Method for screening for substance having desired aroma characteristics

Abstract

Provided is a method for screening for a substance having desired aroma characteristics. A substance having desired aroma characteristics is screened by utilizing an olfactory receptor associated with the desired aroma characteristics.

Description

Screening method for substances with desired aroma characteristics

[0001] The present invention relates to a method for screening for substances having desired odor characteristics.

[0002] Aroma is an important factor that influences the palatability of foods, cosmetics, etc. Therefore, techniques for screening aroma components necessary for reproducing a specific aroma, techniques for reproducing a specific aroma by combining aroma components, and techniques for screening components that suppress (i.e., mask) a specific undesirable aroma are industrially important techniques for developing foods, cosmetics, etc.

[0003] Conventionally, screening for compounds that affect specific aromas has been carried out by humans evaluating the aroma of test substances through sensory testing, but sensory testing has problems such as the need to train experts who can evaluate aromas and low throughput.

[0004] In mammals such as humans, odors are perceived when molecules of odor components bind to olfactory receptors on olfactory nerve cells present in the olfactory epithelium in the upper part of the nasal cavity, and the receptor's response to the molecules is transmitted to the central nervous system. In recent years, methods have been reported for screening substances that exhibit or suppress specific odors using the response of olfactory receptors as an indicator (Patent Documents 1 to 4, etc.).

[0005] Patent Publication No. 2019-037197, WO2021 / 064201, Patent Publication No. 2019-129773, Patent Publication No. 2019-129772

[0006] An object of the present invention is to provide a method for screening for substances having desired aroma characteristics.

[0007] As a result of extensive research to solve the above problems, the present inventors discovered a correlation between various odor descriptors and the activity of various olfactory receptors, thereby completing the present invention.

[0008] That is, the present invention can be exemplified as follows. [1] A method for screening for a substance having a desired aroma characteristic, comprising the following steps (A) to (C): (A) contacting an olfactory receptor with a test substance; (B) measuring the response of the olfactory receptor to the test substance; and (C) identifying the test substance as a substance having the desired aroma characteristic based on the response, wherein the aroma characteristic is a property of exhibiting the desired aroma or a property of suppressing the desired aroma, and the olfactory receptor is an olfactory receptor associated with the aroma characteristic. [2] The method wherein the response is activation of the olfactory receptor. [3] The method wherein the response is inactivation of the olfactory receptor, and wherein step (A) is carried out in the presence of a substance that activates the olfactory receptor. [4] The method, wherein the combination of the aroma and the olfactory receptor is selected from the following combinations: a combination of meaty and OR2C1; a combination of meaty and OR4S2; a combination of bready and OR6B1; a combination of creamy and OR10A3; a combination of guaiacol and OR10G4; a combination of raw and OR5K1; a combination of peanut roasted peanut and OR5K1; a combination of hazelnut and OR5K1; a combination of potato and OR5K1; a combination of popcorn and OR5K1; a combination of corn chip and OR5K1; a combination of beany and OR5K1; a combination of cocoa and OR5K1; a combination of roasted and OR5K1; a combination of coffee and OR5K1; a combination of burnt and OR5K1; a combination of musty and OR5K1; a combination of horseradish and OR2C1; a combination of rubbery and OR4S2; a combination of maple and OR8D1; a combination of lactonic and OR8D1; a combination of sharp and OR51E1; a combination of natural and OR1D5; a combination of fruit tropical Combination of fruit and OR6C65; Combination of solvent and OR4C3; Combination of tomato and OR2K2; Combination of eggy and OR1R1P;Magnolia and OR4K5; cooling and OR8K3; lemon and OR2W3; mushroom and OR2W1; caraway and OR1A1; jasmin and OR1A1; floral and OR1A1; banana and OR2L8; warm and OR10G7; musk and OR5AN1; heliotrope and OR5AN1; pepper bell and OR2AG2; bois de rose and OR1C1; apricot and OR1D2; cucumber and OR2J2; cinnamyl and OR2J2; melon and OR2J2; acacia and OR5P3; hawthorn and OR5P3; naphthyl and OR5P3; bacon and OR10G7; bacon and OR10G4; clove and OR10G7; clove and OR10G4 Combination of clove and OR10D3; Combination of clove and OR2J2; Combination of carnation and OR10G7; Combination of carnation and OR10G4; Combination of carnation and OR10D3; Combination of carnation and OR2J2; Combination of vanilla and OR10G4; Combination of vanilla and OR10G7; Combination of vanilla and OR2J2; Combination of vanilla and OR10D3; Combination of vanilla and OR10G3; Combination of smoky and OR10G4; Combination of smoky and OR10G7; Combination of phenolic and OR10G4; Combination of phenolic and OR9Q2; Combination of phenolic and OR10G7; Combination of medicinal and OR10G4; Combination of medicinal and OR9Q2; Combination of nutty and OR5K1; Combination of nutty and OR6B1; Combination of nutty and OR51E2; Combination of sulfurous and OR2C1;Combination of sulfurous and OR4S2; Combination of alliaceous and OR2C1; Combination of alliaceous and OR4S2; Combination of onion and OR4S2; Combination of onion and OR2C1; Combination of cabbage and OR4S2; Combination of cabbage and OR2C1; Combination of garlic and OR4S2; Combination of garlic and OR2C1; Combination of caramellic and OR8D1; Combination of caramellic and OR6B1; Combination of pungent and OR6B1; Combination of pungent and OR51E1; Combination of mustard and OR2A4; Combination of mustard and OR10H2; Combination of sweet and OR8B3; Combination of sweet and OR2J2; Combination of sweet and OR5P3; Combination of sweet and OR1D2; Combination of sweet and OR11H4; Combination of fresh and OR1D2; Combination of fresh and OR10A3; Combination of fresh and OR9A4; Combination of woody and OR7A17; Combination of woody and OR4X1; Combination of minty and OR2B11; Combination of minty and OR10T2; Combination of honey and OR51L1; Combination of honey and OR1A1; Combination of fruity and OR2L8; Combination of fruity and OR1D2; Combination of pineapple and OR2L8; Combination of pineapple and OR1D2; Combination of spicy and OR2J2; Combination of spicy and OR10G7; Combination of spicy and OR10G4; Combination of spicy and OR8B3; Combination of spicy and OR11H4; Combination of apple skin and OR1F1; Combination of apple skin and OR4D1; Combination of apple skin and OR7D2; Combination of vegetable and OR5K1; Combination of vegetable and OR4S2; Combination of peach and OR1D2; Combination of peach and OR10A3; Combination of peach and OR10A6; Combination of coconut and OR8B3; Combination of coconut and OR10A3;Combination of coconut and OR10A6; Combination of coconut and OR1A1; Combination of sour and OR51E1; Combination of sour and OR51V1; Combination of sour and OR51L1; Combination of sour and OR51I2; Combination of acidic and OR51E1; Combination of acidic and OR51I2; Combination of acidic and OR51V1; Combination of cheesy and OR51E1; Combination of cheesy and OR51I2; Combination of cheesy and OR51L1; Combination of cheesy and OR51V1; Combination of sweaty and OR51E1; Combination of sweaty and OR51I2; Combination of sweaty and OR51L1; Combination of cinnamon and OR2J2; Combination of cinnamon and OR8B3; Combination of cinnamon and OR2B11; Combination of cinnamon and OR2Y1; Combination of cinnamon and OR11H4; Combination of cherry and OR5P3; Combination of cherry and OR2J2; Combination of cherry and OR8B3; Combination of cherry and OR2B11; Combination of almond bitter almond and OR5P3; Combination of almond bitter almond and OR2B11; Combination of almond bitter almond and OR8B3; Combination of almond bitter almond and OR2J2; Combination of almond bitter almond and OR6P1; Combination of tonka and OR8B3; Combination of tonka and OR5P3; Combination of tonka and OR8D1; Combination of coumarinic and OR8B3; Combination of coumarinic and OR5P3; Combination of coumarinic and OR8D1; Combination of mimosa and OR5P3; Combination of mimosa and OR8B3; Combination of lilac and OR5P3; Combination of lilac and OR8D4; Combination of powdery and OR5P3; Combination of powdery and OR5AN1; Combination of orangeflower and OR5P3; Combination of orangeflower and OR1D2;a combination of orangeflower and OR8B12; a combination of orangeflower and OR4P4; a combination of orangeflower and OR7A3P; a combination of anisic and OR5P3; a combination of anisic and OR1A1; a combination of licorice and OR5P3; a combination of licorice and OR1A1; a combination of anise and OR5P3; a combination of anise and OR1A1; a combination of anise and OR8B3. [5] The method, wherein the test substance is identified as exhibiting a target odor when the olfactory receptor is activated by the test substance, or the test substance is identified as suppressing a target odor when the olfactory receptor is inactivated by the test substance. [6] The method described above, wherein the combination of the aroma and the olfactory receptor is selected from the following combinations: a combination of peanut roasted peanut and OR4H12P; a combination of meaty and OR2W1; a combination of sulfurous and OR1D2; a combination of sulfurous and OR13F1; a combination of sulfurous and OR52E4; a combination of sulfurous and OR10D3; a combination of sulfurous and OR6B2; a combination of sulfurous and OR5L2; a combination of sulfurous and OR2T10; a combination of sulfurous and OR5AU1; a combination of sulfurous and OR10G6; a combination of sulfurous and OR6C75; a combination of soapy and OR51E2; a combination of soapy and OR56A5; a combination of orchid and OR51E2; a combination of waxy and OR5K1; a combination of waxy and OR51E2; a combination of waxy and OR10G4; a combination of waxy and OR2B11; a combination of gardenia and OR6J1; a combination of fruity and OR5P3; Combination of fruity and OR5K1; Combination of fruity and OR10G4; Combination of fruity and OR4S2; Combination of cherry and OR6B1;A combination of floral and OR6B1. [7] The method, wherein the test substance is identified as suppressing the target odor when the olfactory receptor is activated by the test substance, or the test substance is identified as exhibiting the target odor when the olfactory receptor is inactivated by the test substance. [8] 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 the target substance based on the degree of activation D1. [9] The method, wherein step (C1) is carried out by the following step (C2): (C2) identifying the test substance as the target substance based on the difference between the degree of activation D1 and the degree of activation D2 of the olfactory receptor under control conditions.

[10] The method, wherein the response is activation of the olfactory receptor, and the control condition is the following condition (C2-1) or (C2-2): (C2-1) a condition in which the olfactory receptor is not contacted with the test substance; (C2-2) a condition in which the olfactory receptor is contacted with the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in step (A).

[11] The method, wherein the response is inactivation of the olfactory receptor, and the control condition is the following condition (C2-1) or (C2-2): (C2-1) a condition in which the olfactory receptor is not contacted with the test substance;(C2-2) Conditions for contacting the olfactory receptor with the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in step (A).

[12] The method described above, further comprising a step of measuring the degree of activation D2.

[13] The method described above, wherein activation of the olfactory receptor by the test substance is determined to have been observed if the degree of activation D1 is higher than the degree of activation D2.

[14] The method described above, wherein inactivation of the olfactory receptor by the test substance is determined to have been observed if the degree of activation D1 is lower than the degree of activation D2.

[15] The method described above, 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.

[16] The method described above, wherein the olfactory receptor is used in a form supported by the cell.

[17] The method described above, wherein the cell is an animal cell.

[18] The method described above, wherein the response is measured using intracellular cAMP concentration or intracellular calcium concentration as an indicator.

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

[20] The method described above, further comprising a step of evaluating the aroma characteristics of the target substance identified in step (C).

[21] The method described above, wherein the evaluation is performed by sensory evaluation.

[22] The method described above, wherein the olfactory receptor is a human olfactory receptor.

[23] The method described above, wherein the substance having the target aroma characteristics is a substance that exhibits a meaty aroma, and the olfactory receptor is OR2C1 and / or OR4S2.

[24] The method described above, wherein the substance that exhibits a meaty aroma is a substance that exhibits a meaty flavor or aroma.

[25] The method described above, wherein the substance that exhibits a meaty aroma is a substance that imparts a meaty flavor or aroma to the target object.

[26] The method described above, wherein the target object is a meat substitute or cultured meat.

[0009] This figure shows the difference in olfactory receptor activity values ​​depending on whether or not an odor descriptor is present. For 201 pairs of descriptor and olfactory receptor combinations for which there was a significant difference in olfactory receptor activity depending on whether or not a descriptor was present, and for which a significant correlation was also observed between the IR-IOF of the descriptor and olfactory receptor activity, the difference in average olfactory receptor activity between the groups with and without the descriptor is shown in a heat map. The descriptors and olfactory receptors are arranged on the vertical and horizontal axes of the heat map according to the results of hierarchical clustering. Euclidean distance was used to calculate distance prior to hierarchical clustering, and Ward's method was used for clustering. The difference in average activity values ​​between groups, ranging from -6 to 6, is shown using a black to white gradation.

