Method for screening for substance that suppresses amino acid odor and method for suppressing amino acid odor

Abstract

The present invention provides a technique for suppressing amino acid odors. Olfactory receptors OR4S2 and / or OR4E2 are used to screen for a substance that suppresses amino acid odors. The substance that has been screened is used to suppress amino acid odors in food products.

Description

Screening method for substances that suppress amino acid odor and method for suppressing amino acid odor

[0001] The present invention relates to a technique for suppressing amino acid odor. Specifically, the present invention can relate to a method for screening for a substance that suppresses amino acid odor and a method for suppressing amino acid odor.

[0002] Amino acids are widely used as ingredients in foods such as health foods and nutritional supplements. However, increasing the amino acid content in foods can lead to off-flavors such as bitterness and amino acid odor (also known as "heat-deteriorated odor" or "muddy odor"). Food ingredients have been developed to suppress such off-flavors, but their effectiveness is insufficient.

[0003] Additionally, methods for screening for substances that suppress specific odors using olfactory receptors have been reported, including a method for screening for substances that suppress the odor of sulfide compounds using the olfactory receptor OR4S2 (Patent Document 1), a method for screening for substances that suppress the odor of methyl mercaptan using the olfactory receptor OR4S2 (Patent Document 2), and a method for screening for substances that suppress the odor of indole using the olfactory receptor OR4S2 or OR1A1 (Patent Document 3).

[0004] Furthermore, techniques for suppressing unpleasant odors such as those caused by proteins or their degradation products and the light-degradation odor of fruit juice-containing products have been reported (Patent Documents 4 and 5). Patent Documents 4 and 5 exemplify components such as methional as a causative agent of such unpleasant odors. Patent Document 5 exemplifies components such as sclareolide as a masking agent for (E,E)-2,4-nonadienal, which is one of the causative agents of the light-degradation odor of fruit juice-containing products.

[0005] Patent Publication No. 2017-006122, Patent Publication No. 2020-010629, Patent Publication No. 2021-069330, WO2019 / 039490, Patent Publication No. 2019-170375

[0006] An object of the present invention is to provide a technique for suppressing amino acid odor. Specifically, an object of the present invention is to provide a method for screening for a substance that suppresses amino acid odor and a method for suppressing amino acid odor.

[0007] As a result of intensive research to solve the above problems, the inventors discovered that the substance responsible for the amino acid odor is methional, that the olfactory receptors OR4S2 and OR4E2 respond to methional, and that the methional odor can be suppressed by various components screened using the inhibition of the response of the olfactory receptors as an indicator, thereby completing the present invention.

[0008] That is, the present invention can be exemplified as follows. [1] A method for screening for a substance that suppresses amino acid odor, comprising the following steps (A) to (C): (A) contacting an olfactory receptor with an olfactory receptor activator in the presence of a test substance; (B) measuring the response of the olfactory receptor to the olfactory receptor activator; and (C) identifying the test substance as a substance that suppresses amino acid odor based on the response; wherein the test substance is identified as a substance that suppresses amino acid odor when the response is inhibited by the test substance, and the olfactory receptor is OR4S2 and / or OR4E2. [2] The method wherein the response is activation of the olfactory receptor. [3] The method wherein the olfactory receptor is used in a form supported by a cell, a cell membrane, an artificial lipid bilayer vesicle, or an artificial lipid bilayer membrane. [4] The method wherein the olfactory receptor is used in a form supported by a cell. [5] The method wherein the cell is an animal cell. [6] The method, wherein steps (B) and (C) are carried out by the following steps (B1) and (C1), respectively: (B1) measuring the degree of activation D1 of the olfactory receptor when step (A) is carried out; (C1) identifying the test substance as a substance that suppresses amino acid odor based on the degree of activation D1. [7] The method, wherein step (C1) is carried out by the following step (C2): (C2) identifying the test substance as a substance that suppresses amino acid odor based on the difference between the degree of activation D1 and the degree of activation D2 of the olfactory receptor under control conditions. [8] The method described above, wherein the control condition is the following condition (C2-1) or (C2-2): (C2-1) a condition in which the olfactory receptor is contacted with the olfactory receptor activator in the absence of the test substance; or (C2-2) a condition in which the olfactory receptor is contacted with the olfactory receptor activator in the presence of the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in step (A). [9] The method described above, further comprising a step of measuring the degree of activation D2.

[10] The method described above, wherein the test substance is identified as a substance that suppresses amino acid odor when the degree of activation D1 is lower than the degree of activation D2.

[11] The method, wherein the test substance is identified as a substance that suppresses amino acid odor when the ratio of the degree of activation D1 to the degree of activation D2 is less than 60%.

[12] The method, wherein the response is measured using intracellular cAMP concentration as an index.

[13] The method, wherein the intracellular cAMP concentration is measured by a reporter assay.

[14] The method, wherein the olfactory receptor is a human olfactory receptor.

[15] The method, wherein the OR4S2 is a protein described in (a), (b), or (c) below: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence set forth in SEQ ID NO: 2, and which is responsive to the olfactory receptor activator; (c) a protein comprising an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, and which is responsive to the olfactory receptor activator.

[16] The method, wherein the OR4E2 is a protein described in (a), (b), or (c) below: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 4; (b) a protein comprising an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4, but with a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues, and which is responsive to the olfactory receptor activator; (c) a protein comprising an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 4, and which is responsive to the olfactory receptor activator.

[17] The method, further comprising a step of evaluating whether the identified substance that suppresses amino acid odors has the function of suppressing amino acid odors.

[18] The method, wherein the evaluation is performed by sensory evaluation.

[19] The method, wherein the amino acid odor is methional odor.

[20] A composition for suppressing amino acid odors in foods, the composition comprising the following component (A): (A) a component that inactivates the olfactory receptors OR4S2 and / or OR4E2, the component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[21] A composition for use in producing a food product, comprising the following component (A): (A) a component that inactivates the olfactory receptor OR4S2 and / or OR4E2, the component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[22] The composition, wherein the food product has a suppressed amino acid odor.

[23] The composition, wherein the food product contains a component that exhibits an amino acid odor and / or a component that can produce a component that exhibits an amino acid odor.

[24] The composition, wherein the component exhibiting an amino acid odor is methional.

[25] The composition, wherein the component capable of producing the component exhibiting an amino acid odor is methionine.

[26] The composition, wherein the amino acid odor is methional.

[27] A method for suppressing an amino acid odor in food, the method comprising the step of adding the following component (A) to a raw material of the food: (A) a component that inactivates the olfactory receptor OR4S2 and / or OR4E2, the component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[28] A method for producing a food product, the method comprising the step of adding the following component (A) to a raw material of the food product: (A) a component that inactivates the olfactory receptor OR4S2 and / or OR4E2, the component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[29] The method as described above, wherein the food product has a suppressed amino acid odor.

[30] The method described above, wherein the food or raw material contains a component that exhibits an amino acid odor and / or a component that can produce a component that exhibits an amino acid odor.

[31] The method described above, wherein the component that exhibits an amino acid odor is methional.

[32] The method described above, wherein the component that can produce a component that exhibits an amino acid odor is methionine.

[33] The method described above, wherein the amino acid odor is a methional odor.

[34] The method described above, wherein the food or raw material contains a component that can produce the component that exhibits an amino acid odor.

[35] The method described above, wherein component (A) is added so that its consumption concentration is 0.001 ppt (w / w) to 10 ppm (w / w).

[36] The aforementioned method, wherein 2-methyl-3-furanthiol is added to give an ingestible concentration of 0.001 ppt (w / w) to 100 ppt (w / w),

[37] The aforementioned method, wherein bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, 2,4-decadienal, or 2,4-nonadienal is added to give an ingestible concentration of 0.01 ppt (w / w) to 1 ppb (w / w),

[38] The aforementioned method, wherein furfuryl mercaptan or E-β-damascone is added to give an ingestible concentration of 0.1 ppt (w / w) to 10 ppb (w / w).

[39] The aforementioned method, wherein 2,5-dihydroxy-1,4-dithiane, 2-phenyl-2-butenal, or 2-thiophenethiol is added to give an ingestible concentration of 1 ppt (w / w) to 100 ppb (w / w).

[40] The aforementioned method, wherein nootkatone, allyl isothiocyanate, or cinnamaldehyde is added to give an ingestible concentration of 10 ppt (w / w) to 1 ppm (w / w).

[41] The aforementioned method, wherein sclareolide is added to give an ingestible concentration of 100 ppt (w / w) to 10 ppm (w / w).

[0009] The present invention will be described in detail below.

[0010] <1> Screening for Substances That Inhibit Amino Acid Odors A first aspect of the method of the present invention is a screening method for substances that inhibit amino acid odors using olfactory receptors. This first aspect of the method of the present invention is also referred to as the "screening method of the present invention." In the screening method of the present invention, olfactory receptors can be used to identify substances that inhibit amino acid odors (i.e., to identify whether a test substance is a substance that inhibits amino acid odors). Specifically, in the screening method of the present invention, substances that inhibit amino acid odors can be identified (i.e., to identify whether the test substance is a substance that inhibits amino acid odors) based on the response of olfactory receptors to an olfactory receptor activator in the presence of the test substance. More specifically, in the screening method of the present invention, substances that inhibit amino acid odors can be identified (i.e., to identify whether the test substance is a substance that inhibits amino acid odors) based on the inhibition of the response of olfactory receptors to an olfactory receptor activator by the test substance. That is, the screening method of the present invention may specifically comprise the steps of: (A) contacting an olfactory receptor with an olfactory receptor activator in the presence of a test substance; (B) measuring the response of the olfactory receptor to the olfactory receptor activator; and (C) identifying the test substance as a substance that suppresses amino acid odor based on the response, and if the response is inhibited by the test substance, the test substance is identified as a substance that suppresses amino acid odor, thereby being a screening method for substances that suppress amino acid odor.

[0011] <1-1> Substances that Suppress Amino Acid Odor "Amino acid odor" may refer to an unpleasant odor derived from amino acids. Amino acid odor is also referred to as "heat-deteriorated odor" or "muffled odor." Amino acid odor can be generated, for example, by the oxidation of amino acids. That is, "amino acid odor" may refer to, for example, an unpleasant odor that can be generated by the oxidation of amino acids. In other words, "amino acid odor" may refer to, for example, an unpleasant odor exhibited by a substance that can be generated by the oxidation of amino acids. Examples of amino acid oxidation include oxidation by heating of amino acids and natural oxidation of amino acids. Specific examples of amino acid oxidation include Strecker degradation, which is an oxidative degradation. Examples of amino acid odors include methional odor (i.e., the odor exhibited by methional). Methional can be generated, for example, by the oxidation of methionine (e.g., oxidation by heating or natural oxidation).

[0012] The substance that suppresses amino acid odor is not particularly limited as long as it can suppress amino acid odor (i.e., has the function of suppressing amino acid odor). The function of suppressing amino acid odor is also referred to as a "masking function." The substance that suppresses amino acid odor may consist of a single component (i.e., a pure substance) or a combination of two or more components (i.e., a mixture). A "mixture" is also referred to as a "composition." When the substance that suppresses amino acid odor is a mixture, the number of types and composition ratio of the components that make up the mixture are not particularly limited. When the substance that suppresses amino acid odor is a mixture, as long as the mixture suppresses amino acid odor, each component that makes up the mixture may or may not suppress amino acid odor individually.

[0013] <1-2> Test Substances "Test substance" refers to a substance used in the screening method of the present invention as a candidate substance for suppressing amino acid odor. The test substance is not particularly limited. The test substance may consist of a single component (i.e., a pure substance) or a combination of two or more components (i.e., a mixture). When the test substance is a mixture, the number and composition ratio of the components constituting the mixture are not particularly limited. The test substance may be a known substance or a novel substance. The test substance may be a natural product or an artificial product. The test substance may be, for example, a compound library created using combinatorial chemistry techniques. Examples of test substances include alcohols, ketones, aldehydes, ethers, esters, hydrocarbons, sugars, organic acids, nucleic acids, amino acids, peptides, lipids, and various other organic or inorganic components. Furthermore, test substances particularly include existing food additives. "Existing food additives" refer to substances already approved for use as food additives. The test substance may be a single test substance or a combination of two or more test substances. The test substance may be selected to include, for example, the substances exemplified above, such as existing food additives. That is, the test substance may be, for example, a single existing food additive, a combination of two or more food additives, or a combination of one or more food additives and one or more other ingredients. By contacting two or more ingredients together with an olfactory receptor and carrying out the screening method of the present invention, it is possible to identify whether the combination of ingredients as a whole suppresses the amino acid odor. Examples of "contacting two or more ingredients together with an olfactory receptor" include contacting a mixture of test substances with an olfactory receptor, and contacting two or more test substances together with an olfactory receptor.

[0014] <1-3> Olfactory Receptor Activators "Olfactory receptor activators" refers to substances that activate olfactory receptors. The olfactory receptor activators are not particularly limited as long as they can activate olfactory receptors. The olfactory receptor activators may be composed of a single component (i.e., a pure substance) or a combination of two or more components (i.e., a mixture). When the olfactory receptor activator is a mixture, each component of the mixture may or may not activate the olfactory receptor individually, as long as the mixture activates the olfactory receptor. When the olfactory receptor activator is a mixture, the number of components constituting the mixture and their composition ratios are not particularly limited. The olfactory receptor activators may be known substances or novel substances. The olfactory receptor activators may be natural products or artificial products. The olfactory receptor activators may or may not be known to be capable of activating olfactory receptors. As the olfactory receptor activator, for example, one capable of activating an olfactory receptor may be selected from the test substances exemplified above and used. An example of an olfactory receptor activator is methional.

[0015] <1-4> Olfactory Receptors OR4S2 and / or OR4E2 are used as olfactory receptors. In other words, unless otherwise specified, "olfactory receptor" refers to OR4S2 and / or OR4E2. Either OR4S2 or OR4E2 alone or a combination of OR4S2 and OR4E2 may be used as olfactory receptors. Using OR4S2 and OR4E2 in combination is expected to enable highly accurate screening of substances that suppress amino acid odors, for example. When OR4S2 and OR4E2 are used in combination, the response of the olfactory receptor to a test substance may be measured, for example, for each of OR4S2 and OR4E2 individually. For example, the responses of OR4S2 and OR4E2 to a test substance can be measured individually using cells expressing OR4S2 and OR4E2, respectively. Genes encoding olfactory receptors are also referred to as "receptor genes."

[0016] "OR4S2" refers to an olfactory receptor classified as Olfactory Receptor Family 4 Subfamily S Member 2. The gene encoding OR4S2 is also referred to as the "OR4S2 gene." OR4S2 is responsive to olfactory receptor activators such as methional. As OR4S2, one type of OR4S2 may be used, or two or more types of OR4S2 may be used in combination.

[0017] "OR4E2" refers to an olfactory receptor classified as Olfactory Receptor Family 4 Subfamily E Member 2. The gene encoding OR4E2 is also referred to as the "OR4E2 gene." OR4E2 is responsive to olfactory receptor activators such as methional. As OR4E2, one type of OR4E2 may be used, or two or more types of OR4E2 may be used in combination.

[0018] "An olfactory receptor is responsive to an olfactory receptor activator" may mean that the olfactory receptor is activated by an olfactory receptor activator.

