Method for searching for soybean off-flavor suppressants and soybean off-flavor suppressants

Abstract

To provide a method for searching for soybean off-flavor suppressants, and to provide soybean off-flavor suppressants.SOLUTION: Provided is a method for searching for soybean off-flavor suppressants, comprising the following steps (1) to (3): (1) contacting the olfactory receptor OR5K1 with a test substance and a soybean off-flavor-causing substance; (2) measuring the olfactory receptor response to the test substance and the soybean off-flavor-causing substance after the contact; and (3) selecting, based on the results of the measurement, a test substance that suppresses the olfactory receptor response compared to a control as a candidate soybean off-flavor suppressant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for searching for a soybean off-flavor suppressing agent and a soybean off-flavor suppressing agent. [Background technology]

[0002] Soybean protein has been attracting attention as a plant protein with excellent digestibility and absorbability, along with the increasing health consciousness of the general public. It has also been reported to have nutritional and physiological effects, such as promoting lipid metabolism and relieving muscle fatigue. However, when soybeans are used in food products, the soybean off-flavor, or “bean smell,” becomes a problem. If soybean off-flavor is detected in food products, consumers will not like them.

[0003] Humans have just under 400 types of olfactory receptors, and each odorant has a specific receptor that responds to it. A method has been proposed to search for odor suppressants using odorants and the olfactory receptors that respond specifically to them (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent No. 6104058 Summary of the Invention [Problem to be solved by the invention]

[0005] Components such as n-hexanal and 2-acetyl-2-thiazoline have been identified as causative components of soybean off-flavor. However, the olfactory receptors that respond to these causative components are unknown, and it has not been possible to search for soybean off-flavor suppressants using olfactory receptors. In view of the above problems, an object of the present invention is to provide a method for searching for a soybean off-flavor suppressing agent and a soybean off-flavor suppressing agent. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have discovered an olfactory receptor that responds to the components that cause soybean off-flavor, and have completed the present invention. The present invention includes the following aspects. [1] A method for discovering a soybean off-flavor suppressant, comprising the following steps (1) to (3): (1) contacting the olfactory receptor OR5K1 with a test substance and a soybean off-flavor-causing substance; (2) measuring the response of olfactory receptors to the test substance and the soybean off-flavor-causing substance based on the contact; (3) A step of selecting test substances that suppress the response of olfactory receptors compared to the control based on the results of the measurement as candidates for soybean off-flavor suppressants. [2] The screening method according to [1], wherein the soybean off-flavor-causing substance is 2-acetyl-2-thiazoline. [3] The searching method described in [1] or [2], wherein the olfactory receptor is an olfactory receptor expressed on the cell membrane of a cell that expresses an olfactory receptor derived from a living organism, or on the cell membrane of a recombinant cell that has been genetically engineered to express an olfactory receptor. [4] The screening method according to any one of [1] to [3], wherein the response of an olfactory receptor is measured by a reporter gene assay. [5] The screening method according to any one of [1] to [4], wherein the control is the response of an olfactory receptor to a soybean off-flavor-causing substance to which no test substance is added. [6] The screening method according to any one of [1] to [4], wherein the control is the response of an olfactory receptor to β-caryophyllene and a substance causing a soybean off-flavor. [7] Soy off-flavor suppressant containing beta-caryophyllene. [8] A food composition containing soybean-derived food ingredients and β-caryophyllene. [Effects of the Invention]

[0007] The present invention provides a method for searching for a soybean off-flavor suppressing agent and a soybean off-flavor suppressing agent. The search method of the present invention allows the discovery of new soybean off-flavor suppressing agents, which can be applied to soybean-derived foods and the like, thereby providing foods with improved palatability. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of evaluating the responsiveness of the olfactory receptor OR5K1 to 2-acetyl-2-thiazoline. [Figure 2] Figure 2 shows the results of the sensory evaluation (n=7, mean ± standard deviation, *: p<0.05). DETAILED DESCRIPTION OF THE INVENTION

[0009] [Method for searching for soybean off-flavor suppressants] The method of the present invention for searching for soybean off-flavor suppressors comprises the following steps (1) to (3). (1) contacting the olfactory receptor OR5K1 with a test substance and a soybean off-flavor-causing substance; (2) measuring the response of olfactory receptors to the test substance and the soybean off-flavor-causing substance based on the contact; (3) A step of selecting test substances that suppress the response of olfactory receptors compared to the control based on the results of the measurement as candidates for soybean off-flavor suppressants.

