Method for screening substance for masking off-taste / off-flavor in plant-derived-protein-containing food, and composition for masking off-taste / off-flavor

Abstract

The present invention addresses the problem of providing a technique for masking bitterness in a plant-derived-protein-containing food. The present invention provides, inter allia, a composition for masking bitterness in a plant-derived-protein-containing food, the composition containing component (a): at least one component selected from the group consisting of cysteine or salts thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or salts thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

Description

Method for screening substances that mask off-tastes and off-flavors in foods containing plant-derived proteins and compositions for masking off-tastes and off-flavors

[0001] The present invention relates to a technique for masking off-tastes and off-flavors (particularly bitterness). Specifically, the present invention relates to a method for screening substances that mask off-tastes and off-flavors in plant-derived protein-containing foods, and a composition for masking off-tastes and off-flavors that contains the substances.

[0002] Plant-based proteins are widely used as ingredients in foods such as health foods and nutritional supplements. However, increasing the plant-based protein content in foods can result in bitterness. Food ingredients that mask (or suppress) this bitterness have been developed, but their effectiveness is insufficient. Because plant-based proteins are useful as food ingredients, further development of technologies is needed to suppress their bitterness and enable daily and continuous intake.

[0003] In humans, bitter taste perception begins with binding to the bitter taste receptor, Taste type 2 receptor (TAS2R, T2R), which is expressed on taste cells in taste buds located on the soft palate and tongue. TAS2R is a type of G protein-coupled receptor (GPCR), and approximately 26 types of receptors are thought to function in humans. When bitter substances bind to TAS2R, they couple with gustducin, which is classified as Gαi, and transmit a signal by increasing intracellular calcium concentration.

[0004] Methods for screening for substances that suppress specific bitterness using bitter taste receptors and substances that suppress specific bitterness have been reported. For example, Patent Document 1 reports a method for screening for substances that suppress the bitterness of citrus fruit products using the bitter taste receptor TAS2R and bitter taste-suppressing compounds obtained by the method. Patent Document 2 reports a method for screening for substances that suppress the bitterness of acesulfame K using the bitter taste receptors TAS2R43 and TAS2R44 and bitter taste-suppressing compounds obtained by the method. Patent Document 3 reports a method for screening for substances that suppress the bitterness of catechins using TAS2R14 and TAS2R40. In addition, bitter taste receptors activated by soyasaponin I have been reported to be T2R1, T2R3, T2R5, T2R8, T2R13, T2R14, T2R16, T2R38, T2R41, T2R42, and T2R46 (Non-Patent Document 1).

[0005] Furthermore, as a method for suppressing a specific bitterness using a substance that suppresses a specific bitterness, for example, Patent Document 4 reports a method for reducing the bitterness of whey peptides using shiitake mushroom extract, sodium inosinate, and sodium guanylate. Patent Document 5 reports a method for reducing the bitterness of potassium chloride (potassium chloride), phosphates, monoglycerides, etc., using a yeast extract composition containing sodium 5'-inosinate and sodium 5'-guanylate. Patent Document 6 reports a method for masking the bitterness of polyphenols such as catechins, enzyme-treated isoquercitrin, bayberry extract, naringin, chlorogenic acid, tannins, soy isoflavones, or anthocyanins using advantame. Patent Document 7 reports a method for masking the bitterness of polyphenols such as catechins, enzyme-treated isoquercitrin, bayberry extract, naringin, or chlorogenic acid using advantame.

[0006] Patent application No. 2022-546204, Patent application No. 2017-165730, Patent application No. 2013-063049, Patent application No. 2009-261299, Patent application No. 62-289161, Patent application No. 2016-077292, Patent application No. 2015-023871

[0007] Food Chem. 2023 Nov 15:426:136548.

[0008] In view of the above, an object of the present invention is to provide a technique for masking the bitterness of plant-derived protein-containing foods. Specifically, an object of the present invention is to provide a method for screening substances that mask the bitterness of plant-derived protein-containing foods, a composition for masking the bitterness, and the like.

[0009] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that bitter taste receptors such as T2R43, T2R44, T2R49, and T2R50 respond to soyasaponin I contained in plant-derived protein-containing foods, and that various components screened using inhibition of the response of these bitter taste receptors as an indicator can mask the bitterness of plant-derived protein-containing foods. Based on these findings, the present invention was completed. Specifically, the gist of the present invention relates to the following.

[0010] [1] A composition for masking off-tastes and off-flavors in plant-derived protein-containing foods, comprising an antagonist of at least one bitter taste receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50.

[0011] "Off-flavor" may mean an off-taste and / or an off-odor. That is, the use of the composition may mask an off-taste and / or an off-odor. The use of the composition may particularly mask both an off-taste and an off-odor. "Off-taste" may mean an unusual or undesirable taste. Off-tastes include unpleasant tastes. "Off-taste" may mean a taste that may be perceived as unpleasant by the eater. Off-tastes (e.g., unpleasant tastes) include bitterness, astringency, sourness, and acridness. Off-tastes (e.g., unpleasant tastes) particularly include bitterness.

[0012] [1'] A composition for masking bitterness in plant-derived protein-containing foods, comprising an antagonist of at least one bitter taste receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50. [2] A composition for masking off-tastes and off-flavors in plant-derived protein-containing foods, comprising the following component (a): (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate. [2'] A composition for masking bitterness in plant-derived protein-containing foods, comprising the following component (a): (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate. [3] The composition, wherein the component (a) is at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, and guanylic acid or a salt thereof. [4] The composition, wherein the component (a) is selected from the group consisting of: a combination of advantame and cysteine ​​or a salt thereof; a combination of advantame and nootkatone; a combination of cysteine ​​or a salt thereof and guanylic acid or a salt thereof; a combination of advantame and acesulfame potassium; a combination of advantame and 3-(methylthio)propyl isothiocyanate; a combination of 3-(methylthio)propyl isothiocyanate and guanylic acid or a salt thereof; a combination of vitamin B6 and 3-(methylthio)propyl isothiocyanate; and a combination of vitamin B6 and guanylic acid or a salt thereof.[5] The composition wherein the component (a) is selected from the group consisting of: a combination of advantame, 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; a combination of 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; a combination of acesulfame potassium, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; a combination of acesulfame potassium, 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; a combination of vitamin B6, advantame, acesulfame potassium, 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; and a combination of vitamin B6, acesulfame potassium, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof. [6] The composition wherein the cysteine ​​or a salt thereof is cysteine ​​hydrochloride. [7] The composition wherein the composition is for use in the production of a food product. [8] The composition, wherein the food is a food in which a bitter taste has been masked. [9] The composition, wherein the food contains a bitter component.

[10] The composition, wherein the bitter component is saponin.

[11] The composition, wherein the bitter component is soyasaponin.

[12] The composition, wherein the food is selected from veggie meat, veggie milk, plant-based seafood, and plant-based eggs.

[13] A plant-derived protein-containing food, wherein the following component (a) has been added to the food ingredients to mask an off-taste or off-flavor: (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.[13'] A plant-derived protein-containing food, wherein the following component (a) is added to the raw materials of the food to mask its bitterness: (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

[14] A method for screening for a substance that masks off-tastes and off-flavors in plant-derived protein-containing foods, comprising the following steps (A) to (C): (A) contacting a bitter receptor with a bitter receptor activator in the presence of a test substance; (B) measuring the response of the bitter receptor to the bitter receptor activator; and (C) identifying the test substance as a substance that masks the bitter taste in plant-derived protein-containing foods based on the response; wherein, when the response is inhibited by the test substance, the test substance is identified as a substance that masks the bitter taste in plant-derived protein-containing foods, and the bitter receptor comprises at least one bitter receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50. [14'] A method for screening for a substance that masks the bitter taste in a plant-derived protein-containing food, comprising the following steps (A) to (C): (A) contacting a bitter receptor with a bitter receptor activator in the presence of a test substance; (B) measuring the response of the bitter receptor to the bitter receptor activator; and (C) identifying the test substance as a substance that masks the bitter taste in a plant-derived protein-containing food based on the response; wherein the test substance is identified as a substance that masks the bitter taste in a plant-derived protein-containing food when the response is inhibited by the test substance, and the bitter receptor comprises at least one bitter receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50.

[15] The method above, wherein the bitter receptor further comprises at least one bitter receptor selected from the group consisting of T2R1, T2R5, T2R14, and T2R46.

[16] The method as described above, wherein the response is activation of the bitter taste receptor.

[17] The method as described above, wherein the bitter taste receptor is used in a form supported on a cell, a cell membrane, an artificial lipid bilayer vesicle, or an artificial lipid bilayer membrane.

[18] The method as described above, wherein the bitter taste receptor is used in a form supported on a cell.

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

[20] The method as described above, wherein steps (B) and (C) are carried out by the following steps (B1) and (C1), respectively: (B1) a step of measuring the degree of activation D1 of the bitter taste receptor when step (A) is carried out; (C1) a step of identifying the test substance as a substance that masks the bitter taste in a plant-derived protein-containing food based on the degree of activation D1.

[21] The method as described above, wherein step (C1) is carried out by the following step (C2): (C2) a step of identifying the test substance as a substance that masks the bitter taste in a plant-derived protein-containing food based on the difference between the degree of activation D1 and the degree of activation D2 of the bitter taste receptor under control conditions.

[22] The method as described above, wherein the control conditions are the following conditions (C2-1) or (C2-2): (C2-1) a condition in which the bitter taste receptor is contacted with the bitter taste receptor activator in the absence of the test substance; or (C2-2) a condition in which the bitter taste receptor is contacted with the bitter taste receptor activator in the presence of the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in step (A).

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

[24] The method as described above, wherein the test substance is identified as a substance that masks bitterness in a plant-derived protein-containing food when the degree of activation D1 is lower than the degree of activation D2.

[25] The method as described above, wherein the test substance is identified as a substance that masks bitterness in a plant-derived protein-containing food when the ratio of the degree of activation D1 to the degree of activation D2 is less than 70%.

[26] The method as described above, wherein the response is measured using intracellular calcium concentration as an index.

[27] The method as described above, wherein the intracellular calcium concentration is measured by a luminescence assay.

[28] The method as described above, wherein the bitter receptor is a human bitter receptor.

[29] The method as described above, further comprising a step of evaluating whether the identified substance that masks the bitterness in a plant-derived protein-containing food has the function of masking the bitterness in the plant-derived protein-containing food.

[30] The method as described above, wherein the evaluation is carried out by sensory evaluation.

[31] A method for masking off-tastes / off-flavors in a plant-derived protein-containing food, comprising a step of adding the following component (a) to a food ingredient: (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate. [31'] A method for masking bitterness in a plant-derived protein-containing food, comprising the step of adding the following component (a) to a food ingredient: (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

[32] A method for producing a plant-derived protein-containing food, comprising the step of adding the following component (a) to a food ingredient: (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

[33] The method as described above, wherein the food is a food in which an off-taste or an unusual flavor has been masked. [33'] The method as described above, wherein the food is a food whose bitterness has been masked.

[34] The method as described above, wherein the food contains a component that imparts a bitter taste.

[35] The method as described above, wherein the component that imparts a bitter taste is saponin.

[36] The method as described above, wherein the component that imparts a bitter taste is soyasaponin.

[37] The method as described above, wherein the food is selected from veggie meat, veggie milk, plant-based seafood, and plant-based eggs.

[38] The aforementioned method, wherein the component (a) is added to the plant-derived protein-containing food so that the concentration of the component (a) in the food is 0.0001 ppm (w / w) to 1000 ppm (w / w).

[0013] The present invention provides a technique for masking the bitterness of plant-derived protein-containing foods. Specifically, the present invention provides a method for screening for substances that mask the bitterness of plant-derived protein-containing foods, and a composition for masking the bitterness containing the substance.

[0014] The present invention will be described below.

[0015] <1> Screening Method for Substances that Mask Bitterness in Plant-Derived Protein-Containing Foods One aspect of the present invention is a method for screening for substances that mask bitterness in plant-derived protein-containing foods. This aspect is also referred to as the "screening method of the present invention." In the screening method of the present invention, a bitter receptor can be used to identify substances that mask bitterness in plant-derived protein-containing foods (i.e., to identify whether a test substance masks bitterness in plant-derived protein-containing foods). Specifically, in the screening method of the present invention, a substance that masks bitterness in plant-derived protein-containing foods can be identified based on the response of the bitter receptor to a bitter receptor activator in the presence of the test substance (i.e., to identify whether the test substance masks bitterness in plant-derived protein-containing foods). More specifically, in the screening method of the present invention, a substance that masks bitterness in plant-derived protein-containing foods can be identified based on the inhibition of the response of the bitter receptor to a bitter receptor activator by the test substance (i.e., to identify whether the test substance masks bitterness in plant-derived protein-containing foods).

[0016] That is, the screening method of the present invention specifically comprises the steps of: (A) contacting a bitter receptor with a bitter receptor activator in the presence of a test substance; (B) measuring the response of the bitter receptor to the bitter receptor activator; and (C) identifying the test substance as a substance that masks the bitter taste in plant-derived protein-containing foods based on the response, and may be a screening method for substances that mask the bitter taste in plant-derived protein-containing foods, in which, if the response is inhibited by the test substance, the test substance is identified as a substance that masks the bitter taste in plant-derived protein-containing foods.