[0019] Olfactory receptor genes and olfactory receptors include those of various organisms. Examples of organisms include mammals and other animals. Specific examples of mammals include Homo sapiens (humans), Mus musculus (mice), Rattus norvegicus (rat), Canis lupus familiaris (dogs), Felis catus (cats), Bos taurus (cattle), Sus scrofa (pigs), Pan troglodytes (chimpanzees), Macaca fascicularis (cynomolgus monkeys), and Equus caballus (horses). Mammals and other animals particularly include humans. Nucleotide sequences of olfactory receptor genes and amino acid sequences of olfactory receptors of various organisms can be obtained from public databases such as NCBI and Ensembl. The nucleotide sequence of the human OR4S2 gene and the amino acid sequence of OR4S2 are shown in SEQ ID NOs: 1 and 2, respectively. The nucleotide sequence of the human OR4E2 gene and the amino acid sequence of OR4E2 are shown in SEQ ID NOs: 3 and 4, respectively.

[0020] That is, the olfactory receptor gene may be, for example, a gene having the known or natural nucleotide sequence of the olfactory receptor gene as described above (e.g., the nucleotide sequence of the olfactory receptor gene of the above-mentioned organisms registered with NCBI, or the nucleotide sequences of SEQ ID NOs: 1 and 3 for the OR4S2 gene and OR4E2 gene, respectively). Furthermore, the olfactory receptor may be, for example, a protein having the known or natural amino acid sequence of the above-mentioned olfactory receptor (e.g., the amino acid sequence of the olfactory receptor of the above-mentioned organisms registered with NCBI, or the amino acid sequences of SEQ ID NOs: 2 and 4 for OR4S2 and OR4E2, respectively). The phrase "a gene has a nucleotide sequence" means that the gene contains the nucleotide sequence, unless otherwise specified, and also encompasses cases where the gene consists of the nucleotide sequence. Furthermore, the phrase "a protein has an amino acid sequence" means that the protein contains the amino acid sequence, unless otherwise specified, and also encompasses cases where the protein consists of the amino acid sequence.

[0021] The olfactory receptor may be, for example, a chimeric protein of two or more olfactory receptors of different origins. That is, OR4S2 also encompasses, for example, chimeric proteins of two or more OR4S2s of different origins. Furthermore, OR4E2 also encompasses, for example, chimeric proteins of two or more OR4E2s of different origins. Such chimeric proteins are also referred to as "chimeric olfactory receptors." In other words, a "chimeric olfactory receptor" refers to a protein having a chimeric sequence of an olfactory receptor (i.e., a protein having a chimeric sequence of two or more olfactory receptors of different origins). A "chimeric olfactory receptor sequence" refers to a chimeric sequence of an olfactory receptor amino acid sequence (i.e., a chimeric sequence of the amino acid sequences of two or more olfactory receptors of different origins). A "chimeric olfactory receptor sequence" specifically refers to an amino acid sequence of an olfactory receptor in which a partial sequence is replaced with a partial sequence of the amino acid sequence of one or more olfactory receptors of other origins. The substitution of amino acid sequences in the construction of a chimeric olfactory receptor can be carried out between corresponding positions in the amino acid sequences of the olfactory receptors. "Corresponding positions in the amino acid sequences of the olfactory receptors" refers to positions that are aligned at corresponding positions in the alignment of the amino acid sequences of the olfactory receptors. Examples of chimeric olfactory receptors include chimeric proteins of olfactory receptors from the organisms listed above (i.e., chimeric proteins of olfactory receptors from two or more organisms selected from the organisms listed above). Specific examples of chimeric olfactory receptors include mammalian chimeric olfactory receptors (i.e., chimeric proteins of olfactory receptors from two or more mammals). That is, the olfactory receptor may be, for example, a protein having a chimeric sequence of the amino acid sequence of the olfactory receptor from the organism listed above (specifically, a chimeric sequence of the amino acid sequence of the olfactory receptor from two or more organisms selected from the organisms listed above). Chimeric olfactory receptors that are responsive to olfactory receptor activators, such as methional, can be selected.

[0022] The number of organisms from which the olfactory receptors constituting the chimeric olfactory receptor are derived is not particularly limited, and the number of organisms from which the olfactory receptors constituting the chimeric olfactory receptor are derived may be two, three, or more.

[0023] The composition ratio of the olfactory receptors derived from each organism in the chimeric olfactory receptor is not particularly limited. The composition ratio of the olfactory receptors derived from each organism can be appropriately set within a range in which the total composition ratio of the olfactory receptors derived from each organism constituting the chimeric olfactory receptor does not exceed 100%. The composition ratio of the olfactory receptors derived from each organism may be, for example, 1% or more, 3% or more, 5% or more, 10% or more, 20% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, or 99% or less, 97% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, or 1% or less, or a compatible combination thereof. "Constituent ratio of olfactory receptors derived from each organism" means the ratio of the number of amino acid residues in the olfactory receptor derived from each organism to the total number of amino acid residues constituting the chimeric olfactory receptor. Note that if the amino acid residues constituting the chimeric olfactory receptor correspond to a conserved sequence in the olfactory receptors derived from each of the organisms constituting the chimeric olfactory receptor, the amino acid residues may be considered to be derived from any of those organisms.

[0024] The distribution pattern of the olfactory receptors derived from each organism in the chimeric olfactory receptor is not particularly limited. In the chimeric olfactory receptor, the olfactory receptors derived from each organism may be present in one location, or may be present in two or more locations. For example, when a chimeric olfactory receptor is designed by replacing the internal amino acid sequence of an olfactory receptor derived from one organism (olfactory receptor A) with the amino acid sequence of an olfactory receptor derived from another organism (olfactory receptor B), the amino acid sequence of olfactory receptor A remains dispersed at the N-terminus and C-terminus of the chimeric olfactory receptor.

[0025] Similarly, examples of olfactory receptor genes include chimeric olfactory receptor genes. The descriptions regarding chimeric olfactory receptors can also be applied mutatis mutandis to chimeric olfactory receptor genes.

[0026] The olfactory receptor gene may be a variant of the olfactory receptor gene exemplified above (e.g., a variant of a gene having the nucleotide sequence of the olfactory receptor gene of the exemplified organism or a chimeric sequence thereof), so long as the original function is maintained. Similarly, the olfactory receptor may be a variant of the olfactory receptor gene exemplified above (e.g., a variant of a protein having the amino acid sequence of the olfactory receptor of the exemplified organism or a chimeric sequence thereof), so long as the original function is maintained. Such variants that maintain the original function may be referred to as "conservative variants." The terms "OR4S2 gene" and "OR4E2 gene" are intended to encompass the OR4S2 gene and OR4E2 gene exemplified above, as well as their conservative variants, respectively. Similarly, the terms "OR4S2" and "OR4E2" are intended to encompass the OR4S2 and OR4E2 genes exemplified above, as well as their conservative variants, respectively. Conservative variants include, for example, homologs and artificially modified versions of the olfactory receptor genes and olfactory receptors exemplified above.

[0027] Furthermore, the olfactory receptor gene identified in the originating biological species is not limited to the olfactory receptor gene itself found in that biological species, but also includes genes having the nucleotide sequence of the olfactory receptor gene found in that biological species and conservative variants thereof. Similarly, the olfactory receptor identified in the originating biological species is not limited to the olfactory receptor itself found in that biological species, but also includes proteins having the amino acid sequence of the olfactory receptor found in that biological species and conservative variants thereof. These conservative variants may or may not be found in that biological species. For example, the term "mammalian olfactory receptor" is intended to include proteins having the amino acid sequence of an olfactory receptor found in a mammal and conservative variants thereof. Furthermore, for example, the term "mammalian chimeric olfactory receptor" is intended to include proteins having a chimeric sequence of the amino acid sequence of an olfactory receptor found in a mammal and conservative variants thereof. In other words, the olfactory receptor constituting a "mammalian chimeric olfactory receptor" is not limited to the olfactory receptor itself found in a mammal, but may also be a conservative variant thereof.

[0028] "Maintaining the original function" means that a gene or protein variant has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. "Maintaining the original function" with respect to a gene means that a gene variant encodes a protein that maintains the original function. In other words, "maintaining the original function" with respect to each olfactory receptor gene may mean that a gene variant encodes an olfactory receptor that is responsive to an olfactory receptor activator such as methional. Furthermore, "maintaining the original function" with respect to each olfactory receptor may mean that an olfactory receptor variant is responsive to an olfactory receptor activator such as methional.

[0029] The responsiveness of an olfactory receptor to an olfactory receptor activator can be confirmed, for example, by measuring the response (e.g., activation) of the olfactory receptor when the olfactory receptor is contacted with an olfactory receptor activator.

[0030] Examples of conservative variants are shown below.

[0031] Olfactory receptor gene homologs or olfactory receptor homologs can be easily obtained from public databases, for example, by BLAST or FASTA searches using the nucleotide sequences of the above-exemplified olfactory receptor genes or the amino acid sequences of the above-exemplified olfactory receptors as query sequences. Alternatively, olfactory receptor gene homologs can be obtained, for example, by PCR using the chromosomes of various organisms as templates and oligonucleotides prepared based on the nucleotide sequences of these known olfactory receptor genes as primers.

[0032] As long as the original function is maintained, the olfactory receptor gene may be a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions in the above-mentioned amino acid sequence (e.g., the amino acid sequence of an olfactory receptor of an organism exemplified above or a chimeric sequence thereof) have been substituted, deleted, inserted, and / or added. For example, the encoded protein may have its N-terminus and / or C-terminus extended or shortened. Note that the term "one or several" above varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0033] The above-mentioned substitution, deletion, insertion, and / or addition of one or several amino acids is a conservative mutation that maintains normal protein function. A typical conservative mutation is a conservative substitution. A conservative substitution is a mutation in which Phe, Trp, and Tyr are substituted for each other when the substitution site is an aromatic amino acid; Leu, Ile, and Val are substituted for each other when the substitution site is a hydrophobic amino acid; Gln and Asn are substituted for each other when the substitution site is a polar amino acid; Lys, Arg, and His are substituted for each other when the substitution site is a basic amino acid; Asp and Glu are substituted for each other when the substitution site is an acidic amino acid; and Ser and Thr are substituted for each other when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Il Examples of substitutions include substitutions of Lys with Leu, Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. The above-mentioned amino acid substitutions, deletions, insertions, or additions also include those resulting from naturally occurring mutations (mutants or variants) based on individual differences or differences in species of the organism from which the gene is derived.

[0034] Furthermore, the olfactory receptor gene may be a gene encoding a protein having an amino acid sequence that has, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity to the entire amino acid sequence, as long as the original function is maintained.

[0035] Furthermore, the olfactory receptor gene may be a gene, such as DNA, that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned base sequence (e.g., the base sequence of the olfactory receptor gene of the above-mentioned organisms or a chimeric sequence thereof), such as a sequence complementary to all or part of the above-mentioned base sequence, so long as the original function is maintained. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. One example of such conditions is a condition under which DNAs with high identity, for example, DNAs with an identity of 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more, hybridize with each other, while DNAs with lower identity do not hybridize with each other; or a condition in which washing is performed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0036] As mentioned above, the probe used in the hybridization may be a portion of the complementary sequence of the gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on a known gene sequence as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. When a DNA fragment of about 300 bp in length is used as the probe, washing conditions for the hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0037] Furthermore, since codon degeneracy differs depending on the host, the olfactory receptor gene may be one in which any codon has been replaced with an equivalent codon. That is, the olfactory receptor gene may be, for example, a variant of the olfactory receptor gene exemplified above due to the degeneracy of the genetic code. For example, the olfactory receptor gene may be modified to have optimal codons depending on the codon usage frequency of the host used.

[0038] In the present invention, the term "gene" is not limited to DNA and may include any polynucleotide as long as it encodes a protein of interest. In other words, "olfactory receptor gene" may refer to any polynucleotide encoding an olfactory receptor. An olfactory receptor gene may be DNA, RNA, or a combination thereof. An olfactory receptor gene may be single-stranded or double-stranded. An olfactory receptor gene may be single-stranded DNA or single-stranded RNA. An olfactory receptor gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. An olfactory receptor gene may contain both DNA residues and RNA residues in a single polynucleotide strand. When an olfactory receptor gene contains RNA, the descriptions regarding DNA, such as the nucleotide sequence exemplified above, may be interpreted appropriately to refer to RNA. An olfactory receptor gene may or may not contain an intron. The form of the olfactory receptor gene can be selected appropriately depending on various conditions, such as its usage mode.

[0039] The "identity" between amino acid sequences refers to the identity between amino acid sequences calculated by blastp using default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated by blastn using default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0040] Furthermore, the olfactory receptor may contain other amino acid sequences in addition to the amino acid sequence of the olfactory receptor described above. That is, the olfactory receptor may be a fusion protein of the amino acid sequence of the olfactory receptor described above with other amino acid sequences. The other amino acid sequences are not particularly limited as long as the olfactory receptor is responsive to an olfactory receptor activator. Examples of other amino acid sequences include tag sequences such as His tags and V5 epitope tags. The other amino acid sequences may be linked, for example, to the N-terminus, C-terminus, or both of the olfactory receptor.

[0041] The olfactory receptor can be used in any form that can be used to screen for substances that suppress amino acid odors. Specifically, the olfactory receptor can be used in any form as long as the olfactory receptor can be contacted with a test substance and is responsive to an olfactory receptor activator. The form in which the olfactory receptor is used can be appropriately determined depending on various conditions, such as the embodiment of the screening method of the present invention.

[0042] The olfactory receptor may be used in a form isolated to a desired degree, such as a purified or crude product, or in a form contained in a material. Specifically, the olfactory receptor may be used, for example, in a form supported by a structure. Examples of the structure include cells, cell membranes, artificial lipid bilayer vesicles, and artificial lipid bilayer membranes. Examples of the structure include cells, in particular. In other words, the olfactory receptor may be used in the form of a structure having (supporting) an olfactory receptor, such as a cell having an olfactory receptor, a cell membrane having an olfactory receptor, an artificial lipid bilayer vesicle having an olfactory receptor, or an artificial lipid bilayer membrane having an olfactory receptor. These structures having an olfactory receptor may also be used, for example, in a form isolated to a desired degree, or in a form contained in a material. The olfactory receptor may also constitute a part of an instrument. That is, the olfactory receptor may also be used in the form of an instrument equipped with an olfactory receptor. Examples of devices equipped with olfactory receptors include devices with immobilized olfactory receptors and devices equipped with structures (such as lipid bilayer membranes) having olfactory receptors. Examples of devices equipped with lipid bilayer membranes include chips with arrayed lipid bilayer membranes (WO2005 / 000558; Watanabe R. et al., Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity. Nat Commun. 2014 Jul 24;5:4519.; Kamiya K. et al., Preparation of artificial cell membrane and single ion channel measurement, Electrochemistry, 83, 1096-1100 (2015)) and ion channel measurement devices equipped with lipid bilayer membranes prepared by the droplet contact method (Kawano R. et al., Automated Parallel Recordings of Topologically Identified Single Ion Channels, Scientific Reports, 3, No. 1995 (2013)).All of these forms of olfactory receptors are included in the scope of olfactory receptors used in the screening method of the present invention.

