Method for evaluating action of test substance on quercitrin reception by bitter taste receptor, screening method for modulating substance having modulating ability on quercitrin reception by bitter taste receptor, kit for evaluating action of test substance on quercitrin reception by bitter taste receptor, and bitterness suppressant of quercitrin

Abstract

To provide a method that evaluates the action of a test substance on quercitrin reception by the bitter taste receptors TAS2R8 or TAS2R38.SOLUTION: A method for evaluating the action of a test substance on quercitrin reception by a bitter taste receptor, comprising: allowing coexistence of the bitter taste receptor and quercitrin in the presence of the test substance; detecting an interaction between the bitter taste receptor and quercitrin in the presence of the test substance; and evaluating the action of the test substance on the reception of quercitrin by the bitter taste receptor based on the detected interaction in the presence of the test substance, wherein the bitter taste receptor is TAS2R8 or TAS2R38.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the effect of a test substance on the reception of quercitrin by bitter taste receptors, a method for screening for regulatory substances capable of regulating the reception of quercitrin by bitter taste receptors, a kit for evaluating the effect of a test substance on the reception of quercitrin by bitter taste receptors, and a quercitrin bitterness inhibitor. [Background technology]

[0002] Green peppers are a food rich in vitamin C, vitamin K, and beta-carotene. However, because they have a bitter taste, they are a food that children in particular dislike. It has been revealed that the bitterness of green peppers is related to quercitrin, a type of polyphenol. It is also known that the bitterness of green peppers is perceived when quercitrin combines with pyrazines contained in green peppers (Non-Patent Document 1).

[0003] Taste 2 receptors (TAS2R) are a type of G protein-coupled receptor, and 25 types are known in humans. However, the bitter receptor that responds to quercitrin has not been identified. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Report of the Horticultural Culture Research Institute, Keisen University: Horticultural Culture, 2015, No. 11, pp. 25-30 Summary of the Invention [Problem to be solved by the invention]

[0005] As described in the Examples below, the present inventors have identified TAS2R8 and TAS2R38 as bitter receptors that respond to quercitrin. The present invention is based on this novel finding and aims to provide a method for evaluating the effect of a test substance on quercitrin reception by the bitter receptor TAS2R8 or TAS2R38, a method for screening for modulators capable of regulating quercitrin reception by the bitter receptor TAS2R8 or TAS2R38, a kit for evaluating the effect of a test substance on quercitrin reception by the bitter receptor TAS2R8 or TAS2R38, and a quercitrin bitterness suppressor. [Means for solving the problem]

[0006] The present invention includes, for example, the following inventions. [1] A method for evaluating the effect of a test substance on the reception of quercitrin by a bitter taste receptor, comprising: Coexistence of bitter taste receptors and quercitrin in the presence of a test substance; Detecting an interaction between a bitter taste receptor and quercitrin in the presence of the test substance; and and evaluating the effect of the test substance on the reception of quercitrin by the bitter taste receptor based on the detected interaction in the presence of the test substance. The method, wherein the bitter taste receptor is TAS2R8 or TAS2R38. [2] A method for screening for a modulator having an ability to modulate quercitrin reception by a bitter taste receptor, comprising: Coexistence of bitter taste receptors and quercitrin in the presence of a test substance; Detecting an interaction between a bitter taste receptor and quercitrin in the presence of the test substance; and determining whether the test substance is the modulator based on the detected interaction in the presence of the test substance; The method, wherein the bitter taste receptor is TAS2R8 or TAS2R38. [3] allowing a bitter taste receptor and quercitrin to coexist in the absence of the test substance; Detecting an interaction between a bitter taste receptor and quercitrin in the absence of the test substance; and The method according to [1] or [2], further comprising comparing the detected interaction in the presence of the test substance with the detected interaction in the absence of the test substance. [4] The coexistence of the bitter receptor and quercitrin is achieved by contacting a cell expressing the bitter receptor with a solution containing quercitrin; and Detecting the interaction is a method for detecting Ca in the cell. 2+ The method according to any one of [1] to [3], comprising detecting a change in concentration. [5] The method according to any one of [1] to [4], wherein the bitter taste receptor TAS2R8 comprises an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1. [6] The method according to any one of [1] to [5], wherein the bitter taste receptor TAS2R38 comprises an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:2. [7] A kit for evaluating the effect of a test substance on the reception of quercitrin by a bitter taste receptor, comprising: Contains a bitter taste receptor or a nucleic acid encoding the bitter taste receptor, and / or quercitrin, A kit in which the bitter taste receptor is TAS2R8 or TAS2R38. [8] The kit described in [7], comprising a bitter taste receptor or a nucleic acid encoding the bitter taste receptor, and quercitrin. [9] A quercitrin bitterness suppressant containing egg yolk protein as an active ingredient. [Effects of the Invention]