[0010] The present invention will be described in detail below.

[0011] The method of the present invention is a screening method for a substance having a desired odor characteristic using an olfactory receptor. A substance having a desired odor characteristic is also referred to as a "target substance." In the method of the present invention, the target substance can be identified using an olfactory receptor (i.e., whether a test substance is a target substance can be identified). Specifically, the method of the present invention can identify the target substance (i.e., whether the test substance is a target substance) based on the response of the olfactory receptor to the test substance. That is, the method of the present invention may be a screening method for a target substance, specifically comprising: (A) contacting the olfactory receptor with a test substance; (B) measuring the response of the olfactory receptor to the test substance; and (C) identifying the test substance as a target substance based on the response.

[0012] <1> Target substance The term "target substance" refers to a substance having a desired aroma characteristic.

[0013] The term "aroma characteristic" refers to the property of exhibiting an aroma or the property of suppressing an aroma. The aroma characteristic may particularly be the property of exhibiting an aroma. An aroma corresponding to a target aroma characteristic (i.e., an aroma exhibited or suppressed by a substance having the target aroma characteristic) is also referred to as a "target aroma." There are no particular limitations on the type of target aroma. Target aromas include absinthe, acacia, acai, acerola, acetic, acetone, acidic, acorn, acrylate, agarwood, alcoholic, aldehydic, alfalfa, algae, alliaceous, allspice, almond, almond bitter almond, almond roasted almond, almond toasted. almond, amber, ambergris, ambrette, ammoniacal, angelica, animal, anise, anisic, apple, apple cooked apple, apple dried apple, apple green apple, apple red apple, apple skin, apricot, aromatic, arrack, artichoke, asafetida, asparagus, astringent, autumn, avocado, bacon, baked, balsamic, banana, banana peel, banana ripe banana, banana unripe banana, barley roasted barley, basil, bay, bean green bean, beany, beef juice, beefy, beefy roasted beefy, beer, beeswax, benzoin, bergamot, berry, berry ripe berry, bitter, blackberry, bloody, blueberry, bois de rose, boronia, bouillon, boysenberry, brandy, bread baked, bread crust, bread rye bread, bready, broccoli, brothy, brown, bubble gum, buchu, burnt, butterrancid、buttermilk、butterscotch、buttery、cabbage、calamus、camphoreous、cananga、candy、cantaloupe、capers、caramellic、caraway、cardamom、carnation、carrot、carrot seed、carvone、cascarilla、cashew、cassia、castoreum、catty、cauliflower、cedar、cedarwood、celery、cereal、chamomile、charred、cheesy、cheesy bleu cheese、cheesy cheddar cheese、cheesy feta cheese、cheesy gorgonzola cheese、cheesy gouda cheese、cheesy limburger cheese、cheesy parmesan cheese、cheesy roquefort cheese、chemical、cherry、cherry maraschino cherry、chervil、chestnut、chicken、chicken coup、chicken fat、chicken roasted chicken、chicory、chive、chocolate、chocolate dark chocolate、chocolate white chocolate、chrysanthemum、cider、cilantro、ciltrano、cinnamon、cinnamyl、cistus、citronella、citrus、citrus peel、citrus rind、civet、clam、clean、cloth laundered cloth、clove、clover、cocoa、coconut、coffee、coffee roasted coffee、cognac、cologne、cooked、cookie、cooling、copaiba、coriander、corn、corn chip、cornmeal、cornmint、cortex、costus、cottoncandy、coumarinic、cranberry、creamy、cubeb、cucumber、cucumber skin、cumin、currant black currant、currant bud black currant bud、currant red currant、curry、custard、cyclamen、cypress、dairy、date、davana、deertongue、dewy、dill、dirty、dragon fruit、dry、durian、dusty、earthy、egg nog、egg yolk、eggy、elderberry、elderflower、elemi、estery、ethereal、eucalyptus、fatty、fecal、fennel、fenugreek、fermented、fig、filbert、fir needle、fishy、fleshy、floral、foliage、forest、fougere、frankincense、freesia、fresh、fresh outdoors、fried、fruit dried fruit、fruit overripe fruit、fruit ripe fruit、fruit tropical fruit、fruity、fudge、fungal、fusel、galanga、galbanum、gardenia、garlic、gasoline、gassy、genet、geranium、ginger、ginseng、goaty、goji berry、gooseberry、gourmand、graham cracker、grain、grain toasted grain、grape、grape skin、grapefruit、grapefruit peel、grassy、gravy、greasy、green、grilled、guaiacol、guaiacwood、guava、hairy、ham、harsh、hawthorn、hay、hay new mown hay、hazelnut、hazelnut roastedhazelnut、heather、heliotrope、herbal、hibiscus、honey、honeydew、honeysuckle、hops、horehound、horseradish、huckleberry、humus、hyacinth、hyssop、immortelle、incense、jackfruit、jammy、jasmin、jonquil、juicy、juicy fruit、juniper、ketonic、kimchi、kiwi、kokumi、kumquat、labdanum、lachrymatory、lactonic、lamb、lard、lavandin、lavender、lavender spike lavender、leafy、leathery、leek、lemon、lemon peel、lemongrass、lettuce、licorice、licorice black licorice、lilac、lily、lily of the valley、lime、linden flower、lingonberry、liver、lobster、loganberry、lovage、lychee、macadamia、mace、magnolia、mahogany、malty、mandarin、mango、maple、marigold、marine、marjoram、marshmallow、marzipan、mastic、meaty、meaty roasted meaty、medicinal、melon、melon rind、melon unripe melon、mentholic、metallic、milky、mimosa、minty、molasses、moldy、mossy、muguet、mulberry、mushroom、musk、mustard、musty、mutton、myrrh、naphthyl、narcissus、nasturtium、natural、neroli、noni fruit、nut flesh、nut skin、nutmeg、nutty、oakmoss、oatmeal、oats、ocean、oily、onion、onion cooked onion、onion greenonion、opoponax、orange、orange bitter orange、orange peel、orange rind、orangeflower、orchid、oriental、origanum、orris、osmanthus、oyster、ozone、painty、palmarosa、papaya、paper、parsley、passion fruit、patchouli、pea green pea、peach、peanut、peanut butter、peanut roasted peanut、pear、pear skin、pecan、peely、pennyroyal、peony、pepper bell pepper、pepper black pepper、peppermint、peppery、peru balsam、petal、petitgrain、petroleum、phenolic、pimenta、pine、pineapple、pistachio、plastic、plum、plum skin、pomegranate、popcorn、pork、potato、potato baked potato、potato chip、potato raw potato、powdery、praline、privet、privetblossom、prune、pulpy、pumpkin、pungent、quince、radish、rain、raisin、rancid、raspberry、raw、reseda、resinous、rhubarb、rindy、ripe、roasted、root beer、rooty、rose、rose dried rose、rose red rose、rose tea rose、rose white rose、rosemary、rubbery、rue、rummy、saffron、sage、sage clary sage、salmon、salty、sandalwood、sandy、sappy、sarsaparilla、sassafrass、sauerkraut、sausage、sausage smokedsausage、savory、sawdust、scallion、seafood、seashore、seaweed、seedy、sesame、sharp、shellfish、shrimp、skunk、smoky、soapy、soft、solvent、soup、sour、spearmint、spicy、spinach、spruce、starchy、starfruit、storax、strawberry、stringent、styrene、sugar、sugar brown sugar、sugar burnt sugar、sulfurous、sweaty、sweet、sweet pea、taco、tagette、tallow、tamarind、tangerine、tansy、tarragon、tart、tea、tea black tea、tea green tea、tea rooibos tea、tea white tea、tequila、terpenic、thujonic、thyme、toasted、tobacco、toffee、tolu balsam、tomato、tomato leaf、tonka、tropical、truffle、tuberose、tuna、turkey、turmeric、turnup、tutti frutti、umami、urine、valerian root、vanilla、vegetable、verbena、vetiver、vinegar、violet、violet leaf、walnut、warm、wasabi、watercress、watermelon、watermelon rind、watery、waxy、weedy、wet、whiskey、winey、wintergreen、woody、woody burnt wood、woody oak wood、woody old wood、wormwood、yeasty、ylang、yogurt、yuzu、zedoary、zesty、bark、birch bark、blood、raw meat、burnt candle、burnt milk、burnt pepper、burnt rubber、cadaverous (dead animal)、cardboard、catExamples of aromas of interest include urine, chalky, cleaning fluid, cooked vegetables, cork, creosote, crushed grass, crushed weeds, dirty linen, disinfectant, carbolic, fermented (rotten) fruit, fragrant, fresh green vegetables, fresh tobacco smoke, fried chicken, heavy, household gas, kerosene, kippery (smoked fish), laurel leaves, light, mothballs, mouse, nail polish remover, new rubber, peanut butter, perfumery, putrid, four, decayde, rope, seasoning (for meat), seminal, sperm-like, sewer, sickening, sooty, sour milk, stale, stale tobacco smoke, tab, tea leaves, turpentine (pine oil), varnish, wet paper, wet wool, and wet dog. The target aroma may be a single aroma or a combination of two or more aromas.

[0014] The target substance may be composed 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 target substance is a mixture, as long as the mixture has the desired aroma characteristics, each component constituting the mixture may or may not have the desired aroma characteristics on its own.

[0015] <2> Test Substance The term "test substance" refers to a substance used in the method of the present invention as a candidate for a target substance. 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, and various other organic or inorganic components. In particular, test substances 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, substances such as those 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 substances. By contacting two or more components together with an olfactory receptor and carrying out the method of the present invention, it is possible to identify whether the combination of components as a whole is a target substance. Examples of "contacting two or more components together with an olfactory receptor" include contacting a test substance that is a mixture with an olfactory receptor, and contacting two or more test substances together with an olfactory receptor.

[0016] <3> Olfactory Receptor An olfactory receptor associated with a target aroma characteristic is used as the olfactory receptor. "An olfactory receptor associated with a target aroma characteristic" may refer to an olfactory receptor whose activation is correlated with an aroma (specifically, an odor descriptor) corresponding to the target aroma characteristic. The correlation between olfactory receptor activation and an aroma (specifically, an odor descriptor) can be confirmed, for example, by the method described in the Examples. The correlation between olfactory receptor activation and an aroma may be a positive correlation or a negative correlation. The correlation between olfactory receptor activation and an aroma may particularly be a positive correlation. The correlation between olfactory receptor activation and an aroma is also referred to as "the correlation between olfactory receptor and an aroma."

