefore, previous research to identify an odorant that exhibits a deodorant effect has not been easy to implement.
    [0004]
    In mammals such as humans, the sense of smell works through a mechanism where odorant molecules bind to olfactory receptors on the olfactory nerve cells present in the olfactory epithelium, located in the upper region of the nasal cavity, in order to transmit the receptor responses thereto to the central nervous system. In humans, approximately 400 olfactory receptors have been identified and the genes encoding these receptors account for approximately 3% of all human genes. In general, a set of olfactory receptors is associated with a set of odorant molecules. This means that individual olfactory receptors may be responsive to a plurality of structurally similar odorant molecules with different affinities, while individual odorant molecules may be recognized by a plurality of olfactory receptors. According to another report, a scent molecule that activates a given olfactory receptor functions as an antagonist that inhibits the activation of a different olfactory receptor. These combined responses of a set of olfactory receptors allow the perception of individual odors.
    [0005]
    Accordingly, when a given odorant molecule coexists with a different odorant molecule, the different odorant molecule may inhibit the response of a receptor corresponding to the given odorant molecule, resulting in the final perception of a different odor. Such a mechanism is called antagonism of an olfactory receptor. Alteration of an odor caused by this receptor antagonism is a preferred deodorization approach since this approach, unlike deodorization processes which involve adding another odor, for example, a perfume, and fragrance, can specifically cancel the perception of a bad smell and causes no discomfort derived from such a fragrance.
    [0006]
    Bronzing agents (also called self-tanning or bronzing without sun) are cosmetic skin products that color the skin. Primarily, dihydroxyacetone (DHA) is used alone or in combination with erythrulose or the like as an ingredient that causes browning of the skin color. Such an ingredient reacts with the top layer of the skin to color the skin brown. Although it is believed that this coloration occurs through a browning reaction, the details of the underlying mechanism of this reaction have barely been elucidated. The browning reaction is also called the Maillard reaction in the field of food chemistry. This term refers to the reaction by which a nitrogen-containing compound, such as an amino acid or protein, is polymerized with a reduced sugar to form a brown polymer called melanoidin. The Maillard reaction is involved in the coloring of foods or the formation of flavor components caused by warming, etc. food.
    [0007]
    2,5-Dimethyl-4-hydroxy-3 (2H) -furanone (furanoleol) is a substance known to possess an odor expressed as a "strong aroma of fruity caramel", a "burnt caramel aroma", "Grilled sugar", "curry-type flavor", "cotton candy smell", etc. (Non-Patent Documents 1 to 4). Patent Document 1 discloses a beer-flavored beverage having a taste and flavor, or a scented odor, which is enhanced by the formation of maltol and furanol in undiluted fermentation solution.
    [List of citations] [Patent documents] [0008] [Patent document 1] WO 2009/078360 [Non-patent documents] [0009] [Non-patent document 1] Gosei Koryo - Kagaku To Shohin Chishiki - (Synthetic Flavor and Fragrance - Chemistry and Product Knowledge - 2005, The Chemical Daily Co., Ltd.
    [Non-Patent Document 2] J. Agric. Food Chem., 1997, 45 (6): 2217-2224 [Non-Patent Document 3] ACS Symp Ser., 2002, 836: 108-123 [Non-Patent Document 4] Anal Chim Acta., 2010, 657 (2): 198-203 [Summary of the Invention] [0010]
    The present invention provides: a method of searching for an odor inhibitor caused by 2,5-dimethyl-4-hydroxy-3 (2H) -furanone (furanoleol), comprising: adding an test substance and 2,5-dimethyl-4-hydroxy-3 (2H) -furanone (furanoleol) to an OR5K1 olfactory receptor or to a polypeptide having at least 80% amino acid sequence identity with it this ; measuring the response of the olfactory receptor or the polypeptide to 2,5-dimethyl-4-hydroxy-3 (2H) -furanone (furanoleol); and identifying a test substance inhibiting the olfactory receptor or polypeptide response, based on the measured response.
    [Brief description of the drawings] [0011] [Figure 1] Figure 1 shows the responses of olfactory receptors to furanol. The x-axis represents individual olfactory receptors. The y-axis represents the intensity of the response.