[0043] Olfactory receptors can be produced, for example, by expressing an olfactory receptor gene. Expression of the olfactory receptor gene may be carried out, for example, using cells or a cell-free protein synthesis system. For expression of the olfactory receptor gene using cells, see the description of cells having olfactory receptors below. The expressed olfactory receptor can be obtained in the form described above as appropriate and used in the screening method of the present invention.

[0044] A cell having an olfactory receptor is also referred to as a "cell of the present invention." An olfactory receptor can function by being localized, for example, in the cell membrane. Thus, a cell of the present invention may have an olfactory receptor, for example, in the cell membrane.

[0045] Olfactory receptors are expressed from olfactory receptor genes. Thus, the cells of the present invention have an olfactory receptor gene. Specifically, the cells of the present invention have an olfactory receptor gene in an expressible manner. It is sufficient for the cells of the present invention to have the olfactory receptor gene until the olfactory receptor is expressed. In other words, the cells of the present invention may or may not have the olfactory receptor gene after the olfactory receptor is expressed. In other words, the cells of the present invention are cells that have expressed an olfactory receptor gene, and are also cells that have expressed an olfactory receptor. It is to be noted that "expression of an olfactory receptor gene" and "expression of an olfactory receptor" can be used synonymously.

[0046] The cells of the present invention may have one copy of the olfactory receptor gene, or may have two or more copies of the olfactory receptor gene.

[0047] The cells of the present invention may inherently have an olfactory receptor gene, or may be modified to have an olfactory receptor gene.

[0048] Cells that inherently have an olfactory receptor gene include cells of organisms from which the above-mentioned olfactory receptor genes are derived, such as taste cells of mammals such as humans. Cells that inherently have an olfactory receptor gene can be obtained, for example, from organisms or tissues that contain the cells.

[0049] Cells modified to have an olfactory receptor gene include cells into which an olfactory receptor gene has been introduced.

[0050] The cells of the present invention and cells used to obtain them (for example, cells into which or into which an olfactory receptor gene is introduced) are also collectively referred to as "host cells."

[0051] The host cells are not particularly limited as long as they can express functional olfactory receptors and can be used to screen for substances that suppress amino acid odors. Examples of host cells include bacterial cells, fungal cells, plant cells, insect cells, and animal cells. Preferred host cells include eukaryotic cells such as fungal cells, plant cells, insect cells, and animal cells. More preferred host cells include animal cells. Examples of animals include mammals, birds, and amphibians. Examples of mammals include rodents and primates. Examples of rodents include Chinese hamsters, hamsters, mice, rats, and guinea pigs. Examples of primates include humans, monkeys, and chimpanzees. Examples of birds include chickens. Examples of amphibians include Xenopus laevis. Furthermore, the tissues or cells from which the host cells are derived are not particularly limited. Examples of tissues or cells from which the host cells are derived include the ovary, kidney, adrenal gland, tongue epithelium, olfactory epithelium, pineal gland, thyroid gland, and melanocytes. Examples of Chinese hamster cells include Chinese hamster ovary cell lines (CHO). Specific examples of CHO include CHO-DG44 and CHO-K1. Examples of human cells include human embryonic kidney cell-derived cell lines (HEK). Specific examples of HEK include HEK293 and HEK293T. Examples of monkey cells include African green monkey kidney cell-derived cell lines (COS). Specific examples of COS include COS-1. Examples of Xenopus cells include Xenopus oocytes. Examples of insect cells include Spodoptera frugiperda-derived cells such as Sf9, Sf21, and SF+, and Trichoplusia ni-derived cells such as High-Five. Host cells may be individual, independent cells (e.g., free cells) or may form aggregates such as tissues.

[0052] Olfactory receptor genes can be obtained by cloning from organisms that have the olfactory receptor genes. Nucleic acids such as genomic DNA and cDNA containing the genes can be used for cloning. Olfactory receptor genes can also be obtained by chemical synthesis (Gene, 60(1), 115-127 (1987)).

[0053] The obtained olfactory receptor gene can be used as is or after appropriate modification. That is, by modifying the olfactory receptor gene, its variant can be obtained. Gene modification can be performed by known techniques. For example, a desired mutation can be introduced into a target site in DNA by site-directed mutagenesis. That is, for example, site-directed mutagenesis can be used to modify the coding region of a gene so that the encoded protein contains substitutions, deletions, insertions, and / or additions of amino acid residues at specific sites. Site-directed mutagenesis methods include PCR-based methods (Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, H.J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. in Enzymol., 154, 367 (1987)). Variants of olfactory receptor genes can also be obtained directly by chemical synthesis.

[0054] The manner in which an olfactory receptor gene is introduced into a host cell is not particularly limited. The olfactory receptor gene may be expressibly maintained in the host cell. Specifically, for example, when an olfactory receptor gene is introduced in a form requiring transcription of DNA or the like, the olfactory receptor gene may be expressibly maintained in the host cell under the control of a promoter that functions in the host cell. In the host cell, the olfactory receptor gene may be present extrachromosomally or may be introduced onto the chromosome. When two or more genes are introduced, each gene may be expressibly maintained in the host cell.

[0055] The promoter for expressing the olfactory receptor gene is not particularly limited as long as it functions in the host cell. A "promoter functional in the host cell" refers to a promoter that has promoter activity in the host cell. The promoter may be a promoter native to the host cell or a heterologous promoter. The promoter may be the native promoter of the olfactory receptor gene or a promoter of another gene. The promoter may be stronger than the native promoter of the olfactory receptor gene. For example, promoters that function in animal cells include the SV40 promoter, EF1a promoter, RSV promoter, CMV promoter, and SRalpha promoter. Furthermore, highly active versions of native promoters may be obtained and used by using various reporter genes. Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0056] An olfactory receptor gene can be introduced into a host cell using, for example, a vector containing the gene. A vector containing an olfactory receptor gene is also referred to as an "olfactory receptor gene expression vector." An olfactory receptor gene expression vector can be constructed, for example, by ligating a DNA fragment containing the olfactory receptor gene to a vector. The olfactory receptor gene expression vector can be introduced into a host cell by introducing the olfactory receptor gene into the host cell. The vector may comprise a marker such as a drug resistance gene. The vector may also comprise an expression regulatory sequence such as a promoter for expressing the inserted gene. The vector can be selected appropriately depending on various conditions, such as the type of host cell and the mode of introduction of the olfactory receptor gene. For example, vectors that can be used to introduce genes into animal cells include plasmid vectors and viral vectors. Examples of viral vectors include retroviral vectors and adenoviral vectors. Examples of plasmid vectors include pcDNA series vectors (pcDNA3.1, etc.; Thermo Fisher Scientific), pBApo-CMV series vectors (Takara Bio), and pCI-neo (Promega). Depending on the type and configuration of the vector, the vector can be integrated into the chromosome of the host cell, can autonomously replicate extrachromosomally, or can be temporarily maintained extrachromosomally in the host cell. For example, vectors having a viral replication origin such as the SV40 replication origin can autonomously replicate extrachromosomally in animal cells. Specifically, for example, the pcDNA series vectors have the SV40 replication origin and can autonomously replicate extrachromosomally in host cells (e.g., COS-1 and HEK293T) that express the SV40 large T antigen.

[0057] Alternatively, the olfactory receptor gene can be introduced into a host cell by, for example, introducing a nucleic acid fragment containing the gene into the host cell. A nucleic acid fragment containing the olfactory receptor gene is also referred to as an "olfactory receptor gene fragment." Such fragments include linear DNA and linear RNA. Examples of linear RNA include mRNA and cRNA.

[0058] The method for introducing nucleic acids such as vectors and nucleic acid fragments into host cells can be selected appropriately depending on various conditions such as the type of host cells. For example, methods for introducing nucleic acids such as vectors and nucleic acid fragments into host cells such as animal cells include the DEAE-dextran method, calcium phosphate method, lipofection, electroporation, and microinjection. Furthermore, when the vector is a viral vector, the vector can be introduced into the host cells by infecting the host cells with the vector (virus).

[0059] Alternatively, cells inherently containing an olfactory receptor gene may be modified to increase expression of the olfactory receptor gene. "Increased gene expression" means that the expression level of the gene per cell is increased compared to unmodified cells. "Unmodified cells" herein refer to control cells that have not been modified to increase expression of the target gene. Examples of unmodified cells include wild-type cells and the original cells. Techniques for increasing expression of an olfactory receptor gene include increasing the copy number of the olfactory receptor gene and improving the transcription efficiency or translation efficiency of the olfactory receptor gene. The copy number of the olfactory receptor gene can be increased by introducing the olfactory receptor gene into host cells. Introduction of the olfactory receptor gene can be carried out as described above. The introduced olfactory receptor gene may be derived from the host cell or from a heterologous source. The transcription efficiency or translation efficiency of the olfactory receptor gene can be improved by modifying the gene expression regulatory sequence, such as a promoter. For example, the transcription efficiency of the olfactory receptor gene can be improved by replacing the promoter of the olfactory receptor gene with a stronger promoter.

[0060] The cells of the present invention may have any other properties as long as they can be used to screen for substances that suppress amino acid odors. Examples of such properties include properties that are useful for measuring the response of olfactory receptors to olfactory receptor activators. The description of the properties of the cells of the present invention can also be applied mutatis mutandis to cases where olfactory receptors are used in other embodiments. Examples of cases where olfactory receptors are used in other embodiments include cases where artificial lipid bilayer vesicles, cell membranes, or artificial lipid bilayer membranes containing olfactory receptors are used.

[0061] The cells of the present invention may or may not have, for example, olfactory receptors other than the selected olfactory receptor (also referred to as "other olfactory receptors"). It may be preferable that the cells of the present invention do not have other olfactory receptors. Examples of cells that do not have other olfactory receptors include cells that do not have genes encoding other olfactory receptors, and cells that have genes encoding other olfactory receptors but do not express said genes. The cells of the present invention may, for example, not inherently have other olfactory receptors, or may be modified so that they do not have other olfactory receptors. Modifying cells so that they do not have other olfactory receptors can be achieved, for example, by knocking out genes encoding other olfactory receptors.

[0062] Furthermore, the cells of the present invention may contain, for example, a protein involved in signal transduction. In other words, the cells of the present invention may contain a gene encoding a protein involved in signal transduction. Examples of proteins involved in signal transduction include G proteins (e.g., Golf), G protein activators (e.g., Ric8B), adenylate cyclase, and calcium channels. Examples of Golf include animal Golf such as human Golf (GenBank accession No. NP_892023). Examples of Ric8B include animal Ric8B such as rat Ric8B (GenBank accession No. NP_783188). The cells of the present invention may contain, for example, components corresponding to the parameter to be measured. Examples of such components include a probe such as a calcium indicator and a reporter gene such as a luciferase gene. When a probe such as a calcium indicator is expressed from a gene, the cells of the present invention may contain a gene encoding the probe.

[0063] Furthermore, the cells of the present invention may contain, for example, a protein that promotes membrane expression of an olfactory receptor. In other words, the cells of the present invention may contain a gene encoding such a protein. Examples of such proteins include RTP1s (Zhuang H and Matsunami H, J Biol Chem 282, 15284-15293 (2007)). Examples of RTP1s include animal RTP1s such as human RTP1s (GenBank accession no. AAT70680), mouse RTP1s (GenBank accession no. ABU23737), and bat RTP1s (the amino acid sequence from the methionine residue at position 37 to the C-terminus of GenBank accession no. XP_006765914). The amino acid sequence of mouse RTP1s shares 93.3% identity with the amino acid sequence of human RTP1s. The amino acid sequence of bat RTP1s (the partial sequence shown above) shares 90.7% identity with the amino acid sequence of human RTP1s.

[0064] The cells of the present invention may inherently possess the properties exemplified above, or may be modified to have the properties exemplified above. Regarding cell modification, the description of cell modification related to olfactory receptor genes, such as the introduction of olfactory receptor genes, can be applied mutatis mutandis. The genes exemplified above may be genes derived from the host cell or genes derived from a different species. Furthermore, the genes exemplified above may or may not be derived from the same source as the olfactory receptor gene. When two or more genes are introduced, it is sufficient that each gene is retained in the host cell in an expressible manner. For example, all of the genes may be retained on a single expression vector, or all of the genes may be retained on a chromosome. Furthermore, the genes may be retained separately on multiple expression vectors, or may be retained separately on a single or multiple expression vectors and on a chromosome. The genes exemplified above and the proteins encoded thereby may have, for example, the nucleotide sequences and amino acid sequences of known genes and proteins, respectively. Furthermore, the genes exemplified above and the proteins encoded thereby may be, for example, conservative variants of known genes and proteins, respectively. For conservative variants of genes and proteins, the descriptions regarding conservative variants of olfactory receptor genes and olfactory receptors can be applied mutatis mutandis.

[0065] Cells having an olfactory receptor gene can be used as cells having an olfactory receptor (cells of the present invention) either as is or after appropriate expression of the olfactory receptor gene. That is, if cells having an olfactory receptor gene already express the olfactory receptor gene, the cells may be used as cells having an olfactory receptor (cells of the present invention) as is. Alternatively, cells having an olfactory receptor (cells of the present invention) can be obtained by expressing the olfactory receptor gene in cells having an olfactory receptor gene. For example, the olfactory receptor gene can be expressed by culturing cells having an olfactory receptor gene, thereby obtaining cells having an olfactory receptor (cells of the present invention). Specifically, for example, after introduction (e.g., transfection) of the olfactory receptor gene, the host cells can be continuously cultured to express the olfactory receptor gene. The medium composition and culture conditions are not particularly limited as long as the cells having the olfactory receptor gene can be maintained (e.g., proliferated) and the olfactory receptor gene is expressed. During culture, cells having an olfactory receptor gene may or may not proliferate. The medium composition and culture conditions can be appropriately set depending on various conditions, such as the type of host cell. Culturing can be performed using, for example, a conventional medium and conditions used for culturing cells such as animal cells, either as is or with appropriate modifications. Specific examples of media that can be used for culturing animal cells include Opti-MEM medium (Thermo Fisher Scientific), DMEM medium, RPMI 1640 medium, and CD293 medium. Culturing can be performed, for example, at 36°C to 38°C under 5% CO ２ CO etc. ２ The culture can be carried out by static culture in a culture-containing atmosphere. If necessary, a selective drug or an expression inducer can be used.

[0066] Expression of an olfactory receptor can be confirmed by measuring the response of the olfactory receptor to an olfactory receptor activator (for example, activation of the olfactory receptor by an olfactory receptor activator). Expression of an olfactory receptor can also be confirmed by measuring the amount of mRNA transcribed from the olfactory receptor gene or by detecting the olfactory receptor by Western blotting using an antibody.

[0067] The cells of the present invention can be used in the screening method of the present invention, for example, as they are (contained in the culture) or after being recovered from the medium. Furthermore, the culture or cells recovered therefrom may be used in the screening method of the present invention after, for example, appropriate treatment such as washing, concentration, dilution, or fixation. Thus, the cells of the present invention may be used, for example, in a form isolated to a desired degree, or in a form contained in a material such as a culture. The same applies to other structures having olfactory receptors.

[0068] The cell membrane having an olfactory receptor can be prepared, for example, from the cells of the present invention. Specifically, the cell membrane having an olfactory receptor can be obtained, for example, as a membrane fraction obtained by disrupting the cells of the present invention. The cell membrane having an olfactory receptor may be used, for example, as is or dispersed in an artificial lipid bilayer membrane. The cell membrane having an olfactory receptor may also be used in the form of vesicles (i.e., by preparing vesicles from the cell membrane).