[0010] Steps (1), (2) and (3) are carried out in this order.

[0011] <Soybean off-flavor suppressant> The screening method of the present invention is used to screen for candidates for soybean off-flavor suppressing agents. A "soy off-flavor suppressor" suppresses soy off-flavors so that they are partially or completely undetectable. Suppressing soy off-flavors is also referred to as masking soy off-flavors. "Off-flavor" refers to a different flavor that is not inherent in a product or sample. For example, it is defined in JIS Z8144:2004. Soybean off-flavor refers to an odor that is also expressed as “beany smell.” In the present invention, the substance causing soybean off-flavor is preferably 2-acetyl-2-thiazoline. Soybeans are the seeds of the legume Glycine max.

[0012] <Process (1)> In step (1), the olfactory receptor OR5K1 is contacted with a test substance and a substance causing a soybean off-flavor. The contact of the olfactory receptor with the test substance and the substance causing a soybean off-flavor is carried out, for example, in an aqueous medium, a buffer solution, or a culture medium. The specific means for contacting the olfactory receptor with the above-mentioned substance can be appropriately selected from known means. The conditions for contact, such as the amount of the test substance and the soybean off-flavor-causing substance, temperature, etc., can also be appropriately set.

[0013] (olfactory receptor OR5K1) In the present invention, the olfactory receptor OR5K1 is used. Olfactory receptors are G protein-coupled receptor proteins expressed in olfactory cells that bind to odorant molecules. When an olfactory receptor binds to an odorant molecule, a response is elicited. Specifically, when an olfactory receptor binds to an odorant molecule, it couples with a G protein to promote cAMP synthesis by adenylate cyclase, increasing the amount of intracellular cAMP and activating downstream signal transduction pathways. The amino acid sequence of the native human (Homo sapiens) olfactory receptor OR5K1 is registered in GenBank, provided by the National Center for Biotechnology Information (NCBI), under the following accession number (if multiple revisions are registered, the latest revision is understood to be the one referred to): Human olfactory receptor OR5K1: NP_001004736

[0014] In this specification, the olfactory receptor OR5K1 is not limited to olfactory receptors having a natural sequence, but also includes olfactory receptors that have an amino acid sequence that is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more identical to the natural amino acid sequence, and that respond to soybean off-flavors.

[0015] The olfactory receptor OR5K1 is preferably expressed on the cell membrane of a cell that expresses an olfactory receptor derived from a living organism, or on the cell membrane of a recombinant cell that has been genetically engineered to express an olfactory receptor. Recombinant cells can be produced by known methods, for example, by transforming cells with a vector incorporating a gene encoding the olfactory receptor.

[0016] (Test substance) The test substance is not particularly limited. The test substance may be a naturally occurring substance or a substance artificially synthesized by chemical or biological methods. The test substance may be a single compound, or a composition or mixture containing two or more compounds. When the soybean off-flavor suppressant is applied to food, etc., the test substance is preferably an approved food additive.

[0017] <Process (2)> In step (2), the response of the olfactory receptors to the test substance and the soybean off-flavor-causing substance resulting from the contact in step (1) is measured. The response of olfactory receptors to the test substance and the soybean off-flavor-causing substances can be measured by known means.