    [Figure 2] Figure 2 shows the response of an OR5K1 olfactory receptor to various concentrations of furanol. n = 3, error bar = ± ES.
    [Figure 3] Figure 3 shows the results of a sensory evaluation of the inhibitory effects of various compounds on furanol odor. n = 3, error bar = ± ES.
    [Detailed description of the invention] [0012]
    The term "masking" in relation to odors, as used herein, refers to a general approach to cancel or weaken the perception of the odor of interest. "Masking" can include chemical approaches, physical approaches, biological approaches, and sensory approaches. Examples of masking include: an arbitrary approach to remove an odorant molecule, responsible for the odor of interest, from the environment (for example, adsorption and chemical decomposition of the odor molecule); an approach to prevent the odor of interest from being released into the environment (eg, confinement); and a method that involves adding a different smell to an aroma, fragrance, or the like, to reduce the perception of the odor of interest.
    [0013]
    The term "olfactory receptor antagonistic masking" as used herein refers to a form of the aforementioned "masking" in the broad sense. This approach uses both an odor scent molecule of interest and a different scent molecule to thereby inhibit the response of a receptor to the scent molecule of interest by the different scent molecule, resulting in a modification of the smell that is perceived by the individuals. Olfactory receptor antagonistic masking is distinct from the approach of canceling the odor of interest by a strong odor different from a fragrance or the like, even though both approaches employ an odoriferous molecule different. An example of masking based on olfactory receptor antagonism is the case of the use of a substance, such as an antagonist, which inhibits the response of an olfactory receptor. A substance that inhibits the response of a receptor to an odorant molecule responsible for a particular odor can be applied to the receptor to thereby inhibit the receptor response to the odorant molecule. The odor that is ultimately perceived by individuals can be modified.
    [0014]
    The term "furanol" as used herein refers to 2,5-dimethyl-4-hydroxy-3 (2H) -furanone. The term "odor caused by furanole" may be an odor caused by 2,5-dimethyl-4-hydroxy-3 (2H) -furanone. "The odor caused by furanol" or "the odor caused by 2,5-dimethyl-4-hydroxy-3 (2H) -furanone" as used herein can typically be expressed, for example , as a caramel aroma or a smell of toasted sugar. In addition, "the odor caused by furanol" or "the odor caused by 2,5-dimethyl-4-hydroxy-3 (2H) -furanone", as used herein, may be an odor unpleasantness caused by the application of a conventional self-tanning agent to the skin, more specifically an unpleasant odor, expressed as "roasted sugar odor" or the like, generated by the application of a tanning agent containing dihydroxyacetone ( DHA) on the skin.
    [0015]
    The found problem of commercially available bronzing agents (self-tanning agents or bronzers without sun) is their unique unpleasant odor which is expressed as earthy smell, toasty sugar, etc. during the application (D. M. Hindenlang and M. E. McDonnell, Cosmetics & Toiletries magazine, 2008, 123, No. 7, pp. 67-74). Therefore, an improvement of these odors has been requested. Following an examination of the cause of the unpleasant odor, 2,5-dimethyl-4-hydroxy-3 (2H) -furanone (furanoleol), produced via methylglyoxal from the dihydroxyacetone (DHA) contained in the agents bronzers, turned out to be a causative substance. In order to reduce such an unpleasant odor attributed to furanol, it was necessary to control the smell of furanol.
    [0016]
    The present inventors have searched for an olfactory receptor that was sensitive to furanole and successfully identified the receptor. The present inventor has also discovered that a substance that inhibits the olfactory receptor response could inhibit the perception of odor caused by furanol by masking based on the antagonism of an olfactory receptor. Based on these findings, the present inventor has discovered that it is possible to search for a substance that controls an odor caused by furanol using the olfactory receptor response as indicated.
    [0017]
    In this specification, sequence identity between nucleotide sequences or amino acid sequences is calculated according to the Lipman-Pearson method (Science, 1985, 227: 1435-41). Specifically, the sequence identity is computed by an analysis using the Geneticsx-Win Genetic Information Processing Software Geneticsx-Win (Figure 5.1.1; Software Development Co., Ltd.).