[0069] In addition, artificial lipid bilayer vesicles or artificial lipid bilayer membranes having olfactory receptors can be produced using olfactory receptors. For example, artificial lipid bilayer vesicles or artificial lipid bilayer membranes having olfactory receptors can be prepared by incorporating olfactory receptors into pre-prepared artificial lipid bilayer vesicles or artificial lipid bilayer membranes. In addition, artificial lipid bilayer vesicles or artificial lipid bilayer membranes having olfactory receptors can be prepared by using olfactory receptors as raw materials to prepare olfactory receptors in artificial lipid bilayer vesicles or artificial lipid bilayer membranes. To prepare artificial lipid bilayer vesicles or artificial lipid bilayer membranes having olfactory receptors, olfactory receptors in an appropriate form, such as a membrane fraction having olfactory receptors, can be used. Artificial lipid bilayer vesicles and artificial lipid bilayer membranes can be produced, for example, by known means. For example, methods for producing artificial lipid bilayer membranes include the Montal-Mueller method and the droplet contact method (Kawano R. et al., Automated Parallel Recordings of Topologically Identified Single Ion Channels, Scientific Reports, 3, No. 1995 (2013)). For example, US2018-0095071 discloses the preparation of artificial lipid bilayer membranes using crudely purified membrane fractions obtained from cultured cells. Artificial lipid bilayer membrane vesicles may have, for example, olfactory receptors in their membranes. Examples of lipid bilayer membrane vesicles include liposomes.

[0070] A membrane such as a cell membrane or an artificial lipid bilayer membrane can be used, for example, to generate a space separated by the membrane. Such a membrane can be used, for example, to separate two spaces, such as two wells. That is, such a membrane can be used to provide a reaction system having two spaces, such as two wells, where the two spaces are separated from each other by the membrane. Such two spaces only need to have at least a portion of their boundary separated by the membrane. Such a reaction system can be provided, for example, as the device described above.

[0071] <1-5> Screening Method of the Present Invention The screening method of the present invention can be carried out in vitro.

[0072] Step (A) is a step of contacting an olfactory receptor with an olfactory receptor activator in the presence of a test substance. That is, first, the olfactory receptor can be contacted with an olfactory receptor activator in the presence of a test substance. In other words, the olfactory receptor can be contacted with a test substance in the presence of an olfactory receptor activator. In other words, the olfactory receptor can be contacted with an olfactory receptor activator and a test substance. That is, the expressions "contacting an olfactory receptor with an olfactory receptor activator in the presence of a test substance," "contacting an olfactory receptor with a test substance in the presence of an olfactory receptor activator," and "contacting an olfactory receptor with an olfactory receptor activator and a test substance" can be used interchangeably. Hereinafter, the olfactory receptor activator and the test substance will be collectively referred to as "both substances."

[0073] The system in which the olfactory receptors come into contact with both substances is also called a "reaction system."

[0074] The contact between the olfactory receptor and both substances can be carried out in an appropriate liquid. The liquid in which the contact between the olfactory receptor and both substances is carried out is also referred to as a "reaction liquid." In other words, a reaction liquid can be used as a reaction system. For example, the olfactory receptor and both substances can be contacted by coexisting them in an appropriate reaction liquid. Specifically, for example, the olfactory receptor (e.g., in the form exemplified above, such as a cell having an olfactory receptor) and both substances can be contacted by dissolving, suspending, dispersing, or the like in an appropriate liquid medium. Examples of the liquid medium include aqueous media such as water and aqueous buffer solutions. Note that when two or more components, such as both substances, are contacted together with the olfactory receptor, the contact between these components and the olfactory receptor may or may not begin simultaneously. That is, for example, after contact between a certain component and the olfactory receptor has begun, another component may be added to the reaction system. Specifically, for example, the olfactory receptor activator may be added to the reaction system after the contact between the test substance and the olfactory receptor has begun, or the test substance may be added to the reaction system after the contact between the olfactory receptor activator and the olfactory receptor has begun. Typically, the test substance and the olfactory receptor activator are premixed and then contacted with the olfactory receptor. The reaction conditions (conditions for contacting the olfactory receptor with both substances) are not particularly limited as long as they enable screening for substances that suppress amino acid odors. The reaction conditions can be appropriately set depending on various conditions, such as the mode of use of the olfactory receptor, the type of test substance, and the method for measuring the olfactory receptor response. Known reaction conditions for measuring interactions between substances, such as interactions between proteins and ligands, may be used as is, or modified as appropriate. The concentration of the test substance may be, for example, 0.01 nM to 500 mM, 10 nM to 100 mM, 1 μM to 10 mM, or 3 μM to 1 mM. The concentration of the olfactory receptor activator may be, for example, 0.01 nM to 500 mM, 10 nM to 100 mM, 1 μM to 10 mM, or 3 μM to 1 mM. The concentration of the olfactory receptor may be, for example, 1 pg / mL to 10 mg / mL.Furthermore, when cells having olfactory receptors are used, the concentration of the cells having olfactory receptors may be, for example, 10 cells / mL to 10,000,000 cells / mL. The contact between the olfactory receptor and both substances may or may not be terminated at an appropriate time point. The contact between the olfactory receptor and both substances may generally be continued until the response of the olfactory receptor to the olfactory receptor activator is measured. The duration of contact between the olfactory receptor and both substances may be, for example, 0.1 seconds or more, 0.5 seconds or more, 1 second or more, 5 seconds or more, 10 seconds or more, 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, or 2 hours or more; or 24 hours or less, 12 hours or less, 6 hours or less, 2 hours or less, or 1 hour or less, or any combination thereof that is compatible. Specifically, the duration of contact between the olfactory receptor and both substances may be, for example, 1 hour to 6 hours. The reaction system may contain other components in addition to the olfactory receptor (e.g., in the form of cells having olfactory receptors, as exemplified above) and both substances, as long as it is possible to screen for substances that suppress amino acid odors. The other components can be appropriately selected depending on various conditions, such as the form in which the olfactory receptor is used, the type of test substance, and the method for measuring the olfactory receptor response. Examples of other components include salts such as calcium salts, carbon sources such as glucose, other medium components, and pH buffers.

[0075] Step (B) is a step of measuring the response of the olfactory receptor to the olfactory receptor activator. That is, the response of the olfactory receptor to the olfactory receptor activator can then be measured. The response of the olfactory receptor to the olfactory receptor activator is also referred to as "the olfactory receptor activator eliciting a response of the olfactory receptor." The response of the olfactory receptor to the olfactory receptor activator serves as an index for evaluating response inhibition by a test substance, as described below. Therefore, "measuring the response of the olfactory receptor to the olfactory receptor activator" may specifically mean measuring response inhibition by the test substance. "Response inhibition by the test substance" means that the response of the olfactory receptor to the olfactory receptor activator is inhibited by the test substance.

[0076] The response of the olfactory receptor to an olfactory receptor activator includes activation of the olfactory receptor by the olfactory receptor activator.

[0077] The timing for measuring the olfactory receptor response to an olfactory receptor activator is not particularly limited, as long as it is the time point at which the test substance inhibits the amino acid odor to a measurable extent. The timing for measuring the olfactory receptor response to a test substance can be appropriately set depending on various conditions, such as the form of use of the olfactory receptor, the types of both substances, and the method for measuring the olfactory receptor response. Specifically, the timing for measuring the olfactory receptor response to an olfactory receptor activator may be any appropriate time point from the time when contact between the olfactory receptor and both substances begins to the time when response inhibition by the test substance disappears. The timing for measuring the olfactory receptor response to an olfactory receptor activator may be, for example, the time point at which response inhibition by the test substance is maximized. Furthermore, the timing for measuring the response of the olfactory receptor to an olfactory receptor activator may be, for example, 0.1 seconds or later, 0.5 seconds or later, 1 second or later, 5 seconds or later, 10 seconds or later, 30 seconds or later, 1 minute or later, 5 minutes or later, 10 minutes or later, 30 minutes or later, 1 hour or later, or 2 hours or later from the time when contact between the olfactory receptor and both substances begins, or up to 24 hours, 12 hours, 6 hours, 2 hours, or 1 hour, or any combination thereof that is not inconsistent. Specifically, the timing for measuring the response of the olfactory receptor to an olfactory receptor activator may be, for example, from 1 hour to 6 hours from the time when contact between the olfactory receptor and both substances begins.

[0078] Step (C) is a step of identifying the test substance as a substance that suppresses amino acid odor based on the response of the olfactory receptor to the olfactory receptor activator. That is, the test substance can then be identified as a substance that suppresses amino acid odor based on the response of the olfactory receptor to the olfactory receptor activator. That is, the test substance can be identified as a substance that suppresses amino acid odor based on the response of the olfactory receptor to the olfactory receptor activator.

[0079] Specifically, response inhibition by a test substance can be evaluated based on the response of the olfactory receptor to an olfactory receptor activator, and the test substance can be identified as a substance that suppresses amino acid odors based on response inhibition by the test substance. More specifically, if response inhibition by a test substance is observed, i.e., if the response of the olfactory receptor to an olfactory receptor activator is inhibited by the test substance, the test substance can be identified as a substance that suppresses amino acid odors. That is, for example, if activation of the olfactory receptor by an olfactory receptor activator is inhibited by the test substance, the test substance can be identified as a substance that suppresses amino acid odors. Inhibition of olfactory receptor activation by an olfactory receptor activator by a test substance is also referred to as "inhibition of activation by the test substance" or "inactivation of the olfactory receptor by the test substance." Note that when both OR4S2 and OR4E2 are used in screening, "response inhibition by the test substance is observed" means that response inhibition by the test substance is observed for at least one of OR4S2 and OR4E2.

[0080] The inactivation of the olfactory receptor by the test substance can be determined using as an indicator the degree of activation of the olfactory receptor (degree of activation D1) when the step (A) is carried out (i.e., under conditions in which the olfactory receptor is contacted with an olfactory receptor activator in the presence of the test substance). That is, the step (B) may be, for example, (B1) a step of measuring the degree of activation D1. Furthermore, the step (C) may be, for example, (C1) a step of identifying whether the test substance is a substance that suppresses amino acid odor based on the degree of activation D1.

[0081] Specifically, the inactivation of the olfactory receptor by the test substance can be determined by comparing the degree of activation of the olfactory receptor (degree of activation D1) when the above step (A) is performed (i.e., under conditions in which the olfactory receptor is contacted with an olfactory receptor activator in the presence of the test substance) with the degree of activation of the olfactory receptor under control conditions (degree of activation D2). That is, the above step (C1) may be, for example, (C2) a step of identifying whether the test substance is a substance that suppresses amino acid odor based on the difference between the degree of activation D1 and the degree of activation D2.

[0082] "Control conditions" refers to the following conditions (C2-1) or (C2-2): (C2-1) conditions in which an olfactory receptor is contacted with an olfactory receptor activator in the absence of a test substance; (C2-2) conditions in which an olfactory receptor is contacted with an olfactory receptor activator in the presence of a test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in step (A) above.

[0083] In other words, the inactivation of olfactory receptors by a test substance can be determined, for example, using as an index the difference in the degree of olfactory receptor activation due to the presence or absence or different concentrations of the test substance in the presence of an olfactory receptor activator.

[0084] The above condition (C2-1) includes conditions before contacting the olfactory receptor with the test substance, and conditions under which the olfactory receptor is contacted with an olfactory receptor activator. The above condition (C2-1) also includes conditions after contacting the olfactory receptor with the olfactory receptor activator and the test substance, and conditions under which the test substance is substantially (e.g., completely) removed from the reaction system, and response inhibition by the test substance is substantially (e.g., completely) eliminated. The concentration of the test substance under the above condition (C2-2) is not particularly limited, as long as a measurable difference is observed between the activation level D1 and the activation level D2. The concentration of the test substance under the above condition (C2-2) may be, for example, 90% or less, 70% or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the concentration of the test substance in the above step (A). Other than the presence or absence or concentration of the test substance, the control condition is not particularly limited, as long as it allows evaluation of response inhibition by the test substance. The control conditions may be, for example, the same as the conditions in step (A) above, except for the presence or absence or concentration of the test substance.

[0085] The screening method of the present invention may include a step of measuring the degree of activation D2. The degree of activation D1 and the degree of activation D2 may be measured in a single reaction system with a time lag, or may be measured simultaneously or with a time lag in separate reaction systems. The degree of activation D2 may be measured before or after the degree of activation D1. For example, after measuring the degree of activation D2, a test substance may be added to the reaction system and the degree of activation D1 may be measured.

[0086] When the level of activation D1 is low, it may be determined that the inactivation of the olfactory receptor by the test substance has been observed. Specifically, when the level of activation D1 is lower than the level of activation D2, it may be determined that the inactivation of the olfactory receptor by the test substance has been observed. For example, when the ratio of the level of activation D1 to the level of activation D2 is less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, it may be determined that the inactivation of the olfactory receptor by the test substance has been observed. This ratio is also referred to as the "residual activity rate." The value obtained by subtracting the residual activity rate from 100% is also referred to as the "inhibition rate of activity." Specific examples of the inhibition rate of activity include the inhibition rates described in the Examples.

[0087] The above description regarding the determination of olfactory receptor inactivation by a test substance can also be applied mutatis mutandis to the determination of response inhibition by a test substance based on other indicators. In such cases, the "degree of activation" in the above description can be appropriately replaced with a term corresponding to the other indicator.

[0088] The method for measuring the response of an olfactory receptor to an olfactory receptor activator is not particularly limited. The method for measuring the response of an olfactory receptor to an olfactory receptor activator can be appropriately selected depending on various conditions, such as the form of use of the olfactory receptor and the type of response to be measured. That is, the response of an olfactory receptor to an olfactory receptor activator can be measured, for example, by an appropriate method that can measure the activation of the olfactory receptor by an olfactory receptor activator.

[0089] The method for measuring the activation of olfactory receptors by olfactory receptor activators is not particularly limited. The activation of olfactory receptors by olfactory receptor activators can be measured, for example, by known methods for measuring the activity of receptors such as olfactory receptors. Examples of such methods include methods for measuring intracellular calcium concentration and intracellular cAMP concentration. That is, the activation of olfactory receptors by olfactory receptor activators can be measured using, for example, intracellular calcium concentration or intracellular cAMP concentration as an indicator. Specifically, the activation of olfactory receptors by olfactory receptor activators can be measured, for example, using cells having olfactory receptors and using intracellular calcium concentration or intracellular cAMP concentration as an indicator. For example, in HEK293T cells, when olfactory receptors are activated by odorants, they couple with intracellular G proteins (such as Golf) to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP (Kajiya K. et al., Molecular bases of odor discrimination: Reconstitution of olfactory receptors that recognize overlapping sets of odorants. Journal of Neuroscience, 2001, 21:6018-6025). Techniques for measuring intracellular cAMP levels include, for example, ELISA and reporter assays. An example of a reporter assay is luciferase assay. Reporter assays can measure intracellular cAMP levels using a reporter gene (such as a luciferase gene) whose expression is configured to depend on the cAMP concentration. An example of a technique for measuring intracellular calcium levels is calcium imaging. In calcium imaging, intracellular calcium levels can be measured using calcium indicators. Examples of calcium indicators include calcium-sensitive fluorescent dyes and calcium-sensitive fluorescent proteins. Examples of calcium-sensitive fluorescent dyes include Fura 2 and Fluo 4.Furthermore, examples of calcium-sensitive fluorescent proteins include Cameleon, TN-XL, GCaMP, and G-GECO. "Calcium concentration" may refer to the concentration of free calcium ions.