[0007] The present invention can provide a method for evaluating the effect of a test substance on quercitrin reception by the bitter taste receptor TAS2R8 or TAS2R38. The present invention can also provide a method for screening for a modulator capable of regulating quercitrin reception by the bitter taste receptor TAS2R8 or TAS2R38. The present invention can further provide a kit for evaluating the effect of a test substance on quercitrin reception by the bitter taste receptor TAS2R8 or TAS2R38. The present invention still further provides an agent for suppressing the bitterness of quercitrin. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a graph showing the results of luminescence measurement in Test Example 1. [Figure 2] 1 is a graph showing the results of luminescence measurement in Test Example 2. [Figure 3] 10 is a graph showing the results of absorption spectrum measurement in Test Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0010] [Method for evaluating the effects of test substances] The method for evaluating the effect of a test substance on quercitrin reception by a bitter taste receptor according to this embodiment (hereinafter simply referred to as the "evaluation method") includes coexisting a bitter taste receptor and quercitrin in the presence of the test substance (coexistence step), detecting an interaction between the bitter taste receptor and quercitrin in the presence of the test substance (detection step), and evaluating the effect of the test substance on quercitrin reception by the bitter taste receptor based on the detected interaction in the presence of the test substance (evaluation step). The bitter taste receptor used in this evaluation method is TAS2R8 or TAS2R38.

[0011] Quercitrin (CAS Registry Number: 522-12-3) is a compound also known as 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyl-2-tetrahydropyranyl]oxy]-4-chromenone.

[0012] The bitter taste receptors TAS2R8 and TAS2R38 are types of G protein-coupled receptors and perceive quercitrin. As used herein, "perceive quercitrin" refers to a conformational change in the bitter taste receptor TAS2R8 or TAS2R38 upon interaction with quercitrin. This conformational change typically results in conversion of the coupled G protein (e.g., conversion of a GDP-type G protein to a GTP-type G protein). Furthermore, within the cell, the conversion of the G protein further induces corresponding downstream reactions.

[0013] The bitter taste receptor TAS2R8 used in the evaluation method according to this embodiment may contain an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, so long as it accepts quercitrin. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the human bitter taste receptor TAS2R8. The sequence identity may be 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or even 100%.

[0014] The bitter taste receptor TAS2R38 used in the evaluation method according to this embodiment may contain an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, so long as it accepts quercitrin. The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of human bitter taste receptor TAS2R38. The sequence identity may be 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or even 100%.

[0015] As used herein, sequence identity refers to the percentage (%) of matching bases or residues when the base sequences or amino acid sequences being compared are aligned (multiple alignment). Multiple alignment refers to an alignment of base sequences with appropriate gaps inserted so that corresponding base or amino acid sequence portions are aligned, making the base sequences or amino acid sequences comparable to each other. For multiple alignment, known multiple alignment creation programs can be used. For example, Clustal W, Clustal X, BLAST, etc., can be suitably used.

[0016] The test substance is not particularly limited and may be any compound. Specific examples of test substances include inorganic compounds, low-molecular-weight (molecular weight of 1000 g / mol or less) organic compounds, and high-molecular-weight (molecular weight of more than 1000 g / mol) organic compounds. Test substances may also be biomolecules, such as nucleic acids (DNA, RNA, PNA, etc.), oligopeptides, polypeptides (including proteins), lipids, and molecules formed by combining two or more of these.

[0017] The coexistence step in the evaluation method according to this embodiment is a step in which the bitter taste receptor and quercitrin are allowed to coexist in the presence of a test substance.

[0018] The bitter receptor may be the protein itself, but since the protein is a seven-transmembrane membrane protein, it is preferable to subject it to the coexistence step together with a lipid bilayer membrane in order to form an accurate three-dimensional structure. The lipid bilayer membrane may be the lipid bilayer membrane that constitutes a cell, or may be an in vitro replica of the lipid bilayer membrane possessed by a cell. Since this makes it possible to more easily carry out the evaluation method according to this embodiment, it is preferable to subject the bitter receptor to the coexistence step as a cell expressing the bitter receptor (i.e., a cell having a bitter receptor on the cell membrane).