[0018] Specific measurement means include, for example, techniques for measuring the amount of intracellular cAMP, such as ELISA and reporter gene assay. Among these, reporter gene assays are preferred, and more preferred are those that use a luciferase gene as a reporter gene and measure by the luciferin-luciferase reaction. The luciferase gene as a reporter is, for example, placed under the control of a cAMP response element (CRE), so that the luciferase gene is transcribed in response to the amount of intracellular cAMP. Another measurement method is an electrophysiological technique such as a patch clamp method.

[0019] <Process (3)> In step (3), based on the results of the measurement in step (2), test substances that suppress the response of the olfactory receptors compared to the control are selected as candidates for soybean off-flavor suppressants.

[0020] (Control) The control can be selected appropriately. In one embodiment, the control is the response of the olfactory receptor to a soybean off-flavor-causing substance without the addition of a test substance. The measurement is preferably carried out under the same conditions as in step (2), except that the test substance is not added. In another embodiment, the control is the response of the olfactory receptor to other soybean off-flavor suppressants and soybean off-flavor causative substances. The conditions are preferably the same as those for the measurement in step (2), except that other soybean off-flavor suppressants are used instead of the test substance. Other soy off-flavor suppressants include, for example, β-caryophyllene.

[0021] Comparison with a control can be performed by known means. For example, as in the Examples described below, the fold increase value calculated by dividing the measured value of the test substance by the measured value of the control can be used as an index. Alternatively, the difference calculated by subtracting the measured value of the control from the measured value of the test substance can also be used as an index.

[0022] Soybean off-flavor suppressors are screened by selecting test substances that suppress the response of olfactory receptors compared to the control as candidates for soybean off-flavor suppressors. According to the present invention, it is possible to search for soybean off-flavor suppressants without relying on human sensory evaluation. The obtained soybean off-flavor suppressant candidates can be subjected to, for example, human sensory testing to verify their effectiveness as soybean off-flavor suppressants.

[0023] [Soybean off-flavor suppressant] The present invention also provides a soybean off-flavor suppressing agent. In one embodiment, the soybean off-flavor suppressor is a soybean off-flavor suppressor obtained by the method of the present invention for exploring soybean off-flavor suppressors. In another embodiment, the soybean off-flavor suppressor is β-caryophyllene.

[0024] The soy off-flavor suppressant can be contained in products containing soy-derived ingredients, such as foods, beverages, supplements, etc. The products may be in any of solid, gel, or liquid form. Soybean-derived food ingredients include whole soybeans, processed soybeans, and extracted soybean protein.

[0025] Soybean processing methods include heating, steaming, drying, fermentation, etc. Processed soybean products include soy milk, which is the supernatant obtained by adding water to soybeans and then grinding them, and soybean lees, which is the residue; soybean oil, which is obtained by squeezing soybeans, and soybean meal, which is the residue; Specific examples of products containing processed soybeans include tofu, miso, soy sauce, yuba (tofu skin), soybean flour, natto, and soy milk.

[0026] Soy protein can be extracted from soybeans or soybean plants by known extraction and / or separation methods. Soy protein can be obtained in either dry or wet form. Dry soy protein can be used, for example, by mixing with water to form a paste. Specific examples of products containing soy protein include substitute foods that use soy protein. More specifically, these include artificial meat products in which animal protein is replaced with soy protein, and products in which grain ingredients such as wheat and rice are replaced with soy protein. Other examples include processed products such as tofu bars and protein bars. Products in which soy protein is substituted for grain ingredients such as wheat and rice are, for example, dried ingredients for instant noodles (instant cup noodles) and instant cup rice.

[0027] The amount of soybean off-flavor suppressant used can be set appropriately. For example, 100 parts by mass of a product containing soybean-derived food ingredients can be used. -12 parts by mass to 1 part by mass.

