Method for screening raw material for inhibiting malodor

Abstract

Provided are a method and the like for efficiently screening for a candidate substance for a starting material that suppresses hexanal odor. The screening method according to the present invention comprises the steps of: adding a test substance and hexanal to a specific olfactory receptor polypeptide; a step for measuring the response of the olfactory receptor polypeptide to hexanal; and identifying, on the basis of the measured response, a test substance that inhibits the response of the olfactory receptor polypeptide as a candidate substance for a raw material that inhibits hexanal odor.

Description

Technical Field

[0001] The present invention relates to a method for screening candidate substances for raw materials for suppressing bad odor. Background Art

[0002] In recent years, the number of people who are sensitive to the odors and bad smells around them has increased due to the diversification of consumer preferences and living environments. This ranges from odors in living spaces to subtle off-flavors contained in food, and in order to improve the quality of life, technologies that can more effectively reduce and eliminate unpleasant odors are required.

[0003] As representative causes of unpleasant odors in living spaces, aldehyde odors such as hexanal contained in body odor, semi-dry odor, bathroom odor, and nursing odor can be cited (Patent Documents 1 to 3, Non-Patent Document 1). In addition, aldehyde odor, which is one of the representative odors in the living room of an average family, is considered to be an odor group with a high contribution to the odor in the living room and is considered to be an important deodorization target (Non-Patent Document 2). It is generally believed that aldehydes such as hexanal contained in body odor are derived from unsaturated fatty acids and are generated by peroxidation reactions and decompositions catalyzed by enzymes such as lipoxygenases based on metabolism in the body and resident microorganisms present on the surface of the body. In particular, in the perfume industry, hexanal is also an important unpleasant odor component in perfumes used to reproduce sweat odor, body odor, and scalp odor (Patent Documents 4, 5). Furthermore, aldehydes such as hexanal are not only produced by the decomposition / metabolism of sebum shed from the human body and oils used for conditioning by microorganisms resident in the living space, but are also naturally produced by oxidation reactions with oxygen in the air (Non-Patent Document 2).

[0004] It is difficult to completely suppress the generation of aldehydes such as hexanal that are naturally generated in living spaces through metabolism and oxidation reactions, and there is a widespread demand for odor control (masking) technology that selectively makes unpleasant odors in living spaces using fragrances less perceptible.

[0005] In addition, hexanal is known as a "grassy smell" and is also a foreign smell produced when soybeans and dairy products deteriorate, and is also one of the factors that reduce the palatability of food (Non-Patent Literature 3, 4). In soybeans, soybean lipoxygenase acts on unsaturated fatty acids to produce lipid peroxides, and lipid hydroperoxide lyases (hydroperoxidelyas) act on the produced lipid peroxides to generate aldehydes such as n-hexanal, which is the cause of the grassy smell. In dairy products, during the processing / storage of raw milk, the linoleic acid contained in the milk fat is subjected to enzyme reactions and photooxidation to produce aldehydes represented by n-hexanal, thereby producing a different odor from the usual. Therefore, since fatty acids are contained, it is difficult to completely eliminate the production of hexanal in food, and it is important to explore flavor raw materials that do not make people feel the hexanal odor that is particularly felt when eating.

[0006] Thus, the search for fragrance raw materials that are less likely to smell hexanal is very important for improving the quality of life in terms of food, clothing, housing and transportation. However, in the conventional method of searching for unpleasant odor reduction / deodorizing substances, it is necessary for odor evaluation experts to confirm a large number of candidate substances for raw materials that suppress malodor one by one based on sensory evaluation, which is inefficient. On the other hand, in the molecular biology method established in recent years, by searching for olfactory receptors that respond to malodor source substances and simultaneously screening in vitro for malodor suppressing raw materials that suppress the response of the olfactory receptors, it is possible to quickly identify malodor suppressing raw materials.

[0007] There are about 400 kinds of human olfactory receptors. It is known that a variety of olfactory receptors respond to one odorant, but most of the responding receptors are so-called broad-spectrum olfactory receptors, which recognize a wide range of odorants indiscriminately. Non-patent document 5 mentions / exemplifies broad-spectrum olfactory receptors such as OR1A1 and OR2W1 that respond widely regardless of the fragrance tone. Patent document 6 discloses that broad-spectrum olfactory receptors OR1A1, OR2W1, and OR10A6 are responsible for adjusting the sensitivity of odors, and that by enhancing or inhibiting the response of the olfactory receptors, the sensitivity of odors can be enhanced or inhibited.