    [0018]
    As shown in Figure 1, the present inventor has identified an OR5K1 olfactory receptor as the sole furaneol-sensitive receptor among many olfactory receptors. OR5K1 is a new furanol receptor that, until now, has not been identified as being sensitive to furanole. As shown in Figure 2, OR5K1 is furaneol-sensitive in a concentration-dependent manner. Therefore, a substance that inhibits the OR5K1 response may alter the perception of furanol-induced odor in the central nervous system by olfactory receptor antagonism, resulting in selective inhibition of the smell caused by furanol.
    [0019]
    According to the present invention, an odor inhibitor caused by furanol can be effectively sought. The furanol odor inhibitor identified by the present invention can selectively deodorize the odor caused by furanol by masking based on the antagonism of an olfactory receptor. Therefore, the odor inhibitor caused by furanol, identified by the present invention, can deodorize the odor caused by furanole, for example an unpleasant odor generated by the application of conventional self-tanning agents (also known as furanols). bronzers without sun), without causing problems such as a nuisance deriving from the scent of a fragrance in conventional deodorizing processes using a deodorant or a fragrance.
    [0020]
    Therefore, the present invention provides a method of searching for an odor inhibitor caused by furanol. This method comprises: adding a test substance and furanol to an OR5K1 olfactory receptor; measuring the response of the olfactory receptor to furanole; and identifying a test substance that inhibits the olfactory receptor response, based on the measured response. The identified test substance is selected as an odor inhibitor caused by furanole. The method of the present invention may be a method performed in vitro or ex vivo.
    [0021]
    In the method of the present invention, a test substance and the furanol substance responsible for the odor are added to an OR5K1 olfactory receptor. A commercially available product (eg, FURANEOL (R), Nihon Firmenich K.K.) may be purchased and used as furanol.
    [0022]
    The test substance used in the process of the present invention is not particularly limited as long as it is desirable to use the substance as an odor inhibitor caused by furanole. The test substance may be a naturally occurring substance or may be a substance artificially synthesized by a chemical or biological process or the like. Alternatively, the test substance may be a compound, a composition, or a mixture.
    [0023]
    The OR5K1 olfactory receptor used in the method of the present invention refers to an olfactory receptor expressed on a human olfactory cell that is registered as Gl: 115270955 in GenBank. OR5K1 is encoded by a gene having the nucleotide sequence represented by SEQ ID NO: 1. This protein consists of the amino acid sequence represented by SEQ ID NO: 2.
    [0024]
    In the method of the present invention, the olfactory receptor OR5K1 may be used in an arbitrary form unless the form loses its sensitivity to furanol. For example, the olfactory receptor may be used in a form comprising: tissues or cells naturally expressing the olfactory receptor, such as an osmoreceptor or olfactory cells isolated from an organism, or cultures thereof; olfactory cell membranes comprising the olfactory receptor; recombinant cells genetically engineered to express the olfactory receptor, or cultures thereof; membranes of recombinant cells comprising the olfactory receptor; and artificial lipid bilayers having the olfactory receptor. These forms are all included in the scope of the olfactory receptor used in the present invention.
    [0025]
    In a preferred aspect, a cell naturally expressing the olfactory receptor, such as an olfactory cell, or a recombinant cell genetically engineered to express the olfactory receptor, or cultures of any of these cells, are used as the olfactory receptor OR5K1 . The recombinant cell may be prepared by transforming a cell with a vector having an insert of the gene encoding the olfactory receptor.
    [0026]
    Preferably, a gene for a receptor transport protein (RTP), with the olfactory receptor gene, is transferred into the cell to promote expression of the olfactory receptor on the cell membrane. Preferably, an RTP1S gene, more preferably RTP1S and RTP2 genes, with the olfactory receptor gene, are transferred into the cell. Examples of RTP1S and RTP2 include human RTP1S and human RTP2, respectively. Human RTP1S is registered as Gl: 50234917 in GenBank. This protein is encoded by a gene having the gene sequence represented by SEQ ID NO: 3 and consists of the amino acid sequence represented by SEQ ID NO: 4. Human RTP2 is registered as Gl: 258547120 in GenBank. This protein is encoded by a gene having the gene sequence represented by SEQ ID NO: 5 and consists of the amino acid sequence represented by SEQ ID NO: 6.