[0019] The cells having bitter taste receptors on their cell membranes are not particularly limited as long as they are isolated cells, and may be, for example, cells derived from prokaryotes or eukaryotes. The cells are preferably mammalian cells, and more preferably human cells.

[0020] The coculture step can be carried out by any method as long as the test substance, bitter receptor, and quercitrin can interact with each other. From the viewpoint of easily ensuring that the test substance, bitter receptor, and quercitrin can contact each other and interact with each other, the coculture step may include preparing a solution or suspension containing the test substance, bitter receptor, and quercitrin. The type of solvent used for the solution or suspension is not particularly limited, as long as it can dissolve or suspend the test substance, bitter receptor, and quercitrin. Examples of the solvent include aqueous solvents (e.g., water, saline, buffer solution, culture medium), organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and alcohols such as methanol and ethanol, as well as mixtures thereof.

[0021] When cells expressing a bitter receptor are used as the bitter receptor, the coculture step can be carried out, for example, by adding a solution or suspension of the test substance and a solution or suspension of quercitrin to a culture medium in which the cells are suspended. Note that the order in which the cells, test substance, and quercitrin are added to the culture medium is arbitrary.

[0022] In the coexistence step, it is preferable to further coexist a G protein that couples with the bitter taste receptor, since this facilitates detection in the detection step. Examples of such G proteins include G15, G16, and chimeric G proteins. Examples of chimeric G proteins include G16gust44 (Gα16 in which the C-terminal 44 amino acid residues are substituted with the amino acid sequence of the taste bud-specific G protein gustducin) and G16t2 ​​(Gα16 in which the C-terminal 44 amino acid residues are substituted with the amino acid sequence of the zebrafish-derived G protein Gαt2).

[0023] When cells expressing a bitter taste receptor are used as the bitter taste receptor, the cells may further express a G protein that couples with the bitter taste receptor.

[0024] The detection step in the evaluation method according to this embodiment is a step of detecting the interaction between the bitter taste receptor and quercitrin in the presence of a test substance.

[0025] When the bitter taste receptor receives (interacts with) quercitrin, a conformational change occurs in the bitter taste receptor. The detection step can be carried out by detecting this conformational change. There are no particular limitations on the method for detecting the conformational change, and examples include a method in which two types of dye molecules are bound to the bitter taste receptor and fluorescence resonance energy transfer (FRET) associated with the conformational change is detected, and a method in which a G protein coupled to the bitter taste receptor is further coexisted and G protein conversion associated with the conformational change (e.g., conversion of a GDP-type G protein to a GTP-type G protein) is detected. The method for detecting G protein conversion may directly detect G protein conversion, or may detect downstream reactions triggered by G protein conversion within the cell.

[0026] The detection may be performed by directly detecting the structural change of the bitter receptor, the conversion of the G protein associated with the structural change, downstream reactions associated with the conversion, etc., or by converting the structural change of the bitter receptor, the conversion of the G protein associated with the structural change, downstream reactions associated with the conversion, etc. into a signal and detecting the signal. The signal may be, for example, luminescence, fluorescence, etc. The detection of the signal may be performed by detecting a signal at a specific time, or by detecting the integrated value of the signal within a specific time. When the bitter receptor TAS2R38 is used, since a weak response persists, the integrated value of the signal within a specific time may be detected. This improves the detection sensitivity.

[0027] When cells expressing bitter taste receptors and G proteins coupled with the bitter taste receptors are used in the coculture step, quercitrin is received by the bitter taste receptors, which then converts the G protein, activates phospholipase Cβ (PLCβ), and increases the concentration of inositol triphosphate (IP3), resulting in the increase of intracellular Ca 2+ The detection step can be performed more easily, so in the detection step, intracellular Ca concentration is increased. 2+ It is preferable to detect intracellular Ca. 2+ For example, intracellular Ca can be detected by incorporating a fluorescent or luminescent indicator into cells. 2+ Fluorescence or luminescence occurs depending on the concentration, and this can be measured. When using the bitter taste receptor TAS2R38, the response is weak and sustained, so the integrated value of fluorescence or luminescence within a certain period of time may be measured. This improves detection sensitivity.