[0017] <1-1> Substances that Mask Bitterness in Plant-Derived Protein-Containing Foods In the present invention, "plant protein-containing food" refers to a food containing plant protein. The plant protein-containing food may contain one type of plant protein, or two or more types of plant proteins. The type of plant protein-containing food is not particularly limited as long as it is desired to suppress bitterness. That is, examples of plant protein-containing foods include plant protein-containing foods that exhibit bitterness. Plant protein-containing foods also include beverages. Examples of plant proteins include legume proteins, cereal proteins, and seed proteins. Examples of legumes include soybeans, peas, broad beans, mung beans, chickpeas, lupin beans, and peanuts. Examples of cereals 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. Examples of plant proteins particularly include legume proteins. More particularly, soy protein is an example of a plant protein. Plant protein-containing foods can be produced using raw materials containing plant protein. Examples of plant protein-containing raw materials include plants from which plant proteins are derived (e.g., beans, grains, seeds) and plant proteins isolated therefrom. Plant protein-containing raw materials may be used, for example, as is or after appropriate processing, to produce plant protein-containing foods. Processing methods include heating, concentration, drying, fractionation, shaping, and seasoning. Specific examples of plant protein-containing raw materials include veggie meat (also known as plant-based meat), veggie milk (also known as analog milk or imitation milk), plant-based seafood, and plant-based eggs. "Vegetarian meat" may refer to food ingredients made from plant protein-containing raw materials processed to resemble meat. Examples of veggie meat include those produced using raw materials containing soy protein (also known as "soy meat"). Examples of soy meat include granular soy protein (i.e., minced soy meat). Similarly, "veggie milk" may refer to a food ingredient made by processing raw materials containing plant protein into a milk-like substance."Plant-based seafood" may refer to a food ingredient in which a raw material containing plant protein is processed to resemble seafood. "Plant-based egg" may refer to a food ingredient in which a raw material containing plant protein is processed to resemble an egg. The plant protein content of the plant protein-containing raw material may be, for example, 40% (w / w) or more, 45% (w / w) or more, or 50% (w / w) or more, based on the dry weight of the plant protein-containing raw material. Specific examples of plant protein-containing foods include processed plant products such as veggie meat, veggie milk, plant-based seafood, and plant-based eggs; processed legume products such as tofu, yuba (bean curd skin), fried tofu, and soy milk; processed grain products such as noodles and bread; and foods produced using these as ingredients. That is, for example, veggie meat may be a raw material containing plant protein, but it may also be a plant protein-containing food. Foods made using veggie meat include hamburger steaks (patties), sausages, bacon, nuggets, minced meat cutlets, meatballs, fried chicken, gyoza dumplings, shumai, and meat sauce. Similarly, foods made using veggie milk include dairy products (plant-based dairy products (also called plant-based dairy)) made using veggie milk, such as yogurt, cheese, butter, cream, and ice cream.

[0018] The form in which the plant protein-containing food is provided is not particularly limited. For example, the plant protein-containing food may be provided in a form that can be consumed as is, or in a form that requires preparation before or at the time of consumption, such as a concentrated product or a dried product. The plant protein-containing food may also be provided in any container, such as a retort pouch, a paper pack, a plastic bottle such as a PET bottle, a metal can such as a steel can or an aluminum can, or a glass bottle. Plant protein-containing foods are not limited to general foods, but also include so-called health foods and medical foods such as nutritional supplements, nutritional functional foods, and foods for specified health uses. For example, the plant protein-containing foods exemplified above may be provided as general foods, or as health foods or medical foods.

[0019] "Ingredients for plant protein-containing foods" refers to food materials used to produce plant protein-containing foods. The ingredients for plant protein-containing foods are not particularly limited as long as they can produce plant protein-containing foods. The ingredients for plant protein-containing foods can be selected appropriately depending on various conditions, such as the type of plant protein-containing food. Examples of ingredients for plant protein-containing foods include ingredients that are commonly used in the production of plant protein-containing foods, such as those exemplified above. As ingredients for plant protein-containing foods, ingredients containing at least plant protein are used. The plant protein-containing ingredients are as described above. In addition to the plant protein-containing ingredients, other ingredients (hereinafter also referred to as "additional ingredients") may also be used as ingredients for plant protein-containing foods. Specific examples of additional ingredients include ingredients such as vegetables, meat, seafood, and eggs; milk such as milk; seasoning ingredients such as sugars, inorganic salts, organic acids, nucleic acids, amino acids, and protein hydrolysates; spices; flavorings; oils and fats; and alcohol. For example, a combination of veggie meat and meat may be used to produce a plant protein-containing food (e.g., a food produced using the veggie meat ingredients exemplified above). In this case, the ratio of the amount of veggie meat to the total amount of veggie meat and meat may be, for example, 20% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, or 90% (w / w) or more. Note that the "amount of veggie meat" referred to here may refer to the amount of veggie meat adjusted to a normal moisture content (e.g., in the case of dried meat, rehydrated in water beforehand). Examples of meat include animal and poultry meat. Examples of animals include livestock such as cows, pigs, horses, sheep, goats, and rabbits; wild animals such as boars, deer, and bears; and marine mammals such as whales, dolphins, and sea lions. Examples of birds include chickens, turkeys, ducks, geese, guinea fowl, quails, and ostriches. Examples of amino acids include aspartic acid. Unless otherwise specified, amino acids may be D-, L-, or a combination thereof. The amino acids may particularly be L-. When the raw material of the plant protein-containing food is capable of forming a salt, the raw material may be used in its free form, as a salt, or as a combination thereof.Furthermore, when the raw materials for the plant protein-containing food can form hydrates, the raw materials may be used as non-hydrates, hydrates, or a combination thereof. The descriptions of the salts and hydrates of the raw materials for the plant protein-containing food can be applied mutatis mutandis to the salts and hydrates of the active ingredients. For example, a specific example of a salt of aspartic acid is sodium aspartic acid. Examples of sodium salts of aspartic acid include monosodium aspartate and disodium aspartate. Examples of sodium salts of aspartic acid include monosodium aspartate. The raw materials for the plant protein-containing food may be appropriately processed in advance.

[0020] Examples of "bitterness" in plant protein-containing foods include bitterness derived from raw materials containing plant protein (e.g., plants from which the plant protein is derived or plant proteins isolated therefrom), and bitterness derived from components contained in or derived from raw materials containing plant protein. Components contained in or derived from raw materials containing plant protein that impart bitterness to plant protein-containing foods include plant proteins, peptides, amino acids, aldehydes, ketones, flavonoids, saponins, lecithin, alkaloids, amygdalin, etc. The use of an active ingredient may suppress one type of bitterness, or two or more types of bitterness.

[0021] Saponins are particularly cited as components that impart bitterness to plant protein-containing foods. Saponins are glycosides with aglycones such as triterpenes and steroids. Examples of saponin compounds in the present invention include soyasaponins. Soyasaponins are compounds contained in legumes such as soybean (scientific name: Glycine max). Soyasaponins are glycosides with soyasapogenols such as soyasapogenol A and soyasapogenol B as the aglycones. Examples of soyasaponin compounds include group A soyasaponins and group B soyasaponins.

[0022] Examples of Group A soyasaponins include soyasaponin Aa (soyasaponin A4), soyasaponin Ab (soyasaponin A1), soyasaponin Ac, soyasaponin Ad, soyasaponin Ae (soyasaponin A5), soyasaponin Af (soyasaponin A2), soyasaponin Ag (soyasaponin A6), and soyasaponin Ah (soyasaponin A3).

[0023] Examples of Group B soyasaponins include soyasaponin Ba (soyasaponin V), soyasaponin Bb (soyasaponin I), soyasaponin Bc (soyasaponin II), soyasaponin Bb' (soyasaponin III), and soyasaponin Bc' (soyasaponin IV).

[0024] The component that imparts bitterness to the plant-derived protein-containing food of the present invention may be a group B soyasaponin, or may be soyasaponin Bb (soyasaponin I).

[0025] The substance that masks the bitterness in a plant-derived protein-containing food is not particularly limited as long as it can mask the bitterness in the plant-derived protein-containing food (i.e., has the function of masking the bitterness in the plant-derived protein-containing food). Masking here means that the bitterness in the plant-derived protein-containing food can be suppressed (or reduced) when evaluated by a human sensory test. The substance that masks the bitterness in a plant-derived protein-containing food 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 a substance that masks the bitterness in a plant-derived protein-containing food is a mixture, the number of components constituting the mixture and their composition ratios are not particularly limited. When a substance that masks the bitterness in a plant-derived protein-containing food is a mixture, each component constituting the mixture may or may not be able to mask the bitterness in the plant-derived protein-containing food by itself, as long as the mixture masks the bitterness in the plant-derived protein-containing food.

[0026] <1-2> Test Substance The term "test substance" refers to a substance used in the screening method of the present invention as a candidate substance for masking the bitterness of plant-derived protein-containing foods. The test substance is not particularly limited. The test substance may consist of a single component (i.e., a pure substance) or a combination of two or more components (i.e., a mixture). When the test substance is a mixture, the number and composition ratio of the components constituting the mixture are not particularly limited. The test substance may be a known substance or a novel substance. The test substance may be a natural product or an artificial product. The test substance may be, for example, a compound library created using combinatorial chemistry techniques. Examples of test substances include alcohols, ketones, aldehydes, ethers, esters, hydrocarbons, sugars, organic acids, nucleic acids, amino acids, peptides, lipids, and various other organic or inorganic components. Furthermore, test substances particularly include existing food-approved flavorings and food additives. The term "existing food-approved flavoring" refers to a substance already approved for use as a food flavoring. "Existing food additive" refers to a substance that has already been approved for use as a food additive. A single test substance may be used, or two or more test substances may be used in combination. Test substances may be selected to include, for example, existing food-approved flavorings and food additives, such as those exemplified above. That is, the test substance may be, for example, a substance selected from one existing food-approved flavoring and food additive, a combination of two or more substances selected from food-approved flavorings and food additives, or a combination of one or more substances selected from one or more food-approved flavorings and food additives with one or more other ingredients. By conducting the screening method of the present invention by contacting two or more ingredients together with a bitter taste receptor, it is possible to identify whether the combination of ingredients as a whole masks the bitter taste in plant-derived protein-containing foods.Examples of "contacting two or more components together with the bitter receptor" include contacting a mixture of test substances with the bitter receptor, and contacting two or more test substances together with the bitter receptor.

[0027] <1-3> Bitter Receptor Activators "Bitter receptor activators" refer to substances that activate bitter receptors. The bitter receptor activators are not particularly limited as long as they can activate bitter receptors. Bitter receptor activators may consist of a single component (i.e., a pure substance) or a combination of two or more components (i.e., a mixture). When a bitter receptor activator is a mixture, each component of the mixture may or may not activate the bitter receptor individually, as long as the mixture activates the bitter receptor. When a bitter receptor activator is a mixture, the number of components constituting the mixture and their composition ratios are not particularly limited. Bitter receptor activators may be known substances or novel substances. Bitter receptor activators may be natural products or artificial products. Bitter receptor activators may or may not be known to be able to activate bitter receptors. For example, a bitter receptor activator capable of activating bitter receptors may be selected from the test substances exemplified above and used. Examples of bitter taste receptor activators include the bitter components in the plant-derived protein-containing foods mentioned above, particularly saponins, particularly soyasaponins, and particularly soyasaponin I.

[0028] <1-4> Bitter taste receptor At least one bitter taste receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50 is used. That is, unless otherwise specified, "bitter taste receptor" means at least one bitter taste receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50. As the bitter taste receptor, one bitter taste receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50 may be used, or two or more bitter taste receptors may be used in combination, or three or more bitter taste receptors may be used in combination, or all four bitter taste receptors may be used in combination. Furthermore, at least one bitter receptor selected from the group consisting of T2R1, T2R3, T2R5, T2R8, T2R13, T2R14, T2R16, T2R38, T2R41, T2R42, and T2R46 (preferably at least one bitter receptor selected from the group consisting of T2R1, T2R5, T2R14, and T2R46) may be used in combination. By using multiple bitter receptors in combination, it is expected that substances that mask the bitterness of plant-derived protein-containing foods can be screened with high accuracy. When multiple bitter receptors are used in combination, the response of the bitter receptor to a test substance can be measured individually for each of the multiple bitter receptors. For example, the response of multiple bitter receptors to a test substance can be measured individually using cells that express each of the multiple bitter receptors. A gene encoding a bitter receptor is also referred to as a "receptor gene."

[0029] Genes encoding T2Rs are also referred to as "T2R genes." T2Rs are responsive to bitter taste receptor activators such as soyasaponin I. As the T2R, one type of T2R may be used, or two or more types of T2Rs may be used in combination.

[0030] "A bitter receptor is responsive to a bitter receptor activator" may mean that the bitter receptor is activated by a bitter receptor activator.

[0031] Bitter taste receptor genes and bitter taste receptors include bitter taste receptor genes and bitter taste receptors of various organisms. Examples of organisms include animals such as mammals. Specific examples of animals such as 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 and other animals particularly include humans. The nucleotide sequences of bitter taste receptor genes and amino acid sequences of bitter taste receptors of various organisms can be obtained from public databases such as NCBI and Ensembl.

[0032] That is, the bitter receptor gene may be, for example, a gene having the known or natural nucleotide sequence of the bitter receptor gene as described above (for example, the nucleotide sequence of the bitter receptor gene of the above-mentioned organism registered with NCBI). The bitter receptor may also be, for example, a protein having the known or natural amino acid sequence of the bitter receptor as described above (for example, the amino acid sequence of the bitter receptor of the above-mentioned organism registered with NCBI). The expression "a gene has a nucleotide sequence" means that the gene contains the nucleotide sequence, unless otherwise specified, and also encompasses cases where the gene consists of the nucleotide sequence. The expression "a protein has an amino acid sequence" means that the protein contains the amino acid sequence, unless otherwise specified, and also encompasses cases where the protein consists of the amino acid sequence.

[0033] The bitter receptor gene may be a variant of the bitter receptor gene exemplified above (e.g., a variant of a gene having the nucleotide sequence of the bitter receptor gene of the organism exemplified above or a chimeric sequence thereof), so long as the original function is maintained. Similarly, the bitter receptor may be a variant of the bitter receptor exemplified above (e.g., a variant of a protein having the amino acid sequence of the bitter receptor of the organism exemplified above or a chimeric sequence thereof), so long as the original function is maintained.