    [0027]
    Alternatively, a polypeptide which consists of an amino acid sequence having a sequence identity of at least 78%, e.g. 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more, with the amino acid sequence (SEQ ID NO: 4) of human RTP1S or the amino acid sequence (SEQ ID NO: 6) of human RTP2, which promotes the expression of the olfactory receptor on the cell membrane, as with human RTP1S or RTP2, can be used in place of RTP1S or Human RTP2. For example, a variant of human RTP1S which is encoded by a gene having the gene sequence represented by SEQ ID NO: 7 and consists of the amino acid sequence represented by SEQ ID NO: 8, has 78.9% of sequence identity with the amino acid sequence of human RTP1S represented by SEQ ID NO: 4, and has the function of promoting the expression of the olfactory receptor on the cell membrane. Alternatively, murine RTP1S (Sei Signal., 2009, 2: ra9) also has 89% sequence identity with the amino acid sequence of human RTP1S represented by SEQ ID NO: 4 and has the function of promoting the expression of the olfactory receptor on the cell membrane. Such a variant of human RTP1S and murine RTP1S can be used in place of human RTP1S in the preparation of the recombinant cell expressing the olfactory receptor as mentioned above. Alternatively, a variant RTP1S polypeptide which has an amino acid sequence identity of at least 80%, for example 80% or more, preferably 85% or more, more preferably 90% or more, always more preferably 95% or more, even more preferably 98% or more, still more preferably 99% or more, with the variant of human RTP1S consisting of the amino acid sequence represented by SEQ ID NO : 8 or murine RTP1S, which promotes the expression of the olfactory receptor on the cell membrane, can also be used in place of human RTP1S in the preparation of the recombinant cell expressing the olfactory receptor as mentioned above.
    [0028]
    According to the method of the present invention, the response of the OR5K1 olfactory receptor to furanoleol is measured following the addition of the test substance and furanol to the olfactory receptor. This measurement can be performed by an arbitrary method known in the art, as a method for measuring the response of the olfactory receptor, for example, measuring an intracellular cAMP level. For example, it is known that the olfactory receptor, when activated by an odor molecule, is conjugated with an intracellular gas to activate adenylate cyclase, thereby increasing intracellular cAMP levels (Mombaerts P. Nat Neurosci 5, 263-278). Therefore, the level of intracellular cAMP following the addition of the odorant molecule can be used as an indicator for measuring the olfactory receptor response. Examples of methods for measuring cAMP level include an ELISA and reporter gene assay. Another example of the method for measuring the olfactory receptor response comprises calcium imaging.
    [0029]
    Next, the effect of the test substance on the furanole response is evaluated based on the measured olfactory receptor response to identify a test substance that inhibits the response. The evaluation of the effect of the test substance can be performed, for example, by comparing the furanol receptor responses measured in the presence of various concentrations of the test substance. As a more specific example, furanol receptor responses are compared between a group exposed to a higher concentration of the test substance and a group exposed to a lower concentration of the test substance, between a group exposed to the test substance and a group not exposed to the test substance, between a group exposed to the test substance and a group exposed to a control substance, or between before and after the addition of the test substance. When the addition of the test substance or the addition of a higher concentration of the test substance inhibits the response of the receptor, that test substance may be identified as a substance that inhibits the response of the olfactory receptor to furanol.
    [0030]
    In the method of the present invention, a polypeptide having a function equivalent to OR5K1 can be used as an olfactory receptor instead of OR5K1. Examples of the polypeptide include a polypeptide which consists of an amino acid sequence having a sequence identity of at least 80%, e.g. 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more, with the OR5K1 amino acid sequence (SEQ ID NO: 2) , and who is sensitive to furanole.
    [0031]
    Alternatively, in the process of the present invention, IOR5K1 mentioned above as an olfactory receptor and polypeptides having a function equivalent thereto may each be used alone or may be used in a combination of both thereof or more.