[0028] Intracellular Ca 2+ Examples of fluorescent indicators for detecting intracellular Ca include Fura-2, Fura2-AM, and red fluorescent protein (pDsRed2-N1). 2+ Examples of luminescent indicators for detecting calcium include calcium-binding photoproteins (aequorin, clytin 2, etc.).

[0029] The evaluation step in the evaluation method according to this embodiment is a step of evaluating the effect of the test substance on the reception of quercitrin by the bitter taste receptor based on the detected interaction in the presence of the test substance.

[0030] The evaluation method according to this embodiment is based on the novel finding that bitter receptors TAS2R8 and TAS2R38 perceive quercitrin. The coexistence of a test substance with a bitter receptor (TAS2R8 or TAS2R38) and quercitrin can alter the degree of interaction between the bitter receptor and quercitrin. In this case, a test substance that strengthens the interaction can be evaluated as a substance that enhances quercitrin perception by the bitter receptor (e.g., a bitterness enhancer). A test substance that weakens the interaction can be evaluated as a substance that inhibits quercitrin perception by the bitter receptor (e.g., a bitterness suppressor). Furthermore, a test substance that does not affect the interaction can be evaluated as a substance that does not affect quercitrin perception by the bitter receptor.

[0031] In addition to the steps described above, the evaluation method according to this embodiment may further include allowing the bitter receptor and quercitrin to coexist in the absence of a test substance, detecting an interaction between the bitter receptor and quercitrin in the absence of the test substance, and comparing the detected interaction in the presence of the test substance with the detected interaction in the absence of the test substance. This additional step is equivalent to determining the background level, and allows for more sensitive evaluation of the effect of the test substance on the interaction between the bitter receptor and quercitrin. This additional step can be performed in the same manner as the steps described above, except that the test substance is not used.

[0032] [Method for screening regulators] The evaluation method according to the present embodiment described above can also be considered as a screening method for a regulator capable of regulating the reception of quercitrin by bitter taste receptors (hereinafter also simply referred to as a "screening method").

[0033] The screening method of this embodiment includes coexisting a bitter receptor and quercitrin in the presence of a test substance (coexistence step), detecting the interaction between the bitter receptor and quercitrin in the presence of the test substance (detection step), and determining whether the test substance is a modulator based on the detected interaction in the presence of the test substance (determination step). The bitter receptor used in this screening method is also TAS2R8 or TAS2R38.

[0034] As specific aspects of the coexistence step and detection step in the screening method according to this embodiment, the aspects explained in the evaluation method according to this embodiment can be applied without any restrictions.

[0035] The discrimination step in the screening method of this embodiment can be applied without limitation to the aspects described in the evaluation method of this embodiment above, except that a test substance that strengthens or weakens the interaction between the bitter receptor and quercitrin is discriminated to be a modulatory substance, and a test substance that does not affect the interaction between the bitter receptor and quercitrin is discriminated to be a non-modulatory substance.

[0036] The screening method of this embodiment may include, instead of or in addition to the discrimination step, selecting a regulatory substance based on an interaction in the presence of a detected test substance (selection step).

[0037] The modulator may be a substance that has the effect of enhancing the reception of quercitrin by the bitter taste receptor, or a substance that has the effect of suppressing the reception of quercitrin by the bitter taste receptor.

[0038] In addition to the steps described above, the screening method of this embodiment may further include allowing the bitter receptor and quercitrin to coexist in the absence of the test substance, detecting the interaction between the bitter receptor and quercitrin in the absence of the test substance, and comparing the detected interaction in the presence of the test substance with the detected interaction in the absence of the test substance. This additional step is synonymous with determining the background level, and allows for more sensitive evaluation of the effect of the test substance on the interaction between the bitter receptor and quercitrin.

[0039] Other specific aspects of the screening method according to this embodiment can be applied without limitation to the aspects described above in relation to the evaluation method according to this embodiment.

[0040] 〔kit〕 The kit for evaluating the effect of a test substance on quercitrin reception by a bitter taste receptor according to this embodiment (hereinafter simply referred to as the "kit") includes a bitter taste receptor or a nucleic acid encoding the bitter taste receptor, and / or quercitrin. The bitter taste receptor used in the kit is also TAS2R8 or TAS2R38.