[0017] The olfactory receptors are OR1A1, OR1A2, OR1B1, OR1C1, OR1D2, OR1D4, OR1D5, OR1E1, OR1E2, OR1F1, OR1F12, OR1G1, OR1I1, OR1J1, OR1J2, OR1J4, OR1K1, OR1L1, OR1L3, OR1L4, OR1L6, OR1L8, OR1M1, OR1N1, OR1N2, OR1Q1, OR1R1P, OR1S1, OR1S2, OR2A1, OR2A2, OR2A4, OR2A5, OR2A7, OR2A12, OR2A14, OR2A25, OR2AE1, and OR2AG. 1, OR2AG2, OR2AJ1P, OR2AK2, OR2AP1, OR2AT4, OR2B2, OR2B3, OR2B6, OR2B11, OR2C1, OR2C3, OR2D2, OR2D3, OR2F1, OR2F2, OR2G2, OR2G3, OR2G6, OR2H1, O R2H2, OR2J1P, OR2J2, OR2J3, OR2K2, OR2L2, OR2L3, OR2L5, OR2L8, OR2L13, OR2M2, OR2M3, OR2M4, OR2M5, OR2M7, OR2S2, OR2T1, OR2T2, OR2T3, OR2T4, OR2T 5, OR2T6, OR2T7, OR2T8, OR2T10, OR2T11, OR2T12, OR2T27, OR2T29, OR2T33, OR2T34, OR2T35, OR2V1, OR2V2, OR2W1, OR2W3, OR2Y1, OR2Z1, OR3A1, OR3A2, OR3A3, OR3A4, OR4A4P, OR4A5, OR4A15, OR4A16, OR4A47, OR4B1, OR4C3, OR4C5, OR4C6, OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4C45, OR4C46, OR4D1, OR4D2, OR4D5, OR4D6, OR4D9, OR4D10, OR4D11, OR4E2, OR4F3, OR4F4, OR4F5, OR4F6, OR4F14P, OR4F15, OR4F17, OR4F21, OR4G11P, OR4H12P, OR4K1, OR4K2, OR4K5, OR4K13, OR4K14, OR4K15, OR4K17, OR4L1, OR4M1, OR4M2, OR4N2, OR4N4, OR4N5, OR4P4, OR4Q3, OR4S1, OR4S2, OR4X1, OR4X2, OR5A1, OR5A2, OR5AC2,OR5AK2、OR5AK3P、OR5AN1、OR5AP2、OR5AR1、OR5AS1、OR5AU1、OR5B2、OR5B3、OR5B12、OR5B17、OR5B21、OR5C1、OR5D13、OR5D14、OR5D16、OR5D18、OR5F1、OR5H1、OR5H2、OR5H6、OR5H14、OR5H15、OR5I1、OR5J2、OR5K1、OR5K2、OR5K3、OR5K4、OR5L1、OR5L2、OR5M1、OR5M3、OR5M8、OR5M9、OR5M10、OR5M11、OR5P2、OR5P3、OR5R1、OR5T1、OR5T2、OR5T3、OR5V1、OR5W2、OR6A2、OR6B1、OR6B2、OR6B3、OR6C1、OR6C2、OR6C3、OR6C4、OR6C6、OR6C65、OR6C66P、OR6C68、OR6C70、OR6C74、OR6C75、OR6C76、OR6F1、OR6J1、OR6K2、OR6K3、OR6K6、OR6M1、OR6N1、OR6N2、OR6P1、OR6Q1、OR6S1、OR6T1、OR6V1、OR6X1、OR6Y1、OR7A3P、OR7A5、OR7A10、OR7A17、OR7C1、OR7C2、OR7D2、OR7D4、OR7E24、OR7G1、OR7G2、OR7G3、OR8A1、OR8B2、OR8B3、OR8B4、OR8B8、OR8B12、OR8D1、OR8D2、OR8D4、OR8G1、OR8G2、OR8G5、OR8H1、OR8H2、OR8H3、OR8I2、OR8J1、OR8J3、OR8K1、OR8K3、OR8K5、OR8S1、OR8U1、OR8U8、OR9A2、OR9A4、OR9G1、OR9G4、OR9I1、OR9K2、OR9Q1、OR9Q2、OR10A2、OR10A3、OR10A4、OR10A5、OR10A6、OR10A7、OR10AD1、OR10AG1、OR10C1、OR10D3、OR10D4P、OR10G2、OR10G3、OR10G4、OR10G6、OR10G7、OR10G8、OR10G9、OR10H1、OR10H2、OR10H3、OR10H4、OR10H5、OR10J1、OR10J3、OR10J5、OR10K1、OR10K2、OR10P1、OR10Q1、OR10R2、OR10S1、OR10T2、OR10V1、OR10W1、OR10X1, OR10Z1, OR11A1, OR11G2, OR11H1, OR11H2, OR11H4, OR11H6, OR11H12, OR11L1, OR12D2, OR12 D3, OR13A1, OR13C2, OR13C3, OR13C4, OR13C5, OR13C8, OR13C9, OR13D1, OR13F1, OR13G1, OR13H1, OR1 3J1, OR14A2, OR14A16, OR14C36, OR14I1, OR14J1, OR14K1, OR14L1P, OR51A1P, OR51A2, OR51A4, OR51 A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E2, OR51F1, OR51F2, OR51F5P, OR51G1, OR 51G2, OR51H1, OR51I1, OR51I2, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1 , OR52I2, OR52J3, OR52K1, OR52K2, OR52L1, OR52L2P, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52P2P, OR52R1, OR52W1, OR52Z1P, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B2P, and OR56B4.

[0018] Olfactory receptors include OR1A1, OR1A2, OR1B1, OR1C1, OR1D2, OR1D5, OR1E1, OR1F1, OR1F12, OR1G1, OR1I1, OR1J1, OR1J2, OR1J4, OR1K1, OR1L1, OR1L3, OR1L4, OR1L8, OR1M1, OR1N1, OR1N2, OR1Q1, OR1R1P, OR1S1, OR2A1, OR2A2, OR2A4, OR2A5, OR2A12, OR2A14, OR2A25, OR2AE1, OR2AG1, OR2AG2, OR2AJ1P, OR2AK2, OR 2AP1, OR2AT4, OR2B2, OR2B3, OR2B6, OR2B11, OR2C1, OR2C3, OR2D2, OR2D3, OR2F1, OR2G2, OR2G3, OR2G6, OR2H1, OR2H2, OR2J2, OR2J3, OR2K2, OR2L2, OR2 L8, OR2L13, OR2M2, OR2M4, OR2M7, OR2S2, OR2T1, OR2T2, OR2T5, OR2T6, OR2T8, OR2T10, OR2T11, OR2T27, OR2T34, OR2V2, OR2W1, OR2W3, OR2Y1, OR2Z1, OR3 A1, OR3A2, OR3A3, OR3A4, OR4A5, OR4A15, OR4A16, OR4A47, OR4B1, OR4C3, OR4C5, OR4C6, OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4C46, OR4D1, OR4D 2, OR4D5, OR4D6, OR4D9, OR4D10, OR4D11, OR4E2, OR4F3, OR4F5, OR4F6, OR4F14P, OR4F15, OR4G11P, OR4H12P, OR4K1, OR4K2, OR4K5, OR4K13, OR4K14, OR4K 15, OR4K17, OR4L1, OR4M1, OR4N2, OR4N4, OR4N5, OR4P4, OR4Q3, OR4S1, OR4S2, OR4X1, OR4X2, OR5A1, OR5A2, OR5AC2, OR5AK2, OR5AK3P, OR5AN1, OR5AP2, OR5AR1, OR5AS1, OR5AU1, OR5B2, OR5B3, OR5B12, OR5B17, OR5B21, OR5C1, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1, OR5H1, OR5H2, OR5H6, OR5H14, OR5I1,OR5J2、OR5K1、OR5K3、OR5K4、OR5L2、OR5M3、OR5M8、OR5M9、OR5M10、OR5M11、OR5P3、OR5R1、OR5T1、OR5T2、OR5T3、OR5V1、OR5W2、OR6A2、OR6B1、OR6B2、OR6C1、OR6C2、OR6C3、OR6C4、OR6C6、OR6C65、OR6C66P、OR6C68、OR6C70、OR6C74、OR6C75、OR6C76、OR6F1、OR6J1、OR6K2、OR6K3、OR6K6、OR6M1、OR6N1、OR6N2、OR6P1、OR6Q1、OR6S1、OR6T1、OR6V1、OR6X1、OR6Y1、OR7A3P、OR7A5、OR7A10、OR7A17、OR7C1、OR7C2、OR7D2、OR7D4、OR7E24、OR7G1、OR7G2、OR7G3、OR8A1、OR8B3、OR8B4、OR8B8、OR8B12、OR8D1、OR8D2、OR8D4、OR8G2、OR8G5、OR8H3、OR8I2、OR8J1、OR8J3、OR8K1、OR8K3、OR8K5、OR8S1、OR8U1、OR9A4、OR9G1、OR9G4、OR9I1、OR9K2、OR9Q1、OR9Q2、OR10A3、OR10A4、OR10A5、OR10A6、OR10A7、OR10AD1、OR10AG1、OR10C1、OR10D3、OR10D4P、OR10G2、OR10G3、OR10G4、OR10G6、OR10G7、OR10G9、OR10H2、OR10H4、OR10J1、OR10J3、OR10J5、OR10K1、OR10K2、OR10P1、OR10Q1、OR10R2、OR10S1、OR10T2、OR10V1、OR10W1、OR10X1、OR10Z1、OR11A1、OR11G2、OR11H4、OR11H6、OR11H12、OR11L1、OR12D2、OR12D3、OR13A1、OR13C2、OR13C3、OR13C4、OR13C8、OR13D1、OR13F1、OR13G1、OR13H1、OR13J1、OR14A2、OR14A16、OR14C36、OR14I1、OR14J1、OR14K1、OR14L1P、OR51A1P、OR51A4、OR51A7、OR51B2、OR51B4、OR51B5、OR51B6、OR51D1、OR51E1、OR51E2, OR51F1, OR51F2, OR51F5P, OR51G1, OR51G2, OR51H1, OR51I1, OR51I2, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, O R52E5, OR52E8, OR52H1, OR52I2, OR52J3, OR52K2, OR52L2P, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52P2P, OR52R1, OR52W1, OR52Z1P, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B2P, and OR56B4.

[0019] Olfactory receptors, more particularly OR1A1, OR1C1, OR1D2, OR1D5, OR1F1, OR1R1P, OR2A4, OR2AG2, OR2B11, OR2C1, OR2J2, OR2K2, OR2L8, OR2T10, OR2W1, OR2W3, OR2Y1, OR4C3, OR4D1, OR4H12P, OR4K5, OR4P4, OR4S2, OR4X1, OR5AN1, OR5AU1, OR5K1, OR5L2, OR5P3, OR6B1, OR6B2, OR6C6 5, OR6C75, OR6J1, OR6P1, OR7A3P, OR7A17, OR7D2, OR8B3, OR8B12, OR8D1, OR8D4, OR8K3, OR9A4, OR9Q2, OR10A3, OR10A6, OR10D3, OR10G3, OR10G4, OR10G6, OR10G7, OR10H2, OR10T2, OR11H4, OR13F1, OR51E1, OR51E2, OR51I2, OR51L1, OR51V1, OR52E4, and OR56A5.

[0020] Examples of olfactory receptors include those listed in Tables 3 to 8. Each of these olfactory receptors may exhibit a positive correlation with any of the aromas (specifically, odor descriptors) listed in Tables 3 to 8. Examples of combinations of target aromas and olfactory receptors (specifically, combinations of target aromas and olfactory receptors that exhibit a positive correlation) include combinations of aromas (specifically, odor descriptors) and olfactory receptors that exhibit a positive correlation listed in Tables 3 to 8. Combinations of target aromas and olfactory receptors (specifically, combinations of target aromas and olfactory receptors that show a positive correlation) include the following: combination of guaiacol and OR10G4; combination of raw and OR5K1; combination of peanut roasted peanut and OR5K1; combination of hazelnut and OR5K1; combination of potato and OR5K1; combination of popcorn and OR5K1; combination of corn chip and OR5K1; combination of beany and OR5K1; combination of cocoa and OR5K1; combination of roasted and OR5K1; combination of coffee and OR5K1; combination of burnt and OR5K1; combination of musty and OR5K1; combination of horseradish and OR2C1; combination of rubbery and OR4S2; combination of maple and OR8D1; combination of lactonic and OR8D1; combination of bready and OR6B1; combination of sharp and OR51E1; combination of creamy and OR10A3; combination of natural and OR1D5; combination of fruit tropical fruit and OR6C65; Solvent and OR4C3; Tomato and OR2K2; Eggy and OR1R1P; Magnolia and OR4K5; Cooling and OR8K3; Lemon and OR2W3; Mushroom and OR2W1; Caraway and OR1A1; Jasmin and OR1A1; Floral and OR1A1; Banana and OR2L8; Warm and OR10G7;Combination of musk and OR5AN1; Combination of heliotrope and OR5AN1; Combination of pepper bell pepper and OR2AG2; Combination of bois de rose and OR1C1; Combination of apricot and OR1D2; Combination of cucumber and OR2J2; Combination of cinnamyl and OR2J2; Combination of melon and OR2J2; Combination of acacia and OR5P3; Combination of hawthorn and OR5P3; Combination of naphthyl and OR5P3; Combination of bacon and OR10G7; Combination of bacon and OR10G4; Combination of clove and OR10G7; Combination of clove and OR10G4; Combination of clove and OR10D3; Combination of clove and OR2J2; Combination of carnation and OR10G7; Combination of carnation and OR10G4; Combination of carnation and OR10D3; Combination of carnation and OR2J2; Combination of vanilla and OR10G4; Combination of vanilla and OR10G7; Combination of vanilla and OR2J2 Combination of vanilla and OR10D3; Combination of vanilla and OR10G3; Combination of smoky and OR10G4; Combination of smoky and OR10G7; Combination of phenolic and OR10G4; Combination of phenolic and OR9Q2; Combination of phenolic and OR10G7; Combination of medicinal and OR10G4; Combination of medicinal and OR9Q2; Combination of nutty and OR5K1; Combination of nutty and OR6B1; Combination of nutty and OR51E2; Combination of meaty and OR2C1; Combination of meaty and OR4S2; Combination of sulfurous and OR2C1; Combination of sulfurous and OR4S2; Combination of alliaceous and OR2C1; Combination of alliaceous and OR4S2; Combination of onion and OR4S2; Combination of onion and OR2C1; Combination of cabbage and OR4S2; Combination of cabbage and OR2C1;Combination of garlic and OR4S2; Combination of garlic and OR2C1; Combination of caramellic and OR8D1; Combination of caramellic and OR6B1; Combination of pungent and OR6B1; Combination of pungent and OR51E1; Combination of mustard and OR2A4; Combination of mustard and OR10H2; Combination of sweet and OR8B3; Combination of sweet and OR2J2; Combination of sweet and OR5P3; Combination of sweet and OR1D2; Combination of sweet and OR11H4; Combination of fresh and OR1D2; Combination of fresh and OR10A3; Combination of fresh and OR9A4; Combination of woody and OR7A17; Combination of woody and OR4X1; Combination of minty and OR2B11; Combination of minty and OR10T2; Combination of honey and OR51L1; Combination of honey and OR1A1; Combination of fruity and OR2L8; Combination of fruity and OR1D2; Combination of pineapple and OR2L8; Combination of pineapple and OR1D2; Combination of spicy and OR2J2 Combination of spicy and OR10G7; Combination of spicy and OR10G4; Combination of spicy and OR8B3; Combination of spicy and OR11H4; Combination of apple skin and OR1F1; Combination of apple skin and OR4D1; Combination of apple skin and OR7D2; Combination of vegetable and OR5K1; Combination of vegetable and OR4S2; Combination of peach and OR1D2; Combination of peach and OR10A3; Combination of peach and OR10A6; Combination of coconut and OR8B3; Combination of coconut and OR10A3; Combination of coconut and OR10A6; Combination of coconut and OR1A1; Combination of sour and OR51E1; Combination of sour and OR51V1; Combination of sour and OR51L1; Combination of sour and OR51I2; Combination of acidic and OR51E1;Combination of acidic and OR51I2; Combination of acidic and OR51V1; Combination of cheesy and OR51E1; Combination of cheesy and OR51I2; Combination of cheesy and OR51L1; Combination of cheesy and OR51V1; Combination of sweaty and OR51E1; Combination of sweaty and OR51I2; Combination of sweaty and OR51L1; Combination of cinnamon and OR2J2; Combination of cinnamon and OR8B3; Combination of cinnamon and OR2B11; Combination of cinnamon and OR2Y1; Combination of cinnamon and OR11H4; Combination of cherry and OR5P3; Combination of cherry and OR2J2; Combination of cherry and OR8B3; Combination of cherry and OR2B11; Combination of almond bitter almond and OR5P3; Combination of almond bitter almond and OR2B11; Combination of almond bitter almond and OR8B3; Combination of almond bitter almond and OR2J2; Combination of almond bitter Combination of almond and OR6P1; Combination of tonka and OR8B3; Combination of tonka and OR5P3; Combination of tonka and OR8D1; Combination of coumarinic and OR8B3; Combination of coumarinic and OR5P3; Combination of coumarinic and OR8D1; Combination of mimosa and OR5P3; Combination of mimosa and OR8B3; Combination of lilac and OR5P3; Combination of lilac and OR8D4; Combination of powdery and OR5P3; Combination of powdery and OR5AN1; Combination of orangeflower and OR5P3; Combination of orangeflower and OR1D2; Combination of orangeflower and OR8B12; Combination of orangeflower and OR4P4; Combination of orangeflower and OR7A3P; Combination of anisic and OR5P3; Combination of anisic and OR1A1; Combination of licorice and OR5P3;A combination of licorice and OR1A1; a combination of anise and OR5P3; a combination of anise and OR1A1; a combination of anise and OR8B3. These combinations may be used alone or in combination.