[0034] "Maintaining the original function" means that a gene or protein variant has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. "Maintaining the original function" with respect to a gene means that a gene variant encodes a protein that maintains the original function. In other words, "maintaining the original function" with respect to each bitter receptor gene may mean that a gene variant encodes a bitter receptor that is responsive to a bitter receptor activator such as soyasaponin I. Furthermore, "maintaining the original function" with respect to each bitter receptor may mean that a bitter receptor variant is responsive to a bitter receptor activator such as soyasaponin I.

[0035] The responsiveness of a bitter receptor to a bitter receptor activator can be confirmed, for example, by measuring the response (e.g., activation) of the bitter receptor when the bitter receptor is contacted with a bitter receptor activator.

[0036] The bitter receptor can be used in any form that can be used to screen for substances that mask the bitterness of plant-derived protein-containing foods. That is, specifically, the bitter receptor can be used in any form as long as the bitter receptor can be contacted with the test substance and is responsive to a bitter receptor activator. The form of use of the bitter receptor can be appropriately determined depending on various conditions, such as the embodiment of the screening method of the present invention.

[0037] The bitter receptor may be used in a form isolated to a desired extent, such as a purified or crude product, or in a form contained in a material. Specifically, the bitter receptor may be used 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 bitter receptor may be used in the form of a structure having (supporting) a bitter receptor, such as a cell having a bitter receptor, a cell membrane having a bitter receptor, an artificial lipid bilayer vesicle having a bitter receptor, or an artificial lipid bilayer membrane having a bitter receptor. These structures having a bitter receptor may also be used in a form isolated to a desired extent, or in a form contained in a material. The bitter receptor may also constitute a part of an appliance. That is, the bitter receptor may also be used in the form of an appliance equipped with a bitter receptor. Examples of devices equipped with bitter receptors include devices with immobilized bitter receptors and devices equipped with structures (such as lipid bilayer membranes) having bitter 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 bitter receptors are included in the scope of bitter receptors used in the screening method of the present invention.

[0038] Bitter receptors can be produced, for example, by expressing a bitter receptor gene. Expression of the bitter receptor gene may be carried out, for example, using cells or a cell-free protein synthesis system. For expression of the bitter receptor gene using cells, see the description of cells having bitter receptors below. The expressed bitter receptor can be obtained appropriately in the form described above and used in the screening method of the present invention.

[0039] Cells that have bitter taste receptors and can be used in the screening method of the present invention are also referred to as "cells used in the present invention." Bitter taste receptors can function by localizing, for example, in the cell membrane. Therefore, cells used in the present invention may have bitter taste receptors, for example, in the cell membrane. Cells used in the present invention can be produced based on conventional genetic engineering techniques.

[0040] Bitter receptors are expressed from bitter receptor genes. Therefore, the cells used in the present invention have a bitter receptor gene. Specifically, the cells used in the present invention have a bitter receptor gene in an expressible state. It is sufficient for the cells used in the present invention to have the bitter receptor gene until the bitter receptor is expressed. In other words, the cells used in the present invention may or may not have the bitter receptor gene after the bitter receptor is expressed. In other words, the cells used in the present invention are cells that have expressed a bitter receptor gene, and are also cells that have expressed a bitter receptor. It is to be noted that "expression of a bitter receptor gene" and "expression of a bitter receptor" can be used synonymously.

[0041] The cells used in the present invention may have one copy of the bitter taste receptor gene, or may have two or more copies of the bitter taste receptor gene.

[0042] The cells used in the present invention may be cells that inherently have a bitter taste receptor gene, or may be cells that have been modified to have a bitter taste receptor gene.

[0043] Examples of cells that inherently have a bitter taste receptor gene include cells of organisms from which the bitter taste receptor gene is derived, such as taste cells of mammals such as humans. Cells that inherently have a bitter taste receptor gene can be obtained, for example, from organisms or tissues that contain the cells.

[0044] Cells modified to have a bitter taste receptor gene include cells into which a bitter taste receptor gene has been introduced.

[0045] The cells used in the present invention and the cells used to obtain them (e.g., cells into which or into which a bitter taste receptor gene has been introduced) are collectively referred to as "host cells."

[0046] The host cell is not particularly limited as long as it can express a functional bitter taste receptor and can be used to screen for substances that mask the bitter taste in plant-derived protein-containing foods. Examples of host cells include bacterial cells, fungal cells, plant cells, insect cells, and animal cells. Preferred host cells include eukaryotic cells such as fungal cells, plant cells, insect cells, and animal cells. More preferred host cells include animal cells. Examples of animals include mammals, birds, and amphibians. Examples of mammals include rodents and primates. Examples of rodents include Chinese hamsters, hamsters, mice, rats, and guinea pigs. Examples of primates include humans, monkeys, and chimpanzees. Examples of birds include chickens. Examples of amphibians include Xenopus laevis. Furthermore, the tissues or cells from which the host cells are derived are not particularly limited. Examples of tissues or cells from which host cells are derived include ovaries, kidneys, adrenal glands, lingual epithelium, olfactory epithelium, pineal gland, thyroid gland, and melanocytes. Examples of Chinese hamster cells include Chinese hamster ovary-derived cell lines (CHO). Specific examples of CHO include CHO-DG44 and CHO-K1. Examples of human cells include PEAKrapid cells (ATCC CRL-2828). 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.

[0047] Bitter taste receptor genes can be obtained by cloning from organisms that have bitter taste receptor genes. For cloning, nucleic acids such as genomic DNA or cDNA containing the genes can be used.

[0048] The obtained bitter taste receptor gene can be used as is or after appropriate modification. That is, by modifying the bitter taste 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 substitution, deletion, insertion, and / or addition of amino acid residues at a specific site. Site-directed mutagenesis methods include PCR-based methods (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). Variants of bitter receptor genes may also be obtained directly by chemical synthesis.

[0049] The manner in which the bitter receptor gene is introduced into a host cell is not particularly limited. The bitter receptor gene may be retained in the host cell in an expressible manner. Specifically, for example, when the bitter receptor gene is introduced in a form requiring transcription of DNA or the like, the bitter receptor gene may be retained in the host cell in an expressible manner under the control of a promoter that functions in the host cell. In the host cell, the bitter receptor gene may be present extrachromosomally or may be introduced onto the chromosome. When two or more genes are introduced, each gene may be retained in the host cell in an expressible manner.

[0050] The promoter for expressing the bitter taste receptor gene is not particularly limited as long as it functions in host cells. A "promoter functional in host cells" refers to a promoter that has promoter activity in host cells. The promoter may be a promoter native to the host cell or a heterologous promoter. The promoter may be the bitter taste receptor gene's native promoter or a promoter of another gene. The promoter may be stronger than the bitter taste receptor gene's native promoter. 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)).

[0051] The bitter taste receptor gene can be introduced into a host cell using, for example, a vector containing the gene. A vector containing the bitter taste receptor gene is also referred to as a "bitter taste receptor gene expression vector." An expression vector for the bitter taste receptor gene can be constructed, for example, by ligating a DNA fragment containing the bitter taste receptor gene to a vector. The bitter taste receptor gene can be introduced into a host cell by introducing the expression vector 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 appropriately selected depending on various conditions, such as the type of host cell and the mode of introduction of the bitter taste receptor gene. For example, vectors that can be used for gene introduction 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.

[0052] Alternatively, the bitter taste 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 bitter taste receptor gene is also referred to as a "bitter taste receptor gene fragment." Such fragments include linear DNA and linear RNA. Examples of linear RNA include mRNA and cRNA.

[0053] 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).

[0054] Alternatively, cells inherently containing a bitter receptor gene may be modified to increase the expression of the bitter receptor gene. "Increased gene expression" means that the expression level of the gene per cell is increased compared to unmodified cells. "Unmodified cells" as used herein refer to control cells that have not been modified to increase the expression of a target gene. Examples of unmodified cells include wild-type cells and the original cells. Techniques for increasing the expression of a bitter receptor gene include increasing the copy number of the bitter receptor gene and improving the transcription efficiency or translation efficiency of the bitter receptor gene. The copy number of the bitter receptor gene can be increased by introducing the bitter receptor gene into a host cell. Introduction of the bitter receptor gene can be carried out as described above. The introduced bitter receptor gene may be derived from the host cell or from a heterologous source. The transcription efficiency or translation efficiency of the bitter receptor gene can be improved by modifying the gene's expression regulatory sequence, such as a promoter. For example, the transcription efficiency of the bitter receptor gene can be improved by replacing the bitter receptor gene promoter with a stronger promoter.

[0055] The cells used in the present invention may have any other properties as long as they can be used to screen for substances that mask the bitter taste in plant-derived protein-containing foods. Such properties include, for example, properties that are useful for measuring the response of bitter taste receptors to bitter taste receptor activators. The description of the properties of the cells used in the present invention can also be applied mutatis mutandis to the use of bitter taste receptors in other embodiments. Examples of the use of bitter taste receptors in other embodiments include the use of artificial lipid bilayer vesicles, cell membranes, or artificial lipid bilayer membranes containing bitter taste receptors.

[0056] The cells used in the present invention may or may not have bitter receptors other than the selected bitter receptor (also referred to as "other bitter receptors"). It may be preferable that the cells used in the present invention do not have other bitter receptors. Examples of cells that do not have other bitter receptors include cells that do not have genes encoding other bitter receptors, and cells that have genes encoding other bitter receptors but do not express the genes. The cells used in the present invention may, for example, not inherently have other bitter receptors, or may be modified so that they do not have other bitter receptors. Modifying cells so that they do not have other bitter receptors can be achieved, for example, by knocking out genes encoding other bitter receptors.

[0057] Furthermore, the cells used in the present invention may have, for example, a protein involved in signal transduction. In other words, the cells used in the present invention may have a gene encoding a protein involved in signal transduction. Examples of proteins involved in signal transduction include G proteins (such as Gαgust (gustducin)), G protein activators (such as Ric8B), phosphodiesterase, and calcium channels. Furthermore, the cells used in the present invention may have, for example, components corresponding to the parameter to be measured. Examples of such components include probes such as calcium indicators and reporter genes such as luciferase genes. When a probe such as a calcium indicator is expressed from a gene, the cells used in the present invention may have a gene encoding the probe.

[0058] Furthermore, the cells used in the present invention may contain, for example, a protein that promotes membrane expression of bitter taste receptors. In other words, the cells used in the present invention may contain a gene encoding such a protein. Examples of such proteins include RTP1s (Zhuang H and Matsunami H, J Biol Chem 282, 15284-15293 (2007)). Examples of RTP1s include animal RTP1s such as human RTP1s (GenBank accession no. AAT70680), mouse RTP1s (GenBank accession no. ABU23737), and bat RTP1s (the amino acid sequence from the methionine residue at position 37 to the C-terminus of GenBank accession no. XP_006765914). The amino acid sequence of mouse RTP1s shares 93.3% identity with the amino acid sequence of human RTP1s. The amino acid sequence of bat RTP1s (the partial sequence shown above) shares 90.7% identity with the amino acid sequence of human RTP1s.

[0059] The cells used in the present invention may inherently have the properties exemplified above, or may be modified to have the properties exemplified above. Regarding cell modification, the description of cell modification related to bitter taste receptor genes, such as the introduction of a bitter taste receptor gene, can be applied mutatis mutandis. The genes exemplified above may be genes derived from host cells or genes derived from heterologous species. Furthermore, the genes exemplified above may or may not be derived from the same source as the bitter taste 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 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.

[0060] Cells having a bitter receptor gene can be used as cells having a bitter receptor (cells used in the present invention) either as is or after appropriately expressing the bitter receptor gene. That is, if cells having a bitter receptor gene already express the bitter receptor gene, the cells may be used as cells having a bitter receptor (cells used in the present invention) as is. Alternatively, cells having a bitter receptor (cells used in the present invention) can be obtained by expressing the bitter receptor gene in cells having a bitter receptor gene. For example, cells having a bitter receptor gene can be expressed by culturing the cells having the bitter receptor gene, thereby obtaining cells having a bitter receptor (cells used in the present invention). Specifically, for example, after introducing (e.g., transfection) the bitter receptor gene, the culture of the host cells can be continued to express the bitter receptor gene. The medium composition and culture conditions are not particularly limited as long as the cells having the bitter receptor gene can be maintained (e.g., grown) and the bitter receptor gene is expressed. During culture, cells having a bitter receptor gene may or may not grow. 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 agent or an expression inducer can be used.

[0061] Expression of bitter receptors can be confirmed by measuring their response to bitter receptor activators (e.g., activation of bitter receptors by bitter receptor activators). Expression of bitter receptors can also be confirmed by measuring the amount of mRNA transcribed from the bitter receptor gene or by detecting the bitter receptor by Western blotting using an antibody.

[0062] The cells used in the present invention can be used in the screening method of the present invention, for example, as they are (as they are contained in the culture) or after being recovered from the medium. Furthermore, the culture or the cells recovered therefrom may be used in the screening method of the present invention after, for example, appropriate treatment such as washing, concentration, dilution, or fixation. Thus, the cells used in the present invention may be used, for example, in a form isolated to a desired extent, or in a form contained in a material such as a culture. The same applies to other structures having bitter receptors.

[0063] Cell membranes having bitter receptors can be prepared, for example, from the cells used in the present invention. Specifically, cell membranes having bitter receptors can be obtained, for example, as membrane fractions obtained by disrupting the cells used in the present invention. Cell membranes having bitter receptors can be used, for example, as they are or dispersed in an artificial lipid bilayer membrane. Cell membranes having bitter receptors can also be used in the form of vesicles (i.e., vesicles prepared from the cell membrane).

[0064] Furthermore, bitter receptors can be used to produce artificial lipid bilayer vesicles or artificial lipid bilayer membranes having bitter receptors. For example, artificial lipid bilayer vesicles or artificial lipid bilayer membranes having bitter receptors can be prepared by incorporating bitter receptors into pre-prepared artificial lipid bilayer vesicles or artificial lipid bilayer membranes. Furthermore, artificial lipid bilayer vesicles or artificial lipid bilayer membranes having bitter receptors can be prepared by using bitter receptors as a raw material to prepare bitter receptors in artificial lipid bilayer vesicles or artificial lipid bilayer membranes. To prepare artificial lipid bilayer vesicles or artificial lipid bilayer membranes having bitter receptors, bitter receptors in an appropriate form, such as a membrane fraction having bitter 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 bitter taste receptors, for example, in their membranes. Examples of lipid bilayer membrane vesicles include liposomes.