[0041] The bitter taste receptor TAS2R8 may comprise an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, so long as it accepts quercitrin. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of human bitter taste receptor TAS2R8. The sequence identity may be 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or even 100%.

[0042] The bitter taste receptor TAS2R38 may comprise an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, so long as it accepts quercitrin. The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of human bitter taste receptor TAS2R38. The sequence identity may be 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or even 100%.

[0043] The kit according to this embodiment may contain the bitter receptor protein itself, or may contain a nucleic acid encoding the bitter receptor. The nucleic acid is preferably in the form of an expression vector, as this facilitates handling. The expression vector comprises a nucleic acid sequence encoding the bitter receptor and one or more regulatory sequences operably linked to the nucleic acid sequence. The regulatory sequence is a sequence (e.g., promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.) that controls expression in cells into which the expression vector is introduced, and can be selected appropriately depending on the type of cell. The type of expression vector can be selected appropriately depending on the type of cell, such as a plasmid vector, viral vector, cosmid vector, or artificial chromosome vector.

[0044] The kit according to this embodiment may further include a G protein coupled with a bitter taste receptor, or a nucleic acid encoding the G protein. The nucleic acid encoding the G protein is preferably in the form of an expression vector, as this facilitates handling. The expression vector comprises a nucleic acid sequence encoding the G protein and one or more regulatory sequences operably linked to the nucleic acid sequence. The regulatory sequence is a sequence (e.g., promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.) that controls expression in cells into which the expression vector is introduced, and can be selected appropriately depending on the type of cell. The type of expression vector can be selected appropriately depending on the type of cell, such as a plasmid vector, viral vector, cosmid vector, or artificial chromosome vector.

[0045] The kit according to this embodiment may further include cells for introducing and expressing a nucleic acid encoding a bitter receptor and a nucleic acid encoding a G protein coupled to the bitter receptor. The cells may be, for example, cells derived from prokaryotes or eukaryotes. The cells are preferably mammalian cells, and more preferably human cells.

[0046] The kit according to this embodiment may or may not contain quercitrin. When the kit contains quercitrin, the quercitrin may be the quercitrin itself (e.g., in the form of a solid powder) or a solution in which the quercitrin is dissolved (e.g., a solution in which the quercitrin is dissolved in a mixed solvent of DMSO and a buffer solution).

[0047] The kit according to this embodiment can be suitably used in the evaluation method and screening method according to this embodiment described above.

[0048] [Bitterness suppressant] The quercitrin bitterness inhibitor of this embodiment contains egg yolk protein as an active ingredient. As described in the Examples below, the bitterness inhibitor of this embodiment is based on the finding that egg yolk protein has the effect of weakening the interaction between the bitter taste receptor TAS2R8 and quercitrin, i.e., inhibiting quercitrin reception by the bitter taste receptor TAS2R8. The bitter taste receptor TAS2R8 is the bitter taste receptor that contributes most to quercitrin reception, and egg yolk protein inhibits quercitrin reception by the bitter taste receptor TAS2R8, so egg yolk protein can inhibit the bitterness of quercitrin. The mechanism by which egg yolk protein inhibits the bitterness of quercitrin is based, at least in part, on the fact that, when egg yolk protein binds to quercitrin, the quercitrin bound to egg yolk protein cannot interact with the bitter taste receptor TAS2R8. Based on this inhibitory mechanism, egg yolk proteins are thought to also inhibit the reception of quercitrin by the taste receptor TAS2R38.

[0049] In this specification, containing "as an active ingredient" means containing an ingredient that has the effect of suppressing the bitterness of quercitrin, or an ingredient that exerts this effect.

[0050] Egg yolk proteins are composed of some or all of the protein components contained in egg yolk. The egg yolk proteins according to this embodiment include not only proteins but also lipoproteins in which proteins are bound to lipids. Egg yolk proteins can be obtained, for example, by defatting and dehydrating egg yolk (for example, spray drying or freeze drying).

[0051] The bitterness suppressant of this embodiment may consist solely of the above-mentioned active ingredient, or may contain, in addition to the above-mentioned active ingredient, other ingredients acceptable for use in foods, quasi-drugs, or pharmaceuticals.

[0052] Examples of other ingredients include excipients, binders, lubricants, disintegrants, surfactants, bases, solubilizers, colorants, flavorings, sweeteners, bittering agents, salts, acidulants, preservatives, anti-mold agents, antioxidants, emulsifiers, pH adjusters, thickening stabilizers, etc.