[0008] Non-Patent Document 6 discloses a broad-spectrum olfactory receptor OR2W1 as an olfactory receptor involved in the perception of hexanal, which is the cause of the grassy smell of soybeans, and identifies α-pinene and the like as a masking agent that inhibits the response of OR2W1.

[0009] Thus, although olfactory receptors that are thought to respond to hexanal are known, in in vitro tests, the only olfactory receptor that shows a concentration-dependent hexanal response and is actually involved in the recognition of hexanal odor is the broad-spectrum olfactory receptor OR2W1.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent Application Laid-Open No. 11-286423

[0013] Patent Document 2: Japanese Patent No. 4950927

[0014] Patent Document 3: Japanese Patent Application Publication No. 2007-14749

[0015] Patent Document 4: Japanese Patent Application Publication No. 2004-263102

[0016] Patent Document 5: Japanese Patent Application Publication No. 2011-184469

[0017] Patent Document 6: Japanese Patent Application Publication No. 2018-121623

[0018] Patent Document 7: WO2019 / 230903

[0019] Patent Document 8: WO2021 / 193429

[0020] Non-patent literature

[0021] Non-patent document 1: Ogata Noriya, "Design of deodorization function for comfortable space in nursing care", Kyoto City Industrial Technology Research Institute Research Report, No. 9, p. 73-76 (2014)

[0022] Non-patent document 2: Yuko Nakamura et al., Investigation of actual status of indoor odor components in homes with different living environments, Indoor Environment, Vol. 17, No. 1, pp. 1-9 (2014)

[0023] Non-patent literature 3: Food Sci. Nutr., 2020 Jan 14; 8(2): 1139-1149.

[0024] Non-patent document 4: Milk Science, Vol. 57, No. 3, 2008

[0025] Non-patent document 5: Flavour Fragr. J., 2015, 30, 342-361

[0026] Non-patent document 6: Yuko Terada et al., Identification of olfactory receptors related to the perception of n-hexanal, which is the cause of the grassy smell of soybeans, Soybean Protein Research, Vol. 23, p. 159-164 (2020)

[0027] Non-patent literature 7: Christophe Verbeurgt et al, PLoS One., 2014.9(5):e96333 (https: / / doi.org / 10.1371 / journal.pone.0096333) Summary of the invention

[0028] Problem that the invention aims to solve

[0029] For the use of masking agents that target broad-spectrum olfactory receptors that respond to a wide range of odors and adjust the sensitivity of odors, in order to suppress a wide range of odors indiscriminately, it is undeniable that there is a possibility that the preferred odors contained in foods and spices are even masked, and the overall odor is weakly felt. In such a case, it is desired to identify the olfactory receptor group that is truly important for recognizing hexanal odor itself, rather than the broad-spectrum olfactory receptors that recognize a wide range of odors indiscriminately, and to develop a method for efficiently screening candidate substances for raw materials that suppress hexanal odor.

[0030] Solutions for solving problems

[0031] The present invention has been made in consideration of the above circumstances, and provides a method for screening candidate substances for a raw material that suppresses hexanal odor as shown below.

[0032] [1] A method for screening candidate substances for raw materials that inhibit hexanal odor, the method comprising the following steps:

[0033] A step of adding a test substance and hexanal to at least one olfactory receptor polypeptide selected from the group consisting of olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1, and polypeptides comprising an amino acid sequence having 80% or more homology to the amino acid sequence of each of the aforementioned olfactory receptors and showing responsiveness to hexanal, a malodor-derived substance;

[0034] a step of determining the response of the aforementioned olfactory receptor polypeptide to hexanal; and

[0035] A step of identifying a test substance that inhibits the response of the aforementioned olfactory receptor polypeptide as a candidate substance for a raw material for inhibiting hexanal odor based on the measured response.

[0036] [2] The method according to [1], wherein the hexanal odor is an odor derived from hexanal, or an odor that at least partially contains this odor, and is an odor that causes unpleasantness to people, and is further an odor that is one or more of body odor, sweat odor, body odor, scalp odor, semi-dry odor, bathroom odor, nursing odor, and spoiled odor of soybeans or dairy products.