[0065] 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.

[0066] <1-5> Screening Method of the Present Invention The screening method of the present invention can be carried out in vitro.

[0067] Step (A) is a step of contacting a bitter receptor with a bitter receptor activator in the presence of a test substance. That is, first, the bitter receptor can be contacted with a bitter receptor activator in the presence of a test substance. In other words, the bitter receptor can be contacted with a test substance in the presence of a bitter receptor activator. In other words, the bitter receptor can be contacted with a bitter receptor activator and a test substance. That is, the expressions "contacting a bitter receptor with a bitter receptor activator in the presence of a test substance," "contacting a bitter receptor with a test substance in the presence of a bitter receptor activator," and "contacting a bitter receptor with a bitter receptor activator and a test substance" can be used interchangeably. Hereinafter, the bitter receptor activator and the test substance are collectively referred to as "both substances."

[0068] The system in which the bitter taste receptor and both substances come into contact is also called a "reaction system."

[0069] The bitter receptor and both substances can be contacted in a suitable liquid. The liquid in which the bitter receptor and both substances are contacted is also referred to as a "reaction liquid." In other words, a reaction liquid can be used as a reaction system. For example, the bitter receptor and both substances can be contacted by coexisting them in a suitable reaction liquid. Specifically, the bitter receptor and both substances can be contacted by dissolving, suspending, dispersing, or the like the bitter receptor (e.g., in the form exemplified above, such as cells having a bitter receptor) and both substances in a suitable liquid medium. Examples of the liquid medium include aqueous media such as water and aqueous buffer solutions. When two or more components, such as both substances, are contacted together with the bitter receptor, the contact between these components and the bitter receptor may or may not begin simultaneously. For example, after contact between a certain component and the bitter receptor has begun, another component may be added to the reaction system. Specifically, for example, the bitter receptor activator may be added to the reaction system after the contact between the test substance and the bitter receptor has begun, or the test substance may be added to the reaction system after the contact between the bitter receptor activator and the bitter receptor has begun. Typically, the test substance and the bitter receptor activator are premixed and then contacted with the bitter receptor. The reaction conditions (conditions for contacting the bitter receptor with both substances) are not particularly limited as long as they enable screening for substances that mask the bitterness of plant-derived protein-containing foods. The reaction conditions can be appropriately set depending on various factors, such as the use form of the bitter receptor, the type of test substance, and the method for measuring the bitter receptor response. Known reaction conditions for measuring interactions between substances, such as interactions between proteins and ligands, may be used as is, or modified as appropriate. The concentration of the test substance may be, for example, 0.01 nM to 500 mM, 10 nM to 100 mM, 1 μM to 10 mM, or 3 μM to 1 mM. The concentration of the bitter taste receptor activator may be, for example, 0.01 nM to 500 mM, 10 nM to 100 mM, 1 μM to 10 mM, or 3 μM to 1 mM. The concentration of the bitter taste receptor may be, for example, 1 pg / mL to 10 mg / mL.Furthermore, when cells having a bitter receptor are used, the concentration of the cells having the bitter receptor may be, for example, 10 cells / mL to 10,000,000 cells / mL. The contact between the bitter receptor and both substances may or may not be terminated at an appropriate time point. The contact between the bitter receptor and both substances may generally be continued until the response of the bitter receptor to the bitter receptor activator is measured. The duration of contact between the bitter receptor and both substances may be, for example, 0.1 seconds or more, 0.5 seconds or more, 1 second or more, 5 seconds or more, 10 seconds or more, 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, or 2 hours or more; or 24 hours or less, 12 hours or less, 6 hours or less, 2 hours or less, or 1 hour or less, or any combination thereof that is compatible. Specifically, the duration of contact between the bitter receptor and both substances may be, for example, 1 minute to 6 hours. The reaction system may contain other components in addition to the bitter receptor (e.g., cells having bitter receptors, such as those exemplified above) and both substances, as long as it is possible to screen for substances that mask the bitterness of plant-derived protein-containing foods. The other components can be appropriately selected depending on various conditions, such as the form of use of the bitter receptor, the type of test substance, and the method for measuring the bitter receptor response. Examples of other components include salts such as calcium salts, carbon sources such as glucose, other medium components, and pH buffers.

[0070] Step (B) is a step of measuring the response of the bitter receptor to the bitter receptor activator. That is, the response of the bitter receptor to the bitter receptor activator can then be measured. The response of the bitter receptor to the bitter receptor activator is also referred to as "the bitter receptor activator eliciting a response of the bitter receptor." The response of the bitter receptor to the bitter receptor activator serves as an index for evaluating response inhibition by a test substance, as described below. Therefore, "measuring the response of the bitter receptor to the bitter receptor activator" may specifically mean measuring response inhibition by the test substance. "Response inhibition by the test substance" means that the bitter receptor response to the bitter receptor activator is inhibited by the test substance.

[0071] The response of the bitter receptor to a bitter receptor activator includes activation of the bitter receptor by the bitter receptor activator.

[0072] The timing for measuring the bitter receptor response to a bitter receptor activator is not particularly limited, as long as the test substance is a substance that masks the bitterness of a plant-derived protein-containing food and the test substance inhibits the response to a measurable extent. The timing for measuring the bitter receptor response to a test substance can be appropriately set depending on various conditions, such as the form of bitter receptor use, the types of both substances, and the method for measuring the bitter receptor response. Specifically, the timing for measuring the bitter receptor response to a bitter receptor activator may be any appropriate time from the time when contact between the bitter receptor and both substances begins to the time when response inhibition by the test substance disappears. The timing for measuring the bitter receptor response to a bitter receptor activator may be, for example, the time when response inhibition by the test substance is maximized. Furthermore, the timing for measuring the response of the bitter receptor to a bitter receptor activator may be, for example, 0.1 seconds or later, 0.5 seconds or later, 1 second or later, 5 seconds or later, 10 seconds or later, 30 seconds or later, 1 minute or later, 5 minutes or later, 10 minutes or later, 30 minutes or later, 1 hour or later, or 2 hours or later after the start of contact between the bitter receptor and both substances, or up to 24 hours, 12 hours, 6 hours, 2 hours, or 1 hour, or any combination thereof that is not inconsistent. Specifically, the timing for measuring the response of the bitter receptor to a bitter receptor activator may be, for example, from 1 minute to 6 hours after the start of contact between the bitter receptor and both substances.

[0073] Step (C) is a step of identifying the test substance as a substance that masks the bitter taste in a plant-derived protein-containing food based on the response of the bitter taste receptor to the bitter taste receptor activator. That is, it is then possible to identify whether the test substance is a substance that masks the bitter taste in a plant-derived protein-containing food based on the response of the bitter taste receptor to the bitter taste receptor activator. That is, it is possible to identify the test substance as a substance that masks the bitter taste in a plant-derived protein-containing food based on the response of the bitter taste receptor to the bitter taste receptor activator.

[0074] Specifically, response inhibition by a test substance can be evaluated based on the response of the bitter receptor to a bitter receptor activator, and the test substance can be identified as a substance that masks the bitter taste in a plant-derived protein-containing food based on the response inhibition by the test substance. More specifically, if response inhibition by a test substance is observed, i.e., if the response of the bitter receptor to a bitter receptor activator is inhibited by the test substance, the test substance can be identified as a substance that masks the bitter taste in a plant-derived protein-containing food. That is, for example, if the activation of the bitter receptor by a bitter receptor activator is inhibited by the test substance, the test substance can be identified as a substance that masks the bitter taste in a plant-derived protein-containing food. Inhibition of bitter receptor activation by a bitter receptor activator by a test substance is also referred to as "inhibition of activation by the test substance" or "inactivation of the bitter receptor by the test substance." In addition, when at least one selected from the group consisting of T2R43, T2R44, T2R49, and T2R50 is used for screening, "response inhibition by the test substance is observed" means that response inhibition by the test substance is observed for at least one selected from the group consisting of T2R43, T2R44, T2R49, and T2R50.

[0075] The inactivation of bitter receptors by a test substance can be determined using the degree of bitter receptor activation (degree of activation D1) when step (A) is performed (i.e., under conditions in which the bitter receptor is contacted with a bitter receptor activator in the presence of the test substance) as an indicator. That is, step (B) may be, for example, (B1) a step of measuring the degree of activation D1. Furthermore, step (C) may be, for example, (C1) a step of identifying whether the test substance is a substance that masks the bitterness of a plant-derived protein-containing food based on the degree of activation D1.

[0076] Specifically, the inactivation of the bitter receptor by the test substance can be determined by comparing the degree of bitter receptor activation (degree of activation D1) when step (A) is performed (i.e., under conditions in which the bitter receptor is contacted with a bitter receptor activator in the presence of the test substance) with the degree of bitter receptor activation 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 that masks the bitterness in a plant-derived protein-containing food based on the difference between the degree of activation D1 and the degree of activation D2.

[0077] "Control conditions" means the following conditions (C2-1) or (C2-2): (C2-1) A condition in which a bitter receptor is contacted with a bitter receptor activator in the absence of a test substance; (C2-2) A condition in which a bitter receptor is contacted with a bitter receptor activator in the presence of a test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in step (A) above.

[0078] In other words, the inactivation of bitter receptors by a test substance can be determined, for example, using as an indicator the difference in the degree of activation of bitter receptors between the presence and absence or different concentrations of the test substance in the presence of a bitter receptor activator.

[0079] The above condition (C2-1) includes a condition before contacting the bitter receptor with a test substance, and a condition under which the bitter receptor is contacted with a bitter receptor activator. The above condition (C2-1) also includes a condition after contacting the bitter receptor with a bitter receptor activator and a test substance, and in which the test substance is substantially (e.g., completely) removed from the reaction system, and response inhibition by the test substance is substantially (e.g., completely) eliminated. The concentration of the test substance under the above condition (C2-2) is not particularly limited, as long as a measurable difference is observed between the activation level D1 and the activation level D2. The concentration of the test substance under the above condition (C2-2) may be, for example, 90% or less, 70% or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the concentration of the test substance in the above step (A). Other than the presence or absence or concentration of the test substance, the control condition is not particularly limited, as long as it allows evaluation of response inhibition by the test substance. The control conditions may be, for example, the same as the conditions in step (A) above, except for the presence or absence or concentration of the test substance.

[0080] The screening method of the present invention may include a step of measuring the degree of activation D2. The degree of activation D1 and the degree of activation D2 may be measured in a single reaction system with a time lag, or may be measured simultaneously or with a time lag in separate reaction systems. The degree of activation D2 may be measured before or after the degree of activation D1. For example, after measuring the degree of activation D2, a test substance may be added to the reaction system and the degree of activation D1 may be measured.

[0081] When the activation level D1 is low, it may be determined that the bitter receptor has been inactivated by the test substance. Specifically, when the activation level D1 is lower than the activation level D2, it may be determined that the bitter receptor has been inactivated by the test substance. For example, when the ratio of the activation level D1 to the activation level D2 is less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%, it may be determined that the bitter receptor has been inactivated by the test substance. This ratio is also referred to as the "residual activity rate." The value obtained by subtracting the residual activity rate from 100% is also referred to as the "inhibition rate of activity." Specific examples of the inhibition rate of activity include the inhibition rates described in the Examples.

[0082] The above description of determining the inactivation of bitter taste receptors by a test substance can also be applied mutatis mutandis to determining response inhibition by a test substance based on other indicators. In such cases, the "degree of activation" in the above description can be appropriately replaced with a term corresponding to the other indicator.

[0083] The method for measuring the response of a bitter receptor to a bitter receptor activator is not particularly limited. The method for measuring the response of a bitter receptor to a bitter receptor activator can be appropriately selected depending on various conditions, such as the form of use of the bitter receptor and the type of response to be measured. That is, the response of a bitter receptor to a bitter receptor activator can be measured, for example, by an appropriate method that can measure the activation of the bitter receptor by a bitter receptor activator.

[0084] The method for measuring bitter receptor activation by a bitter receptor activator is not particularly limited. Activation of a bitter receptor by a bitter receptor activator can be measured, for example, by known methods for measuring the activity of receptors such as bitter receptors. Examples of such methods include measuring intracellular calcium concentration and intracellular cAMP concentration. That is, activation of a bitter receptor by a bitter receptor activator can be measured, for example, using intracellular calcium concentration as an indicator. Specifically, activation of a bitter receptor by a bitter receptor activator can be measured, for example, using intracellular calcium concentration as an indicator, using cells containing bitter receptors. For example, it is known that when bitter receptors are activated by bitter components, they couple with the intracellular G protein gustducin to increase intracellular calcium concentration. Examples of methods for measuring intracellular calcium concentration include calcium luminescence assays. In calcium luminescence assays, intracellular calcium concentration can be measured using calcium indicators. Examples of calcium indicators include calcium-sensitive luminescent dyes, calcium-sensitive fluorescent dyes, and calcium-sensitive fluorescent proteins. Examples of calcium-sensitive luminescent dyes include coelenterazine. Examples of calcium-sensitive fluorescent dyes include Fura 2 and Fluo 4. 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.

[0085] The description of measuring bitter receptor activation using cells having bitter receptors can also be applied to other embodiments using bitter receptors. Other embodiments using bitter receptors include artificial lipid bilayer vesicles, cell membranes, or artificial lipid bilayer membranes having bitter receptors. Other embodiments using bitter receptors include, in particular, when the bitter receptor is used in a form having an internal space.

[0086] That is, for example, bitter receptor activation can be measured using artificial lipid bilayer vesicles having bitter receptors in the same manner as when cells having bitter receptors are used. In such cases, the term "cells" in the description of measuring bitter receptor activation using cells having bitter receptors can be read as "artificial lipid bilayer vesicles."