[0028] [Food composition] The present invention also provides food compositions. The food composition of the present invention comprises a soy-derived food material and the above-described soy off-flavor suppressor. Accordingly, in one embodiment, the food composition comprises a soy-derived food material and a soy off-flavor suppressor obtained by the above-described method of the present invention for discovering soy off-flavor suppressors. In another embodiment, the food composition comprises a soy-derived food material and β-caryophyllene. Food compositions include solid foods, supplements, gel products, liquid beverages, etc. Specific examples include processed soy products such as tofu, miso, soy sauce, yuba (tofu skin), soybean flour, natto, and soy milk; imitation meat in which animal protein is replaced with soy protein; instant noodles (instant cup noodles) and instant cup rice in which grain ingredients such as wheat and rice are replaced with soy protein; and processed products such as tofu bars and protein bars. Specific embodiments of the soybean-derived food material include those described above in the section "Soybean off-flavor suppressor."

[0029] The content of the soybean off-flavor suppressor in the food composition is, for example, 100 parts by mass of the food composition. -12 parts by mass to 1 part by mass. In food compositions, the soy off-flavor suppressant is preferably an added ingredient, i.e., an ingredient that is not derived from soy-derived food ingredients or other food raw materials. The food composition of the present invention is a food composition in which soybean off-flavor is suppressed.

[0030] The food composition of the present invention comprises a step of adding a soybean off-flavor suppressor during the production process of a food composition containing various known soybean-derived food materials. Therefore, the present invention also provides a method for suppressing soy off-flavor in a food composition containing a soy-derived food material. [Example]

[0031] Reference example 1 (1) Generation of HEK293T cells expressing olfactory receptors A reaction solution having the composition shown in Table 1 below was prepared and added to wells of a 96-well plate. After leaving the mixture in a clean bench for 20 minutes, HEK293T cells (1.4 × 10 5 100 μL of each of the cells / ml was seeded into each well and cultured for 24 hours in an incubator at 37°C and in a 5% CO2 environment.

[0032] [Table 1]

[0033] The pME18S-RTP1S vector encodes the chaperone protein RTP1S (Receptor Transporting Protein 1S) on the eukaryotic expression vector pME18S. The pME18S-Rho OR5K1 vector encodes the olfactory receptor OR5K1 with an N-terminal Rho tag.

[0034] The resulting HEK293T cells express the olfactory receptor OR5K1 on the cell membrane due to the function of the chaperone protein RTP1S. In addition, the olfactory receptors bound to the odor molecules as ligands were expressed by the Gαs This activates adenylate cyclase by conjugating with , thereby increasing intracellular cAMP levels. Cells express firefly luciferase (luc2P) under the control of the cAMP response element (CRE) via the pGL4.29 vector, resulting in expression of firefly luciferase in response to intracellular cAMP levels. In addition, cells express Renilla luciferase (hRluc) under the control of the HSV-TK promoter via the pGL4.74 vector, which is used as an internal control.

[0035] (2) Luciferase assay The medium was removed from each well containing the HEK293T cells expressing the olfactory receptors obtained in (1) above, and 60 μL of a 2-acetyl-2-thiazoline solution with the composition shown in Table 2 below was added to the well. The cells were cultured for 3 hours in an incubator at 37°C in a 5% CO2 environment, allowing sufficient expression of firefly luciferase and Renilla luciferase in the cells.

[0036] [Table 2]

[0037] Luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. The relative activity of firefly luciferase when 2-acetyl-2-thiazoline was added at each concentration was divided by the relative activity of firefly luciferase when 2-acetyl-2-thiazoline was not added, and the fold increase at that concentration was calculated as an index of the strength of the cell response to 2-acetyl-2-thiazoline. The relative activity value of firefly luciferase is a value obtained by standardizing the measured activity value (amount of luminescence) of firefly luciferase with the activity value (amount of luminescence) of hRluc. As a control, a similar test was performed using cells transfected with the pME18S-Rho vector, which does not encode an olfactory receptor (OR), instead of the pME18S-Rho OR5K1 vector (mock). The results are shown in Figure 1.

[0038] The olfactory receptor OR5K1 responds to 2-acetyl-2-thiazoline in a concentration-dependent manner.