[0037] [3] The method according to [1] or [2], wherein the response of the olfactory receptor polypeptide to the hexanal odor-derived substance is measured in cells isolated from a living organism that express the olfactory receptor, or in cells artificially expressing the olfactory receptor through genetic manipulation.

[0038] [4] The method according to any one of [1] to [3], wherein the response of the olfactory receptor polypeptide is measured by reporter gene assay or calcium imaging.

[0039] Effects of the Invention

[0040] By using the method of the present invention, candidate substances for raw materials for inhibiting hexanal odor that can inhibit the binding of hexanal to the olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1 can be screened. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a diagram showing the results of measuring the response of the olfactory receptor OR2C1 to hexanal.

[0042] Figure 2 This is a diagram showing the results of measuring the response of the olfactory receptor OR2J2 to hexanal.

[0043] Figure 3 This is a diagram showing the results of measuring the response of the olfactory receptor OR2J3 to hexanal.

[0044] Figure 4 This is a diagram showing the results of measuring the response of the olfactory receptor OR5K1 to hexanal.

[0045] Figure 5 This is a diagram showing the results of measuring the response of the olfactory receptor OR5P3 to hexanal.

[0046] Figure 6 This is a diagram showing the results of measuring the response of the olfactory receptor OR51E1 to hexanal.

[0047] Figure 7 This is a graph showing the effect of suppressing the response of the olfactory receptor OR2C1 to hexanal by adding β-caryophyllene oxide, a raw material for suppressing hexanal odor.

[0048] Figure 8 This is a graph showing the effect of suppressing the response of the olfactory receptor OR2J2 to hexanal by adding menthyl lactate, a raw material for suppressing the hexanal odor.

[0049] Fig. 9 This is a graph showing the effect of suppressing the response of the olfactory receptor OR2J3 to hexanal by adding nootkatone, a raw material for suppressing the hexanal odor.

[0050] Fig.10 This is a graph showing the effect of suppressing the response of the olfactory receptor OR5K1 to hexanal by adding β-caryophyllene oxide, a raw material for suppressing hexanal odor.

[0051] Fig.11 This is a graph showing the effect of suppressing the response of the olfactory receptor OR5P3 to hexanal by adding menthyl lactate, a raw material for suppressing the hexanal odor.

[0052] Fig.12This is a graph showing the effect of suppressing the response of the olfactory receptor OR51E1 to hexanal by adding β-caryophyllene oxide, a raw material for suppressing hexanal odor. DETAILED DESCRIPTION

[0053] SUMMARY OF THE INVENTION

[0054] This specification includes the entirety of the Japanese Patent Application No. 2022-144633 (filed on September 12, 2022), which is the basis for the priority claim of this application. In addition, all publications cited in this specification, such as prior art documents, public gazettes, patent gazettes, and other patent documents, are incorporated into this specification as references.

[0055] The present invention provides a method for efficiently screening candidate substances for suppressing the hexanal odor by searching for the true olfactory receptor group that responds to hexanal in a concentration-dependent manner, and further provides a composition for suppressing the hexanal odor that reduces the unpleasant feeling of the hexanal odor.

[0056] The present inventors have newly discovered that among the approximately 400 existing human olfactory receptors, specific receptors, namely, OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1, respond to the malodor source substance hexanal in a concentration-dependent manner. In addition, a new insight has been discovered that the known raw materials for inhibiting hexanal odor, which have been proven to have the ability to inhibit hexanal odor in sensory tests based on human olfaction, can inhibit the above-mentioned specific olfactory receptors, and the above-mentioned specific olfactory receptor group has been determined to be a real olfactory receptor group that is important for the recognition of hexanal odor. As a result, it is possible to evaluate and select the raw materials for inhibiting hexanal odor based on the masking effect of olfactory receptor antagonists. The present invention was discovered based on such new insights.

[0057] Hereinafter, the method for screening candidate substances of a raw material for suppressing hexanal odor of the present invention (hereinafter referred to as the screening method of the present invention) will be specifically described.