[0053] Examples of excipients include lactose, sucrose, starch, dextrin, etc. Examples of binders include gum arabic, tragacanth, gelatin, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, etc. Examples of lubricants include magnesium stearate, calcium stearate, talc, etc. Examples of disintegrants include crystalline cellulose, agar, gelatin, calcium carbonate, sodium bicarbonate, etc. Examples of surfactants include Tween 60, Tween 80, Span 80, glyceryl monostearate, etc. Examples of bases include cetostearyl alcohol, lanolin, polyethylene glycol, olive oil, etc. Examples of solubilizers include polyethylene glycol, propylene glycol, etc. Examples of coloring agents include beta-carotene, caramel, red koji pigment, etc. Examples of sweeteners include sugars, sugar alcohols, and high-intensity sweeteners, etc. Examples of bittering agents include caffeine, etc. Examples of salts include sodium chloride, etc. Acidulants include acetic acid, lactic acid, gluconic acid, etc. Preservatives include methylparaben, propylparaben, etc. Antifungals include imazalil, orthophenylphenol, thiabendazole, fludioxonil, etc. Antioxidants include tocopherol, etc. Emulsifiers include glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, glycerin organic acid fatty acid esters, polyglycerin fatty acid esters, calcium stearoyl lactylate, sodium stearoyl lactylate, polyoxyethylene sorbitan fatty acid esters, etc. pH adjusters include citric acid, malic acid, phosphoric acid, etc. Thickening stabilizers include locust bean gum, carrageenan, alginic acids, pectin, xanthan gum, crystalline cellulose, carboxymethylcellulose, methylcellulose, agar, glucomannan, gelatin, starch, etc.

[0054] The bitterness suppressant according to this embodiment may be in any form, such as a solid, liquid (including a solution and a suspension), paste, etc. Furthermore, the bitterness suppressant according to this embodiment may be in any dosage form, such as a tablet (orally disintegrating tablet, chewable tablet, film-coated tablet, etc.), capsule, powder, granule, liquid (syrup, jelly, etc.), etc.

[0055] The bitterness suppressor according to this embodiment can be prepared as, for example, a food composition (drinks and foods), a quasi-drug, or a pharmaceutical. That is, one embodiment of the present invention provides a bitterness-suppressing food composition (drinks and foods), a quasi-drug, or a pharmaceutical, which contains egg yolk protein as an active ingredient.

[0056] The content of the active ingredient in the bitterness suppressor according to this embodiment can be appropriately determined depending on the specific form of the bitterness suppressor so as to achieve a content (effective amount) that can suppress the bitterness of quercitrin. An example of an effective amount is an amount in which the quercitrin:egg yolk protein ratio is 1:1 (weight ratio) to 1:20 (weight ratio). Further examples of effective amounts include a quercitrin:egg yolk protein ratio range of 1:1 (weight ratio) to 1:15 (weight ratio), a quercitrin:egg yolk protein ratio range of 1:2 (weight ratio) to 1:11 (weight ratio), or a quercitrin:egg yolk protein ratio range of 1:3 (weight ratio) to 1:8 (weight ratio).

[0057] The bitterness suppressant according to this embodiment may be orally ingested after being premixed with a food composition containing quercitrin, or may be orally ingested together with a food composition containing quercitrin. The mechanism by which the bitterness suppressant according to this embodiment suppresses the bitterness of quercitrin is at least partly due to the binding of egg yolk protein with quercitrin, and therefore, the bitterness suppression effect can be achieved whether the bitterness suppressant is orally ingested by any of the methods described above. [Example]

[0058] The present invention will be described in more detail below with reference to examples, etc. However, the present invention is not limited to the following examples.

[0059] Test Example 1: Identification of human bitter taste receptors that perceive quercitrin We screened all 25 human bitter taste receptors and identified the bitter receptor that responds to quercitrin. When quercitrin is received by the bitter receptor, it activates phospholipase Cβ (PLCβ), increases inositol triphosphate (IP3) concentration, and then increases intracellular Ca 2+ The concentration of this Ca 2+ By detecting the change in concentration, the interaction between the bitter taste receptor and quercitrin can be detected.