[0090] The description of measuring olfactory receptor activation using cells having olfactory receptors can also be applied mutatis mutandis to cases where olfactory receptors are used in other embodiments. Examples of cases where olfactory receptors are used in other embodiments include cases where artificial lipid bilayer vesicles, cell membranes, or artificial lipid bilayer membranes having olfactory receptors are used. Examples of cases where olfactory receptors are used in other embodiments include cases where the olfactory receptors are used in a form having an internal space.

[0091] That is, for example, using artificial lipid bilayer vesicles having olfactory receptors, activation of olfactory receptors can be measured using the same method as when cells having olfactory receptors are used. In such cases, the term "cells" in the description of measuring activation of olfactory receptors using cells having olfactory receptors can be read as "artificial lipid bilayer vesicles."

[0092] Furthermore, for example, membranes such as cell membranes or artificial lipid bilayer membranes having olfactory receptors can be used to generate a space separated by the membrane, and the activation of olfactory receptors can be measured using the same method as when cells having olfactory receptors are used. Specifically, for example, when such a membrane is used to separate two spaces, that is, when such a membrane is used to provide a reaction system having two spaces separated from each other by the membrane, the activation of olfactory receptors can be measured using the same method as when cells having olfactory receptors are used. In such cases, the space separated by the membrane can be considered the interior of the cell (also referred to as the "internal space"). Specifically, one of the two spaces can be considered the interior of the cell (also referred to as the "internal space"), and the other can be considered the exterior of the cell (also referred to as the "external space"). Of these spaces, the one containing both substances can be considered the external space. In such cases, the terms "intracellular calcium concentration" and "intracellular cAMP concentration" in the description of measuring olfactory receptor activation using cells having olfactory receptors can be read as "calcium concentration within the internal space" and "cAMP concentration within the internal space," respectively.

[0093] In either case, measurable parameters can be selected depending on the manner in which the olfactory receptor is used.

[0094] Note that "measuring a certain parameter and using it as an index for measuring the response of an olfactory receptor to an olfactory receptor activator" means that as long as the response can be measured, specifically, as long as response inhibition by a test substance can be evaluated based on the response (i.e., as long as it is possible to determine whether response inhibition by the test substance is observed), it is sufficient to obtain and use data reflecting the parameter, and it is not necessary to obtain the value of the parameter itself. In other words, when data reflecting a certain parameter is obtained, it is not necessary to calculate the value of the parameter itself from the data. Specifically, for example, when measuring intracellular cAMP concentration by luciferase assay and using the data as an index for measuring olfactory receptor activation by an olfactory receptor activator, it is sufficient to obtain and use data reflecting the intracellular cAMP concentration (e.g., luminescence intensity) as long as the activation can be measured, specifically, as long as it is possible to evaluate olfactory receptor inactivation by a test substance based on the activation (i.e., as long as it is possible to determine whether olfactory receptor inactivation by the test substance is observed), and it is not necessary to calculate the intracellular cAMP concentration itself from the data.

[0095] Furthermore, "measuring the response of an olfactory receptor to an olfactory receptor activator" means obtaining data reflecting the response that can be used to evaluate response inhibition by a test substance. Similarly, the "olfactory receptor response to an olfactory receptor activator" used as an index for evaluating response inhibition by a test substance means data reflecting the response that can be used to evaluate response inhibition by a test substance. Such data can be, for example, data obtained by implementing a method for measuring the response of an olfactory receptor to an olfactory receptor activator (e.g., parameters such as those exemplified above and data reflecting them), either as is or after appropriate processing.

[0096] In this way, substances that suppress amino acid odor can be identified. The screening method of the present invention may further include a step of evaluating the masking function of the identified substance that suppresses amino acid odor (i.e., evaluating whether the identified substance that suppresses amino acid odor has a masking function). In other words, by evaluating the masking function of the identified substance that suppresses amino acid odor, it is possible to confirm whether the substance actually suppresses amino acid odor. The method for evaluating the masking function of the identified substance that suppresses amino acid odor is not particularly limited. The masking function of the identified substance that suppresses amino acid odor can be evaluated, for example, by known methods for evaluating the aroma of a substance. Such methods include sensory evaluation (evaluation by sensory testing). Specifically, the masking function of the identified substance that suppresses amino acid odor can be evaluated, for example, by comparing the amino acid odor in the presence of a substance that suppresses amino acid odor (e.g., a food containing a component that exhibits an amino acid odor, such as methional) with the amino acid odor in the absence of the substance that suppresses amino acid odor.

[0097] In conventional screening methods, in order to screen for amino acid odor inhibitors, it is necessary to confirm the masking function of a huge number of substances or their combinations one by one through sensory tests, etc., and select substances that suppress amino acid odor, which requires a lot of time and cost to develop a substance that suppresses amino acid odor. However, according to the screening method of the present invention, by utilizing olfactory receptors, substances that suppress amino acid odor can be efficiently screened. Therefore, according to the screening method of the present invention, the efficiency of developing amino acid odor inhibitors can be greatly improved.

[0098] The use of the screened substance that suppresses amino acid odor is not particularly limited. Substances that suppress amino acid odor can be used, for example, by blending them into objects in which suppression of amino acid odor is desired. Such objects include those that already exhibit an amino acid odor (e.g., foods containing components that exhibit an amino acid odor, such as methional) and those that may exhibit an amino acid odor in the future (e.g., foods containing components that may produce components that exhibit an amino acid odor, such as methionine). By blending a substance that suppresses amino acid odor, the amino acid odor in the object can be suppressed. Regarding the suppression of amino acid odor using a substance that suppresses amino acid odor, the description regarding the suppression of amino acid odor using active ingredients in "<2> Suppression of Amino Acid Odor" below can be applied mutatis mutandis. Furthermore, substances that suppress amino acid odor can also be used, for example, as raw materials for the development of new substances that suppress amino acid odor.

[0099] <2> Suppression of Amino Acid Odor <2-1> Active Ingredient In suppressing the amino acid odor, the following component (A) is used as an active ingredient: (A) At least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[0100] Component (A) is also referred to as the “active ingredient.” As component (A), one type of component may be used, or two or more types of components may be used in combination.

[0101] Any of the active ingredients may be an ingredient that inactivates the olfactory receptors OR4S2 and / or OR4E2. That is, the active ingredient may specifically be the following ingredient (A): (A) An ingredient that inactivates the olfactory receptors OR4S2 and / or OR4E2, which is at least one ingredient selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[0102] The phrase "a certain component inactivates the olfactory receptor OR4S2 and / or OR4E2" means that the inactivation of the olfactory receptor OR4S2 and / or OR4E2 by the component is observed under appropriate conditions. The olfactory receptors OR4S2 and OR4E2 and their inactivation are as described above in "<1> Screening for substances that suppress amino acid odor." Suitable conditions include those described in "<1> Screening for substances that suppress amino acid odor." Specific examples of suitable conditions include those described in the Examples. In other words, the phrase "a certain component inactivates the olfactory receptor OR4S2 and / or OR4E2" may mean, for example, that the inactivation of the olfactory receptor OR4S2 and / or OR4E2 by the component is observed at least under the conditions described in the Examples.

[0103] The use of an active ingredient can suppress the amino acid odor in foods, i.e., the effect of suppressing amino acid odor in foods can be achieved. This effect is also referred to as the "masking effect." The suppression of amino acid odor in foods is also simply referred to as "amino acid odor suppression." "Suppression of amino acid odor" can also be referred to as "reducing amino acid odor" or "masking amino acid odor." Note that "suppression of amino acid odor" encompasses both the suppression of amino acid odor that may occur in the future and the suppression of amino acid odor that has already occurred. "Suppression of amino acid odor" also encompasses the complete disappearance of amino acid odor. Amino acid odor is as described above in "<1> Screening for substances that suppress amino acid odor." Specifically, the use of an active ingredient can suppress amino acid odor in foods compared to when the active ingredient is not used. Therefore, the masking effect can be determined by measuring and comparing the amino acid odor in foods when the active ingredient is used and when the active ingredient is not used. In other words, a masking effect can be determined if the intensity of the amino acid odor in foods when the active ingredient is used is lower than when the active ingredient is not used. Measurement and comparison of amino acid odor can be performed, for example, by sensory evaluation by a specialist panel.

[0104] The active ingredient may be a commercially available product or may be obtained by appropriate manufacturing. The method for manufacturing the active ingredient is not particularly limited. The active ingredient can be manufactured, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. The active ingredient may be purified to a desired degree or may not be purified. That is, the active ingredient may be a purified product, or a material containing the active ingredient. For example, the active ingredient may be a material containing the active ingredient at a content of 1% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.

[0105] In addition, when a material containing an active ingredient is used, the amount of the active ingredient (for example, the content (concentration) or amount used) is calculated based on the amount of the active ingredient itself in the material.

[0106] <2-2> Composition of the Present Invention The composition of the present invention is a composition containing an active ingredient.

[0107] That is, the composition of the present invention is a composition containing the following component (A): (A) at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[0108] Specifically, the composition of the present invention may be a composition containing the following component (A): (A) a component that inactivates the olfactory receptor OR4S2 and / or OR4E2, said component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[0109] By using the composition of the present invention, the amino acid odor in food can be suppressed, i.e., a masking effect can be obtained. Therefore, the composition of the present invention can be used to suppress the amino acid odor in food. That is, the composition of the present invention can be, for example, a composition for suppressing the amino acid odor in food.

[0110] Furthermore, by utilizing the composition of the present invention, it is possible to produce a food product in which the amino acid odor is suppressed. Therefore, the composition of the present invention may be utilized in the production of a food product (specifically, the production of a food product in which the amino acid odor is suppressed). That is, the composition of the present invention may be, for example, a composition for use in the production of a food product (specifically, the production of a food product in which the amino acid odor is suppressed).

[0111] The composition of the present invention may be, for example, a seasoning. Specifically, the composition of the present invention may be, for example, a seasoning for suppressing amino acid odor in food, or a seasoning for food production (specifically, production of food with suppressed amino acid odor).

[0112] The composition of the present invention may be used to suppress amino acid odor in food or to produce food in the manner described in the second aspect of the method of the present invention below.

[0113] The composition of the present invention may consist of an active ingredient, or may contain ingredients other than the active ingredient. The composition of the present invention may exclude a composition consisting of an active ingredient. As the ingredients other than the active ingredient, one kind of ingredient may be used, or two or more kinds of ingredients may be used in combination.

[0114] The components other than the active ingredient are not particularly limited as long as they do not impair the masking effect. The components other than the active ingredient can be appropriately selected depending on various conditions such as the type of food. Examples of the components other than the active ingredient include ingredients that are blended into foods or pharmaceuticals. Specific examples of the components other than the active ingredient include the raw materials of foods described below.

[0115] The composition of the present invention can be produced, for example, by appropriately mixing the active ingredient and, optionally, other ingredients.

[0116] The composition of the present invention may be formulated as appropriate, for example. When formulating, additives may be used as appropriate. Examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, and solvents. The additives can be selected as appropriate depending on various conditions, such as the shape of the composition of the present invention.

[0117] The form of the composition of the present invention is not particularly limited, and the composition of the present invention may be in any form, such as powder, flakes, tablets, paste, liquid, etc.

[0118] The content and content ratio of each component (i.e., the active ingredient and optionally other ingredients) in the composition of the present invention are not particularly limited as long as a masking effect is obtained. The content and content ratio of each component in the composition of the present invention can be appropriately set depending on various conditions such as the mode of use of the composition of the present invention.

[0119] The content of the active ingredient in the composition of the present invention is greater than 0% (w / w) and less than 100% (w / w). The content of the active ingredient in the composition of the present invention is, for example, 1 ppt (w / w) or more, 10 ppt (w / w) or more, 100 ppt (w / w) or more, 1 ppb (w / w) or more, 10 ppb (w / w) or more, 100 ppb (w / w) or more, 1 ppm (w / w) or more, 10 ppm (w / w) or more, 100 ppm (w / w) or more, 1000 ppm (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, or 10% (w / w) or more. The active ingredient concentration in the composition of the present invention may be 100% (w / w) or less, less than 100% (w / w), 99.9% (w / w) or less, 90% (w / w) or less, 50% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 1000 ppm (w / w) or less, 100 ppm (w / w) or less, 10 ppm (w / w) or less, or 1 ppm (w / w) or less, or any compatible combination thereof. Specific examples of the active ingredient content in the composition of the present invention may be 1 ppt (w / w) to 10% (w / w), 1 ppt (w / w) to 1% (w / w), or 1 ppt (w / w) to 1000 ppm (w / w). When the composition of the present invention contains two or more active ingredients, the "content of the active ingredients in the composition of the present invention" refers to the total content of those two or more active ingredients in the composition of the present invention, unless otherwise specified. However, in one embodiment, when the composition of the present invention contains two or more active ingredients, the contents of those two or more active ingredients in the composition of the present invention may each independently be within the range of the content of the active ingredients in the composition of the present invention exemplified above (provided that the total content is 100% (w / w) or less).

[0120] The content of each component (i.e., the active ingredient and optionally other components) in the composition of the present invention can be set, for example, so as to obtain the amount of each component added in the second embodiment of the method of the present invention described below.

[0121] The components (i.e., the active ingredient and optional other ingredients) contained in the composition of the present invention may be mixed together and contained in the composition of the present invention, or may be contained separately or in any combination. For example, the composition of the present invention may be provided as a set of components each packaged separately. In such a case, the components contained in the set can be used together as appropriate when used.

[0122] <2-3> Second Aspect of the Method of the Present Invention The second aspect of the method of the present invention is a method comprising a step of utilizing an active ingredient.

[0123] That is, a second embodiment of the method of the present invention is a method comprising a step of utilizing the following component (A): (A) at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[0124] Specifically, the second aspect of the method of the present invention may be a method comprising a step of utilizing the following component (A): (A) a component that inactivates the olfactory receptor OR4S2 and / or OR4E2, said component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

[0125] According to the second aspect of the method of the present invention, specifically by utilizing an active ingredient, the amino acid odor in food can be suppressed, i.e., a masking effect can be obtained. Therefore, the second aspect of the method of the present invention may be implemented to suppress the amino acid odor in food. That is, the second aspect of the method of the present invention may be, for example, a method for suppressing the amino acid odor in food. This method is also referred to as the "masking method of the present invention."

[0126] Furthermore, according to the second aspect of the method of the present invention, specifically by utilizing the active ingredient, it is possible to produce a food product with a suppressed amino acid odor. Therefore, the second aspect of the method of the present invention may be carried out for the production of a food product (specifically, the production of a food product with a suppressed amino acid odor). That is, the second aspect of the method of the present invention may be, for example, a method for producing a food product (specifically, the production of a food product with a suppressed amino acid odor). This method is also referred to as the "food production method of the present invention."