[0087] Furthermore, for example, bitter receptor activation can be measured using a membrane such as a cell membrane or an artificial lipid bilayer membrane having a bitter receptor, which creates a space separated by the membrane, in a manner similar to that used when cells having a bitter receptor are used. Specifically, for example, when such a membrane is used to separate two spaces, i.e., when such a membrane is used to provide a reaction system with two spaces separated from each other by the membrane, bitter receptor activation can be measured using a manner similar to that used when cells having a bitter receptor are used. In such cases, the space separated by the membrane can be considered the interior of the cell (also referred to as the "internal space"). Specifically, one of the two spaces can be considered the interior of the cell (also referred to as the "internal space"), and the other can be considered the exterior of the cell (also referred to as the "external space"). Of these spaces, the one containing both substances can be considered the external space. In such cases, the "intracellular calcium concentration" in the description of measuring bitter receptor activation using cells having a bitter receptor can be read as the "calcium concentration in the internal space."

[0088] In either case, the measurable parameters can be selected depending on the manner in which the bitter taste receptor is used.

[0089] Note that "measuring a certain parameter and using it as an index for measuring the response of a bitter taste receptor to a bitter taste receptor activator" means that as long as the response can be measured, specifically, as long as the inhibition of the response by a test substance can be evaluated based on the response (i.e., as long as it is possible to determine whether the inhibition of the response by the test substance is observed), it is sufficient to obtain and use data reflecting the parameter, and it is not necessary to obtain the value of the parameter itself. In other words, when data reflecting a certain parameter is obtained, it is not necessary to calculate the value of the parameter itself from the data. Specifically, for example, when measuring intracellular calcium concentration by a calcium luminescence assay and using the data as an index for measuring bitter taste receptor activation by a bitter taste receptor activator, it is sufficient to obtain and use data reflecting intracellular calcium concentration (e.g., luminescence intensity) as long as the activation can be measured, specifically, as long as it is possible to evaluate the inactivation of the bitter taste receptor by a test substance based on the activation (i.e., as long as it is possible to determine whether the inactivation of the bitter taste receptor by the test substance is observed), and it is not necessary to calculate the intracellular calcium concentration itself from the data.

[0090] Furthermore, "measuring the response of a bitter receptor to a bitter receptor activator" means obtaining data reflecting the response that can be used to evaluate response inhibition by a test substance. Similarly, the "response of a bitter receptor to a bitter receptor activator" used as an index for evaluating response inhibition by a test substance means data reflecting the response that can be used to evaluate response inhibition by a test substance. Such data can be, for example, data obtained by implementing a technique for measuring the response of a bitter receptor to a bitter receptor activator (e.g., parameters such as those exemplified above and data reflecting them), either as is or after appropriate processing.

[0091] In this way, a substance that masks the bitterness in a plant-derived protein-containing food can be identified. The screening method of the present invention may further include a step of evaluating the masking function of the identified substance that masks the bitterness in a plant-derived protein-containing food (i.e., evaluating whether the identified substance that masks the bitterness in a plant-derived protein-containing food has a masking function). In other words, by evaluating the masking function of the identified substance that masks the bitterness in a plant-derived protein-containing food, it can be confirmed whether the substance that masks the bitterness in the plant-derived protein-containing food actually masks the bitterness in the plant-derived protein-containing food. The method for evaluating the masking function of the identified substance that masks the bitterness in a plant-derived protein-containing food is not particularly limited. The masking function of the identified substance that masks the bitterness in a plant-derived protein-containing food can be evaluated, for example, by a known method for evaluating the bitterness of a substance. Such a method includes sensory evaluation (evaluation by sensory testing). Specifically, the masking function of an identified substance that masks the bitterness in plant-derived protein-containing foods can be evaluated, for example, by comparing the bitterness of a plant-derived protein-containing food in the presence of a substance that masks the bitterness in plant-derived protein-containing foods (e.g., a food containing a component that imparts bitterness in plant-derived protein-containing foods, such as soyasaponin I) with the bitterness of the plant-derived protein-containing food in the absence of a substance that masks the bitterness in plant-derived protein-containing foods.

[0092] In conventional screening methods, in order to screen for a masking agent for bitterness in plant-derived protein-containing foods, the masking function of a huge number of substances or combinations thereof must be confirmed one by one through sensory testing or the like to select a substance that masks the bitterness in plant-derived protein-containing foods, which requires a lot of time and cost to develop a substance that masks the bitterness in plant-derived protein-containing foods. However, the screening method of the present invention utilizes bitterness receptors to efficiently screen for substances that mask the bitterness in plant-derived protein-containing foods. Therefore, the screening method of the present invention can greatly improve the efficiency of developing a masking agent for bitterness in plant-derived protein-containing foods.

[0093] The use of the screened substance that masks the bitterness of plant-derived protein-containing foods is not particularly limited. The substance that masks the bitterness of plant-derived protein-containing foods can be used, for example, by blending it with an object for which masking of the bitterness of plant-derived protein-containing foods is desired. Such objects include those that already exhibit the bitterness of plant-derived protein-containing foods (e.g., foods containing components that exhibit the bitterness of plant-derived protein-containing foods, such as soyasaponin I) and those that may exhibit the bitterness of plant-derived protein-containing foods in the future. By blending a substance that masks the bitterness of plant-derived protein-containing foods, the bitterness of plant-derived protein-containing foods can be masked in the object. For masking the bitterness of plant-derived protein-containing foods using a substance that masks the bitterness of plant-derived protein-containing foods, the description regarding masking the bitterness of plant-derived protein-containing foods using an active ingredient in "<2> Masking the bitterness of plant-derived protein-containing foods" described below can be applied mutatis mutandis. Furthermore, substances that mask the bitterness of plant-derived protein-containing foods can also be used, for example, as raw materials for developing new substances that mask the bitterness of plant-derived protein-containing foods.

[0094] <2> Masking Bitterness in Plant-Derived Protein-Containing Foods <2-1> Active Ingredients In masking bitterness in plant-derived protein-containing foods, the following ingredient (a) is used as an active ingredient: (a) at least one ingredient selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone (CAS No: 54814-64-1) (Mebetaoia Lactone natural), guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

[0095] Component (a) is also referred to as an “active ingredient.” As component (a), one type of ingredient may be used, or two or more types of ingredients may be used in combination.

[0096] Any of the active ingredients may be a component that inactivates at least one selected from the group consisting of bitter taste receptors T2R43, T2R44, T2R49, and T2R50. That is, the active ingredient may specifically be the following ingredient (a): (a) A component that inactivates at least one selected from the group consisting of bitter taste receptors T2R43, T2R44, T2R49, and T2R50, which is at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

[0097] "A certain component inactivates at least one bitter receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50" means that the component inactivates at least one bitter receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50 under appropriate conditions. The at least one bitter receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50 and their inactivation are as described above in "<1> Method for screening for substances that mask the bitterness of plant-derived protein-containing foods." Suitable conditions include those described in the above-mentioned "<1> Method for screening for substances that mask the bitterness of plant-derived protein-containing foods." Specific examples of suitable conditions include those described in the Examples. In other words, "a certain component inactivates at least one selected from the group consisting of bitter receptors T2R43, T2R44, T2R49, and T2R50" may mean, for example, that the component inactivates at least one selected from the group consisting of bitter receptors T2R43, T2R44, T2R49, and T2R50 under at least the conditions described in the Examples.

[0098] By using an active ingredient, the bitterness in a plant-derived protein-containing food can be masked, i.e., the effect of masking the bitterness in a plant-derived protein-containing food can be achieved. This effect is also referred to as the "suppression effect." Masking the bitterness in a plant-derived protein-containing food is also simply referred to as "bitterness masking." "Masking the bitterness" is also referred to as "bitterness reduction" or "bitterness suppression." Note that "bitterness masking" encompasses both masking bitterness that may occur in the future and masking bitterness that has already occurred. "Bitterness masking" also encompasses the complete disappearance of bitterness. Bitterness is as described above in "<1> Method for screening substances that mask the bitterness in plant-derived protein-containing foods." Specifically, by using an active ingredient, the bitterness in a food can be masked compared to when the active ingredient is not used. Therefore, the masking effect can be determined by measuring and comparing the bitterness in a food when the active ingredient is used and when the active ingredient is not used. In other words, it can be determined that a masking effect has been achieved if the intensity of the bitterness in a food product when the active ingredient is used is lower than when the active ingredient is not used. Measurement and comparison of the bitterness can be carried out, for example, by sensory evaluation by a specialist panel.

[0099] The active ingredient may be a commercially available product or may be obtained by appropriate manufacturing. The method for manufacturing the active ingredient is not particularly limited. The active ingredient can be manufactured, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. The active ingredient may be purified to a desired degree or may not be purified. That is, the active ingredient may be a purified product, or a material containing the active ingredient. For example, the active ingredient may be a material containing the active ingredient at a content of 1% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.

[0100] In addition, when a material containing an active ingredient is used, the amount of the active ingredient (for example, the content (concentration) or amount used) is calculated based on the amount of the active ingredient itself in the material.

[0101] <2-2> Composition of the Present Invention The composition of the present invention is a composition containing an active ingredient.

[0102] The composition of the present invention contains an antagonist of at least one bitter receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50. The "bitter receptor antagonist" can be screened using the screening method of the present invention. Antagonists of bitter receptors other than T2R43, T2R44, T2R49, and T2R50 can also be used in combination.

[0103] Specifically, the composition of the present invention may be a composition containing the following component (a): (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate. More preferably, the composition may be a composition containing the following component (a'): (a') at least one component selected from the group consisting of cysteine ​​or a salt thereof, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate. More preferably, the composition may be a composition containing the following component (a''): (a'') at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, and guanylic acid or a salt thereof.

[0104] Specifically, the composition of the present invention may be a composition containing the following component (a): (a) a component that inactivates at least one component selected from the group consisting of bitter taste receptors T2R43, T2R44, T2R49, and T2R50, and is at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate. More preferably, the composition may contain the following component (a'): (a') a component that inactivates at least one selected from the group consisting of bitter taste receptors T2R43, T2R44, T2R49, and T2R50, the component being at least one component selected from the group consisting of cysteine ​​or a salt thereof, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate. Also, more preferably, the composition may contain the following component (a"): (a") a component that inactivates at least one selected from the group consisting of bitter taste receptors T2R43, T2R44, T2R49, and T2R50, the component being at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, and guanylic acid or a salt thereof.

[0105] The active ingredient of the present invention may be one component selected from component (a), component (a'), and component (a''), or may be a combination of two or more components. When the active ingredient is a mixture of two or more components, the number of types and composition ratio of the components constituting the mixture are not particularly limited.

[0106] The salt of the active ingredient is not particularly limited as long as it can be orally ingested.For example, the salt of an acidic group such as a carboxyl group specifically includes ammonium salt, salt with alkali metal such as sodium, potassium, salt with alkaline earth metal such as calcium, magnesium, aluminum salt, zinc salt, salt with organic amine such as triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine, dicyclohexylamine, and salt with basic amino acid such as arginine, lysine. Furthermore, examples of salts of basic groups such as amino groups include salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid; salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hybenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, malic acid, methylmalonic acid, and adipic acid; and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. One type of salt may be used, or two or more types of salts may be used in combination. A particular example of a salt of cysteine ​​is cysteine ​​hydrochloride (CysHCl). A particular example of a salt of guanylic acid is disodium guanylate (GMP). Unless otherwise specified, cysteine ​​may be in the D- or L-form, or a combination thereof. Cysteine ​​may in particular be in the L-configuration.

[0107] The combinations are not particularly limited, and may include, for example, combinations of vitamin B6 and 3-(methylthio)propyl isothiocyanate; vitamin B6 and guanylic acid or a salt thereof; advantame and acesulfame potassium; advantame and 3-(methylthio)propyl isothiocyanate; advantame and cysteine ​​or a salt thereof; advantame and nootkatone; cysteine ​​or a salt thereof and guanylic acid or a salt thereof; and 3-(methylthio)propyl isothiocyanate and guanylic acid or a salt thereof.

[0108] Also available are combinations of vitamin B6, advantame, acesulfame potassium, 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; acesulfame potassium, 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; advantame, 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; acesulfame potassium, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; vitamin B6, acesulfame potassium, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof. Examples of mixtures of active ingredients include, in particular, combinations containing cysteine ​​or a salt thereof and guanylic acid or a salt thereof, combinations containing advantame and cysteine ​​or a salt thereof, and combinations containing advantame and nootkatone.

[0109] By using the composition of the present invention, the bitterness in a plant-derived protein-containing food can be masked, i.e., a suppression effect can be obtained. Therefore, the composition of the present invention may be used to mask the bitterness in a plant-derived protein-containing food. That is, the composition of the present invention may be, for example, a composition for masking the bitterness in a plant-derived protein-containing food.

[0110] Furthermore, by utilizing the composition of the present invention, it is possible to produce a plant-derived protein-containing food in which the bitterness is masked. Thus, the composition of the present invention may be utilized in the production of a plant-derived protein-containing food (specifically, the production of a plant-derived protein-containing food in which the bitterness is masked). That is, the composition of the present invention may be, for example, a composition for use in the production of a plant-derived protein-containing food (specifically, the production of a plant-derived protein-containing food in which the bitterness is masked).

[0111] The composition of the present invention may be, for example, a seasoning. Specifically, the composition of the present invention may be, for example, a seasoning for masking the bitterness of a plant-derived protein-containing food, or a seasoning for producing a plant-derived protein-containing food (specifically, producing a plant-derived protein-containing food in which the bitterness is masked).

[0112] The composition of the present invention may be used to mask bitterness in a plant-derived protein-containing food or to produce a plant-derived protein-containing food in the embodiment described in the second embodiment of the method of the present invention below.