    [0032]
    The test substance identified by the above procedures is a substance that can inhibit an individual's perception of an odor caused by furanole by inhibiting the olfactory receptor response to furanol. Therefore, the test substance identified by the above procedures is selected as an odor inhibitor caused by furanol. For example, when the response of the receptor, in a group exposed to the test substance and measured by the above procedures, is preferably reduced to 60% or less, more preferably to 50% or less, still preferably 25% or less of that observed in a group not exposed to the test substance (for example, the group mentioned above not exposed to the test substance, a group exposed to a control substance, or before addition of the test substance), the test substance can be selected as an odor inhibitor caused by furanol.
    [0033]
    The substance selected by the method of the present invention can inhibit the odor caused by furanol by olfactory masking based on the inhibition of the olfactory receptor response to furanol.
    [0034]
    Therefore, in one embodiment, the substance selected by the method of the present invention can serve as an active ingredient in an odor inhibitor caused by furanol. Alternatively, the substance selected by the process of the present invention may be contained, as an active ingredient for inhibiting an odor caused by furanole, in a compound or composition for inhibiting an odor caused by furanole. . Alternatively, the substance selected by the method of the present invention can be used for the production of an odor inhibitor caused by furanol or for the production of a compound or composition for inhibiting an odor caused by furanol.
    [0035]
    In one embodiment, the substance selected by the method of the present invention can be used as an active ingredient to inhibit an odor caused by furanol, e.g., a caramel aroma, a sweet toasted odor, or an odor. unpleasant (for example, a smell of roasted sugar), generated by the application of a self-tanning agent on the skin or following the application of a product containing DHA.
    [0036]
    In one embodiment, the substance selected by the method of the present invention can be used as an active ingredient to inhibit an odor caused by furanol in each compound or composition for which it is desired to inhibit an odor caused by the furanole, or in any environment where it is desired to inhibit an odor caused by furanole. Alternatively, the substance selected by the process of the present invention can be used as an active ingredient to inhibit an odor caused by furanole, for the production of a compound or composition for which it is desired to inhibit an odor caused by furanol. Examples of the compound or composition for which it is desired to inhibit an odor caused by furanol include tanning agents (also called sunless tanning or bronzing agents), for example, a tanning agent containing DHA as a tanning agent. coloring agent and other bronzing agents that use a browning reaction, and other products containing DHA. Furanole contained in excess in a food or drink may impart a bad aroma. For example, the presence of excess furanol in powdered milk reduces its flavor or taste. Therefore, other examples of the compound or composition for which it is desired to inhibit an odor caused by furanol include foods or beverages for which it is desired to reduce the odor of furanol, as well as compositions containing food or drink.
    [0037]
    The following composition, method of production, use, or process will be further described herein as an exemplary embodiment of the present invention. In contrast, the present invention is not intended to be limited by these embodiments.
    A method of searching for an odor inhibitor caused by furanole, comprising: adding a test substance and furanol to an OR5K1 olfactory receptor or a polypeptide having at least 80% amino acid sequence identity therewith; measuring the response of the olfactory receptor or the furanoleol polypeptide; and identifying a test substance inhibiting the olfactory receptor or polypeptide response, based on the measured response.
    [0039] <2> Method according to <1>, wherein the olfactory receptor OR5K1 is a protein consisting of the amino acid sequence represented SEQ ID NO: 2.
    [0040] <3> A method according to <1> or <2>, wherein the polypeptide having at least 80% amino acid sequence identity with the OR5K1 olfactory receptor is a polypeptide preferably having at least 85%, more preferably at least 90%, still more preferably at least 95%, still more preferably at least 98%, still more preferably at least 99% amino acid sequence identity with the OR5K1 olfactory receptor.
    A method according to any one of <1> to <3>, wherein the polypeptide having at least 80% amino acid sequence identity with the OR5K1 olfactory receptor is a polypeptide that exhibits preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, still more preferably at least 98%, still more preferably at least 99% sequence identity of amino acids with the olfactory receptor OR5K1 and which is sensitive to furanole.
    A method according to any one of <1> to <4>, wherein, preferably, the OR5K1 olfactory receptor or the polypeptide having at least 80% amino acid sequence identity with that it is expressed on a recombinant cell genetically modified to express the olfactory receptor or the polypeptide.