[0127] The active ingredient can be added to food ingredients during food production to suppress the amino acid odor or be used in food production. That is, an example of using the active ingredient is adding the active ingredient to food ingredients. That is, the second aspect of the method of the present invention may specifically be, for example, a method for suppressing the amino acid odor in food, which includes adding the active ingredient to food ingredients. Furthermore, the second aspect of the method of the present invention may specifically be, for example, a method for producing food (specifically, producing food with a suppressed amino acid odor), which includes adding the active ingredient to food ingredients. "Addition" is also referred to as "blending."

[0128] The active ingredient may be used in the second aspect of the method of the present invention, for example, in the form of a composition of the present invention. That is, "use of an active ingredient" also includes use of a composition of the present invention. For example, "addition of an active ingredient" also includes addition of a composition of the present invention.

[0129] The food obtained by the second aspect of the method of the present invention is also referred to as the "food of the present invention." Specifically, the food of the present invention is a food in which the amino acid odor is suppressed. In other words, the food of the present invention is a food to which an active ingredient has been added.

[0130] The suppression of amino acid odor or the production of food may be carried out in the same manner as the production of ordinary foods, except for the use of an active ingredient. That is, the suppression of amino acid odor or the production of food may be carried out using the same raw materials as ordinary foods under the same production conditions, except for the use of an active ingredient. Furthermore, the raw materials and production conditions of the food may be appropriately modified and used for the suppression of amino acid odor or the production of food.

[0131] The food is not particularly limited as long as it is desired to suppress the amino acid odor. The food may already exhibit an amino acid odor, or may exhibit an amino acid odor in the future. Foods that already exhibit an amino acid odor include foods containing components that exhibit an amino acid odor. Foods that may exhibit an amino acid odor in the future include foods containing components that can produce components that exhibit an amino acid odor. That is, the food may contain components that exhibit an amino acid odor and / or components that can produce components that exhibit an amino acid odor. Components that exhibit an amino acid odor include components that can be produced by oxidation of amino acids (e.g., oxidation by heating or natural oxidation). Components that can be produced by oxidation of amino acids include methional. Components that can produce components that exhibit an amino acid odor include amino acids. Examples of amino acids include methionine. Foods also include beverages. Foods also include seasonings. Foods may be, for example, liquids or solids.Specific examples of foods include beverages such as milk, soft drinks, alcoholic drinks, and soups; sweets such as jelly; processed meat foods such as ham, sausage, dumplings, shumai, hamburger steak, fried chicken, and pork cutlet; processed seafood foods such as salmon flakes, spicy cod roe, salted cod roe, grilled fish, dried fish, salted fish, fish sausage, kamaboko, boiled fish, tsukudani (simmered fish), and canned goods; sweets such as potato chips, potato snacks, corn snacks, wheat snacks, cinnamon cookies, rice crackers, and arare (rice crackers); noodle soups such as udon soup, soba soup, somen soup, ramen soup, champon soup, and pasta sauce; cooked rice dishes such as rice balls, pilaf, fried rice, mixed rice, rice porridge, and ochazuke (tea rice with green tea); curry, stew, chili con carne, feijoata, hemp Examples include stews such as tofu; rouxes such as stew roux and curry roux; processed vegetables such as kimchi and pickles; other processed foods such as bread, noodles, gratin, and croquettes; sauces such as Chinese sauce, oyster sauce, cheese sauce, tomato sauce, white sauce, demi-glace sauce, curry sauce, Genoa sauce, chili sauce, and Tabasco sauce; seasoning oils such as chili oil; basic seasonings such as soy sauce and miso; flavor seasonings such as bonito flavor, chicken flavor, pork flavor, and beef flavor; spicy seasonings such as shichimi togarashi, doubanjiang, and gochujang; menu-specific seasonings (special seasonings tailored to the menu being cooked); and other seasonings such as dressings, miso, mayonnaise, tomato ketchup, and consommé. "Soft drinks" may refer to non-alcoholic beverages (drinks with an alcohol concentration of less than 1%), excluding milk and dairy products. Specific examples of soft drinks include water, fruit juice, vegetable juice, tea (chai, cinnamon tea, etc.), coffee drinks (coffee, coffee-infused milk drinks, etc.), carbonated drinks (ginger ale, lemon carbonated drinks, etc.), sports drinks, and jelly drinks. Specific examples of soups include dal soup, tom yum goong, egg soup, seaweed soup, shark fin soup, Chinese-style soup, consommé soup, curry-flavored soup, clear soup, miso soup, and potage soup. Examples of foods include, in particular, the foods listed above that contain a component that exhibits an amino acid odor and / or a component that can produce a component that exhibits an amino acid odor.

[0132] The manner in which the food is provided is not particularly limited. For example, the food may be provided in a form that can be consumed as is, or in a form that requires preparation before or at the time of consumption, such as a concentrated product or a dried product. The food may also be provided in any container, such as a retort pouch, a paper pack, a plastic bottle such as a PET bottle, a metal can such as a steel can or an aluminum can, or a glass bottle. Foods are not limited to general foods, but also include so-called health foods or medical foods such as nutritional supplements (supplements), nutritionally functional foods, and foods for specified health uses. That is, for example, the foods exemplified above may be provided as general foods, health foods, or medical foods.

[0133] "Food ingredients" refers to food materials used to produce food. There are no particular limitations on food ingredients as long as they can be used to produce food. Food ingredients can be selected appropriately depending on various conditions, such as the type of food. Food ingredients include ingredients that can be commonly used in the production of foods, such as those exemplified above. Specific examples of food ingredients include ingredients such as grains, vegetables, meat, seafood, and eggs; seasoning ingredients such as sugars, inorganic salts, organic acids, nucleic acids, amino acids, and protein hydrolysates; dairy products such as milk and cheese; spices; flavorings; oils and fats; alcohol; dietary fiber; and pH buffers.

[0134] The active ingredient may be added to food ingredients at any stage of the food manufacturing process as long as a masking effect is obtained. In other words, the "food ingredients" to which the active ingredient is added may be those at any stage of the food manufacturing process. For example, the "food ingredients" to which the active ingredient is added may include finished foods before the active ingredient is added. The active ingredient may be added to the food ingredients either as is or after being prepared into a desired form such as a solution. "Addition of an active ingredient" may refer collectively to the process of allowing the active ingredient to coexist with the food ingredients. Ingredients other than the active ingredient (e.g., ingredients that exhibit an amino acid odor or ingredients that can produce an amino acid odor) may also be added to the food ingredients as appropriate. The description of the addition of an active ingredient also applies mutatis mutandis to the addition of ingredients other than the active ingredient. Each ingredient (i.e., the active ingredient and optionally other ingredients) may be added to the food ingredients all at the same time, separately, or in any combination. The order in which each ingredient is added to the food ingredients is not particularly limited.

[0135] The amounts and ratios of the components (i.e., the active ingredient and optionally other ingredients) added in the second embodiment of the method of the present invention are not particularly limited as long as a masking effect is obtained. The amounts and ratios of the components added in the second embodiment of the method of the present invention can be appropriately set depending on various conditions such as the type of raw material of the food and the type of food.

[0136] The active ingredient may be added to the raw materials of the food product so that the ingestible concentration of the active ingredient falls within a desired range (for example, the range of ingestible concentration of the active ingredient described below).

[0137] The ingested concentration of the active ingredient is, for example, 0.001 ppt (w / w) or more, 0.002 ppt (w / w) or more, 0.005 ppt (w / w) or more, 0.01 ppt (w / w) or more, 0.02 ppt (w / w) or more, 0.05 ppt (w / w) or more, 0.1 ppt (w / w) or more, 0.2 ppt (w / w) or more, 0.5 ppt (w / w) or more, 1 ppt (w / w) or more, 2 ppt (w / w) or more, 5 ppt (w / w) or more, 10 ppt (w / w) or more, 20 ppt (w / w) or more, 50 ppt (w / w) or more, 100 ppt (w / w) or more, 200 ppt (w / w) or more, 500 ppt (w / w) or more, 1 ppb (w / w) or more, 2 It may be 10 ppm (w / w) or less, 5 ppm (w / w) or less, 2 ppm (w / w) or less, 1 ppm (w / w) or less, 500 ppb (w / w) or less, 200 ppb (w / w) or less, 100 ppb (w / w) or less, 50 ppb (w / w) or less, 20 ppb (w / w) or less, 10 ppb (w / w) or less, 5 ppb (w / w) or less, 2 ppb (w / w) or less, 1 ppb (w / w) or less, 500 ppt (w / w) or less, 200 ppt (w / w) or less, 100 ppt(w / w) or less, 50 ppt(w / w) or less, 20 ppt(w / w) or less, 10 ppt(w / w) or less, 5 ppt(w / w) or less, 2 ppt(w / w) or less, 1 ppt(w / w) or less, 0.5 ppt(w / w) or less, 0.2 ppt(w / w) or less, or 0.1 ppt(w / w) or less, or any compatible combination thereof.Specifically, the ingested concentration of the active ingredient may be, for example, 0.001 ppt(w / w) to 10 ppm(w / w), 0.01 ppt(w / w) to 1 ppm(w / w), or 0.1 ppt(w / w) to 100 ppb(w / w), 0.001 ppt(w / w) to 100 ppt(w / w), 0.01 ppt(w / w) to 10 ppt(w / w), or 0.1 ppt(w / w) to 1 ppt(w / w), 0.01 ppt(w / w) to 1 ppb(w / w), 0.1 ppt(w / w) to 100 ppt(w / w), or 1 ppt(w / w) to 10 ppt(w / w), 0.1 ppt(w / w) to 10 ppb(w / w), 1 It may be ppt (w / w) to 1 ppb (w / w), or 10 ppt (w / w) to 100 ppt (w / w), or 1 ppt (w / w) to 100 ppb (w / w), or 10 ppt (w / w) to 10 ppb (w / w), or 100 ppt (w / w) to 1 ppb (w / w), or 10 ppt (w / w) to 1 ppm (w / w), or 100 ppt (w / w) to 100 ppb (w / w), or 1 ppb (w / w) to 10 ppb (w / w), or 100 ppt (w / w) to 10 ppm (w / w), or 1 ppb (w / w) to 1 ppm (w / w), or 10 ppb (w / w) to 100 ppb (w / w). When the food product of the present invention contains two or more active ingredients (e.g., when two or more active ingredients are added), the "ingestion concentration of active ingredients" refers to the total ingestion concentration of those two or more active ingredients, unless otherwise specified. However, in one embodiment, when the food product of the present invention contains two or more active ingredients (e.g., when two or more active ingredients are added), the ingestion concentrations of those two or more active ingredients may each independently be within the range of the ingestion concentration of the active ingredients exemplified above.

[0138] The ingested concentration of 2-methyl-3-furanthiol may be, for example, 0.001 ppt(w / w) or more, 0.002 ppt(w / w) or more, 0.005 ppt(w / w) or more, 0.01 ppt(w / w) or more, 0.02 ppt(w / w) or more, 0.05 ppt(w / w) or more, 0.1 ppt(w / w) or more, 0.2 ppt(w / w) or more, 0.5 ppt(w / w) or more, or 1 ppt(w / w) or more, and may be 100 ppt(w / w) or less, 50 ppt(w / w) or less, 20 ppt(w / w) or less, 10 ppt(w / w) or less, 5 ppt(w / w) or less, 2 ppt(w / w) or less, 1 ppt(w / w) or less, 0.5 ppt(w / w) or less, 0.2 The ingested concentration of 2-methyl-3-furanthiol may be, for example, 0.001 ppt (w / w) to 100 ppt (w / w), 0.01 ppt (w / w) to 10 ppt (w / w), or 0.1 ppt (w / w) to 1 ppt (w / w).

[0139] The ingested concentration of bis(2-methyl-3-furyl) disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, 2,4-decadienal, or 2,4-nonadienal may be, for example, 0.01 ppt(w / w) or more, 0.02 ppt(w / w) or more, 0.05 ppt(w / w) or more, 0.1 ppt(w / w) or more, 0.2 ppt(w / w) or more, 0.5 ppt(w / w) or more, 1 ppt(w / w) or more, 2 ppt(w / w) or more, 5 ppt(w / w) or more, or 10 ppt(w / w) or more, and may be 1 ppb(w / w) or less, 500 ppt(w / w) or less, 200 ppt(w / w) or less, 100 ppt(w / w) or less, 50 ppt(w / w) or less, 20 The ingested concentration of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, 2,4-decadienal, or 2,4-nonadienal may be, for example, 0.01 ppt(w / w) to 1 ppb(w / w), 0.1 ppt(w / w) to 100 ppt(w / w), or 1 ppt(w / w) to 10 ppt(w / w).

[0140] The ingested concentration of furfuryl mercaptan or E-β-damascone may be, for example, 0.1 ppt (w / w) or more, 0.2 ppt (w / w) or more, 0.5 ppt (w / w) or more, 1 ppt (w / w) or more, 2 ppt (w / w) or more, 5 ppt (w / w) or more, 10 ppt (w / w) or more, 20 ppt (w / w) or more, 50 ppt (w / w) or more, or 100 ppt (w / w) or more, or 10 ppb (w / w) or less, 5 ppb (w / w) or less, 2 ppb (w / w) or less, 1 ppb (w / w) or less, 500 ppt (w / w) or less, 200 ppt (w / w) or less, or 100 ppt (w / w) or less, or any compatible combination thereof. The ingested concentration of furfuryl mercaptan or E-β-damascone may be, for example, 0.1 ppt (w / w) to 10 ppb (w / w), 1 ppt (w / w) to 1 ppb (w / w), or 10 ppt (w / w) to 100 ppt (w / w).

[0141] The ingested concentration of 2,5-dihydroxy-1,4-dithiane, 2-phenyl-2-butenal, or 2-thiophenethiol may be, for example, 1 ppt (w / w) or more, 2 ppt (w / w) or more, 5 ppt (w / w) or more, 10 ppt (w / w) or more, 20 ppt (w / w) or more, 50 ppt (w / w) or more, 100 ppt (w / w) or more, 200 ppt (w / w) or more, 500 ppt (w / w) or more, or 1 ppb (w / w) or more, or 100 ppb (w / w) or less, 50 ppb (w / w) or less, 20 ppb (w / w) or less, 10 ppb (w / w) or less, 5 ppb (w / w) or less, 2 ppb (w / w) or less, or 1 ppb (w / w) or less, or any compatible combination thereof. The ingested concentration of 2,5-dihydroxy-1,4-dithiane, 2-phenyl-2-butenal, or 2-thiophenethiol may be, for example, 1 ppt (w / w) to 100 ppb (w / w), 10 ppt (w / w) to 10 ppb (w / w), or 100 ppt (w / w) to 1 ppb (w / w).

[0142] The ingested concentration of nootkatone, allyl isothiocyanate, or cinnamaldehyde may be, for example, 10 ppt (w / w) or more, 20 ppt (w / w) or more, 50 ppt (w / w) or more, 100 ppt (w / w) or more, 200 ppt (w / w) or more, 500 ppt (w / w) or more, 1 ppb (w / w) or more, 2 ppb (w / w) or more, 5 ppb (w / w) or more, or 10 ppb (w / w) or more, or 1 ppm (w / w) or less, 500 ppb (w / w) or less, 200 ppb (w / w) or less, 100 ppb (w / w) or less, 50 ppb (w / w) or less, 20 ppb (w / w) or less, or 10 ppb (w / w) or less, or any compatible combination thereof. The ingested concentration of nootkatone, allyl isothiocyanate, or cinnamaldehyde may be, for example, 10 ppt (w / w) to 1 ppm (w / w), 100 ppt (w / w) to 100 ppb (w / w), or 1 ppb (w / w) to 10 ppb (w / w).