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

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

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

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

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

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

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

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

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

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

[0123] That is, a second aspect of the method of the present invention is a method comprising a step of utilizing the following component (a): (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

[0124] Specifically, the second aspect of the method of the present invention may be a method comprising a step of utilizing the following component (a): (a) a component that inactivates at least one component selected from the group consisting of bitter taste receptors T2R43, T2R44, T2R49, and T2R50, and the component is at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

[0125] According to the second aspect of the method of the present invention, specifically by utilizing an active ingredient, the bitterness in a plant-derived protein-containing food can be masked, i.e., a masking effect can be obtained. Thus, the second aspect of the method of the present invention may be carried out to mask the bitterness in a plant-derived protein-containing food. That is, the second aspect of the method of the present invention may be, for example, a method for masking the bitterness in a plant-derived protein-containing food. This method is also referred to as the "masking method of the present invention."

[0126] Furthermore, according to the second aspect of the method of the present invention, specifically by utilizing an active ingredient, a plant-derived protein-containing food product in which the bitterness is masked can be produced. Thus, the second aspect of the method of the present invention may be carried out for the production of a plant-derived protein-containing food product (specifically, the production of a plant-derived protein-containing food product in which the bitterness is masked). That is, the second aspect of the method of the present invention may be, for example, a method for producing a plant-derived protein-containing food product (specifically, the production of a plant-derived protein-containing food product in which the bitterness is masked). This method is also referred to as the "production method of the present invention."

[0127] The active ingredient can be added to the raw materials of a plant-derived protein-containing food during production thereof to mask bitterness or to produce the plant-derived protein-containing food. That is, an example of using the active ingredient is to add the active ingredient to the raw materials of the plant-derived protein-containing food. Specifically, the second aspect of the method of the present invention may be, for example, a method for masking the bitterness of a plant-derived protein-containing food, comprising adding the active ingredient to the raw materials of the plant-derived protein-containing food. Furthermore, the second aspect of the method of the present invention may be, for example, a method for producing a plant-derived protein-containing food (specifically, producing a plant-derived protein-containing food with a masked bitterness), comprising adding the active ingredient to the raw materials of the plant-derived protein-containing food. "Addition" can also be referred to as "blending."

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

[0129] The plant-derived protein-containing food obtained by the second embodiment of the method of the present invention is also referred to as the "food of the present invention." Specifically, the food of the present invention is a plant-derived protein-containing food in which the bitterness is masked. In other words, the food of the present invention is a food to which an active ingredient has been added.

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

[0131] The food is not particularly limited as long as it is a plant-derived protein-containing food for which masking of bitterness is desired. The food may already exhibit a bitter taste or may exhibit a bitter taste in the future. Examples of foods that already exhibit a bitter taste include foods containing bitter components. Examples of foods that may exhibit a bitter taste in the future include foods containing components that can produce bitter components. That is, the food may contain components that exhibit a bitter taste and / or components that can produce bitter components. Examples of components that exhibit a bitter taste include the bitter components in the plant-derived protein-containing food described above. Among these, saponins are particularly preferred, and soyasaponin is particularly preferred, and soyasaponin I is particularly preferred. Foods also include beverages. Foods also include seasonings. Foods may be, for example, liquid or solid. Specific examples of foods are as described above in "<1> Method for screening for substances that mask the bitter taste in plant-derived protein-containing foods." Examples of foods include, in particular, the foods exemplified above that contain components that impart a bitter taste and / or components that can produce a bitter taste.

[0132] The active ingredient may be added to food ingredients at any stage of the food manufacturing process as long as a masking effect is obtained. In other words, the "food ingredients" to which the active ingredient is added may be those at any stage of the food manufacturing process. For example, the "food ingredients" to which the active ingredient is added may include finished foods before the active ingredient is added. The active ingredient may be added to the food ingredients either as is or after being prepared into a desired form such as a solution. "Addition of an active ingredient" may refer collectively to the process of allowing the active ingredient to coexist with the food ingredients. Ingredients other than the active ingredient (e.g., ingredients that impart a bitter taste or ingredients that can produce a bitter taste) may also be added to the food ingredients as appropriate. The description of the addition of an active ingredient also applies mutatis mutandis to the addition of ingredients other than the active ingredient. Each ingredient (i.e., the active ingredient and optionally other ingredients) may be added to the food ingredients all at the same time, separately, or in any combination. The order in which each ingredient is added to the food ingredients is not particularly limited.

[0133] The amounts and ratios of the components (i.e., the active ingredient and optionally other ingredients) added in the second embodiment of the method of the present invention are not particularly limited as long as a masking effect is obtained. The amounts and ratios of the components added in the second embodiment of the method of the present invention can be appropriately set depending on various conditions such as the type of raw material of the food and the type of food.

[0134] The active ingredient may be added to the food ingredients, for example, so that the concentration in the plant-derived protein-containing food is within a desired range (for example, the concentration range of the active ingredient in the plant-derived protein-containing food described below).

[0135] The concentration of the active ingredient added to the plant-derived protein-containing food may be, for example, 1 ppt (w / w) or more, 10 ppt (w / w) or more, 100 ppt (w / w) or more, 1 ppb (w / w) or more, 10 ppb (w / w) or more, 100 ppb (w / w) or more, 1 ppm (w / w) or more, 10 ppm (w / w) or more, 100 ppm (w / w) or more, 1000 ppm (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, or 10% (w / w) or more. The concentration of the active ingredient in the plant-derived protein-containing food may be 100% (w / w) or less, less than 100% (w / w), 99.9% (w / w) or less, 90% (w / w) or less, 50% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 1000 ppm (w / w) or less, 100 ppm (w / w) or less, 10 ppm (w / w) or less, or 1 ppm (w / w) or less, or any combination thereof, or any combination thereof, as long as the active ingredient is present in the plant-derived protein-containing food. When the food product of the present invention contains two or more active ingredients (e.g., when two or more active ingredients are added), the "addition concentration of the active ingredients in the plant-derived protein-containing food" refers to the total concentration of those two or more active ingredients, unless otherwise specified. However, in one embodiment, when the food product of the present invention contains two or more active ingredients (e.g., when two or more active ingredients are added), the addition concentrations of those two or more active ingredients in the plant-derived protein-containing food may each independently be within the range of the addition concentrations of the active ingredients in the plant-derived protein-containing food exemplified above.

[0136] The concentration of vitamin B6 added to a plant-derived protein-containing food may be, for example, 0.01 ppm (w / w) to 500 ppm (w / w), 0.1 ppm (w / w) to 100 ppm (w / w), or 1 ppm (w / w) to 50 ppm (w / w).

[0137] The concentration of advantame added to a plant-derived protein-containing food may be, specifically, for example, 0.001 ppm (w / w) to 1000 ppm (w / w), 0.001 ppm (w / w) to 100 ppm (w / w), 0.01 ppm (w / w) to 10 ppm (w / w), or 0.1 ppm (w / w) to 1 ppm (w / w).

[0138] The concentration of acesulfame potassium added to a plant-derived protein-containing food may be, for example, 0.01 ppm (w / w) to 500 ppm (w / w), 0.1 ppm (w / w) to 100 ppm (w / w), or 1 ppm (w / w) to 50 ppm (w / w).

[0139] The concentration of cysteine ​​or a salt thereof added to a plant-derived protein-containing food may be, for example, 0.01 ppm (w / w) to 1000 ppm (w / w), 0.01 ppm (w / w) to 500 ppm (w / w), 0.1 ppm (w / w) to 200 ppm (w / w), or 1 ppm (w / w) to 100 ppm (w / w).

[0140] The concentration of lauric acid added to a plant-derived protein-containing food may be, for example, 0.001 ppm (w / w) to 100 ppm (w / w), 0.01 ppm (w / w) to 10 ppm (w / w), or 0.1 ppm (w / w) to 1 ppm (w / w).

[0141] The concentration of nootkatone added to a plant-derived protein-containing food may be, for example, 0.0001 ppm (w / w) to 10 ppm (w / w), 0.001 ppm (w / w) to 1 ppm (w / w), or 0.01 ppm (w / w) to 0.1 ppm (w / w).

[0142] The concentration of 3-(methylthio)propyl isothiocyanate added to a plant-derived protein-containing food may be, for example, 0.0001 ppm (w / w) to 1 ppm (w / w), 0.0005 ppm (w / w) to 0.1 ppm (w / w), or 0.001 ppm (w / w) to 0.01 ppm (w / w).

[0143] The concentration of 5-hydroxy-2-decenoic acid lactone added to a plant-derived protein-containing food may be, for example, 0.0001 ppm (w / w) to 10 ppm (w / w), 0.001 ppm (w / w) to 1 ppm (w / w), or 0.01 ppm (w / w) to 0.1 ppm (w / w).

[0144] The concentration of guanylic acid or a salt thereof added to a plant-derived protein-containing food may be, for example, 0.1 ppm (w / w) to 5000 ppm (w / w), 1 ppm (w / w) to 1000 ppm (w / w), or 10 ppm (w / w) to 500 ppm (w / w).

[0145] Furthermore, the concentration of the active ingredient added in the plant-derived protein-containing food may be, for example, less than the threshold concentration of the active ingredient. Specifically, the concentration of the active ingredient added in the plant-derived protein-containing food may be, for example, within the range of the concentrations of the active ingredient added in the plant-derived protein-containing food exemplified above, but less than the threshold concentration of the active ingredient. The "threshold concentration of the active ingredient" refers to the maximum concentration of the active ingredient at which an off-flavor of the active ingredient itself is not detected when consuming an aqueous solution containing the active ingredient alone. Examples of the threshold concentration of the active ingredient include the threshold concentrations described in the Examples.

[0146] The description of the addition of an active ingredient also applies mutatis mutandis to the addition of the composition of the present invention. For example, the composition of the present invention can be added so as to obtain the amount of the active ingredient exemplified above.

[0147] The food of the present invention may contain a bitter component (e.g., a bitter component in the plant-derived protein-containing food described above, particularly saponin, particularly soyasaponin, particularly soyasaponin I). That is, the food of the present invention may be produced to contain a bitter component. The food of the present invention may contain one bitter component, or two or more bitter components. A food containing a bitter component can be produced, for example, by adding a bitter component. That is, the second aspect of the method of the present invention may further include adding a bitter component to the raw materials of the food. A food containing a bitter component may be produced, for example, by adding the bitter component itself, or by adding a material containing the bitter component, such as a seasoning containing the bitter component. The bitter component may be commercially available or may be obtained by appropriate production. The method of producing the bitter component is not particularly limited. The bitter component can be produced by, for example, chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. The bitter component can be added in the same manner as the addition of an active ingredient. The bitter component may be added to a food ingredient, for example, so that the content of the bitter component in the food of the present invention falls within a desired range (for example, the content range described below). Furthermore, a food containing a bitter component can be produced, for example, using a food ingredient containing a bitter component. That is, the food ingredient may contain a bitter component. Furthermore, the bitter component may be generated, for example, during the production process of the food of the present invention.

[0148] When the food of the present invention contains a bitter component (e.g., soyasaponin I), the content of the bitter component in the food of the present invention may be, for example, as an added concentration in a plant-derived protein-containing food, 1 ppb (w / w) or more, 2 ppb (w / w) or more, 5 ppb (w / w) or more, 10 ppb (w / w) or more, 20 ppb (w / w) or more, 50 ppb (w / w) or more, 100 ppb (w / w) or more, 200 ppb (w / w) or more, or 500 ppb (w / w) or more, and may be 5000 ppm (w / w) or less, 1000 ppm (w / w) or less, 100 ppm (w / w) or less, 50 ppm (w / w) or less, 20 ppm (w / w) or less, 10 ppm (w / w) or less, 5 ppm (w / w) or less, 2 ppm (w / w) or less, 1 The bitter-tasting component content in the food of the present invention may be, for example, 1 ppb (w / w) or less, 500 ppb (w / w) or less, or 200 ppb (w / w) or less, or any compatible combination thereof, as an added concentration in a plant-derived protein-containing food. Specifically, the bitter-tasting component content in the food of the present invention may be, for example, 1 ppb (w / w) to 100 ppm (w / w), 10 ppb (w / w) to 10 ppm (w / w), or 50 ppb (w / w) to 2 ppm (w / w). When the food of the present invention contains two or more bitter-tasting components, the "content of the bitter-tasting components in the food of the present invention" refers to the total content of those two or more components in the food of the present invention, unless otherwise specified. However, in one embodiment, when the food of the present invention contains two or more bitter-tasting components, the content of those two or more components in the food of the present invention may each independently be within the range of the bitter-tasting components in the food of the present invention exemplified above.

[0149] <2-4> Use of Active Ingredient The present invention also discloses the use of the active ingredient in the applications exemplified above. That is, the present invention discloses, for example, the use of the active ingredient for masking bitterness in food or for producing food, and the use of the active ingredient in producing a composition for masking bitterness in food or for producing food.

[0150] The present invention also discloses active ingredients for use in the above-mentioned applications, i.e., active ingredients for masking bitterness in foods or for use in the production of foods, and active ingredients for masking bitterness in foods or for use in the production of compositions for the production of foods.

[0151] The present invention will be described in detail below with reference to examples, but these are merely examples of the present invention and the scope of the present invention is not limited thereto. In the following examples, "%" means "wt%" unless otherwise specified.

[0152] Example 1: Identification of Receptors Activated by Soyasaponin I The bitter taste receptor response to soyasaponin I, a bitter substance found in plant-derived protein-containing foods, was identified using the following method. <1> Measurement of Bitter Taste Receptor Response to Soyasaponin I <1-1> Preparation of Cells Expressing Human Bitter Taste Receptors <1-1-1> Preparation of Expression Vectors for Human Bitter Taste Receptors The human bitter taste receptor gene TAS2Rs was synthesized using sequence information registered in the NCBI Reference Sequence Database (RefSeq). The TAS2Rs gene sequence, Kozak sequence, the N-terminal 45 amino acid sequence of rat somatostatin receptor Type 3, and HSV-tag sequence were totally synthesized. Subcloning was performed into the HindIII and XbaI sites of the pcDNA3.1(+) vector to obtain expression vectors for human bitter taste receptors T2Rs (T2R43, T2R44, T2R46, T2R49, or T2R50).