    [0043] <6> A method according to <5>, wherein, preferably, the recombinant cell is the following cell: a cell co-transfected with an olfactory receptor gene or a polypeptide and a RTP1S gene; a cell co-transfected with an olfactory receptor gene or a polypeptide and the RTP1S and RTP2 genes; a cell cotransfected with an olfactory receptor gene or polypeptide and a gene encoding a polypeptide which consists of an amino acid sequence having a sequence identity of at least 78%, preferably 80% or more, of more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more, with the sequence amino acid represented by SEQ ID NO: 4 and which promotes the expression of the olfactory receptor on the membrane, as with human RTP1S; or a cell cotransfected with an olfactory receptor gene or polypeptide and a gene encoding a variant of human RTP1S.
    [0044] <7> A method according to <5> or <6>, wherein, preferably, cultures of the recombinant cell are used as olfactory OR5K1 receptor or polypeptide having at least 80% sequence identity of amino acids with this one.
    A method according to any one of <1> to <7>, further preferably comprising measuring the response of the olfactory receptor or polypeptide in the absence of the test substance.
    [0046] <9> Method according to <8>, furthermore preferably comprising the following elements: when the response of the olfactory receptor or the polypeptide in the presence of the test substance is reduced compared to the response of the olfactory receptor or the polypeptide in the absence of the test substance, the test substance is identified as a substance that inhibits the furaneol receptor or polypeptide response.
    [0047] <10> A process according to <8>, further preferably comprising the following: when the response of the olfactory receptor or the polypeptide in the presence of the test substance is preferably reduced to 60% or less, more preferably preferred to 50% or less, more preferably 25% or less, of the olfactory receptor or polypeptide response in the absence of the test substance, the test substance is identified as a substance that inhibits the receptor response or furanole polypeptide.
    A method according to any one of <1> to <10>, wherein the step of measuring the response of the olfactory receptor or the polypeptide is the measurement of an intracellular cAMP level by a ELISA or reporter gene assay, or calcium imaging.
    [Examples] [0049]
    Hereinafter, the present invention will be described more specifically with reference to the Examples.
    [0050]
    Example 1 Identification of a furanol-sensitive olfactory receptor 1) Cloning of a human olfactory receptor gene
    Based on the sequence information recorded in GenBank, each human olfactory receptor gene was cloned by PCR with a Human Genomic DNA Female kit (G1521: Promega Corp.) as a template. Each gene thus amplified by PCR was inserted into a pENTR vector (Invitrogen Corp.) according to the manual. A NotI-Ascl site present on the pENTR vector was recombined in a NotI-AScl site prepared downstream of a Flag-Rho marker sequence on a pME18S vector.
    [0051] 2) Preparation of the human pME18S-RTP1S vector
    A human RTP1S gene (SEQ ID NO: 3) encoding human RTP1S (SEQ ID NO: 4) was inserted into the EcoRI-XhoI site of the pME18S vector.
    3) Preparation of a cell expressing an olfactory receptor
    HEK293 cells expressing each of the 400 types of human olfactory receptors were prepared. Each reaction solution having the composition shown in Table 1 was prepared, then allowed to stand for 15 minutes on a clean bench, and then added to each well of a 96-well plate (Becton, Dickinson and Company). ). Then, the HEK293 cells (3 × 10 5 cells / cm 2) were inoculated therein at 100 μl / well and then cultured at 37 ° C. for 24 hours in an incubator maintained at 5% CO 2.
    [Table 1]
    [0054] 4) Luciferase assay
    The olfactory receptors expressed on HEK293 cells are conjugated with intracellular gas to activate adenylate cyclase, thereby increasing intracellular cAMP levels. In this study, their responses to furanole were measured using a luciferase reporter gene assay that involved monitoring the increase in intracellular cAMP level as a luminescence intensity derived from a luciferase gene of the present invention. firefly (fluc2P-CRE-hygro). In addition, the cells were co-transfected with the reporter gene and a fusion gene (hRluc-CMV) of a Renilla luciferase gene downstream of a CMV promoter to serve as an internal standard for correcting the disease. transfection efficiency or an error in the number of cells.