[0021] Combinations of target aromas and olfactory receptors (specifically, combinations of target aromas and olfactory receptors that show a positive correlation) include, in particular, the following combinations: meaty and OR2C1; meaty and OR4S2; bready and OR6B1; and creamy and OR10A3.

[0022] More particularly, examples of combinations of target odors and olfactory receptors (specifically, combinations of target odors and olfactory receptors that show a positive correlation) include the following combinations: a combination of meaty and OR2C1; and a combination of meaty and OR4S2.

[0023] Examples of olfactory receptors include those listed in Table 9. Each of these olfactory receptors may exhibit a negative correlation with any of the aromas (specifically, odor descriptors) listed in Table 9. Examples of combinations of aromas and olfactory receptors of interest (specifically, combinations of aromas and olfactory receptors of interest that exhibit a negative correlation) include combinations of aromas (specifically, odor descriptors) and olfactory receptors that exhibit a negative correlation listed in Table 9. Combinations of target aromas and olfactory receptors (specifically, combinations of target aromas and olfactory receptors that show a negative correlation) include the following: peanut roasted peanut and OR4H12P; meaty and OR2W1; sulfurous and OR1D2; sulfurous and OR13F1; sulfurous and OR52E4; sulfurous and OR10D3; sulfurous and OR6B2; sulfurous and OR5L2; sulfurous and OR2T10; sulfurous and OR5AU1; sulfurous and OR10G6; sulfurous and OR6C75; soapy and OR51E2; soapy and OR56A5; orchid and OR51E2; waxy and OR5K1; waxy and OR51E2; waxy and OR10G4; waxy and OR2B11; gardenia and OR6J1; Combination of fruity and OR5P3; combination of fruity and OR5K1; combination of fruity and OR10G4; combination of fruity and OR4S2; combination of cherry and OR6B1; combination of floral and OR6B1. These combinations may be used alone or in combination with multiple combinations.

[0024] As the olfactory receptor, a single type of olfactory receptor may be used, or two or more types of olfactory receptors may be used in combination. For example, two or more types of olfactory receptors associated with a certain odor characteristic may be used in combination. Specifically, for example, when the target odor is meaty, OR2C1 and OR4S2 may be used in combination as olfactory receptors. By using two or more types of olfactory receptors associated with a certain odor characteristic in combination, it is expected that, for example, substances having that odor characteristic can be screened with high accuracy. When two or more types of olfactory receptors are used in combination, the response of the olfactory receptor to a test substance may be measured individually for each of the olfactory receptors. For example, the response of each olfactory receptor to a test substance can be measured individually using cells expressing each olfactory receptor.

[0025] Genes that encode olfactory receptors are also called "olfactory receptor genes."

[0026] Olfactory receptor genes and olfactory receptors include those of various organisms. Examples of organisms include animals such as mammals. 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 (cyn-eating monkeys), and Equus caballus (horses). Mammals, in particular, include humans. The 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. Human OR1A1, OR1C1, OR1D2, OR1D5, OR1F1, OR1R1P, OR2A4, OR2AG2, OR2B11, OR2C1, OR2J2, OR2K2, OR2L8, OR2T10, OR2W1, OR2W3, OR2Y1, OR4C3, OR4D1, OR4H12P, OR4K5, OR4P4, OR4S2, OR4X1, OR5AN1, OR5AU1, OR5K1, OR5L2, OR5P3, OR6B1, OR6B2, OR6C65, OR6C75, OR6J1, OR6 The base sequences of the P1, OR7A3P, OR7A17, OR7D2, OR8B3, OR8B12, OR8D1, OR8D4, OR8K3, OR9A4, OR9Q2, OR10A3, OR10A6, OR10D3, OR10G3, OR10G4, OR10G6, OR10G7, OR10H2, OR10T2, OR11H4, OR13F1, OR51E1, OR51E2, OR51I2, OR51L1, OR51V1, OR52E4, and OR56A5 genes are shown in SEQ ID NOs: 1 to 63, respectively.

[0027] That is, the olfactory receptor gene may be, for example, a gene having a known or naturally occurring nucleotide sequence of an olfactory receptor gene as described above (for example, the nucleotide sequence of an olfactory receptor gene of the above-mentioned organisms registered with NCBI or the nucleotide sequences of SEQ ID NOs: 1 to 63). The olfactory receptor may also be, for example, a protein having a known or naturally occurring amino acid sequence of an olfactory receptor as described above (for example, the amino acid sequence of an olfactory receptor of the above-mentioned organisms registered with NCBI or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NOs: 1 to 63). Unless otherwise specified, the expression "a gene has a nucleotide sequence" means that the gene contains the nucleotide sequence in question, and also encompasses cases where the gene consists of the nucleotide sequence. Unless otherwise specified, the expression "a protein has an amino acid sequence" means that the protein contains the amino acid sequence in question, and also encompasses cases where the protein consists of the amino acid sequence.

[0028] The olfactory receptor may be, for example, a chimeric protein of two or more olfactory receptors of different origins. In other words, the olfactory receptors specified by the above names also include, for example, chimeric proteins of two or more olfactory receptors of different origins specified by those names. Such chimeric proteins are also called "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 amino acid sequence of an olfactory receptor (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 substituted 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 performed between corresponding positions in the amino acid sequence of the olfactory receptor. "Corresponding sites in the amino acid sequence of an olfactory receptor" refers to sites arranged at corresponding positions in an alignment of the amino acid sequences of those olfactory receptors. Examples of chimeric olfactory receptors include chimeric proteins of olfactory receptors from the organisms exemplified above (i.e., chimeric proteins of olfactory receptors from two or more organisms selected from the organisms exemplified above). Specific examples of chimeric olfactory receptors include mammalian chimeric olfactory receptors (i.e., chimeric proteins of olfactory receptors from two or more mammalian organisms). That is, the olfactory receptor may be, for example, a protein having a chimeric sequence of the amino acid sequence of an olfactory receptor from the organisms exemplified above (specifically, a chimeric sequence of the amino acid sequences of olfactory receptors from two or more organisms selected from the organisms exemplified above). Chimeric olfactory receptors can be selected that are associated with the odor characteristics of interest (specifically, that there is a correlation between their activation and the odor of interest (specifically, an odor descriptor)).

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The olfactory receptor gene may be a variant of the olfactory receptor gene exemplified above, for example, 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 exemplified above, for example, 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 are sometimes referred to as "conservative variants." The olfactory receptors specified by the above names include not only the olfactory receptors specified by the above names, but also their conservative variants. Examples of conservative variants include homologs and artificially modified versions of the olfactory receptor genes and olfactory receptors exemplified above.

[0034] 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.

[0035] "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. That is, "maintaining the original function" with respect to each olfactory receptor gene may mean that the gene variant encodes a protein (olfactory receptor) associated with a desired odor characteristic (specifically, a correlation between its activation and the desired odor (specifically, an odor descriptor)). "Maintaining the original function" with respect to each olfactory receptor gene may specifically mean that the gene variant encodes a protein (olfactory receptor) that responds to a target substance. Furthermore, "maintaining the original function" with respect to each olfactory receptor may mean that the olfactory receptor variant is a protein (olfactory receptor) associated with a desired odor characteristic (specifically, a correlation between its activation and the desired odor (specifically, an odor descriptor)). "Maintaining the original function" for each olfactory receptor may specifically mean that the olfactory receptor variant responds to a target substance.

[0036] Examples of conservative variants are shown below.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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).

[0046] 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 a target substance. 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.

[0047] The olfactory receptor can be used in any form that can be used for screening a target substance. 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 the target substance. The form in which the olfactory receptor is used can be appropriately determined depending on various conditions, such as the embodiment of the method of the present invention.

[0048] 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 within the scope of olfactory receptors used in the methods of the present invention.

[0049] 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 appropriately in the form described above and used in the method of the present invention.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] The cells of the present invention may inherently have an olfactory receptor gene, or may be modified to have an olfactory receptor gene.

[0054] 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.

[0055] Cells modified to have an olfactory receptor gene include cells into which an olfactory receptor gene has been introduced.

[0056] 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."

[0057] The host cell is not particularly limited as long as it can express a functional olfactory receptor and can be used to screen for a target substance. 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 tissue or cell from which the host cell is derived is not particularly limited. Examples of tissue or cell from which the host cell is 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.

[0058] 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)).

[0059] 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.

[0060] 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.

[0061] 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)).

[0062] 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.

[0063] 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.

[0064] 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).

[0065] 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.

[0066] The cells of the present invention may have any other properties as long as they can be used to screen for a target substance. Examples of such properties include properties that are useful for measuring the response of olfactory receptors to a target substance. 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.

[0067] 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.

[0068] 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.

[0069] 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). Mouse RTP1s shares 93.3% amino acid identity with the human RTP1s. Bat RTP1s (the partial sequence described above) shares 90.7% amino acid identity with the human RTP1s.

[0070] 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.

[0071] 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.

[0072] Expression of olfactory receptors can be confirmed by measuring the response of the olfactory receptors to substances that induce a response of the olfactory receptors, or by measuring the amount of mRNA transcribed from the olfactory receptor gene or by detecting the olfactory receptors by Western blotting using antibodies.

[0073] The cells of the present invention can be used in the methods of the present invention, for example, as they are (as they are contained in the culture medium) or after being recovered from the medium. Furthermore, the culture or cells recovered therefrom may be used in the methods 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.

[0074] 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).

[0075] 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.

[0076] 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.

[0077] <4> Method of the Present Invention The method of the present invention can be carried out in vitro.