[0058] As mentioned above, the screening method of the present invention comprises the following steps:

[0059] Adding a test substance and hexanal to at least one olfactory receptor polypeptide selected from the group consisting of human olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3, and OR51E1, and polypeptides comprising an amino acid sequence having 80% or more homology to the amino acid sequence of each of the aforementioned olfactory receptors and showing responsiveness to hexanal;

[0060] measuring the response of the aforementioned olfactory receptor polypeptide to hexanal; and

[0061] Based on the measured response, the test substance that inhibits the response of the olfactory receptor polypeptide is identified as a candidate substance for a raw material for inhibiting hexanal odor. The present invention can be implemented, for example, according to the following embodiments.

[0062] As one embodiment, the present invention is a method for screening candidate substances for raw materials that inhibit hexanal odor from test substances using OR2C1, OR2J2, OR2J3, OR5K1, OR5P3, and OR51E1 that are responsive to hexanal, a malodor source substance, characterized in that the method comprises the following steps:

[0063] Step (i), contacting an olfactory receptor selected from the group consisting of olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1, and a protein (polypeptide) comprising an amino acid sequence having 80% or more homology with the amino acid sequence of each of the above olfactory receptors and showing responsiveness to hexanal with hexanal, and measuring a basal activity response (T0) of the olfactory receptor before contact with hexanal and a response (Tx) of the olfactory receptor to hexanal at any time after contact with hexanal;

[0064] step (ii), comparing the measurement result in step (i), i.e., the basal activity response (T0) of the olfactory receptor before contact with hexanal, with the response (Tx) of the olfactory receptor to hexanal at any time after contact with hexanal, and calculating and obtaining a change value of the responsiveness;

[0065] (iii) a step of mixing the test substance with hexanal in the step (i) and calculating and obtaining a change in responsiveness in the same manner as in the step (ii);

[0066] In step (iv), a test substance whose value in step (iii) is smaller than that in step (ii) is selected as a candidate substance for a raw material for suppressing hexanal odor.

[0067] In the screening method of the present invention, the responsiveness of the test substance to an olfactory receptor selected from the group consisting of olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1, and a protein (polypeptide) comprising an amino acid sequence having 80% or more homology with the amino acid sequence of each of the aforementioned olfactory receptors and showing responsiveness to hexanal is used as an index to select a candidate substance for a raw material for inhibiting hexanal odor from the test substances.

[0068] In the in vitro olfactory receptor assay, usually, the evaluation is performed under the premise that the expression levels of the olfactory receptors are all the same, and it is believed that the higher the response value per olfactory receptor unit, the more sensitive the olfactory receptor. However, the expression level of the olfactory receptors actually expressed in the human olfactory epithelium varies according to the type of olfactory receptor, and the olfactory receptors that are strongly responsive in the in vitro assay may not necessarily transmit the odor sensation present in the olfactory nerve cells of the human olfactory epithelium to the brain center strongly. Therefore, it is speculated that even if the olfactory receptors have a small response value to the odorant based on the in vitro olfactory receptor assay, the response based on the odor stimulus in the olfactory nerve cells that actually express the olfactory receptors with a large expression level in the human olfactory epithelium will also become stronger, which greatly contributes to odor recognition. According to the aforementioned non-patent document 7, the actual expression levels of human olfactory receptors are as follows: the average expression level of OR2W1, a broad-spectrum olfactory receptor, is 6±13, which is very low. On the other hand, the average expression level of hexanal olfactory receptors finally determined by the inventors of the present application after in-depth research is disclosed as follows: the average value of OR2C1 is 78, the average value of OR2J2 is 37, the average value of OR2J3 is 82, the average value of OR5K1 is 598, the average value of OR5P3 is 752, and the average value of OR51E1 is 170. It can be seen that among all the determined olfactory receptors, the expression level is generally high, and it is an olfactory receptor with a high contribution to the recognition of hexanal odor in human olfaction. Therefore, by evaluating the responsiveness of the test substance by taking the hexanal olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1 as targets, a candidate substance that can hinder the binding of hexanal to the olfactory receptor can be selected from the test substance. It should be noted that, in this specification, "test substance" is not particularly limited, and refers to the object of investigation of the effect of inhibiting hexanal odor, and refers to a compound, composition or mixture. In addition, in this specification, "raw material for inhibiting hexanal odor" is not particularly limited, and refers to a compound, composition or mixture that can inhibit hexanal odor. Below, each step of the screening method of the present invention (the aforementioned steps (i) to (v)) is described.