[0143] The ingestible concentration of sclareolide may be, for example, 100 ppt (w / w) or more, 200 ppt (w / w) or more, 500 ppt (w / w) or more, 1 ppb (w / w) or more, 2 ppb (w / w) or more, 5 ppb (w / w) or more, 10 ppb (w / w) or more, 20 ppb (w / w) or more, 50 ppb (w / w) or more, or 100 ppb (w / w) or more, or 10 ppm (w / w) or less, 5 ppm (w / w) or less, 2 ppm (w / w) or less, 1 ppm (w / w) or less, 500 ppb (w / w) or less, 200 ppb (w / w) or less, or 100 ppb (w / w) or less, or any compatible combination thereof. The ingested concentration of sclareolide may be, for example, 100 ppt (w / w) to 10 ppm (w / w), 1 ppb (w / w) to 1 ppm (w / w), or 10 ppb (w / w) to 100 ppb (w / w).

[0144] Furthermore, the ingested concentration of the active ingredient may be, for example, less than the threshold concentration of the active ingredient. Specifically, the ingested concentration of the active ingredient may be, for example, within the range of the ingested concentrations of the active ingredient exemplified above, and less than the threshold concentration of the active ingredient. The "threshold concentration of the active ingredient" refers to the maximum concentration of the active ingredient at which the odor of the active ingredient itself is not detected when ingesting an aqueous solution containing the active ingredient alone. Examples of the threshold concentration of the active ingredient include the threshold concentrations described in the Examples.

[0145] The description of the addition of an active ingredient also applies mutatis mutandis to the addition of the composition of the present invention. For example, the composition of the present invention can be added so as to obtain the amount of the active ingredient exemplified above.

[0146] The food of the present invention may contain a component capable of producing a component that exhibits an amino acid odor (e.g., an amino acid such as methionine). That is, the food of the present invention may be manufactured to contain a component capable of producing a component that exhibits an amino acid odor. The food of the present invention may contain one component capable of producing a component that exhibits an amino acid odor, or may contain two or more components capable of producing a component that exhibits an amino acid odor. A food containing a component capable of producing a component that exhibits an amino acid odor can be produced, for example, by adding a component capable of producing a component that exhibits an amino acid odor. That is, the second aspect of the method of the present invention may further include adding a component capable of producing a component that exhibits an amino acid odor to the raw materials of the food. A food containing a component capable of producing a component that exhibits an amino acid odor can be produced, for example, by adding the component itself that exhibits an amino acid odor, or by adding a material containing a component that exhibits an amino acid odor, such as a seasoning containing a component that exhibits an amino acid odor. The component capable of producing a component that exhibits an amino acid odor may be commercially available, or may be obtained by appropriate manufacturing. The method for producing a component capable of producing an amino acid odor is not particularly limited. A component capable of producing an amino acid odor can be produced, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. The addition of a component capable of producing an amino acid odor can be carried out in the same manner as the addition of an active ingredient. A component capable of producing an amino acid odor can be added to a food ingredient, for example, so that the content of the component capable of producing an amino acid odor in the food of the present invention is within a desired range (for example, the content range described below). Furthermore, a food containing a component capable of producing an amino acid odor can be produced, for example, using a food ingredient containing a component capable of producing an amino acid odor. That is, the food ingredient may contain a component capable of producing an amino acid odor.

[0147] If a component capable of producing an amino acid odor can form a salt, the component capable of producing an amino acid odor can be used as a free form, as a salt, or a combination thereof. In other words, the term "component capable of producing an amino acid odor" can refer to the component capable of producing an amino acid odor in its free form, its salt, or a combination thereof, unless otherwise specified. Furthermore, if a component capable of producing an amino acid odor can form a hydrate, the component capable of producing an amino acid odor can be used as a non-hydrate, a hydrate, or a combination thereof. In other words, the term "component capable of producing an amino acid odor" (e.g., "component capable of producing an amino acid odor in its free form" or "salt of a component capable of producing an amino acid odor") can encompass both non-hydrate and hydrate forms, unless otherwise specified. The component capable of producing an amino acid odor can be in any form, such as an ion, when used.

[0148] The salt is not particularly limited as long as it is orally ingestible. For example, specific examples of salts of acidic groups such as carboxyl groups include ammonium salts, salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, aluminum salts, zinc salts, salts with organic amines such as triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine and dicyclohexylamine, and salts with basic amino acids such as arginine and lysine. Specific examples of salts with basic groups such as amino groups include salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid, salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hybenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, malic acid, methylmalonic acid, and adipic acid, and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. One type of salt may be used, or two or more types of salts may be used in combination.

[0149] In addition, when using a material containing a component that can produce a component that exhibits an amino acid odor, the amount of the component that can produce the amino acid odor (e.g., content (concentration) or amount used) is calculated based on the amount of the component itself that can produce the amino acid odor in the material. Furthermore, when the component that can produce the amino acid odor forms a salt or hydrate, the amount of the component that can produce the amino acid odor (e.g., content (concentration) or amount used) is calculated based on the value obtained by converting the mass of the salt or hydrate to the mass of an equimolar unhydrated free form. The same applies to components that exhibit an amino acid odor.

[0150] When the food product of the present invention contains a component that can produce a component that exhibits an amino acid odor (for example, an amino acid such as methionine), the content of the component that can produce a component that exhibits an amino acid odor in the food product of the present invention may be, for example, 20 ppm (w / w) or more, 50 ppm (w / w) or more, 100 ppm (w / w) or more, 200 ppm (w / w) or more, 500 ppm (w / w) or more, 1000 ppm (w / w) or more, 2000 ppm (w / w) or more, 5000 ppm (w / w) or more, or 1% (w / w) or more, and may be 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 5000 ppm (w / w) or less, 2000 ppm (w / w) or less, 1000 ppm (w / w) or less, 500 ppm (w / w) or less, 200 ppm (w / w) or less, or 100 The content of components capable of producing an amino acid odor in the food of the present invention may be, for example, 20 ppm (w / w) to 5% (w / w), 100 ppm (w / w) to 1% (w / w), or 500 ppm (w / w) to 200 ppm (w / w) in terms of ingested concentration. When the food of the present invention contains two or more components capable of producing an amino acid odor, the "content of components capable of producing an amino acid odor in the food of the present invention" refers to the total content of those two or more components in the food of the present invention, unless otherwise specified. However, in one embodiment, when the food of the present invention contains two or more components capable of producing an amino acid odor, the content of those two or more components in the food of the present invention may each independently be within the range of the content of components capable of producing an amino acid odor in the food of the present invention exemplified above. In one aspect, the content of components that can produce components that exhibit an amino acid odor in the foods of the present invention exemplified above can also be read as the sum of the content of components that can produce components that exhibit an amino acid odor in the foods of the present invention and the content of components that exhibit an amino acid odor.

[0151] The food of the present invention may contain a component that exhibits an amino acid odor (e.g., methional). That is, the food of the present invention may be manufactured to contain a component that exhibits an amino acid odor. The food of the present invention may contain one component that exhibits an amino acid odor, or two or more components that exhibit an amino acid odor. A food containing a component that exhibits an amino acid odor can be produced, for example, by adding a component that exhibits an amino acid odor. That is, the second aspect of the method of the present invention may further include adding a component that exhibits an amino acid odor to the raw materials of the food. A food containing a component that exhibits an amino acid odor can be produced, for example, by adding the component that exhibits an amino acid odor itself, or by adding a material that contains a component that exhibits an amino acid odor, such as a seasoning containing a component that exhibits an amino acid odor. The component that exhibits an amino acid odor can be commercially available or can be obtained by appropriate manufacturing. The method for producing the component that exhibits an amino acid odor is not particularly limited. The component that exhibits an amino acid odor can be produced, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. The addition of a component exhibiting an amino acid odor can be carried out in the same manner as the addition of an active ingredient. The component exhibiting an amino acid odor may be added to a food ingredient, for example, so that the content of the component exhibiting an amino acid odor in the food of the present invention is within a desired range (for example, the content range described below). Furthermore, a food containing a component exhibiting an amino acid odor can be produced, for example, using a food ingredient containing a component exhibiting an amino acid odor. That is, the food ingredient may contain a component exhibiting an amino acid odor. Furthermore, the component exhibiting an amino acid odor may be generated, for example, during the production process of the food of the present invention. The component exhibiting an amino acid odor may be generated, for example, by oxidation of a component capable of producing a component exhibiting an amino acid odor (for example, oxidation by heating). Thus, a second aspect of the method of the present invention may include, for example, a step of heating a food ingredient or food containing a component capable of producing a component exhibiting an amino acid odor. Heating may be carried out before or after the addition of the active ingredient.

[0152] The heating conditions are not particularly limited as long as components exhibiting an amino acid odor are produced. The heating temperature may be, for example, 50°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, or 100°C or higher; 200°C or lower, 150°C or lower, 120°C or lower, 100°C or lower, 90°C or lower, or 80°C or lower, or a compatible combination thereof. Specific examples of the heating temperature include 50 to 200°C, or 80 to 150°C. The heating time may be, for example, 1 minute or more, 3 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, or 60 minutes or more; 120 minutes or less, 90 minutes or less, 60 minutes or less, or 30 minutes or less, or a compatible combination thereof. Specific examples of the heating time include 1 to 120 minutes, 10 to 90 minutes, or 30 to 60 minutes. Heating methods include incubating, baking, steaming, boiling, and frying. Heating may be carried out, for example, in conjunction with the food production process, or may be carried out separately from the food production process. Furthermore, heating may be carried out, for example, in part in conjunction with the food production process, and the remainder separately from the food production process. For example, if the food production process inherently includes a heating step, the food production process may also carry out some or all of the heating.

[0153] When the food of the present invention contains a component that exhibits an amino acid odor (e.g., methional), the content of the component that exhibits an amino acid odor in the food of the present invention may be, for example, 1 ppb (w / w) or more, 2 ppb (w / w) or more, 5 ppb (w / w) or more, 10 ppb (w / w) or more, 20 ppb (w / w) or more, 50 ppb (w / w) or more, 100 ppb (w / w) or more, 200 ppb (w / w) or more, or 500 ppb (w / w) or more, as an ingestible concentration, or 100 ppm (w / w) or less, 50 ppm (w / w) or less, 20 ppm (w / w) or less, 10 ppm (w / w) or less, 5 ppm (w / w) or less, 2 ppm (w / w) or less, 1 ppm (w / w) or less, 500 ppb (w / w) or less, or 200 ppm (w / w) or less. The content of the component(s) exhibiting an amino acid odor in the food product of the present invention may be, for example, 1 ppb (w / w) to 100 ppm (w / w), 10 ppb (w / w) to 10 ppm (w / w), or 50 ppb (w / w) to 2 ppm (w / w) as an intake concentration. When the food product of the present invention contains two or more components exhibiting an amino acid odor, the "content of the component(s) exhibiting an amino acid odor in the food product of the present invention" refers to the total content of those two or more components in the food product of the present invention, unless otherwise specified. However, in one embodiment, when the food product of the present invention contains two or more components exhibiting an amino acid odor, the content of those two or more components in the food product of the present invention may each independently be within the range of the content of the component(s) exhibiting an amino acid odor in the food product of the present invention exemplified above.

[0154] <2-4> Use of Active Ingredient The present invention also discloses the use of the active ingredient in the applications exemplified above. That is, the present invention discloses, for example, the use of the active ingredient for suppressing amino acid odor in food or for producing food, and the use of the active ingredient in producing a composition for suppressing amino acid odor in food or for producing food.

[0155] The present invention also discloses active ingredients for use in the above-exemplified applications. That is, the present invention discloses, for example, active ingredients for use in suppressing amino acid odor in foods or in producing foods, and active ingredients for use in producing a composition for suppressing amino acid odor in foods or in producing foods.

[0156] The present invention will be described in more detail below with reference to non-limiting examples.

[0157] Unless otherwise specified, the raw materials, reagents, compounds, etc. used in the present examples are readily available or can be prepared according to methods commonly practiced in the art, or are commercially available.

[0158] Example 1: Screening for methional odor-inhibiting substances using olfactory receptors The present inventors discovered that methional is the causative substance of amino acid odor and that the olfactory receptors OR4S2 and OR4E2 respond to methional (data not shown). Therefore, in this example, we screened for methional odor-inhibiting substances using the olfactory receptors OR4S2 and OR4E2.

[0159] <1> Generation of Human Olfactory Receptor-Expressing Cells <1-1> Generation of Human Olfactory Receptor Expression Vectors The human olfactory receptor genes OR4S2 and OR4E2 were purchased from the TrueClone cDNA Clone Collection (OriGene). Subcloning fragments for the human olfactory receptor genes OR4S2 and OR4E2 were amplified by PCR using primers designed based on the sequence information registered in GenBank and the purchased human olfactory receptor genes as templates. The amplified subcloning fragments for each gene were subcloned downstream of the Rho tag sequence of the Rho-pME18S vector (K. Kajiya et al., Journal of Neuroscience, 15 August 2001, 21 (16) 6018-6025) using the EcoRI and XhoI sites to obtain expression vectors for the human olfactory receptors OR4S2 and OR4E2.

[0160] <1-2> Preparation of cells expressing olfactory receptors (OR4E2 and OR4S2) HEK293T cells expressing two types of olfactory receptors (OR4E2 and OR4S2) were prepared using the following procedure. A mixture of expression vectors with the composition shown in Table 1 was prepared and left to stand in a clean bench for 20 minutes. pcDNA3.1-microbat RTP1s is an expression vector for bat RTP1s, pcDNA3.1-Golf is an expression vector for human Golf, and pcDNA3.1-Ric8B is an expression vector for rat Ric8B (JP Patent Publication No. 2019-037197). HEK293T cells (2.5-3.5 x 10) were seeded in a 10 cm dish the day before. 6 The expression vector mixture was added to the HEK293T cells (2.5 × 10 cells / 10 cm dish). After culturing for 5 hours in an incubator maintained at 37°C and 5% CO2, HEK293T cells (2.5 × 10 cells / 10 cm dish) were plated in each well of a 96-well plate (BD). 5 100 μl of each of the cells (cells / ml) was seeded and cultured overnight in an incubator maintained at 37°C and 5% CO2.

[0161]

[0162] <2> Luciferase Assay Two olfactory receptors (OR4E2 and OR4S2) expressed in HEK293T cells conjugate with Golf to activate adenylate cyclase, thereby increasing intracellular cAMP levels. In this example, a luciferase reporter gene assay was used to measure the response of olfactory receptors to test substances, monitoring increases in intracellular cAMP levels as increases in luminescence levels derived from firefly luciferase. The "luciferase reporter gene assay" is also referred to as the "luciferase assay." Firefly luciferase is expressed from the firefly luciferase gene carried by the pGL4.29[luc2P / CRE / Hygro] Vector in a manner dependent on the amount of intracellular cAMP. Additionally, luminescence levels derived from Renilla luciferase were used as an internal standard to correct for errors in gene transfer efficiency and cell number in each well. Renilla luciferase is constitutively expressed from the Renilla luciferase gene carried in the pGL4.74[hRluc / TK] Vector under the control of the TK promoter.