[0078] Step (A) is a step of contacting an olfactory receptor with a test substance. That is, first, the olfactory receptor and the test substance can be contacted.

[0079] A system in which contact between an olfactory receptor and a test substance is carried out is also referred to as a "reaction system." Contact between an olfactory receptor and a test substance can be carried out in an appropriate liquid. A liquid in which contact between an olfactory receptor and a test substance is carried out is also referred to as a "reaction solution." That is, for example, contact between an olfactory receptor and a test substance can be achieved by coexisting the olfactory receptor and the test substance in an appropriate reaction solution. Specifically, for example, contact between an olfactory receptor and a test substance can be achieved by dissolving, suspending, dispersing, or the like an olfactory receptor (e.g., a form such as one of the above-mentioned examples, such as a cell having an olfactory receptor) and a test substance 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 are contacted together with an olfactory receptor, contact between the 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. The reaction conditions (conditions for contacting the olfactory receptor with the test substance) are not particularly limited as long as they allow screening of the target substance. The reaction conditions can be appropriately set depending on various conditions, such as the manner in which the olfactory receptor is used, the type of test substance, and the method for measuring the olfactory receptor response. For example, 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 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 the olfactory receptors may be, for example, 10 cell / mL to 10,000,000 cell / mL. The contact between the olfactory receptor and the test substance may or may not be terminated at an appropriate time point. Contact between the olfactory receptor and the test substance may generally be continued until the response of the olfactory receptor to the test substance is measured.The duration of contact between the olfactory receptor and the test substance may be, for example, 0.1 second 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 the test substance 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 exemplified above, such as cells having an olfactory receptor) and the test substance, as long as screening of the target substance is possible. The other components can be appropriately selected depending on various conditions, such as the form of use of the olfactory receptor, 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.

[0080] Furthermore, when measuring the inactivation of olfactory receptors by a test substance, the contact between the olfactory receptor and the test substance can be carried out, for example, in the presence of a substance that activates the olfactory receptor (hereinafter also referred to as an "olfactory receptor activator"). The expressions "contacting the olfactory receptor with the test substance in the presence of an olfactory receptor activator," "contacting the olfactory receptor with an olfactory receptor activator in the presence of a test substance," and "contacting the olfactory receptor with an olfactory receptor activator and a test substance" may be used interchangeably. The description of the contact between the olfactory receptor and the test substance can also be applied mutatis mutandis to the contact between the olfactory receptor and the olfactory receptor activator. The contact between the olfactory receptor activator and the test substance and the olfactory receptor may or may not be initiated simultaneously. For example, the test substance and the olfactory receptor activator may be mixed in advance and then contacted with the olfactory receptor. Furthermore, 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. 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. When the contact between the olfactory receptor and the test substance is carried out in the presence of an olfactory receptor activator, the phrase "duration of contact between the olfactory receptor and the test substance" may be interpreted as "duration of contact between the olfactory receptor and the olfactory receptor activator and the test substance."

[0081] The olfactory receptor activator is not particularly limited as long as it can activate the olfactory receptor. The olfactory receptor activator 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, the number of components constituting the mixture and their composition ratios are not particularly limited. The olfactory receptor activator may be a known substance or a novel substance. The olfactory receptor activator may be a natural product or an artificial product. The olfactory receptor activator may or may not be known to be able to activate the olfactory receptor. As the olfactory receptor activator, for example, one capable of activating the olfactory receptor may be selected from the test substances exemplified above and used.

[0082] Step (B) is a step of measuring the response of the olfactory receptor to the test substance. That is, the response of the olfactory receptor to the test substance can then be measured. The response of the olfactory receptor to a substance is also referred to as "the substance eliciting a response of the olfactory receptor."

[0083] The response of the olfactory receptor to the test substance includes activation or inactivation of the olfactory receptor by the test substance.

[0084] The timing for measuring the olfactory receptor response to a test substance is not particularly limited as long as the olfactory receptor response to the test substance is measurable when the test substance is the target substance. 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 type of test substance, and the method for measuring the olfactory receptor response. Specifically, the timing for measuring the olfactory receptor response to a test substance may be any appropriate time from the time when contact between the olfactory receptor and the test substance begins to the time when the olfactory receptor response to the test substance disappears. The timing for measuring the olfactory receptor response to a test substance may be, for example, the time when the maximum olfactory receptor response to the test substance is obtained (e.g., the time when the degree of activation or inactivation of the olfactory receptor by the test substance is maximum). Furthermore, the timing for measuring the olfactory receptor response to the test substance 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 the test substance begins, or up to 24 hours, 12 hours, 6 hours, 2 hours, or 1 hour, or any compatible combination thereof. Specifically, the timing for measuring the olfactory receptor response to the test substance may be, for example, from 1 hour to 6 hours from the time when contact between the olfactory receptor and the test substance begins. When contact between the olfactory receptor and the test substance is carried out in the presence of an olfactory receptor activator, the phrase "the time when contact between the olfactory receptor and the test substance begins" may be interpreted as "the time when contact between the olfactory receptor and the olfactory receptor activator and the test substance begins."

[0085] Step (C) is a step of identifying the test substance as a substance of interest based on the response of the olfactory receptor to the test substance. That is, based on the response of the olfactory receptor to the test substance, it is then possible to identify whether the test substance has the desired odor characteristics, thereby identifying whether the test substance is a substance of interest. That is, based on the response of the olfactory receptor to the test substance, the test substance can be identified as a substance of interest.

[0086] Specifically, when the response of olfactory receptors to a test substance is observed, the test substance can be identified as a target substance.More specifically, the identification can be carried out as follows: for olfactory receptors that show a positive correlation with the target odor, the test substance can be identified as exhibiting the target odor when the olfactory receptor is activated by the test substance; for olfactory receptors that show a positive correlation with the target odor, the test substance can be identified as suppressing the target odor when the olfactory receptor is inactivated by the test substance; for olfactory receptors that show a negative correlation with the target odor, the test substance can be identified as suppressing the target odor when the olfactory receptor is activated by the test substance; for olfactory receptors that show a negative correlation with the target odor, the test substance can be identified as exhibiting the target odor when the olfactory receptor is inactivated by the test substance.

[0087] The activation or inactivation of the olfactory receptor by the test substance may be determined using as an index 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 brought into contact with 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 of interest based on the degree of activation D1.

[0088] Specifically, activation or inactivation of the olfactory receptor by the test substance may be determined by comparing the degree of activation of the olfactory receptor (degree of activation D1) when step (A) is performed (i.e., under conditions in which the olfactory receptor is brought into contact with the test substance) with the degree of activation of the olfactory receptor under control conditions (degree of activation D2). That is, step (C1) may be, for example, (C2) a step of identifying whether the test substance is a substance of interest based on the difference between the degree of activation D1 and the degree of activation D2.

[0089] "Control conditions" refer to the following conditions (C2-1) or (C2-2): (C2-1) conditions in which the olfactory receptor is not contacted with the test substance; (C2-2) conditions in which the olfactory receptor is contacted with the test substance, and the concentration of the test substance is lower than the concentration of the test substance in the above step (A).

[0090] When the contact between the olfactory receptor and the test substance is carried out in the presence of an olfactory receptor activator, the above condition (C2-1) or (C2-2) can be interpreted as follows: (C2-1) A condition in which the olfactory receptor is contacted with the olfactory receptor activator, but not with the test substance (i.e., a condition in which the olfactory receptor is contacted with the olfactory receptor activator in the absence of the test substance); (C2-2) A condition in which the olfactory receptor is contacted with the test substance in the presence of an olfactory receptor activator, wherein the concentration of the test substance is lower than the concentration of the test substance in the above step (A).

[0091] In other words, activation or inactivation of olfactory receptors by a test substance may be determined, for example, using as an index the difference in the degree of activation of olfactory receptors due to the presence or absence or different concentrations of the test substance.

[0092] The above condition (C2-1) includes conditions before contacting the olfactory receptor with the test substance. The above condition (C2-1) also includes conditions after contacting the olfactory receptor with the test substance, in which the test substance is substantially (e.g., completely) removed from the reaction system and the olfactory receptor response to 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 degree of activation D1 and the degree of activation 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 the olfactory receptor response to 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.

[0093] The 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.

[0094] When the degree of activation D1 is high, it may be determined that the test substance has activated the olfactory receptor. Specifically, when the degree of activation D1 is higher than the degree of activation D2, it may be determined that the test substance has activated the olfactory receptor. For example, when the ratio of the degree of activation D1 to the degree of activation D2 (i.e., D1 / D2) is 1.5 or more, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more, it may be determined that the test substance has activated the olfactory receptor. Examples of the ratio of the degree of activation D1 to the degree of activation D2 include the normalized response values ​​described in the Examples.

[0095] When the degree 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 degree of activation D1 is lower than the degree 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 degree of activation D1 to the degree of activation D2 in the presence of an olfactory receptor activator (i.e., D1 / D2) is 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less, it may be determined that the inactivation of the olfactory receptor by the test substance has been observed. Examples of the ratio of the degree of activation D1 to the degree of activation D2 in the presence of an olfactory receptor activator include the normalized response values ​​described in the Examples obtained in the presence of an olfactory receptor activator.

[0096] The method for measuring the response of an olfactory receptor to a test substance is not particularly limited. The method for measuring the response of an olfactory receptor to a test substance 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 a test substance can be measured, for example, by an appropriate method that can measure the activation or inactivation of the olfactory receptor by the test substance.

[0097] The method for measuring the activation or inactivation of olfactory receptors by a test substance is not particularly limited. The activation or inactivation of olfactory receptors by a test substance can be measured, for example, by known methods for measuring the activity of receptors such as olfactory receptors. Such methods include, for example, methods for measuring intracellular calcium concentration and methods for measuring intracellular cAMP concentration. That is, the activation or inactivation of olfactory receptors by a test substance can be measured, for example, using intracellular calcium concentration or intracellular cAMP concentration as an indicator. That is, the activation or inactivation of olfactory receptors by a test substance 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.

[0098] The description of measuring the activation or inactivation of olfactory receptors 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.

[0099] That is, for example, using artificial lipid bilayer vesicles having olfactory receptors, activation or inactivation of olfactory receptors can be measured using techniques similar to those used when cells having olfactory receptors are used. In such cases, the term "cells" in the description of measuring activation or inactivation of olfactory receptors using cells having olfactory receptors can be read as "artificial lipid bilayer vesicles."

[0100] 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 or inactivation of olfactory receptors can be measured using the same method as when using cells having olfactory receptors. 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 with two spaces separated from each other by the membrane, the activation or inactivation of olfactory receptors can be measured using the same method as when using cells having olfactory receptors. In such cases, the space separated by the membrane can be considered the inside of the cell (also referred to as the "internal space"). Specifically, one of the two spaces can be considered the inside of the cell (also referred to as the "internal space"), and the other can be considered the outside of the cell (also referred to as the "external space"). Of these spaces, the one containing the test substance 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 or inactivation using cells having olfactory receptors can be read as "calcium concentration within the internal space" and "cAMP concentration within the internal space," respectively.

[0101] In either case, measurable parameters can be selected depending on the manner in which the olfactory receptor is used.

[0102] Note that "measuring a certain parameter and using it as an index for measuring the response of an olfactory receptor to a test substance" means that as long as the response can be measured (i.e., it can be determined whether the response 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 the activation or inactivation of an olfactory receptor by a test substance, it is sufficient to obtain and use data reflecting the intracellular cAMP concentration (e.g., luminescence intensity) as long as the activation or inactivation can be measured (i.e., it can be determined whether the activation or inactivation is observed), and it is not necessary to calculate the intracellular cAMP concentration itself from the data.

[0103] In this way, the target substance can be identified. The method of the present invention may further include a step of evaluating the aroma characteristics of the identified target substance. That is, by evaluating the aroma characteristics of the identified target substance, it is possible to confirm whether the target substance actually has the desired aroma characteristics. The method for evaluating the aroma characteristics of the identified target substance is not particularly limited. The desired aroma characteristics of the identified target substance can be evaluated, for example, by known methods for evaluating the aroma of substances. Such methods include sensory evaluation (evaluation by sensory testing).

[0104] The use of the screened target substance is not particularly limited. The target substance can be blended into a target substance, such as a food or beverage or a cosmetic product. If the target substance has the property of exhibiting a target aroma, the blending of the target substance can impart the target aroma to the target substance, such as a food or beverage or a cosmetic product. The target substance to which the target aroma is imparted may or may not inherently have the target aroma. In other words, "imparting a target aroma" means imparting the target aroma to a target substance regardless of whether the target substance inherently exhibits the target aroma, and also includes enhancing the target aroma of a target substance that inherently exhibits the target aroma. If the target substance has the property of suppressing the target aroma, the blending of the target substance can suppress (i.e., mask) the target aroma in a target substance, such as a food or beverage or a cosmetic product, that has the target aroma. Furthermore, the target substance can also be used, for example, as a raw material for developing a new substance with the target aroma characteristics.