    An environment was removed from the crops prepared in the previous paragraph 3). 75 μl of a solution containing furanoleol (3 mM) prepared with CD293 medium (Invitrogen Corp.) was added to the resulting cultures. The cells were cultured for 2.5 hours in a CO2 incubator to express the luciferase genes at sufficient levels in the cells. Luciferase activity was measured using a Dual-Glo (TM) luciferase assay system (Promega Corp.) according to the product's instruction manual. The intensity of luminescence derived from firefly luciferase induced by stimulation with furanoleol was divided by the intensity of luminescence in cells without stimulation by furanoleol to calculate a value, expressed as an increase. at times, which in turn was used as an indication of the intensity of the response.
    [0055] 5) Results
    Following measurements of the responses of the 400 furanol olfactory receptor types (3 mM), only the OR5K1 olfactory receptor exhibited a response to furanole (Figure 1). OR5K1 is a new furanol receptor that, until now, has not been identified as being sensitive to furanole.
    [0056]
    Example 2 Concentration-dependent response of OR5K1 to furanole
    The olfactory receptor OR5K1 (SEQ ID NO: 2) was expressed, with human RTP1S (SEQ ID NO: 4), on HEK293 cells by the same procedures as those of Example 1, and then was examined for concentration -dependence of its response to various concentrations of furanol (0, 3, 10, 30, 100, 300, 1000 and 3000 μΜ). As a result, OR5K1 exhibited a concentration-dependent response to furanole (Figure 2).
    [0057]
    Example 3 Identification of an OR5K1 Antagonist 84 types of test substances were examined for their antagonistic activity on the OR5K1 olfactory receptor response to furanole.
    Furanole (3 mM) and each test substance (100 μl) were added to HEK293 cells in which OR5K1 olfactory receptor expression was obtained using the same procedures as in Example 2. The response of Olfactory receptor was measured to evaluate a change in receptor response caused by the addition of the test substance.
    The rate of inhibition of the receptor response by the test substance was calculated as follows: the intensity of luminescence derived from firefly luciferase (Y) in cells expressing the olfactory OR5K1 receptor without stimulation by furanol was subtracted from the luminescence intensity derived from firefly luciferase (X) induced by furanole-only stimulation to determine receptor (X-Y) activity based on stimulation with furaneol only. Similarly, the intensity of luminescence (Y) in cells without stimulation with furanol was subtracted from the intensity of luminescence (Z) induced by stimulation with the furanol mixture and the test substance so to determine the activity of the (Z - Y) receptor in the presence of the test substance. The rate of reduction of receptor (Z - Y) activity in the presence of the test substance with respect to receptor (X - Y) activity based on stimulation with furanoleol only was calculated according to a formula. shown below to determine the rate of inhibition of the receptor response by the test substance. In this test, a plurality of duplicate independent experiments was performed. An average of the experiments in each analysis session was obtained.
    As a result, 17 types of test substances had an inhibition rate of OR5K1 response to furanole of 40% or more (which reduced the response to 60% or less), demonstrating that these test substances exhibit antagonistic activity against OR5K1 (Table 2).
    [Table]
    [0059]
    Example 4 Evaluation of the ability of an OR5K1 antagonist to inhibit furanoleol odor The ability to inhibit the furanol odor of each test substance exhibiting OR5K1 antagonistic activity, identified in Example 3, was confirmed by a sensory test.
    Each panel member felt a tissue containing 0.5 g of furanol (1%) to which 0.5 μΙ of a flavor was added, and then assessed the intensity of the furanol odor compared to a fabric to which no aroma had been added. The sensory evaluation test was performed by 3 members of a panel. The odor of furanol was rated at 1 when it was highly noticeable and at 5 when it was imperceptible.
    As a result, all 17 types of test substances that demonstrated that they inhibited the OR5K1 response to furanol in Example 3 inhibited the furanol odor (Figure 3).
    权利要求:
    Claims (10)
    [1]
    A method of searching for an odor inhibitor caused by 2,5-dimethyl-4-hydroxy-3 (2H) -furanone, comprising: adding a test substance and 2,5-dimethyl-4-hydroxy-3 (2H) -furanone; dimethyl-4-hydroxy-3 (2H) -furanone having an olfactory OR5K1 receptor or a polypeptide having at least 80% amino acid sequence identity therewith; measuring the response of the olfactory receptor or the polypeptide to 2,5-dimethyl-4-hydroxy-3 (2H) -furanone; and identifying a test substance inhibiting the olfactory receptor or polypeptide response, based on the measured response.