[0039] Example 1 (1) Luciferase assay HEK293T cells expressing the olfactory receptor OR5K1 were prepared in the same manner as in Reference Example 1(1). The medium was removed from each well, and 60 μL of a screening solution having the composition shown below was added. The cells were cultured in an incubator at 37°C and 5% CO2 for 3 hours to allow sufficient intracellular expression of firefly luciferase and Renilla luciferase. The test substance was added at a concentration selected for each individual test substance that would not affect HEK293T cells.

[0040] [Table 3]

[0041] The fold increase was calculated for each test substance in the same manner as in (2) of Reference Example 1. The inhibition rate (%) was then calculated according to the following formula. Candidate substances for the next sensory test were selected using inhibition rates (%) of 30% or higher as an index.

[0042] Suppression rate (%)=(1-B / A)×100 A: Fold increase when 2-acetyl-2-thiazoline alone was added B: Fold increase when a combination of 2-acetyl-2-thiazoline and the test substance was added

[0043] The results are shown in Table 4. β-Caryophyllene, d-limonene, furfural, and isopulegol were selected as candidate inhibitors of 2-acetyl-2-thiazoline.

[0044] [Table 4]

[0045] (2) Sensory evaluation The selected compounds, β-caryophyllene, d-limonene, furfural, and isopulegol, were evaluated for their suppressive effect on the odor of 2-acetyl-2-thiazoline by sensory evaluation. The evaluation was carried out by seven trained panelists who are skilled in evaluating odors. Sensory evaluation samples 1 to 4, with the compositions shown in Table 5 below, were prepared using water as the solvent, and each panelist evaluated the intensity of the 2-acetyl-2-thiazoline odor when smelling them with the tip of their nose. The intensity of 2-acetyl-2-thiazoline alone was assigned a rating of 5, and the odor was rated on an 11-point scale from 0 to 10. The concentrations of each 2-acetyl-2-thiazoline inhibitor candidate were set to subthreshold concentrations, which are concentrations at which the odor of the compound itself is not detectable. The sensory evaluation samples were presented to panelists blind.

[0046] [Table 5]

[0047] The results are shown in Figure 2. In the figure, * indicates p<0.05 in Dunnett's test, with a standard value of 5, which is the intensity when 2-acetyl-2-thiazoline is used alone.

[0048] It was revealed that the soybean off-flavor suppressing agent obtained by the screening method of the present invention also exhibited excellent effects in sensory evaluation. Under the conditions of the sensory evaluation carried out, β-caryophyllene showed a particularly high effect. Even if a compound has a high inhibitory effect on olfactory receptors, it may not be suitable as an inhibitor if it has a strong odor itself.

Claims

1. A method for discovering a soybean off-flavor suppressant, comprising the following steps (1) to (3): (1) contacting the olfactory receptor OR5K1 with a test substance and a soybean off-flavor-causing substance; (2) measuring the response of olfactory receptors to the test substance and soybean off-flavor-causing substances based on the contact; (3) A step of selecting test substances that suppress the response of olfactory receptors compared to the control based on the results of the measurement as candidates for soybean off-flavor suppressants.

2. 2. The screening method according to claim 1, wherein the soybean off-flavor-causing substance is 2-acetyl-2-thiazoline.

3. The searching method according to claim 1, wherein the olfactory receptor is an olfactory receptor expressed on the cell membrane of a cell that expresses an olfactory receptor derived from a living organism or on the cell membrane of a recombinant cell that has been genetically engineered to express an olfactory receptor.

4. The screening method according to claim 1, wherein the response of an olfactory receptor is measured by a reporter gene assay.

5. The screening method according to claim 1, wherein the control is the response of the olfactory receptor to a soybean off-flavor-causing substance without the addition of a test substance.

6. The screening method according to claim 1, wherein the control is the response of olfactory receptors to β-caryophyllene and soy off-flavor-causing substances.

7. Soy off-flavor suppressant containing beta-caryophyllene.

8. A food composition comprising a soy-derived food material and β-caryophyllene.

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