[0105] For example, target substances that exhibit a meaty aroma can be screened using OR2C1 and / or OR4S2. In other words, target substances that exhibit a meaty aroma can be screened by using OR2C1 and / or OR4S2 as the olfactory receptors correlated with the meaty aroma as the target aroma.

[0106] The specific method for screening target substances that exhibit a meaty aroma is as described above in the method of the present invention, except that OR2C1 and / or OR4S2 are used as the olfactory receptors.

[0107] A target substance that exhibits a meaty aroma can be used to impart a meaty aroma to a target object such as a food or drink. That is, by adding a target substance that exhibits a meaty aroma to a target object such as a food or drink, the target object can be imparted with a meaty aroma. That is, a target substance that exhibits a meaty aroma is also a substance that imparts a meaty aroma to a target object such as a food or drink.

[0108] The aroma "meaty" may specifically mean a meaty flavor or a meaty aroma. That is, a target substance that exhibits a meaty aroma may specifically be a substance that exhibits a meaty flavor or a meaty aroma. Furthermore, a target substance that exhibits a meaty aroma may specifically be a substance that imparts a meaty flavor or a meaty aroma to an object such as a food or drink. A substance that imparts a meaty flavor to an object is also called a "meat flavor imparting agent." A substance that imparts a meaty aroma to an object is also called a "meat aroma imparting agent."

[0109] "Meat flavor" may refer to the unique flavor that can be sensed from meat-derived ingredients such as meat (e.g., beef, pork, chicken, etc.) and meat extracts (e.g., beef extract, pork extract, chicken extract, etc.), and in particular may refer to the flavor that can be smelled when meat is cooked, such as by heating. "Meat aroma" may refer to the unique aroma that can be sensed from meat-derived ingredients such as meat (e.g., beef, pork, chicken, etc.) and meat extracts (e.g., beef extract, pork extract, chicken extract, etc.), and in particular may refer to the aroma that can be smelled when meat is cooked, such as by heating. "Meat flavor" and "meat aroma" may refer to the flavor and aroma that are excellent in the richness and aged feel that are inherent to meat, respectively.

[0110] "Imparting a meaty aroma" refers to imparting a meaty aroma to an object regardless of whether the object inherently exhibits a meaty aroma, and also encompasses enhancing the desired aroma of an object that inherently exhibits a meaty aroma. Specifically, "imparting a meat flavor" refers to imparting a meat flavor to an object such as a food or beverage, regardless of whether it contains meat-derived ingredients such as meat or meat extract, and also encompasses enhancing the meat flavor of an object such as a food or beverage that contains meat-derived ingredients. Furthermore, "imparting a meat aroma" refers to imparting a meat aroma to an object such as a food or beverage, regardless of whether it contains meat-derived ingredients such as meat or meat extract, and also encompasses enhancing the meat aroma of an object such as a food or beverage that contains meat-derived ingredients. The presence or level of a meaty aroma (specifically, meat flavor or aroma) can be evaluated by sensory evaluation by a specialist panel. Sensory evaluation can be performed, for example, by the method described in the Examples below.

[0111] The object to which a meaty aroma is imparted using a target substance exhibiting a meaty aroma is not particularly limited. A particular example of an object to which a meaty aroma is imparted using a target substance exhibiting a meaty aroma is meat substitute. "Meat substitute" may refer to a processed food prepared by processing raw materials containing plant protein. Examples of plant proteins include bean proteins, cereal proteins, seed proteins, mushroom proteins, and potato proteins. Examples of beans include soybeans, peas, broad beans, mung beans, chickpeas, lupin beans, and peanuts. Examples of grains include wheat, barley, rye, buckwheat, rice, and corn. Examples of seeds include sunflower seeds, pumpkin seeds, rapeseed, sesame seeds, quinoa, chia seeds, hemp seeds, and almonds. Meat substitutes are also called "pseudo-meat" or "veggie meat." Examples of meat substitutes include those prepared by processing raw materials containing soy protein (also called "soy meat"). The protein contained in the meat substitute may be entirely or partially plant protein. That is, the meat substitute may or may not contain, for example, meat.

[0112] Furthermore, cultured meat is a particular example of an object to which a meaty aroma can be imparted using a target substance that exhibits a meaty aroma. "Cultured meat" may refer to meat or meat-like substances obtained by culturing cells, or to their tissues or processed foods. Examples of cultured meat include cultured animal cells such as those of cows, pigs, chickens, and sheep, cultured seafood cells such as fish and shellfish, cultured crustacean cells, and cultured insect cells. Cultured animal cells include, in particular, cultured cows, pigs, or chickens. Cultured meat is also referred to as "clean meat" or "cell-based meat." Cultured meat may contain other ingredients. Cultured meat may be a complex cellular tissue cultured together with fat cells, for example. Cultured meat may also contain other ingredients, such as heme, lipids, collagen, and vitamins, that improve flavor, texture, nutrition, or shelf life.

[0113] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0114] Example 1 <1> Preparation of cells expressing human olfactory receptors <1-1> Preparation of expression vectors for human olfactory receptors As olfactory receptors, 352 types of human olfactory receptors (OR1A1, OR1A2, OR1B1, OR1C1, OR1D2, OR1D5, OR1E1, OR1F1, OR1F12, OR1G1, OR1I1, OR1J1, OR1J2, OR1J4, OR1K1, OR1L1, OR1L3, OR1L4, OR1L8, OR1M1, OR1N1, OR1N2, OR1Q1, OR1R1P, OR1S1, OR2A1, OR2A2, OR2A4, OR2A5, OR2A12, OR2A14, OR2A25, OR2AE1, OR2AG1, OR 2AG2, OR2AJ1P, OR2AK2, OR2AP1, OR2AT4, OR2B2, OR2B3, OR2B6, OR2B11, OR2C1, OR2C3, OR2D2, OR2D3, OR2F1, OR2G2, OR2G3, OR2G6, OR2H1, OR2H2 , OR2J2, OR2J3, OR2K2, OR2L2, OR2L8, OR2L13, OR2M2, OR2M4, OR2M7, OR2S2, OR2T1, OR2T2, OR2T5, OR2T6, OR2T8, OR2T10, OR2T11, OR2T27, OR2T34 , OR2V2, OR2W1, OR2W3, OR2Y1, OR2Z1, OR3A1, OR3A2, OR3A3, OR3A4, OR4A5, OR4A15, OR4A16, OR4A47, OR4B1, OR4C3, OR4C5, OR4C6, OR4C11, OR4C1 2, OR4C13, OR4C15, OR4C16, OR4C46, OR4D1, OR4D2, OR4D5, OR4D6, OR4D9, OR4D10, OR4D11, OR4E2, OR4F3, OR4F5, OR4F6, OR4F14P, OR4F15, OR4G11 P, OR4H12P, OR4K1, OR4K2, OR4K5, OR4K13, OR4K14, OR4K15, OR4K17, OR4L1, OR4M1, OR4N2, OR4N4, OR4N5, OR4P4, OR4Q3, OR4S1, OR4S2, OR4X1, OR 4X2, OR5A1, OR5A2, OR5AC2, OR5AK2, OR5AK3P, OR5AN1, OR5AP2, OR5AR1, OR5AS1, OR5AU1, OR5B2, OR5B3, OR5B12, OR5B17, OR5B21, OR5C1, OR5D13,OR5D14、OR5D16、OR5D18、OR5F1、OR5H1、OR5H2、OR5H6、OR5H14、OR5I1、OR5J2、OR5K1、OR5K3、OR5K4、OR5L2、OR5M3、OR5M8、OR5M9、OR5M10、OR5M11、OR5P3、OR5R1、OR5T1、OR5T2、OR5T3、OR5V1、OR5W2、OR6A2、OR6B1、OR6B2、OR6C1、OR6C2、OR6C3、OR6C4、OR6C6、OR6C65、OR6C66P、OR6C68、OR6C70、OR6C74、OR6C75、OR6C76、OR6F1、OR6J1、OR6K2、OR6K3、OR6K6、OR6M1、OR6N1、OR6N2、OR6P1、OR6Q1、OR6S1、OR6T1、OR6V1、OR6X1、OR6Y1、OR7A3P、OR7A5、OR7A10、OR7A17、OR7C1、OR7C2、OR7D2、OR7D4、OR7E24、OR7G1、OR7G2、OR7G3、OR8A1、OR8B3、OR8B4、OR8B8、OR8B12、OR8D1、OR8D2、OR8D4、OR8G2、OR8G5、OR8H3、OR8I2、OR8J1、OR8J3、OR8K1、OR8K3、OR8K5、OR8S1、OR8U1、OR9A4、OR9G1、OR9G4、OR9I1、OR9K2、OR9Q1、OR9Q2、OR10A3、OR10A4、OR10A5、OR10A6、OR10A7、OR10AD1、OR10AG1、OR10C1、OR10D3、OR10D4P、OR10G2、OR10G3、OR10G4、OR10G6、OR10G7、OR10G9、OR10H2、OR10H4、OR10J1、OR10J3、OR10J5、OR10K1、OR10K2、OR10P1、OR10Q1、OR10R2、OR10S1、OR10T2、OR10V1、OR10W1、OR10X1、OR10Z1、OR11A1、OR11G2、OR11H4、OR11H6、OR11H12、OR11L1、OR12D2、OR12D3、OR13A1、OR13C2、OR13C3、OR13C4、OR13C8、OR13D1、OR13F1、OR13G1、OR13H1、OR13J1、OR14A2、OR14A16、OR14C36、OR14I1、OR14J1、OR14K1、OR14L1P、OR51A1P、OR51A4, OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E2, OR51F1, OR51F2, OR51F5P, OR51G1, OR51G2, OR51H1, OR51I1, OR51I2, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, O R52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E8, OR52H1, OR52I2, OR52J3, OR52K2, OR52L2P, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52P2P, OR52R1, OR52W1, OR52Z1P, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B2P, OR56B4).

[0115] We purchased 352 human olfactory receptor genes from the TrueClone cDNA Clone Collection (OriGene). Using primers designed based on the sequences registered in GenBank, we amplified subcloning fragments for each of the 352 human olfactory receptor genes by PCR using the purchased human olfactory receptor genes as templates. The amplified subcloning fragments for each gene were subcloned downstream of the Rho tag sequence in the Rho-pME18S vector (K. Kajiya et al., Journal of Neuroscience, 15 August 2001, 21 (16) 6018-6025) using the EcoRI and XhoI sites, yielding 352 expression vectors for human olfactory receptors.

[0116] <1-2> Preparation of Olfactory Receptor-Expressing Cells HEK293T cells expressing each of the 352 olfactory receptors were prepared using the following procedure. The gene mixture shown in Table 1 and the transfection reagent mixture shown in Table 2 were prepared and left to stand at room temperature for 5 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). The gene mixture and transfection reagent mixture were mixed and dispensed in 12.5 μL aliquots into each well of a poly-D-lysine-coated 384-well plate. The mixture was left to stand for 15 minutes in a clean bench. HEK293T cells (2.5 x 10 cells) were seeded in 10 cm dishes the day before. 6 1.2 x 10 cells / 10 cm dish 5 The solution was adjusted to a concentration of 1000 cells / mL, and 25 μL of the solution was seeded into each well of a 384-well plate. The cells were then cultured overnight in an incubator maintained at 37°C and 5% CO2. In this manner, 352 HEK293T cell cultures transfected with the expression vectors shown in Table 1 and expressing the genes encoded by those expression vectors were obtained. As a control, the human olfactory receptor expression vector in the gene mixture shown in Table 1 was replaced with the empty vector Rho-pME18S, and the same procedure was repeated to obtain a culture of HEK293T cells transfected with the empty vector Rho-pME18S (hereinafter also referred to as "control cells").

[0117]

[0118]

[0119] <2> Creation of a Human Olfactory Receptor Activity Database <2-1> Luciferase Assay Olfactory receptor-expressing cells were used to measure the response of olfactory receptors to test substances.

[0120] The 352 olfactory receptors 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. This assay monitors the increase in intracellular cAMP levels as an increase in luminescence intensity 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, the luminescence intensity derived from Renilla luciferase was 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.