[0069] <Step (i)>

[0070] In step (i), an olfactory receptor selected from the group consisting of olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1, and a protein (polypeptide) comprising an amino acid sequence having 80% or more homology to the amino acid sequence of each of the aforementioned olfactory receptors and showing responsiveness to hexanal is brought into contact with hexanal, and a basal activity response (T0) of the olfactory receptor before contact with hexanal and a response (Tx) of the olfactory receptor to hexanal at any time after contact with hexanal are measured.

[0071] As the olfactory receptor, an olfactory receptor selected from the group consisting of OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1, and a protein (polypeptide) comprising an amino acid sequence having 80% or more homology with the amino acid sequence of each of the aforementioned olfactory receptors and showing responsiveness to hexanal is used.

[0072] OR2C1 is registered as "Accession number: NM_012368.2" and "Accession number: NP_036500.2" on the NCBI (GenBank) website (http: / / www.ncbi.nlm.nih.gov / ), and is a protein (polypeptide) consisting of an amino acid sequence (SEQ ID NO: 2) encoded by the DNA of base numbers 53 to 991 of the sequence shown in SEQ ID NO: 1.

[0073] OR2J2 is registered as "Accession number: NM_030905.2" and "Accession number: NP_112167.2" on the NCBI (GenBank) website, and is a protein (polypeptide) consisting of an amino acid sequence (SEQ ID NO: 4) encoded by the DNA of base numbers 103 to 1041 of the sequence shown in SEQ ID NO: 3.

[0074] OR2J3 is registered as "Accession number: NM_001005216.3" and "Accession number: NP_001005216.2" on the NCBI (GenBank) website, and is a protein (polypeptide) consisting of an amino acid sequence (SEQ ID NO: 6) encoded by the DNA of base numbers 82 to 1017 of the SEQ ID NO: 5.

[0075] OR5K1 is registered as "Accession number: NM_001004736.3" and "Accession number: NP_001004736.2" on the NCBI (GenBank) website, and is a protein (polypeptide) consisting of an amino acid sequence (SEQ ID NO: 8) encoded by the DNA of base numbers 98 to 1024 of the sequence shown in SEQ ID NO: 7.

[0076] OR5P3 is registered as "Accession number: NM_153445.2" and "Accession number: NP_703146.1" on the NCBI (GenBank) website, and is a protein (polypeptide) consisting of an amino acid sequence (SEQ ID NO: 10) encoded by the DNA of base numbers 39 to 974 of the sequence shown in SEQ ID NO: 9.

[0077] OR51E1 is registered as "Accession number: NM_152430.3" and "Accession number: NP_689643.2" on the NCBI (GenBank) website, and is a protein (polypeptide) consisting of an amino acid sequence (SEQ ID NO: 12) encoded by the DNA of base numbers 145 to 1101 of the sequence shown in SEQ ID NO: 11.

[0078] The olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1 selectively respond to hexanal odor, and therefore, the screening method using OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1 can contribute to the development of raw materials that inhibit hexanal odor.

[0079] As the olfactory receptor, an olfactory receptor selected from the group consisting of OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1, and a protein (polypeptide) comprising an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and particularly preferably 98% or more homology with the amino acid sequence of each of the above olfactory receptors and showing responsiveness to hexanal can be used. It should be noted that the sequence homology of the amino acid sequence in this specification can be calculated according to the BLAST search algorithm (which can be publicly obtained from NCBI).

[0080] The olfactory receptors may be used alone or in combination of two or more.

[0081] In the present invention, the hexanal odor refers to an odor derived from hexanal, or an odor containing at least a part of the odor, and is preferably not limited as long as it is an odor that causes unpleasantness to people, and examples thereof include body odor, sweat odor, body odor, scalp odor, semi-dry odor, bathroom odor, nursing odor, and spoiled odor of soybeans or dairy products.

[0082] In the present invention, the method of contacting the olfactory receptor with hexanal and measuring the response of the olfactory receptor to the hexanal odor is not particularly limited. For example, the cells expressing the olfactory receptor separated from the organism can be contacted with hexanal to measure the response of the olfactory receptor, or the cells artificially expressing the olfactory receptor by genetic manipulation can be contacted with hexanal to measure the response of the olfactory receptor. The time for contacting the olfactory receptor with hexanal also depends on the concentration of hexanal and the measurement method, and therefore cannot be generalized, but in the luciferase measurement method of the present invention, it is 0 to 60 minutes, preferably 0 to 30 minutes, and in the calcium imaging method, it is about several seconds to several minutes.