[0153] <1-1-2> Gene transfection PEAKrapid-derived Gα15-trans48LD stable expressing cells (PRG48 cells) were placed in a 100 mm dish at 3.3 × 10 6Cells were seeded in 10 mL of growth medium (G418-free) and cultured for 16-24 hours in a CO2 incubator (5% CO2, 37°C). The medium was then replaced with transfection medium (DMEM / Ham's F-12 / 5% FBS). The various expression plasmids prepared as described above were transfected using a transfection reagent (Lipofectamine 2000). Opti-MEM and DNA (B) and Opti-MEM and Lipomectamin 2000 (A) were mixed as shown in Table 1 below and allowed to stand for 5 minutes. The two solutions were then mixed, allowed to stand for 20 minutes, and the entire volume was added to the cells. After 4-6 hours of culture in a CO2 incubator, the cells were harvested and collected at 1 x 10 5 The cells were seeded into a 96-well assay plate (Corning) for FDSS at 100 μL / well. The medium used for the transfection (5% FBS, P / S-free) was used for seeding the assay plate, and the cells were cultured for 16-24 hours before use in the assay.

[0154]

[0155] <1-2> Measurement of Intracellular Calcium Concentration Using FDSS μCELL. After removing the medium from the FDSS assay plate containing the cells, 100 μL / well of bitter buffer containing Cal-520 (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.4) was added. The plate was then incubated in a CO2 incubator (37°C) for 60 minutes, then at room temperature for 30 minutes, protected from light. Soyasaponin I (Sigma Chemical Company, catalog number: S9951) was prepared at eight concentrations, each 2x common ratio, to achieve a concentration three times the final concentration in the sample plate. 100 μL / well of the solution was added to the assay plate using an FDSS μCELL (Hamamatsu Photonics). The maximum concentration for evaluation was 450 μM. Changes in intracellular Ca concentration were measured 10 seconds before and 110 seconds after sample addition.

[0156] As a result of receptor evaluation, soyasaponin activated the following receptors (Table 2).

[0157]

[0158] Example 2 Screening of Antagonists Using Bitter Taste Receptors Antagonists of the bitter taste receptor activated by soyasaponin I were screened as bitter taste-masking materials by the following method.

[0159] <2> Antagonist screening <2-1> Preparation of cells expressing human bitter taste receptors <2-1-1> Preparation of expression vectors for human bitter taste receptors The expression vectors for human bitter taste receptors T2Rs (T2R43, T2R44, T2R46, T2R49, or T2R50) prepared in <1-1-1> above were used.

[0160] <2-1-2> Gene transfection 3.3 x 10 PRG48 cells were cultured in a 100 mm dish. 6 Cells were seeded in 10 mL of growth medium (G418-free) and cultured for 16–24 hours in a CO2 incubator (5% CO2, 37°C). The medium was then replaced with transfection medium (DMEM / Ham's F-12 / 5% FBS). The various expression plasmids prepared as described above were transfected using a transfection reagent (Lipofectamine 2000). Opti-MEM and DNA (B) and Opti-MEM and Lipomectamin 2000 (A) were mixed as shown in Table 3 below and allowed to stand for 5 minutes. The two solutions were then mixed, allowed to stand for 20 minutes, and the entire volume was added to the cells. After 4–6 hours of culture in a CO2 incubator, the cells were harvested and collected at 0.6 × 10 5 The cells were seeded at 25 μL / well into a 384-well assay plate (Corning) for FDSS. The medium used for seeding the assay plate was the transfection medium (5% FBS, P / S-free), and the plate was cultured for 16–24 hours before use.

[0161]

[0162] <2-2> Measurement of Intracellular Calcium Concentration Using the FDSS7000 After removing the medium from the FDSS assay plate seeded with cells, 30 μL / well of bitter buffer containing coelenterazine (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.4) was added. The plate was then incubated in a CO2 incubator (37°C) for 60 minutes, and then left at room temperature for 4 hours in the dark. The following material library was used as the sample. The sample solution was prepared at three times the final concentration in the sample plate and added to the assay plate at 15 μL / well using the FDSS7000 (Hamamatsu Photonics). Changes in intracellular Ca concentration were measured 10 seconds before and 110 seconds after sample addition. Luminescence values ​​were analyzed using the software (FDSS7000EX) included with the FDSS / μCELL.

[0163] <2-3> Antagonist Screening <2-3-1> Evaluated Material Library The material library used in the masking material screening consisted of 368 food-approved flavorings and 60 food additives. Food additives were selected and used at concentrations (varies depending on the substance) that did not show a nonspecific response in preliminary receptor evaluation. All flavorings were evaluated at a maximum concentration of 100 μM. All were adjusted to three times the evaluation concentration before use.

[0164] <2-3-2> Agonists Used The agonists used in the screening evaluation were the substances listed in Table 4 below.

[0165]

[0166] <2-3-3> Screening Method Using Bitter Taste Receptors <2-3-3-1> Primary Screening The maximum concentration of flavors evaluated was 100 μM, and a single concentration was used for evaluation. The maximum concentration of food additives was set at a concentration that did not induce a nonspecific response. A 2.5 μM coelenterazine solution (in bitter buffer), a luminescent substrate, was added to cells overexpressing each bitter taste receptor. Flavorings were dissolved in 2.5 μM coelenterazine solution, and both the coelenterazine and flavor were allowed to stand for 4 hours. After 4 hours, agonists (and food additives) for each bitter taste receptor were added, and the intracellular Ca responses were measured using the FDSS7000. Evaluation was performed in duplicate wells. The mean value of the well containing only the agonist without the masking material was set as 100%, and the value of the masking material was calculated as a relative value. The criterion for selection in the primary screening was an average value of 70% or less across two wells (inhibition rate of 30% or more), and hit compounds were selected.

[0167] <2-3-3-2> Secondary Screening The maximum concentration of flavorings evaluated was 100 μM, and four concentrations were evaluated with a common ratio of 2. The maximum concentration of food additives was set at a concentration that did not elicit a nonspecific response, and four concentrations were evaluated with a common ratio of 2. A 2.5 μM coelenterazine solution (in bitter buffer), a luminescent substrate, was added to cells overexpressing each bitter receptor and allowed to stand for four hours. After four hours, agonists for each bitter receptor and flavoring or food additive were simultaneously added, and the intracellular Ca response was measured using the FDSS7000. Evaluations were performed in duplicate wells. The average value of wells without masking material was set to 100%, and the value of the masking material was calculated as a relative value. The criterion was set at 80%, and cells with an average value of 80% or less were selected.

[0168] As a result of receptor screening, the following components were selected as inhibitors of the receptors activated by soyasaponin I (Table 5). That is, the components listed in Table 5 were found to have the effect of inhibiting the receptors activated by soyasaponin I.

[0169]

[0170] Example 3: Evaluation of bitter taste masking effect by bitter taste receptor antagonists The compounds selected as described above that showed inhibitory activity against bitter taste receptor responses were subjected to sensory evaluation using the following method to evaluate their bitter taste masking effect.

[0171] <3> Evaluation of Bitterness-Masking Effect <3-1> Confirmation of Bitterness-Masking Action by Single Component <3-1-1> Preparation of Evaluation Samples Evaluation samples (Table 6) were prepared by adding compounds that exhibited inhibitory activity against bitter taste receptor responses to a soy protein aqueous solution (prepared with a 5 wt% aqueous solution of Fujipro FR, a refined soy protein powder from Fuji Oil Co., Ltd.). The concentration levels were set so that the compound itself did not produce an unpleasant flavor when the soy protein aqueous solution containing each compound alone was placed in the mouth and swallowed.

[0172] <3-1-2> Evaluation of Bitterness Masking Effect The bitterness masking strength was evaluated when each evaluation sample was held in the mouth and swallowed naturally. The bitterness masking strength was evaluated using the bitterness of a soy protein aqueous solution without added compounds as the standard, and was scored on a scale of 0 to 3 points for each of the timings of initial taste (0-2 seconds), middle taste (2-4 seconds), and aftertaste (4-6 seconds) according to the following evaluation criteria. A panel of four experts calculated the average value for each evaluation sample.

[0173] (Evaluation criteria) 0: No inhibitory effect (same bitterness as soy protein aqueous solution) 1: Inhibition of 10% or more but less than 30% 2: Inhibition of 30% or more but less than 50% 3: Inhibition of 50% or more

[0174] As a result of the evaluation, nine compounds were found to have a bitterness-masking effect (Table 6).

[0175]

[0176] <3-2> Confirmation of bitterness-masking effect by two-component combination <3-2-2> Preparation of evaluation samples Evaluation samples (Table 7) were prepared by adding compounds that demonstrated inhibitory activity against bitter taste receptor responses to a soy protein aqueous solution (prepared at a concentration of 5 wt% using FujiPro FR, a refined soy protein powder from Fuji Oil Co., Ltd.). The concentration levels were set so that the component itself did not produce an unpleasant flavor when the soy protein aqueous solution containing each compound was placed in the mouth and swallowed.

[0177] <3-2-3> Evaluation of bitterness masking effect The bitterness masking strength was evaluated when each evaluation sample was held in the mouth and swallowed naturally. The bitterness masking strength was evaluated using the bitterness of a soy protein aqueous solution without added compounds as the standard, and was scored on a scale of 0 to 3 points for each of the timings of initial taste (0-2 seconds), middle taste (2-4 seconds), and aftertaste (4-6 seconds) according to the following evaluation criteria. A panel of four experts calculated the average value for each evaluation sample.

[0178] (Evaluation criteria) 0: No inhibitory effect (same bitterness as soy protein aqueous solution) 1: Inhibition of 10% or more but less than 30% 2: Inhibition of 30% or more but less than 50% 3: Inhibition of 50% or more

[0179]

[0180] As a result of the evaluation, all six combinations of the samples that mixed two types of compounds evaluated (shown in bold in the table below) showed a strong after-bitterness masking effect, with more than half of the panelists observing a 50% or greater reduction in bitterness (Table 8).

[0181]

[0182] <3-3> Confirmation of bitterness-masking effect of 3-type and 4-type combinations <3-3-1> Preparation of evaluation samples Evaluation samples (Table 9) were prepared by adding compounds that showed inhibitory activity against bitter taste receptor responses to a soy protein aqueous solution (Fuji Oil Co., Ltd.'s refined soy protein powder "Fujipro FR" prepared at a concentration of 5 wt %). The concentration levels were set so that the component itself did not produce an unpleasant flavor when the soy protein aqueous solution containing each compound alone was placed in the mouth and swallowed.

[0183] <3-3-2> Evaluation of Bitterness Masking Effect The bitterness masking strength was evaluated when each evaluation sample was held in the mouth and swallowed naturally. The bitterness masking strength was evaluated using the bitterness of a soy protein aqueous solution without added compounds as the standard, and was scored on a scale of 0 to 3 points for each of the timings of initial taste (0-2 seconds), middle taste (2-4 seconds), and aftertaste (4-6 seconds) according to the following evaluation criteria. A panel of four experts calculated the average value for each evaluation sample.

[0184] (Evaluation criteria) 0: No inhibitory effect (same bitterness as soy protein aqueous solution) 1: Inhibition of 10% or more but less than 30% 2: Inhibition of 30% or more but less than 50% 3: Inhibition of 50% or more

[0185] As a result of the evaluation, five of the samples containing three or four ingredients were evaluated, and all of the panelists observed a strong bitterness-masking effect, especially in the aftertaste, of which bitterness was suppressed by 50% or more (Table 10). All of these combinations contained guanylic acid and cysteine ​​hydrochloride.

[0186]

[0187]

[0188] Example 4: Evaluation of Bitterness-Masking Effect in Plant-Derived Protein-Containing Foods The bitterness-masking effect of plant-derived protein-containing foods was evaluated as follows. <4-1> Preparation of Plant-Derived Protein-Containing Foods Using the formulations listed in Table 11, patties were prepared as plant-derived protein-containing foods as follows, and the resulting patties were evaluated. Granular soy protein was rehydrated with water. Canola oil, methylcellulose, and cold water were mixed for 1 minute to prepare curds. These were then mixed with powdered soy protein binders and granular soy protein binders at low speed for 60 seconds. Seasonings and a masking material with the formulations listed in Table 12 were added, and the mixture was further mixed at low speed for 60 seconds. The mixture was taken by hand and formed into patties of 80 g each. The molded product was placed in a mold and baked at 180°C for 1.5 minutes on each side to obtain patties. After baking, the baked patties were frozen, vacuum-pouched, and stored for 1 day. Immediately before evaluation, the sample was heated in a microwave oven at 600W for 1.5 minutes and evaluated by the method described below.

[0189]

[0190] <4-2> Sensory evaluation The bitterness masking strength was evaluated when the putty was eaten and swallowed naturally. The bitterness of the putty without any added masking material was used as the standard, and the strength was scored on a scale of 0 to 4 points for each of the timings of initial taste (0-2 seconds), middle taste (2-4 seconds), and aftertaste (4-6 seconds) according to the following evaluation criteria. Four expert panelists calculated the average value for each evaluation sample (No. 0-5).

[0191] (Evaluation criteria) 0: 0% or more but less than 10% suppression 1: 10% or more but less than 30% suppression 2: 30% or more but less than 50% suppression 3: 50% or more but less than 80% suppression 4: 80% or more suppression

[0192] As a result of the evaluation, all of the products showed a bitterness-masking effect, but all panelists noted that the product containing 0.013% cysteine ​​hydrochloride, 0.0013% cysteine ​​hydrochloride, and 0.02% guanylic acid suppressed the bitterness by 50% or more (Table 12).

[0193]

[0194] Example 5: Evaluation of bitterness-masking effect of bitter taste receptor antagonists 2 Using the following method, sensory evaluation was performed on compounds (cysteine ​​hydrochloride, nootkatone, and advantame) that showed inhibitory activity against bitter taste receptor responses to confirm the concentration range in which they had a bitterness-masking effect. The effects of cysteine ​​hydrochloride and cysteine ​​were also compared.