[0121] A total of 2621 test substances were selected, including those listed at The Good Scents Company (http: / / www.thegoodscentscompany.com / ). The medium was removed from the 352 cultures obtained in <1-2> above, and 15 μL of the 2621 test substance solutions was added to each culture, yielding 352 × 2621 reaction solutions. Each test substance solution was prepared by dissolving the test substance in CD293 (Life Technologies, Inc.). The test substance concentration in the test substance solution was generally 300 μM. However, for test substances that showed cytotoxicity at 300 μM, the test substance concentration in the test substance solution was set to 3 μM, 10 μM, 30 μM, or 100 μM. For a small number of test substances, the test substance concentration in the test substance solution was set to 1000 μM. The reaction solution was placed in an incubator maintained at 37°C and 5% CO2, and the cells were cultured for 4 hours to allow sufficient expression of the firefly luciferase gene within the cells. The luminescence value derived from intracellular firefly luciferase was measured and designated as the "Luc value." The luminescence value derived from intracellular Renilla luciferase was also measured and designated as the "hRLuc value." The luminescence value derived from each luciferase was measured using Dual-Glo TM Measurement was performed using a luciferase assay system (Promega) according to the product's operating manual.

[0122] <2-2> Calculation of olfactory receptor activity The luminescence value (Luc value) derived from firefly luciferase induced by stimulation with the test substance was divided by the luminescence value (hRluc value) derived from Renilla luciferase in the same well to obtain the "Luc / hRluc value." The Luc / hRluc value in cells stimulated with the test substance was divided by the Luc / hRluc value in cells not stimulated with the test substance to obtain the "fold increase." Furthermore, the fold increase in cells transfected with the olfactory receptor expression vector was divided by the fold increase in control cells (cells transfected with the empty vector Rho-pME18S) to obtain the "normalized response." The common logarithm of the normalized response was used as the "olfactory receptor activity," a quantitative index of the response strength of the olfactory receptor to the test substance. Hereinafter, when olfactory receptor activity is expressed as -1, 0, or 1, this refers to the common logarithm of the normalized response being -1, 0, or 1, i.e., the normalized response being 0.1, 1, or 10, meaning that the response of olfactory receptor-introduced cells to test substance stimulation was 1 / 10, 1, or 10 times stronger, respectively, than the response of control cells (cells transfected with the empty vector Rho-pME18S) to test substance stimulation. For simplicity, the possible effect of differences in test substance concentration in the test substance solution on olfactory receptor activity was ignored.

[0123] <2-3> Calculating Descriptor Scores The order in which descriptors are listed in the Organoleptic Properties column for each aroma component in The Good Scents Company tended to correlate with the degree of influence of each descriptor on the overall aroma quality of that aroma component. Furthermore, the frequency of each descriptor's appearance varied across The Good Scents Company samples. Adjective-like descriptors expressing vague odor impressions, such as sweet, fruity, and green, were assigned to approximately 20% of aroma components, while noun-like descriptors describing individual ingredients, such as peach and cucumber, appeared less frequently (approximately 1%). For these two reasons, we concluded that simply representing the aroma quality information of aroma components as a 0 / 1 expression based on the presence or absence of a descriptor is insufficient from the perspective of information preservation. In the field of natural language processing, a method known as Term Frequency-Inverse Document Frequency (TF-IDF) has been commonly used to score the importance of words contained in each document and represent documents as vectors of word importance scores, with the aim of characterizing each document and retrieving similar documents. TF represents the frequency of occurrence of word t in document d and is an index of the dominance of each word in an individual document. IDF represents the proportion (the inverse of the natural logarithm) of documents containing word t out of the total number of documents and is a correction index used to reduce the relative importance of words that appear frequently in various documents. We reinterpreted the TF-IDF in natural language processing as "document = aroma quality (aroma component)" and "word = descriptor," and introduced the Inverse Rank - Inverse Odor Frequency (IR-IOF), a score of the importance of each descriptor to aroma quality, as shown below. Instead of TF, we introduced IR as a dominance index, taking the inverse of the rank order of occurrence of descriptor d in aroma component c as IR. For example, for vanillin (CAS number: 121-33-5), which is expressed as "sweet / vanilla / creamy / chocolate," the IRs for the four descriptors are sweet: 1, vanilla: 0.5, creamy: 0.33, and chocolate: 0.25.We introduced the IOF as an index of relative importance to replace the IDF, and defined the IOF as the reciprocal of the natural logarithm of the ratio of the number of aroma components with descriptor d to the total number of aroma components. The IR-IOF was calculated by multiplying the IR and IOF, and this was used as the importance score of each descriptor for each aroma component.

[0124] All data listed by The Good Scents Company was filtered by the following criteria: presence of CAS numbers, elimination of duplicate CAS numbers, elimination of duplicates of The Good Scents Company's internal IDs, and presence of a descriptor other than odorless in the Organoleptic Properties column. Furthermore, the analysis was limited to aroma compounds that share a descriptor with at least one other aroma compound, and descriptors assigned to aroma compounds that share at least one descriptor. The resulting 4,059 aroma compounds and 455 descriptors were then scored using IR-IOF.

[0125] <3> Comprehensive correlation analysis between olfactory receptor activity and descriptors We examined whether there was a difference in the activity of each olfactory receptor depending on the presence or absence of individual odor descriptors. We used a Welch t-test, which does not assume equal variance, to perform the test. We used the t.test function in the stats package in R, specifying "two.sided" for the alternative argument, FALSE for the paired argument, and FALSE for the var.equal argument.

[0126] Furthermore, we tested for a correlation between the descriptor score (IR-IOF) of each aroma component and olfactory receptor activity. To test for correlation, we used the cor.test function in the same package, specifying "two.sided" as the alternative argument and "pearson" as the method argument.

[0127] Generally, when performing multiple tests as described above, the greater the number of tests, the greater the chance that some of the rejected null hypotheses will be incorrectly rejected (indicating a difference when in fact there is no difference). This probability is called the False Discovery Rate (FDR), and while various correction methods (multiple comparison correction) have been proposed to control the FDR, we adopted the Benjamini-Hochberg correction method here. The p.adjust function in the stats package in R was used to correct the p-value by specifying "BH" as the method argument, and a corrected p-value of less than 0.05 was considered significant.

[0128] There were 201 pairs of olfactory receptors and descriptors (63 unique olfactory receptors and 94 descriptors) in which activity differed depending on the presence or absence of a descriptor (p < 0.05), and there was a significant correlation between the descriptor score and the activity (p < 0.05 after correction for multiple comparisons using the BH method). For these 201 pairs of olfactory receptors and descriptors, the difference in mean olfactory receptor activity between the groups with and without a descriptor was displayed in a heat map (Figure 1). Tables 3 and 4 show combinations in which the activity of a single olfactory receptor was positively correlated with a single odor descriptor. Tables 5-8 show combinations in which the activity of multiple olfactory receptors was positively correlated with a single odor descriptor. Table 9 shows combinations in which the activity of one or more olfactory receptors was negatively correlated with a single odor descriptor.

[0129] By understanding the correlation between these odor descriptors and olfactory receptor activity and combining this with screening techniques for activating and inhibiting components of each olfactory receptor, it will be possible to search for target flavor-enhancing materials and off-flavor-masking materials.

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] Example 2: The aroma characteristics perceived by subjects of 10 different samples were investigated using nine different food-related descriptors based on the CATA (Check-All-That-Apply) method. The CATA method is a method in which all evaluation terms that describe the characteristics of a sample are checked from multiple evaluation terms, and can be used to clarify the characteristics of a sample based on the number of checks for each evaluation term.

[0138] <1> Determination of Sample Concentration: For each sample, the concentration that resulted in the same fragrance intensity as the reference sample (10 ppm Methional) was determined using the following procedure. Specifically, each sample was diluted with propylene glycol to 10,000 ppm to prepare a stock solution. The stock solution was diluted 10 times with milliQ water to obtain sample concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, 10 ppm, 100 ppm, or 1,000 ppm, and 10 ml of the diluted solution was dispensed into a 50 ml brown sample bottle. The solution was left at room temperature for 5 hours, and the resulting solution was used to fully fill the sample bottle. The fragrance intensity was evaluated using the test sample. The sample concentration that was perceived to be the same as the fragrance intensity of the reference sample was determined. If the sample concentration was too high at 10 times the common ratio, it was further diluted to 3 times the common ratio, and the sample concentration that was perceived to be the same as the fragrance intensity of the reference sample was determined. The fragrance intensity evaluation was performed by three or four expert evaluators. The concentrations of each sample that gave the same fragrance intensity as 10 ppm Methional are shown in Table 10, and each sample was used for CATA evaluation at that concentration.

[0139]

[0140] <2> CATA evaluation For 10 types of samples, the evaluators were asked to check all of the nine descriptors that they felt best represented the characteristics of the sample. Seven evaluators evaluated each sample three times on different dates, obtaining a total of 21 pieces of data. The nine descriptors are as follows: Acidic, Bready, Cabbage, Creamy, Eggy, Lemon, Meaty, Pepper, and Tomato.

[0141] <Results> A Cochran's Q test was performed on the frequency at which each descriptor was selected, and the results showed that there was a significant difference in all samples at a significance level of α = 0.05. Multiple comparisons were performed using the McNemar test (FDR correction), and descriptors that showed differences between descriptors in each sample were analyzed.

[0142] The results are shown in Table 11. In the table, the letters (a, b, c) indicate that for each sample, the probability of selecting the descriptor marked with a different letter differs significantly (p<0.05). For Furfural and Propionaldehyde, Bready was significantly selected over all descriptors except Meaty and Cabbage. For delta-Decalactone, gamma-Nonalactone, delta-Nonanolactone, gamma-Undecalactone, and gamma-Octalactone, Creamy was significantly selected over all other descriptors. For 2-methyl-3-furyl propyl disulfide, Allyl mercaptan, 1-isothiocyanate-3-(methylthio)propane, and diallyl trisulfide, Meaty was significantly selected over some or all of the other descriptors.

[0143] Analysis of the correlation between olfactory receptor activity (measured in Example 1) and descriptors for each sample revealed that furfural and propionaldehyde, which were significantly selected for bready, commonly activated OR6B1. Furthermore, delta-decalactone, gamma-nonalactone, delta-nonanolactone, gamma-undecalactone, and gamma-octalactone, which were significantly selected for creamy, commonly activated OR10A3. Furthermore, 2-methyl-3-furyl propyl disulfide, allyl mercaptan, 1-isothiocyanate-3-(methylthio)propane, and diallyl trisulfide, which were significantly selected for meaty, commonly activated OR4S2. In particular, allyl mercaptan and diallyl trisulfide, which were significantly selected for meaty over all other descriptors, activated OR4S2 as well as OR2C1. These results demonstrate a correlation between olfactory receptor activity and descriptors.

[0144]

[0145] According to the present invention, substances having desired aroma characteristics can be efficiently screened.

[0146] <Explanation of the sequence listing> SEQ ID NO: 1: Nucleotide sequence of human OR1A1 gene 2: Nucleotide sequence of human OR1C1 gene 3: Nucleotide sequence of human OR1D2 gene 4: Nucleotide sequence of human OR1D5 gene 5: Nucleotide sequence of human OR1F1 gene 6: Nucleotide sequence of human OR1R1P gene 7: Nucleotide sequence of human OR2A4 gene 8: Nucleotide sequence of human OR2AG2 gene 9: Nucleotide sequence of human OR2B11 gene 10: Nucleotide sequence of human OR2C1 gene 11: Nucleotide sequence of human OR2J2 gene 12: Nucleotide sequence of human OR2K2 gene 13: Nucleotide sequence of human OR2L8 gene 14: Nucleotide sequence of human OR2T10 gene 15: Nucleotide sequence of human OR2W1 gene 16: Nucleotide sequence of human OR2W3 gene 17: Nucleotide sequence of human OR2Y1 gene 18: Nucleotide sequence of the human OR4C3 gene 19: Nucleotide sequence of the human OR4D1 gene 20: Nucleotide sequence of the human OR4H12P gene 21: Nucleotide sequence of the human OR4K5 gene 22: Nucleotide sequence of the human OR4P4 gene 23: Nucleotide sequence of the human OR4S2 gene 24: Nucleotide sequence of the human OR4X1 gene 25: Nucleotide sequence of the human OR5AN1 gene 26: Nucleotide sequence of the human OR5AU1 gene 27: Nucleotide sequence of the human OR5K1 gene 28: Nucleotide sequence of the human OR5L2 gene 29: Nucleotide sequence of the human OR5P3 gene 30: Nucleotide sequence of the human OR6B1 gene 31: Nucleotide sequence of the human OR6B2 gene 32: Nucleotide sequence of the human OR6C65 gene 33: Nucleotide sequence of the human OR6C75 gene 34: Nucleotide sequence of the human OR6J1 gene 35: Nucleotide sequence of human OR6P1 gene 36: Nucleotide sequence of human OR7A3P gene 37: Nucleotide sequence of human OR7A17 gene 38: Nucleotide sequence of human OR7D2 gene 39: Nucleotide sequence of human OR8B3 gene 40: Nucleotide sequence of human OR8B12 gene 41: Nucleotide sequence of human OR8D1 gene 42: Nucleotide sequence of human OR8D4 gene 43: Nucleotide sequence of human OR8K3 gene 44: Nucleotide sequence of human OR9A4 gene 45: Nucleotide sequence of human OR9Q2 gene 46: Nucleotide sequence of human OR10A3 gene 47: Nucleotide sequence of human OR10A6 gene 48: Nucleotide sequence of human OR10D3 gene49: Nucleotide sequence of human OR10G3 gene 50: Nucleotide sequence of human OR10G4 gene 51: Nucleotide sequence of human OR10G6 gene 52: Nucleotide sequence of human OR10G7 gene 53: Nucleotide sequence of human OR10H2 gene 54: Nucleotide sequence of human OR10T2 gene 55: Nucleotide sequence of human OR11H4 gene 56: Nucleotide sequence of human OR13F1 gene 57: Nucleotide sequence of human OR51E1 gene 58: Nucleotide sequence of human OR51E2 gene 59: Nucleotide sequence of human OR51I2 gene 60: Nucleotide sequence of human OR51L1 gene 61: Nucleotide sequence of human OR51V1 gene 62: Nucleotide sequence of human OR52E4 gene 63: Nucleotide sequence of human OR56A5 gene

Claims

1. A method for screening a substance having a target aroma characteristic, comprising: the following steps (A) to (C): (A) a step of bringing an olfactory receptor into contact with a test substance; (B) a step of measuring the response of the olfactory receptor to the test substance; and (C) a step of identifying the test substance as a substance having the target aroma characteristic based on the response wherein the aroma characteristic is a property of exhibiting a target aroma or a property of suppressing a target aroma, and the olfactory receptor is an olfactory receptor associated with the aroma characteristic.