[0163] The medium was removed from the culture obtained in <1-2> above, and 15 μl of a mixed solution of 15 test substances and methional (containing 100 μM test substances and 100 μM methional) or a methional solution (containing 100 μM methional) was added to prepare a reaction solution. Each solution was prepared by dissolving the designated substance in CD293 (Life Technologies, Inc.) containing copper(II) chloride (final concentration 30 μM). The cells were cultured at 37°C for 3 hours in a CO2 incubator to allow sufficient intracellular expression of the firefly luciferase gene. The luminescence value derived from intracellular firefly luciferase was measured and used as the "Luc value." The luminescence value derived from firefly luciferase was measured using Dual-Glo TM Measurement was performed using a luciferase assay system (Promega) according to the product's operating manual.

[0164] The rate of inhibition of olfactory receptor responses by the test substance was calculated as follows: The Luc value (X) of cells stimulated with methional alone was subtracted by the Luc value (Y) of cells expressing the same receptor but without stimulation. Similarly, the Luc value (Z) of cells stimulated with a mixture of methional and the test substance was subtracted by the Luc value (Y) of cells expressing the same receptor but without stimulation. The rate of inhibition of olfactory receptor responses by the test substance was calculated using the increase in luminescence value (XY) due to stimulation with methional alone as the standard using the following formula. The experiment was performed in duplicate, and the average value was obtained. Inhibition rate (%) = {1 - (Z - Y) / (X - Y)} x 100

[0165] The results are shown in Table 2. Fifteen test substances exhibited inhibitory activity against olfactory receptor responses at either or both OR4E2 and OR4S2 (Table 2).

[0166]

[0167] Furthermore, the concentration dependence of the olfactory receptor response inhibitory activity was examined for each test substance. The test substance concentrations were set to 12.5, 25, 50, or 100 μM, and the Luc value was measured using the same procedure as above. The response intensity (Luc value) of the olfactory receptor to methional (100 μM) at each test substance concentration was calculated relative to the response intensity (Luc value) of the olfactory receptor to methional (100 μM) in the absence of the test substance, which was set at 100%. From the results, the 50% inhibitory concentration (IC) of each test substance for the olfactory receptor response was determined. 50 The results are shown in Table 3.

[0168]

[0169] Example 2: Evaluation of methional odor-masking effect In this example, the methional odor-masking effect of 15 compounds that showed inhibitory activity against olfactory receptor responses in Example 1 was evaluated.

[0170] Test Example 1 Preparation of Evaluation Samples Each compound shown in Table 4 was added to a 0.3 ppm methional aqueous solution to three concentration levels (addition concentrations) shown in Table 4 to prepare evaluation samples. The concentration levels were determined as follows: 1 / 10 of the threshold concentration, the threshold concentration, and a concentration 10 times the threshold concentration, with the maximum concentration at which the odor of the compound itself was not detected when an aqueous solution containing each compound alone was held in the mouth and swallowed.

[0171] (Evaluation of methional odor masking effect) Five milliliters of each evaluation sample was placed in the mouth and swallowed naturally to evaluate the methional odor intensity. The methional odor intensity was scored on a scale of 0 to 5 points using the following evaluation criteria, with aqueous methional solutions prepared at 0.3 ppm, 0.1 ppm, or 0 ppm as the standard. Six expert panels were used to calculate the average value for each evaluation sample.

[0172] [Evaluation criteria] 0: Methional odor intensity of 0 ppm methional aqueous solution (water with no methional added) 3: Methional odor intensity of 0.1 ppm methional aqueous solution 5: Methional odor intensity of 0.3 ppm methional aqueous solution

[0173] [Masking effect evaluation criteria] -: Methional odor intensity is 5.2 or more ±: Methional odor intensity is 4.8 or more and less than 5.2 +: Methional odor intensity is 4.0 or more and less than 4.8 ++: Methional odor intensity is 3 or more and less than 4 +++: Methional odor intensity is less than 3 (similar intensity to a 0.1 ppm methional aqueous solution)

[0174] The results are shown in Table 4. All of the 15 compounds evaluated showed a methional odor-masking effect.

[0175]

[0176] Test Example 2 (Preparation of Evaluation Samples) Each compound shown in Table 5 was added to an aqueous solution of 0.35% amino acid mixture and 2.8% sucrose to the three concentrations (addition concentrations) shown in Table 5 to prepare evaluation samples. The amino acid mixture used here was a mixture of multiple amino acids containing 3.27% methionine. The methionine concentration in the 0.35% amino acid mixture aqueous solution was 0.011445%. It is believed that some of the methionine in the amino acid mixture was converted to methional during storage. The concentration levels were determined as the maximum concentration at which the odor of the compound itself was not detected when an aqueous solution containing each compound was held in the mouth and swallowed, and the threshold concentrations were 1 / 10 of the threshold concentration, the threshold concentration, and 10 times the threshold concentration.

[0177] (Evaluation of methional odor masking effect) Five milliliters of each evaluation sample was placed in the mouth and swallowed naturally to evaluate the methional odor intensity. The methional odor intensity was scored on a scale of 0 to 5 using the following evaluation criteria, with a 0.35% aqueous solution of water and amino acid mixture and a 2.8% aqueous solution of sucrose as the standard. Six expert panelists calculated the average score for each evaluation sample.

[0178] [Evaluation criteria] 0: Methional odor intensity of water 5: Methional odor intensity of an aqueous solution of 0.35% amino acid mixture and 2.8% sucrose

[0179] [Masking effect evaluation criteria] -: Methional odor intensity is 5.2 or more ±: Methional odor intensity is 4.8 or more and less than 5.2 +: Methional odor intensity is 4.0 or more and less than 4.8 ++: Methional odor intensity is 3.0 or more and less than 4.0

[0180] The results are shown in Table 5. All three evaluated compounds exhibited a methional odor-masking effect even when an aqueous solution containing an amino acid mixture and sucrose was used as the evaluation system.

[0181]

[0182] Test Example 3 (Preparation of Evaluation Samples) Each compound shown in Table 6 was added to an aqueous solution of 2.5% amino acid mixture and 20% sucrose at three concentrations (addition concentrations) shown in Table 6 to prepare evaluation samples. The amino acid mixture used here was a mixture of multiple amino acids containing 3.27% methionine. The methionine concentration in the 2.5% amino acid mixture aqueous solution was 0.08175%. It is believed that some of the methionine in the amino acid mixture was converted to methional during storage. The concentration levels were determined as follows: 1 / 10 of the threshold concentration, threshold concentration, and 10 times the threshold concentration, with the maximum concentration at which the odor of the compound itself was not detected when an aqueous solution containing each compound was held in the mouth and swallowed.

[0183] (Evaluation of methional odor masking effect) Five milliliters of each evaluation sample was placed in the mouth and swallowed naturally to evaluate the methional odor intensity. Based on a 2.5% aqueous solution of water and amino acid mixture and a 20% aqueous solution of sucrose, the samples were scored on a scale of 0 to 5 using the following evaluation criteria. A panel of six experts calculated the average score for each evaluation sample.

[0184] [Evaluation criteria] 0: Methional odor intensity of water 5: Methional odor intensity of an aqueous solution of 2.5% amino acid mixture and 20% sucrose

[0185] [Masking effect evaluation criteria] -: Methional odor intensity is 5.2 or more ±: Methional odor intensity is 4.8 or more and less than 5.2 +: Methional odor intensity is 4.0 or more and less than 4.8 ++: Methional odor intensity is 3.0 or more and less than 4.0

[0186] The results are shown in Table 6. All three evaluated compounds exhibited a methional odor-masking effect even when an aqueous solution containing a high concentration of an amino acid mixture and sucrose was used as the evaluation system.

[0187]

[0188] In one aspect, the present invention allows for efficient screening of substances that suppress amino acid odor. Also, in another aspect, the present invention allows for suppression of amino acid odor.

[0189] <Explanation of the sequence listing> SEQ ID NO: 1: Nucleotide sequence of human OR4S2 gene 2: Amino acid sequence of human OR4S2 protein 3: Nucleotide sequence of human OR4E2 gene 4: Amino acid sequence of human OR4E2 protein

Claims

1. A method for screening a substance that suppresses amino acid odor, comprising: The following steps (A) to (C): (A) contacting an olfactory receptor with an olfactory receptor activator in the presence of a test substance; (B) measuring the response of the olfactory receptor to the olfactory receptor activator; and (C) identifying the test substance as a substance that suppresses the amino acid odor based on the response. Including, If the response is inhibited by the test substance, the test substance is identified as a substance that suppresses the amino acid odor; A method wherein the olfactory receptor is OR4S2 and / or OR4E2.

2. The method of claim 1 , wherein the response is activation of the olfactory receptor.

3. The method according to claim 1 or 2, wherein the olfactory receptor is used in a form supported on a cell, a cell membrane, an artificial lipid bilayer vesicle, or an artificial lipid bilayer membrane.

4. The method according to claim 1 or 2, wherein the olfactory receptor is used in a cell-borne form.

5. The method of claim 3 , wherein the cell is an animal cell.

6. The method according to claim 1 or 2, wherein steps (B) and (C) are carried out by the following steps (B1) and (C1), respectively: (B1) measuring the degree D1 of activation of the olfactory receptor when the step (A) is carried out; (C1) A step of identifying the test substance as a substance that suppresses amino acid odor based on the degree of activation D1.

7. The method according to claim 6, wherein the step (C1) is carried out by the following step (C2): (C2) A step of identifying the test substance as a substance that suppresses the amino acid odor based on the difference between the degree of activation D1 and the degree of activation D2 of the olfactory receptor under control conditions.

8. The method according to claim 7, wherein the control conditions are the following conditions (C2-1) or (C2-2): (C2-1) a condition in which the olfactory receptor is contacted with the olfactory receptor activator in the absence of the test substance; (C2-2) Conditions under which the olfactory receptor is contacted with the olfactory receptor activator in the presence of the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in step (A).

9. The method of claim 7, further comprising measuring the degree of activation D2.

10. The method according to claim 7, wherein the test substance is identified as a substance that suppresses the amino acid odor if the degree of activation D1 is lower than the degree of activation D2.

11. The method according to claim 7, wherein the test substance is identified as a substance that suppresses the amino acid odor if the ratio of the degree of activation D1 to the degree of activation D2 is less than 60%.

12. The method according to claim 3, wherein the response is measured using intracellular cAMP concentration as an index.

13. The method of claim 12, wherein the intracellular cAMP concentration is measured by a reporter assay.

14. The method of claim 1 or 2, wherein the olfactory receptor is a human olfactory receptor.

15. The OR4S2 is a protein described in (a), (b), or (c) below. Method according to 1 or 2: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, and having responsiveness to the olfactory receptor activator; (c) A protein comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having responsiveness to the olfactory receptor activator.

16. The OR4E2 is a protein described in (a), (b), or (c) below. Method according to 1 or 2: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 4; (b) a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 4, and having responsiveness to the olfactory receptor activator; (c) A protein comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 4 and having responsiveness to the olfactory receptor activator.

17. The method according to claim 1 or 2, further comprising a step of evaluating whether the identified substance that suppresses amino acid odor has the function of suppressing amino acid odor.

18. 18. The method of claim 17, wherein the evaluation is performed by sensory evaluation.

19. The method according to claim 1 or 2, wherein the amino acid odor is methional odor.

20. A composition for suppressing amino acid odor in food, comprising: A composition comprising the following component (A): (A) A component that inactivates the olfactory receptor OR4S2 and / or OR4E2, the component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

21. 1. A composition for use in the manufacture of a food product, comprising: A composition comprising the following component (A): (A) A component that inactivates the olfactory receptor OR4S2 and / or OR4E2, the component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

22. The composition according to claim 21, wherein the food is a food in which the amino acid odor is suppressed.

23. The composition according to any one of claims 20 to 22, wherein the food contains a component that exhibits an amino acid odor and / or a component that can produce a component that exhibits an amino acid odor.

24. The composition according to claim 23, wherein the component exhibiting an amino acid odor is methional.

25. The composition according to claim 23, wherein the component capable of producing the component exhibiting an amino acid odor is methionine.

26. 23. The composition according to claim 20 or 22, wherein the amino acid odor is methional odor.

27. A method for suppressing amino acid odor in food, comprising: A method comprising the step of adding the following component (A) to a food ingredient: (A) A component that inactivates the olfactory receptor OR4S2 and / or OR4E2, the component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

28. 1. A method of producing a food product, comprising: A method comprising the step of adding the following component (A) to a food ingredient: (A) A component that inactivates the olfactory receptor OR4S2 and / or OR4E2, the component being at least one component selected from the group consisting of bis(2-methyl-3-furyl)disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, furfuryl mercaptan, 2,5-dihydroxy-1,4-dithiane, 2,4-decadienal, 2,4-nonadienal, nootkatone, allyl isothiocyanate, cinnamaldehyde, 2-methyl-3-furanthiol, 2-phenyl-2-butenal, 2-thiophenethiol, sclareolide, and E-β-damascone.

29. The method according to claim 28, wherein the food product is a food product in which the amino acid odor is suppressed.

30. The method according to any one of claims 27 to 29, wherein the food or the raw material contains a component that exhibits an amino acid odor and / or a component that can produce a component that exhibits an amino acid odor.

31. The method according to claim 30, wherein the component having an amino acid odor is methional.

32. The method according to claim 30, wherein the component capable of producing the component exhibiting an amino acid odor is methionine.

33. 30. The method of claim 27 or 29, wherein the amino acid odor is methional odor.

34. The method according to claim 30, comprising a step of heating the food or the raw material containing a component capable of producing the component exhibiting the amino acid odor.

35. The component (A) is added so that the ingestible concentration is 0.001 ppt (w / w) to 10 ppm (w / w). The method according to any one of claims 27 to 29,

36. The method according to any one of claims 27 to 29, wherein 2-methyl-3-furanthiol is added so that its ingestible concentration is 0.001 ppt (w / w) to 100 ppt (w / w).

37. 30. The method according to any one of claims 27 to 29, wherein bis(2-methyl-3-furyl) disulfide, ethyl vinyl ketone, 2-methylbenzenethiol, 2,4-decadienal, or 2,4-nonadienal is added so that the ingestible concentration thereof is 0.01 ppt (w / w) to 1 ppb (w / w).

2. The method according to claim 1.

38. The method according to any one of claims 27 to 29, wherein furfuryl mercaptan or E-β-damascone is added so that its ingestible concentration is 0.1 ppt (w / w) to 10 ppb (w / w).

39. The method according to any one of claims 27 to 29, wherein 2,5-dihydroxy-1,4-dithiane, 2-phenyl-2-butenal, or 2-thiophenethiol is added so that its ingestible concentration is 1 ppt (w / w) to 100 ppb (w / w).

40. 30. The method according to claim 27, wherein nootkatone, allyl isothiocyanate, or cinnamaldehyde is added to the food so that the ingestible concentration is 10 ppt (w / w) to 1 ppm (w / w). The method described below.

41. Sclareolide was added to the food at a concentration of 100 ppt (w / w) to 10 ppm (w / w). The method according to any one of claims 27 to 29,