[0195] <5> Evaluation of Bitterness-Masking Effect <5-1> Confirmation of Bitterness-Masking Action by Single Component <5-1-1> Preparation of Evaluation Samples Evaluation samples (Table 13) were prepared by adding compounds that exhibited inhibitory activity against bitter taste receptor responses to a soy protein aqueous solution (prepared with a 5 wt% aqueous solution of purified soy protein powder "Fujipro FR" from Fuji Oil Co., Ltd.). Note that an evaluation sample to which Cys was added was prepared as a comparison with Cys-HCl.

[0196] <5-1-2> Evaluation of bitterness masking effect The bitterness masking strength was evaluated when each evaluation sample was held in the mouth and swallowed naturally. The bitterness masking strength was evaluated using the bitterness of a soy protein aqueous solution without added compounds as the standard, and was scored on a scale of 0 to 5 for each timing of the aftertaste (4 to 6 seconds) according to the following evaluation criteria. Three expert panelists were used to calculate the average value for each evaluation sample.

[0197] (Evaluation criteria) 0: 0% or more but less than 10% suppression 1: 10% or more but less than 30% suppression 2: 30% or more but less than 50% suppression 3: 50% or more but less than 70% suppression 4: 70% or more but less than 90% suppression 5: 90% or more suppression

[0198] As a result of the evaluation, a concentration range in which each compound had a bitterness-masking effect was confirmed (Table 13). That is, the masking effect was observed in the following concentration ranges. (The bitterness-suppressing effect of soybeans of 10% or more was observed, and the off-flavor derived from the active ingredient was not detected.) Cys-HCl: 0.1 ppm or more and less than 1000 ppm Advantame: 0.001 ppm or more and less than 10 ppm Nootkatone: 0.01 ppm or more When comparing Cys-HCl and Cys, Cys-HCl had a higher bitterness-masking effect.

[0199]

[0200] <5-2> Confirmation of bitterness-masking effect by two-component combination <5-2-2> Preparation of evaluation samples Evaluation samples (Table 14) were prepared by adding compounds that showed inhibitory activity against bitter taste receptor responses (cysteine ​​hydrochloride, nootkatone, advantame) to a soy protein aqueous solution (prepared at a concentration of 5 wt% using Fujipro FR, a refined soy protein powder from Fuji Oil Co., Ltd.). The concentration levels were set within the range of concentrations that were effective in <5-1>.

[0201] <5-2-3> Evaluation of bitterness masking effect Each evaluation sample was held in the mouth and swallowed naturally to evaluate the bitterness masking strength. The bitterness was evaluated using the bitterness of a soy protein aqueous solution without added compounds as the standard, and the intensity was scored on a scale of 0 to 5 for each timing of the aftertaste (4 to 6 seconds) according to the following evaluation criteria. Three expert panelists were used to calculate the average value for each evaluation sample.

[0202] (Evaluation criteria) 0: 0% or more but less than 10% suppression 1: 10% or more but less than 30% suppression 2: 30% or more but less than 50% suppression 3: 50% or more but less than 70% suppression 4: 70% or more but less than 90% suppression 5: 90% or more suppression

[0203] As a result of the evaluation, it was confirmed that the two combinations of the evaluated two compounds (shown in bold in the table below) had an enhanced masking effect compared to the addition of either compound alone (Table 14). That is, the combinations of Cys-HCl 50 ppm and advantame 0.25 ppm, and advantame 0.25 ppm and nootkatone 0.01 ppm had an enhanced masking effect compared to the addition of either compound alone.

[0204]

[0205] Example 6: Evaluation of Bitterness-Masking Effect in Plant-Derived Protein-Containing Foods 2 The bitterness-masking effect of cysteine ​​hydrochloride, nootkatone, and advantame in plant-derived protein-containing foods was evaluated as follows. Furthermore, the effects of cysteine ​​hydrochloride and cysteine ​​were compared. <6-1> Preparation of Plant-Derived Protein-Containing Foods <6-1-1> Soy Yogurt Using the formulation described in Table 15, soy yogurt was prepared as a plant-derived protein-containing food as follows, and the resulting soy yogurt was evaluated. 1. The starter was dissolved in soy milk at a concentration of 1.8 wt% to prepare a starter solution. 2. The soy milk was added to a sterilized container. 3. The starter solution was added to the soy milk, and the whole was mixed 30 times with a spatula. 4. The mixture was kept at 44°C for 8-10 hours and fermented until the pH reached 4.6. 5. The mixture was cooled to 20°C by immersion in running water and strained through a 500 μm mesh strainer to prepare a starter. 6. Each masking material was added and the whole was mixed 50 times with a spatula.

[0206]

[0207] <6-1-2> Soybean Patties Soybean patties were prepared as plant-derived protein-containing foods using the formulations listed in Table 16 as follows, and the resulting soybean patties were evaluated. 1. Granular soy protein was rehydrated with water. 2. Canola oil, methylcellulose, and cold water were mixed for 1 minute to create a curd. 3. The curd was mixed with powdered soybean protein binder, granular soybean protein binder, and shortening at low speed for 60 seconds. 4. Seasonings and masking materials were added, and the mixture was mixed at low speed for another 60 seconds. 5. The mixture was taken by hand and formed into 80g patties. 6. The patties were baked at 180°C for 2.5 minutes on each side to obtain patties. 7. After baking, the patties were frozen, vacuum-pouched, and stored for 1 day. 8. Immediately prior to evaluation, the patties were heated in a microwave oven at 600W for 1.5 minutes, and a sensory evaluation was performed.

[0208]

[0209] <6-1-3> Soy Cheese Using the formulation shown in Table 17, soy cheese was produced as a plant-derived protein-containing food product as follows, and the resulting soy cheese was evaluated. 1. All ingredients were placed in a heated mixer (Vorwerk Thermomix TM21) and mixed for 10 minutes at 90°C and speed 2.5. 2. A masking material was added to the mixture, and with heating stopped, the mixture was mixed for 1 minute at speed 2.5. 3. After storing at 5°C for 12 hours, a sensory evaluation was performed.

[0210]

[0211] <6-2> Sensory evaluation The bitterness masking strength was evaluated when the plant-derived protein-containing food was eaten and swallowed naturally. The bitterness was measured based on the bitterness of the plant-derived protein-containing food without added masking material, and the bitterness was scored on a scale of 0 to 5 for each timing of the aftertaste (4 to 6 seconds) according to the following evaluation criteria. Three expert panelists calculated the average value for each evaluation sample.

[0212] (Evaluation criteria) 0: 0% or more but less than 10% suppression 1: 10% or more but less than 30% suppression 2: 30% or more but less than 50% suppression 3: 50% or more but less than 70% suppression 4: 70% or more but less than 90% suppression 5: 90% or more suppression

[0213] <6-2-1> Sensory evaluation of soy yogurt As a result of the evaluation, when comparing Cys-HCl and Cys, Cys-HCl had a higher bitterness masking effect. The combination of Cys-HCl and advantame, and the combination of advantame and nootkatone showed an enhanced bitterness masking effect compared to the addition of a single component (Table 18).

[0214]

[0215] <6-2-2> Sensory evaluation of soybean pate As a result of the evaluation, when comparing Cys-HCl and Cys, Cys-HCl had a higher bitterness masking effect. The combination of Cys-HCl and advantame, and the combination of advantame and nootkatone were found to have an enhanced bitterness masking effect compared to the addition of a single component (Table 19).

[0216]

[0217] <6-2-3> Sensory evaluation of soy cheese As a result of the evaluation, it was found that the combination of Cys-HCl and advantame, and the combination of advantame and nootkatone enhanced the bitterness masking effect compared to the addition of a single component (Table 20).

[0218]

[0219] INDUSTRIAL APPLICABILITY The present invention is useful in the field of food products because it can mask the bitterness of plant-derived protein raw materials without impairing the original flavor and other properties of plant-derived protein-containing foods.

Claims

1. A composition for masking bitterness in plant-derived protein-containing foods, comprising an antagonist of at least one bitter taste receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50.

2. A composition for masking bitterness in plant-derived protein-containing foods, comprising the following component (a): (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

3. The composition according to claim 2, wherein component (a) is at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, and guanylic acid or a salt thereof.

4. The composition of claim 2, wherein component (a) is selected from: a combination of advantame and cysteine ​​or a salt thereof; a combination of advantame and nootkatone; a combination of cysteine ​​or a salt thereof and guanylic acid or a salt thereof; a combination of advantame and acesulfame potassium; a combination of advantame and 3-(methylthio)propyl isothiocyanate; a combination of 3-(methylthio)propyl isothiocyanate and guanylic acid or a salt thereof; a combination of vitamin B6 and 3-(methylthio)propyl isothiocyanate; and a combination of vitamin B6 and guanylic acid or a salt thereof.

5. The composition of claim 2, wherein component (a) is selected from: a combination of advantame, 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; a combination of 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; a combination of acesulfame potassium, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; a combination of acesulfame potassium, 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; a combination of vitamin B6, advantame, acesulfame potassium, 3-(methylthio)propyl isothiocyanate, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof; and a combination of vitamin B6, acesulfame potassium, cysteine ​​or a salt thereof, and guanylic acid or a salt thereof.

6. The composition according to claim 4 or 5, wherein the cysteine ​​or a salt thereof is cysteine ​​hydrochloride.

7. The composition according to claim 1 or 2, which is a composition for the production of food products.

8. The composition according to claim 7, wherein the food is a food whose bitterness is masked.

9. The composition of claim 7, wherein the food product contains a bitter-tasting ingredient.

10. The composition according to claim 9, wherein the bitter-tasting component is saponin.

11. The composition according to claim 9, wherein the bitter component is soyasaponin.

12. The composition of claim 7, wherein the food product is selected from veggie meat, veggie milk, plant-based seafood, and plant-based eggs.

13. A plant-derived protein-containing food, in which the following component (a) is added to the raw materials of the food to mask its bitterness: (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

14. A method for screening for a substance that masks bitterness in plant-derived protein-containing foods, comprising the following steps (A) to (C): (A) contacting a bitter receptor with a bitter receptor activator in the presence of a test substance; (B) measuring the response of the bitter receptor to the bitter receptor activator; and (C) identifying the test substance as a substance that masks bitterness in plant-derived protein-containing foods based on the response; wherein, when the response is inhibited by the test substance, the test substance is identified as a substance that masks bitterness in plant-derived protein-containing foods, and the bitter receptor comprises at least one bitter receptor selected from the group consisting of T2R43, T2R44, T2R49, and T2R50.

15. The method of claim 14, wherein the bitter taste receptors further comprise at least one bitter taste receptor selected from the group consisting of T2R1, T2R5, T2R14, and T2R46.

16. The method of claim 14, wherein the response is activation of the bitter taste receptor.

17. The method according to claim 14, wherein the bitter taste receptor is used in a form supported on a cell, a cell membrane, an artificial lipid bilayer vesicle, or an artificial lipid bilayer membrane.

18. The method of claim 14, wherein the bitter taste receptor is used in a cell-borne form.

19. The method of claim 17, wherein the cell is an animal cell.

20. The method according to claim 14, 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 bitter taste receptor when step (A) is carried out; (C1) identifying the test substance as a substance that masks the bitter taste in plant-derived protein-containing foods based on the degree of activation D1.

21. The method according to claim 20, wherein step (C1) is carried out by the following step (C2): (C2) a step of identifying the test substance as a substance that masks the bitter taste in a plant-derived protein-containing food based on the difference between the degree of activation D1 and the degree of activation D2 of the bitter taste receptor under control conditions.

22. The method of claim 21, wherein the control conditions are the following conditions (C2-1) or (C2-2): (C2-1) a condition in which the bitter receptor is contacted with the bitter receptor activator in the absence of the test substance; (C2-2) a condition in which the bitter receptor is contacted with the bitter receptor activator in the presence of the test substance, wherein the concentration of the test substance is lower than the concentration of the test substance in step (A).

23. The method of claim 21, further comprising the step of measuring the degree of activation D2.

24. The method according to claim 21, wherein the test substance is identified as a substance that masks bitterness in a plant-derived protein-containing food if the degree of activation D1 is lower than the degree of activation D2.

25. The method according to claim 21, wherein the test substance is identified as a substance that masks bitterness in a plant-derived protein-containing food if the ratio of the degree of activation D1 to the degree of activation D2 is less than 70%.

26. The method of claim 14, wherein the response is measured using intracellular calcium concentration as an index.

27. The method of claim 26, wherein the intracellular calcium concentration is measured by a luminescent assay.

28. The method of claim 14, wherein the bitter taste receptor is a human bitter taste receptor.

29. The method according to claim 14, further comprising a step of evaluating whether the identified substance that masks the bitter taste in plant-derived protein-containing foods has the function of masking the bitter taste in plant-derived protein-containing foods.

30. The method of claim 29, wherein the evaluation is performed by sensory evaluation.

31. A method for masking bitterness in a plant-derived protein-containing food, comprising the step of adding the following component (a) to a food ingredient: (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

32. A method for producing a plant-derived protein-containing food, comprising the step of adding the following component (a) to a food ingredient: (a) at least one component selected from the group consisting of cysteine ​​or a salt thereof, lauric acid, nootkatone, 5-hydroxy-2-decenoic acid lactone, guanylic acid or a salt thereof, advantame, acesulfame potassium, vitamin B6, and 3-(methylthio)propyl isothiocyanate.

33. The method of claim 32, wherein the food is a food whose bitterness is masked.

34. The method of claim 32, wherein the food product contains a bitter-tasting ingredient.

35. The method of claim 34, wherein the bitter-tasting component is saponin.

36. The method of claim 34, wherein the bitter-tasting component is soyasaponin.

37. The method of claim 32, wherein the food product is selected from veggie meat, veggie milk, plant-based seafood, and plant-based eggs.

38. The method according to claim 32, wherein component (a) is added to the plant-derived protein-containing food at a concentration of 0.0001 ppm (w / w) to 1000 ppm (w / w).

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