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

3. The method according to claim 1, wherein the response is inactivation of the olfactory receptor, and step (A) is carried out in the presence of a substance that activates the olfactory receptor.

4. The method according to claim 1, wherein the combination of the aroma and the olfactory receptor is selected from the following combinations: combination of meaty and OR2C1; combination of meaty and OR4S2; combination of bready and OR6B1; combination of creamy and OR10A3; combination of guaiacol and OR10G4; combination of raw and OR5K1; combination of peanut roasted peanut and OR5K1; combination of hazelnut and OR5K1; combination of potato and OR5K1; combination of popcorn and OR5K1; combination of corn chip and OR5K1; combination of beany and OR5K1; combination of cocoa and OR5K1; combination of roasted and OR5K1; combination of coffee and OR5K1; combination of burnt and OR5K1; combination of musty and OR5K1; combination of horseradish and OR2C1; combination of rubbery and OR4S2; combination of maple and OR8D1; combination of lactonic and OR8D1; combination of sharp and OR51E1; combination of natural and OR1D5; combination of fruit tropical fruit and OR6C65; combination of solvent and OR4C3; combination of tomato and OR2K2; combination of eggy and OR1R1P; combination of magnolia and OR4K5; combination of cooling and OR8K3; The combination of lemon and OR2W3; The combination of mushroom and OR2W1; The combination of caraway and OR1A1; The combination of jasmine and OR1A1; The combination of floral and OR1A1; The combination of banana and OR2L8; The combination of warm and OR10G7; The combination of musk and OR5AN1; The combination of heliotrope and OR5AN1; The combination of pepper bell pepper and OR2AG2; The combination of bois de rose and OR1C1; The combination of apricot and OR1D2; The combination of cucumber and OR2J2; The combination of cinnamyl and OR2J2; The combination of melon and OR2J2; The combination of acacia and OR5P3; The combination of hawthorn and OR5P3; The combination of naphthyl and OR5P3; The combination of bacon and OR10G7; The combination of bacon and OR10G4; The combination of clove and OR10G7; The combination of clove and OR10G4; The combination of clove and OR10D3; The combination of clove and OR2J2; The combination of carnation and OR10G7; The combination of carnation and OR10G4; The combination of carnation and OR10D3; The combination of carnation and OR2J2; The combination of vanilla and OR10G4; The combination of vanilla and OR10G7; The combination of vanilla and OR2J2; The combination of vanilla and OR10D3; The combination of vanilla and OR10G3; The combination of smoky and OR10G4; The combination of smoky and OR10G7; The combination of phenolic and OR10G4; The combination of phenolic and OR9Q2; The combination of phenolic and OR10G7; The combination of medicinal and OR10G4; The combination of medicinal and OR9Q2; The combination of nutty and OR5K1; The combination of nutty and OR6B1; The combination of nutty and OR51E2; The combination of sulfurous and OR2C1; The combination of sulfurous and OR4S2; The combination of alliaceous and OR2C1; The combination of alliaceous and OR4S2; The combination of onion and OR4S2; Combination of onion and OR2C1; Combination of cabbage and OR4S2; Combination of cabbage and OR2C1; Combination of garlic and OR4S2; Combination of garlic and OR2C1; Combination of caramellic and OR8D1; Combination of caramellic and OR6B1; Combination of pungent and OR6B1; Combination of pungent and OR51E1; Combination of mustard and OR2A4; Combination of mustard and OR10H2; Combination of sweet and OR8B3; Combination of sweet and OR2J2; Combination of sweet and OR5P3; Combination of sweet and OR1D2; Combination of sweet and OR11H4; Combination of fresh and OR1D2; Combination of fresh and OR10A3; Combination of fresh and OR9A4; Combination of woody and OR7A17; Combination of woody and OR4X1; Combination of minty and OR2B11; Combination of minty and OR10T2; Combination of honey and OR51L1; Combination of honey and OR1A1; Combination of fruity and OR2L8; Combination of fruity and OR1D2; Combination of pineapple and OR2L8; Combination of pineapple and OR1D2; Combination of spicy and OR2J2; Combination of spicy and OR10G7; Combination of spicy and OR10G4; Combination of spicy and OR8B3; Combination of spicy and OR11H4; Combination of apple skin and OR1F1; Combination of apple skin and OR4D1; Combination of apple skin and OR7D2; Combination of vegetable and OR5K1; Combination of vegetable and OR4S2; Combination of peach and OR1D2; Combination of peach and OR10A3; Combination of peach and OR10A6; Combination of coconut and OR8B3; Combination of coconut and OR10A3; Combination of coconut and OR10A6; Combination of coconut and OR1A1; Combination of sour and OR51E1; Combination of sour and OR51V1; Combination of sour and OR51L1; Combination of sour and OR51I2; Combination of acidic and OR51E1; The combination of acidic and OR51I2; The combination of acidic and OR51V1; The combination of cheesy and OR51E1; The combination of cheesy and OR51I2; The combination of cheesy and OR51L1; The combination of cheesy and OR51V1; The combination of sweaty and OR51E1; The combination of sweaty and OR51I2; The combination of sweaty and OR51L1; The combination of cinnamon and OR2J2; The combination of cinnamon and OR8B3; The combination of cinnamon and OR2B11; The combination of cinnamon and OR2Y1; The combination of cinnamon and OR11H4; The combination of cherry and OR5P3; The combination of cherry and OR2J2; The combination of cherry and OR8B3; The combination of cherry and OR2B11; The combination of almond bitter almond and OR5P3; The combination of almond bitter almond and OR2B11; The combination of almond bitter almond and OR8B3; The combination of almond bitter almond and OR2J2; The combination of almond bitter almond and OR6P1; The combination of tonka and OR8B3; The combination of tonka and OR5P3; The combination of tonka and OR8D1; The combination of coumarinic and OR8B3; The combination of coumarinic and OR5P3; The combination of coumarinic and OR8D1; The combination of mimosa and OR5P3; The combination of mimosa and OR8B3; The combination of lilac and OR5P3; The combination of lilac and OR8D4; The combination of powdery and OR5P3; The combination of powdery and OR5AN1; The combination of orangeflower and OR5P3; The combination of orangeflower and OR1D2; The combination of orangeflower and OR8B12; The combination of orangeflower and OR4P4; The combination of orangeflower and OR7A3P; The combination of anisic and OR5P3; The combination of anisic and OR1A1; The combination of licorice and OR5P3; The combination of licorice and OR1A1; The combination of anise and OR5P3; a combination of anise and OR1A1; a combination of anise and OR8B3.

5. The method according to claim 4, wherein when the olfactory receptor is activated by the test substance, the test substance is identified as exhibiting the target aroma, or when the olfactory receptor is inactivated by the test substance, the test substance is identified as suppressing the target aroma.

6. The method according to claim 1, wherein the combination of the aroma and the olfactory receptor is selected from the following combinations: a combination of peanut roasted peanut and OR4H12P; a combination of meaty and OR2W1; a combination of sulfurous and OR1D2; a combination of sulfurous and OR13F1; a combination of sulfurous and OR52E4; a combination of sulfurous and OR10D3; a combination of sulfurous and OR6B2; a combination of sulfurous and OR5L2; a combination of sulfurous and OR2T10; a combination of sulfurous and OR5AU1; a combination of sulfurous and OR10G6; a combination of sulfurous and OR6C75; a combination of soapy and OR51E2; a combination of soapy and OR56A5; a combination of orchid and OR51E2; a combination of waxy and OR5K1; a combination of waxy and OR51E2; a combination of waxy and OR10G4; a combination of waxy and OR2B11; a combination of gardenia and OR6J1; a combination of fruity and OR5P3; a combination of fruity and OR5K1; a combination of fruity and OR10G4; a combination of fruity and OR4S2; a combination of cherry and OR6B1; a combination of floral and OR6B1.

7. The method according to claim 6, wherein when the olfactory receptor is activated by the test substance, the test substance is identified as suppressing the target aroma, or when the olfactory receptor is inactivated by the test substance, the test substance is identified as exhibiting the target aroma.

8. The method according to claim 1, wherein the steps (B) and (C) are each carried out by the following steps (B1) and (C1): The method according to claim 1, wherein the steps (B) and (C) are each carried out by the following steps (B1) and (C1): (B1) a step of measuring the degree of activation D1 of the olfactory receptor when the step (A) is carried out; Step (C1) of identifying the test substance as the target substance based on the degree of activation D1. **Claim 9** The method according to claim 8, wherein the step (C1) is carried out by the following step (C2): (C2) Step of identifying the test substance as the target substance based on the difference between the degree of activation D1 and the degree of activation D2 of the olfactory receptor under control conditions. **Claim 10** The method according to claim 9, wherein the response is the activation of the olfactory receptor, and the control condition is the following condition (C2-1) or (C2-2): (C2-1) Condition of not contacting the olfactory receptor with the test substance; (C2-2) Condition of contacting the olfactory receptor with the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in the step (A). **Claim 11** The method according to claim 9, wherein the response is the inactivation of the olfactory receptor, and the control condition is the following condition (C2-1) or (C2-2): (C2-1) Condition of not contacting the olfactory receptor with the test substance; (C2-2) Condition of contacting the olfactory receptor with the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in the step (A). **Claim 12** The method according to claim 9, further comprising the step of measuring the degree of activation D2. **Claim 13** The method according to claim 9, wherein when the degree of activation D1 is higher than the degree of activation D2, it is determined that the activation of the olfactory receptor by the test substance is recognized. **Claim 14** The method according to claim 9, wherein when the degree of activation D1 is lower than the degree of activation D2, it is determined that the inactivation of the olfactory receptor by the test substance is recognized. **Claim 15** The method according to claim 1, wherein the olfactory receptor is used in a form supported by cells, cell membranes, artificial lipid bilayer vesicles, or artificial lipid bilayers. **Claim 16** The method according to claim 15, wherein the olfactory receptor is used in a form supported by the cells. **Claim 17** The method according to claim 15, wherein the cells are animal cells. **Claim 18** The method according to claim 15, wherein the response is measured using the intracellular cAMP concentration or the intracellular calcium concentration as an index. **Claim 19** The method according to claim 18, wherein the intracellular cAMP concentration is measured by a reporter assay. **Claim 20** The method according to claim 1, further comprising a step of evaluating the aroma characteristics of the target substance identified in the step (C).

21. The method according to claim 20, wherein the evaluation is carried out by sensory evaluation.

22. The method according to claim 1, wherein the olfactory receptor is a human olfactory receptor.

23. The substance having the target aroma characteristics is a substance presenting an aroma of meaty, and the olfactory receptor is OR2C1 and / or OR4S2, the method according to any one of claims 1 to 5 and 8 to 22.

24. The method according to claim 23, wherein the substance presenting an aroma of meaty is a substance presenting a livestock meat flavor or a livestock meat aroma. 10]]The method according to claim 2 3.

25. The method according to claim 23, wherein the substance presenting an aroma of meaty is a substance that imparts a livestock meat flavor or a livestock meat aroma to the target object .

26. The method according to claim 25, wherein the target object is alternative meat or cultured meat.