[0083] By transforming cells with a vector into which a gene encoding an olfactory receptor has been inserted, it is possible to produce cells in which an olfactory receptor is artificially expressed by genetic manipulation.

[0084] In a preferred embodiment of the present invention, the N-terminal 20 amino acid residues of bovine rhodopsin may be inserted together with the olfactory receptor. By inserting the N-terminal 20 amino acid residues of bovine rhodopsin, the cell membrane expression of the olfactory receptor can be promoted.

[0085] Bovine rhodopsin is registered as "Accession number: NM_001014890.2" and "Accession number: NP_001014890.1" on the NCBI (GenBank) website. Bovine rhodopsin is a protein (polypeptide) consisting of an amino acid sequence (SEQ ID NO. 14) encoded by DNA of base numbers 1 to 1047 of the sequence shown in SEQ ID NO. 13.

[0086] Alternatively, a protein (polypeptide) comprising an amino acid sequence having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and particularly preferably 98% or more to the amino acid sequence shown in SEQ ID NO: 14 and capable of promoting cell membrane expression of olfactory receptors may be used instead of bovine rhodopsin.

[0087] It should be noted that the amino acid residues are not limited to bovine rhodopsin and may be amino acid residues of other proteins (polypeptides) as long as they can promote the cell membrane expression of olfactory receptors.

[0088] The method for measuring the response of the olfactory receptor is not particularly limited, and any method used in the art can be used. For example, it is known that when an aroma compound binds to an olfactory receptor, the G protein in the cell is activated, and the G protein activates adenylate cyclase, converts ATP into cyclic AMP (cAMP), and increases the amount of cAMP in the cell. Therefore, by measuring the amount of cAMP, the response of the olfactory receptor can be measured. As a method for measuring the amount of cAMP, ELISA, reporter gene analysis, etc. can be used. Among them, it is preferred to measure the response of the olfactory receptor by using a reporter gene analysis method using a luminescent substance such as luciferase.

[0089] <Step (ii)>

[0090] In step (ii), the basal activity response (T0) of the olfactory receptor before contact with hexanal, which is the measurement result in step (i), is compared with the response (Tx) of the olfactory receptor to hexanal at any time after contact with hexanal, and the change value of the responsiveness is calculated and determined.

[0091] According to one embodiment of the present invention, the change in responsiveness can be evaluated using a fold increase value as an index, the fold increase value being obtained by dividing the response (Tx) of the olfactory receptor to hexanal at any time after the contact with hexanal in step (i) by the measurement result of the basal activity response (T0) of the olfactory receptor before the contact with hexanal. For example, when the response of the olfactory receptor is measured by a reporter gene analysis method using a luminescent substance such as luciferase, the odor source substance at a concentration where the fold increase value is preferably 1 or more, more preferably 1.2 or more, and even more preferably 2 or more can be used for evaluation.

[0092] <Process (iii)>

[0093] In step (iii), the test substance is mixed with hexanal in step (i), and the change value of the responsiveness is calculated and determined in the same manner as in step (ii).

[0094] <Step (iv)>

[0095] In step (iv), the test substance whose change value obtained in step (iii) is smaller than the change value obtained in step (ii) is selected as a candidate substance for a raw material for suppressing bad odor.

[0096] In the present invention, when the comparison of the measurement results in steps (ii) and (iii) shows that the change value of the responsiveness is reduced, the test substance used in step (iii) can be evaluated as a candidate substance for a raw material for suppressing bad odor.

[0097] In the present invention, when the measurement results of the change values ​​in steps (ii) and (iii) are compared and a decrease in the change value of the responsiveness is observed, the test substance used in the evaluation step (iii) can be evaluated as a candidate substance for a raw material for suppressing bad odor.

[0098] As described above, candidate substances for raw materials that inhibit hexanal odor can be screened out from test substances. According to the present invention, candidate substances for raw materials that inhibit hexanal odor can be selected from a large number of test substances without causing problems such as olfactory fatigue and individual differences caused by sensory evaluation based on human sense of smell.