[0060] <Preparation of quercitrin solution> The quercitrin solution was prepared by dissolving crystalline solid quercitrin in dimethyl sulfoxide and then diluting with N-(2-hydroxyethyl)-piperazineethanesulfonic acid buffer solution (hereinafter referred to as HEPES buffer solution).

[0061] <Fluorescence measurement> HEK293T cells were seeded onto 12-well plates and cultured at 37°C for 18–20 hours to transiently express the human bitter taste receptor (TAS2R) and chimeric G protein (G16gust44). A fluorescent indicator (Fura2-AM, Thermo Fisher Scientific) was then added and allowed to enter the cells for 30 minutes at 37°C, after which fluorescence measurements were performed. Fluorescence measurements were performed using a fluorescence microscope (IX-70, Olympus Corporation) at excitation wavelengths of 340 nm and 380 nm and an observation (fluorescence) wavelength of 510 nm. Measured fluorescence images were analyzed using METAFLUOR software (Molecular Devices).

[0062] Fluorescence measurements revealed that, of the 25 types of human bitter taste receptors, six (TAS2R3, TAS2R8, TAS2R9, TAS2R38, TAS2R43, and TAS2R46) showed fluorescence responses, although the intensity varied.

[0063] <Luminescence measurement> HEK293T cells were seeded into 12-well plates and cultured at 37°C for 18–20 hours to transiently express the human bitter taste receptor (TAS2R), a chimeric G protein (G16gust44), and a calcium-binding photoprotein (aequorin). After 4 hours, the cells were seeded into 96-well plates and cultured at 37°C for an additional 18 hours. After washing with HEPES buffer, the cells were added with a luminescent substrate (coelenterazine) and cultured at 27°C for 4 hours. Next, a quercitrin solution was added to the culture medium to a final quercitrin concentration of 1 mM. Changes in intracellular calcium concentration associated with bitter taste receptor activation were measured by luminescence intensity. Luminescence intensity was measured using a Flex Station 3 (Molecular Devices) as the integrated value for 120 seconds after quercitrin addition. As a control, luminescence intensity was measured in the same manner as above, except that only a solvent not containing quercitrin (HEPES buffer containing dimethyl sulfoxide) was added instead of the quercitrin solution.

[0064] The results of the luminescence measurements are shown in Figure 1. In the graph shown in Figure 1, the "cellular response intensity" is the relative luminescence intensity for each bitter taste receptor, with the control luminescence intensity set to 100. As shown in Figure 1, responses to quercitrin were observed in multiple bitter taste receptors, and particularly strong responses to quercitrin were confirmed in TAS2R8 and TAS2R38.

[0065] TAS2R8 exhibited a significant response to quercitrin in both fluorescence and luminescence measurements, suggesting it is the bitter taste receptor most responsible for quercitrin reception. TAS2R38 showed a weak response in image analysis of fluorescence measurements, but a strong response was confirmed in luminescence measurements. Because luminescence measurements were performed by measuring the integrated value over 120 seconds after quercitrin addition, it is believed that the weak response of TAS2R38 continued for a relatively long time, resulting in an increased measured value. Because TAS2R38 exhibited a sustained weak response, it is believed that it can be used with high sensitivity for evaluation of quercitrin reception, for example, by measuring the integrated value of the response.

[0066] [Test Example 2: Verification of the effect of egg yolk protein in reducing the bitterness of quercitrin] Using TAS2R8 as a bitter taste receptor, we verified the effect of egg yolk protein in reducing the bitterness of quercitrin.

[0067] <Preparation of egg yolk protein solution> Egg yolk was defatted and spray-dried, then dissolved in 20 w / w% HEPES buffer, and the precipitate was removed by centrifugation. The pH was then adjusted to 7.4 to obtain an egg yolk protein solution.

[0068] <Luminescence measurement> The procedure was the same as in Test Example 1, except that after adding a luminescent substrate (coelenterazine) and culturing at 27°C for 4 hours, the luminescence intensity was measured without adding quercitrin or egg yolk protein (A), with adding quercitrin (final concentration 1 mM) (B), or with adding quercitrin (final concentration 1 mM) and egg yolk protein (final concentration 1.5 mg / mL) (C).

[0069] The results of the luminescence measurements are shown in Figure 2. As shown in Figure 2, the addition of egg yolk protein reduced the bitter taste receptor response to quercitrin. Furthermore, a Turkey test confirmed statistically significant differences between (A), (B), and (C).