    [2]
    The method of claim 1, wherein the olfactory receptor OR5K1 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
    [3]
    The method according to claim 1 or 2, wherein the polypeptide having at least 80% amino acid sequence identity with the OR5K1 olfactory receptor is a polypeptide which consists of an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO: 2 and is sensitive to 2,5-dimethyl-4-hydroxy-3 (2H) -furanone.
    [4]
    The method according to any one of claims 1 to 3, wherein the OR5K1 olfactory receptor or the polypeptide having at least 80% amino acid sequence identity therewith is expressed on a recombinant cell genetically modified to express the olfactory receptor or the polypeptide.
    [5]
    The method of any one of claims 1 to 4, further comprising measuring the response of the olfactory receptor or polypeptide in the absence of the test substance.
    [6]
    The method of claim 5, wherein when the response of the olfactory receptor or polypeptide in the presence of the test substance is reduced to 60% or less of the olfactory receptor or polypeptide response in the absence of the test substance. test, the test substance is identified as a substance that inhibits the olfactory receptor or polypeptide response to 2,5-dimethyl-4-hydroxy-3 (2H) -furanone.
    [7]
    The method of any one of claims 1 to 6, further comprising measuring the response of the olfactory receptor or polypeptide in the absence of the test substance.
    [8]
    The method of claim 7, further comprising the following: when the response of the olfactory receptor or the polypeptide in the presence of the test substance is reduced compared to the response of the olfactory receptor or polypeptide in the absence of the test substance. test substance, the test substance is identified as a substance that inhibits the response of the receptor or polypeptide to 2,5-dimethyl-4-hydroxy-3 (2H) -furanone.
    [9]
    The method according to any one of claims 1 to 8, wherein the step of measuring the response of the olfactory receptor or the polypeptide is the measurement of an intracellular cAMP level by an ELISA or reporter gene assay, or calcium imaging.
    [10]
    The method according to any one of claims 1 to 9, wherein cultures of the recombinant cell that is genetically engineered to express the olfactory receptor or polypeptide are used as an olfactory OR5K1 receptor or polypeptide having at least 80% d amino acid sequence identity with it.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2012169644A1|2011-06-06|2012-12-13|Kao Corporation|Method for identifying a malodor inhibitor|
    JP2012250958A|2011-06-06|2012-12-20|Kao Corp|Malodor inhibiting agent|
    US20130210775A1|2012-02-09|2013-08-15|Kao Corporation|Agent for inhibiting odor of pyrazine derivatives|
    US7344845B2|2001-12-21|2008-03-18|Senomyx, Inc.|Olfactory receptor for isovaleric acid and related malodorants and use thereof in assays for identification of blockers of malodor|
    WO2009078360A1|2007-12-14|2009-06-25|Suntory Holdings Limited|Beer taste drink with high maltol and furaneol contents|
    BR102012002838A2|2012-02-08|2013-10-22|Amaral Assy Jose Roberto Do|SEED SELECTOR AND DRIVER SYSTEM FOR SEED DOSER DEVICE EMPLOYED IN FARMING EQUIPMENT|
    US9057090B2|2012-02-09|2015-06-16|Kao Corporation|Method of identifying an agent for inhibiting odor of pyrazine derivatives|
    JP6243698B2|2013-10-22|2017-12-06|花王株式会社|Odor control by furaneol|
    JP6203598B2|2013-10-22|2017-09-27|花王株式会社|Sotron Smell Suppressor|
    JP6393505B2|2014-04-14|2018-09-19|花王株式会社|Search method for raw dry odor control agent|
    JP6395426B2|2014-04-14|2018-09-26|花王株式会社|Search method for butyl acrylate odor suppressor|
    JP6422665B2|2014-04-14|2018-11-14|花王株式会社|Search method for odor control agent|JP6243698B2|2013-10-22|2017-12-06|花王株式会社|Odor control by furaneol|
    JP6203598B2|2013-10-22|2017-09-27|花王株式会社|Sotron Smell Suppressor|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP2013-219336|2013-10-22|
    JP2013219336A|JP6276555B2|2013-10-22|2013-10-22|Search method of odor control agent by furaneol|
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