[0099] The selected substance can be used as a candidate substance for a raw material for suppressing the hexanal odor. It is also possible to develop a novel compound with the best odor based on the selected substance and to make changes as needed. Furthermore, the selected substance can be blended with other perfume raw materials to suppress the hexanal odor and develop a perfume raw material with the best odor. By using the screening method of the present invention, a new perfume raw material as a raw material for suppressing the hexanal odor can be developed.

[0100] Example

[0101] Hereinafter, the present invention will be described in further detail with reference to Examples, but the present invention is not limited to these Examples at all.

[0102] [Example 1]

[0103] Confirmation of the response of olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3, and OR51E1 to hexanal

[0104] (1) Olfactory receptor gene cloning

[0105] The human olfactory receptor gene was obtained by cloning from Human Genomic DNA: Female (Promega) by PCR based on the base sequence information registered on the NCBI (GenBank) website (see sequence numbers 1, 3, 5, 7, 9 and 11). DNA encoding the N-terminal 20 amino acid residues of bovine rhodopsin (DNA consisting of bases 1 to 60 of the base sequence of sequence number 13) was inserted into the pME18S vector, and the obtained human olfactory receptor gene was further inserted downstream thereof, thereby obtaining a human olfactory receptor gene expression vector.

[0106] (2) Screening of expression of olfactory receptor genes responsive to hexanal

[0107] 0.05 μg of human olfactory receptor gene expression vector, 0.01 μg of RTP1S vector, and pGloSensor containing a CMV promoter were added. TM -22F cAMP Plasmid (Promega) 0.01μg was dissolved in Opti-MEM I (gibco) 10μL to form a gene solution (1 well). 100μL of HEK293T cells were seeded into a 96-well plate (Biocoat, Corning) at a subconfluent cell number after 24 hours. According to the usage of Lipofectamine 3000, the gene solution was added to each well by liposome transfection method, and the gene was introduced into the cells. The cells were cultured at 37°C and 5% carbon dioxide atmosphere for 24 hours. After removing the culture solution, wash once with HEPES buffer, and add GloSensor adjusted to a specified concentration with HEPES buffer containing FBS to each well. TM 100 μL of cAMP Reagent (Promega) was left to stand at room temperature for 2 hours. Afterwards, 10 μL of a 10 mM solution of hexanal prepared with a HEPES buffer containing 300 μM CuCl2 and FBS was added to each well, and the response value of the luciferase activity was measured every 1 minute from the time of addition, thereby obtaining the response of about 400 human olfactory receptors to hexanal. The response intensity of the olfactory receptor is expressed by the maximum response value of the luciferase activity produced after hexanal stimulation divided by the luciferase activity before hexanal stimulation to obtain the multiple value. Thus, OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1 can be determined as candidates for olfactory receptors that respond to hexanal. After that, the aroma compound of the test object was prepared in a HEPES buffer containing 300 μM CuCl2 and FBS in a concentration 10 times the final concentration, and 10 μL of the aroma compound stimulation solution was added to each well. The change in luciferase activity over time was measured every 1 minute from the time of addition, thereby obtaining the response of about 400 human olfactory receptors to the aroma compound. The response intensity of the olfactory receptor was expressed as the multiple value obtained by dividing the maximum value of luciferase activity generated by the stimulation of the aroma compound by the luciferase activity before the stimulation of the aroma compound.

[0108] (3) Identification of olfactory receptors responsive to hexanal

[0109] The above luciferase activity assay was used to measure the responses of olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3, and OR51E1 to hexanal, a malodorous substance, at various concentrations. The results are shown in Figures 1 to 6 . OR2C1, OR2J2, OR2J3, OR5K1, OR5P3, and OR51E1 showed various responses to hexanal in a concentration-dependent manner. On the other hand, no response was observed in the Mock test (using cells that do not express olfactory receptors). That is, OR2C1, OR2J2, OR2J3, OR5K1, OR5P3, and OR51E1 showed responses specifically to hexanal.

[0110] [Example 2]

[0111] Evaluation of the response suppression effect of OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1 based on raw materials for suppressing hexanal odor

[0112] For the known raw materials for inhibiting hexanal odor that are known to mask the hexanal odor in the aforementioned Patent Documents 7 and 8: menthyl lactate, nootkatone, and β-caryophyllene oxide, the response inhibitory effect of the hexanal olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3, and OR51E1 was determined by the following luciferase assay.