[0070] Test Example 3: Verification of the binding between quercitrin and egg yolk protein To investigate the mechanism by which egg yolk proteins reduce the bitterness perception of quercitrin, we evaluated the binding between egg yolk proteins and quercitrin.

[0071] A quercitrin solution (1 mM) and an egg yolk protein solution (2.8 mg / mL) were mixed and allowed to stand at 37°C for 30 minutes to obtain a test solution containing quercitrin and egg yolk protein. For comparison, control solution 1, which contained the same concentration of quercitrin as the test solution, and control solution 2, which contained the same concentration of egg yolk protein as the test solution, were prepared.

[0072] To remove unbound quercitrin (free quercitrin), the test solution, control solution 1, and control solution 2 were subjected to ultrafiltration using a 10 kDa filter. The filtrate was adjusted to the same volume as before ultrafiltration using HEPES buffer to obtain the test solution (after filtration), control solution 1 (after filtration), and control solution 2 (after filtration).

[0073] The absorption spectra of the test solution, control solution 1, and control solution 2, as well as the test solution (after filtration), control solution 1 (after filtration), and control solution 2 (after filtration), were measured by ultraviolet spectroscopy. The residual quercitrin rate after ultrafiltration was calculated from the absorbance at the quercitrin absorption maximum (wavelength 351 nm). The results of the absorption spectrum measurements are shown in Figure 3. The calculated results of the residual quercitrin rate after ultrafiltration are shown in Table 1.

[0074] [Table 1]

[0075] The results for Control Solution 2 (egg yolk protein) and Control Solution 2 (after filtration) (egg yolk protein (after filtration)) shown in Figure 3 confirmed that egg yolk protein does not absorb at a wavelength of 351 nm, which is the absorption maximum of quercitrin.

[0076] The residual rate of quercitrin after ultrafiltration was 35.5% when egg yolk protein was mixed and 18.9% when egg yolk protein was not mixed. This result was thought to be due to quercitrin binding to egg yolk protein and not passing through the ultrafiltration filter. Therefore, it was suggested that egg yolk protein binds to quercitrin.

Claims

1. A method for evaluating the effect of a test substance on the reception of quercitrin by a bitter taste receptor, comprising: Coexistence of bitter taste receptors and quercitrin in the presence of a test substance; Detecting an interaction between a bitter taste receptor and quercitrin in the presence of the test substance; and and evaluating the effect of the test substance on the reception of quercitrin by the bitter taste receptor based on the detected interaction in the presence of the test substance. The method, wherein the bitter taste receptor is TAS2R8 or TAS2R38.

2. A method for screening for a modulator having an ability to modulate quercitrin reception by a bitter taste receptor, comprising: Coexistence of bitter taste receptors and quercitrin in the presence of a test substance; Detecting an interaction between a bitter taste receptor and quercitrin in the presence of the test substance; and determining whether the test substance is the modulator based on the detected interaction in the presence of the test substance; The method, wherein the bitter taste receptor is TAS2R8 or TAS2R38.

3. allowing a bitter taste receptor and quercitrin to coexist in the absence of the test substance; Detecting an interaction between a bitter taste receptor and quercitrin in the absence of the test substance; and The method of claim 1 or 2, further comprising comparing the detected interaction in the presence of the test substance with the detected interaction in the absence of the test substance.

4. The coexistence of the bitter receptor and quercitrin is achieved by contacting a cell expressing the bitter receptor with a solution containing quercitrin; and Detecting the interaction includes detecting Ca in the cell. ２＋ 3. The method of claim 1 or 2, comprising detecting a change in concentration.

5. The method of claim 1 or 2, wherein the bitter taste receptor TAS2R8 comprises an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

6. The method of claim 1 or 2, wherein the bitter taste receptor TAS2R38 comprises an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

7. A kit for evaluating the effect of a test substance on the reception of quercitrin by a bitter taste receptor, comprising: A bitter taste receptor or a nucleic acid encoding the bitter taste receptor, and / or quercitrin, A kit, wherein the bitter taste receptor is TAS2R8 or TAS2R38.

8. The kit according to claim 7, comprising a bitter taste receptor or a nucleic acid encoding the bitter taste receptor, and quercitrin.

9. A quercitrin bitterness suppressant containing egg yolk protein as an active ingredient.

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