[0113] (1) Luciferase assay

[0114] 0.05 μg of each gene expression vector of human olfactory receptor OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1, 0.01 μg of RTP1S vector, and pGloSensor, a firefly luciferase vector containing a CMV promoter, were added. TM -22F cAMPPlasmid (Promega) 0.01μg was dissolved in Opti-MEM I (gibco) 10μL to form a gene solution (1 well). 100μL of HEK293T cells were seeded into a 96-well plate (Biocoat, Corning) at a subconfluent cell number after 24 hours. According to the usage of Lipofectamine 3000, the gene solution was added to each well by liposome transfection method to introduce genes into the cells, and cultured for 24 hours at 37°C and 5% carbon dioxide atmosphere. After removing the culture solution, wash once with HEPES buffer, and add GloSensor adjusted to a specified concentration with HEPES buffer containing FBS to each well. TMcAMP Reagent (Promega) 100 μL, let stand at room temperature for 2 hours. After that, add 10 μL of a solution of 7 mM hexanal solution prepared with HEPES buffer containing 300 μM CuCl2 and FBS mixed with 3 mM of a raw material for inhibiting hexanal odor to each well, and measure the time-dependent change of luciferase activity every 1 minute from the time of addition, so as to obtain the time-dependent response of each human olfactory receptor mentioned above. The response intensity of the olfactory receptor is expressed by the multiple value obtained by dividing the luciferase activity generated by the stimulation of the aroma compound by the luciferase activity before the stimulation of the aroma compound.

[0115] (2) Evaluation of the inhibition of olfactory receptor response based on raw materials that inhibit hexanal odor

[0116] In the above luciferase assay, hexanal and a raw material for suppressing hexanal odor: any of menthyl lactate, nootkatone, or β-caryophyllene oxide were mixed and used in the test subject, and the time-dependent change of the multiplier value in the test without mixing the raw material for suppressing hexanal odor and in the test with mixing the raw material for suppressing hexanal odor was determined. The results are shown in Figure 7 to Figure 12 The raw materials for inhibiting the hexanal odor (menthyl lactate, nootkatone, β-caryophyllene oxide) showed the effect of inhibiting the response of the hexanal olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1 ( Figure 7 to Figure 12 ). These results indicate that the known raw materials for inhibiting hexanal odor, represented by menthyl lactate, nootkatone, and β-caryophyllene oxide, inhibit hexanal odor by inhibiting at least one olfactory receptor in the hexanal olfactory receptor group OR2C1, OR2J2, OR2J3, OR5K1, OR5P3, and OR51E1.

[0117] Industrial Applicability

[0118] According to the present invention, a method for screening candidate substances for raw materials for inhibiting hexanal odor that can inhibit the binding of hexanal to the olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1 can be provided.

Claims

1. A method for screening candidate substances of raw materials for inhibiting hexanal odor, the method comprising the steps of: A step of adding a test substance and hexanal to at least one olfactory receptor polypeptide selected from the group consisting of olfactory receptors OR2C1, OR2J2, OR2J3, OR5K1, OR5P3 and OR51E1, and polypeptides comprising an amino acid sequence having 80% or more homology to the amino acid sequence of each olfactory receptor and showing responsiveness to hexanal; a step of determining the response of the olfactory receptor polypeptide to hexanal; and Based on the measured response, a test substance that inhibits the response of the olfactory receptor polypeptide is identified as a candidate substance for a raw material that inhibits hexanal odor.

2. The method according to claim 1, wherein: The hexanal odor is an odor derived from hexanal, or an odor at least partially containing the odor, and is an odor that causes unpleasantness to people in some cases, and is further one or more of body odor, sweat odor, body odor, scalp odor, semi-dry odor, bathroom odor, nursing odor, and spoiled odor of soybeans or dairy products in some cases.

3. The method according to claim 1 or 2, wherein: The response of the olfactory receptor polypeptide to hexanal is measured on cells expressing the olfactory receptor isolated from an organism, or on cells artificially expressing the olfactory receptor by genetic manipulation.

4. The method according to claim 1 or 2, wherein: The response of the olfactory receptor polypeptides is measured by reporter gene assays or calcium imaging.

Images