Screening method for odor-suppressing material

Abstract

The present invention relates to a screening method for odor-suppressing materials. A screening method according to the present invention includes: causing a test substance and an odor-causing substance to act on at least one olfactory receptor polypeptide selected from the group consisting of olfactory receptor OR2AG2 and polypeptides that include an amino acid sequence having 80% or greater identity with the amino acid sequence of the aforementioned olfactory receptor and exhibit responsiveness to odor-causing substances; measuring the response of the olfactory receptor polypeptide to the odor-causing substance; and, on the basis of the measured response, identifying test substances that suppress a response of the olfactory receptor polypeptide as candidate substances for an odor-suppressing material.

Description

Screening method for odor-controlling materials

[0001] The present invention relates to a method for screening candidate malodor control materials.

[0002] In recent years, as consumer preferences and living environments have become more diverse, an increasing number of people are becoming sensitive to odors and bad smells in their surroundings. These odors range from slight off-flavors found in food to unpleasant odors in living spaces. To improve the quality of life, there is a demand for the development of technologies that can effectively reduce or eliminate these bad odors (off-flavors and malodors).

[0003] One of the most significant odors found in food and in living spaces is "dusty odor." An internet search for "dusty taste" reveals that many people perceive this "dusty odor" as an unpleasant taste or odor in a variety of situations. When this "dusty odor" is present, it can be perceived as dusty, earthy, grassy, ​​powdery, medicinal, or moldy. For example, Patent Document 1 describes the problem of chewing gum and other confectioneries that combine the high-sensitivity sweetener aspartame with certain fruit flavors developing a "grassy odor," or "dusty odor," that develops over time and becomes dusty, earthy, powdery, medicinal, or moldy. This "dusty smell" is described in various ways using a combination of expressions such as earthy, dusty, musty, moldy, smoky, galbanum, bell pepper, vegetable, and green pea, and in literature it is often expressed in a two-word contraction ("dominant note-minor note") such as "Earthy-Dusty," "Earthy-Musty," or "Dusty-Moldy." Although this is understood intuitively, there is no standardized way of describing it because it is a sensory thing.

[0004] For example, in processed foods, pyridines or pyrazines, which are generated by chemical reactions of amino acids, flavorings, and additives present in the food during the processing process, are thought to be one of the main causative substances of the "dusty odor." Furthermore, "dusty odors" can also be generated not only by the processing process but also by the action of environmental microorganisms present in nature. For example, in drinking water, alkylmethoxypyrazines such as 2-isopropyl-3-methoxypyrazine and 2-isobutyl-3-methoxypyrazine, which are generated by metabolic decomposition by environmental microorganisms present in ponds and rivers, are contaminated with drinking water, causing the perception of a dusty, earthy, grassy, ​​fishy, ​​or moldy "dusty odor" (Non-Patent Document 1). Furthermore, in living spaces, many literature cites certain pyrazines contained in cigarette smoke, grilled meat, cooking, kitchen odors, food waste, and river odors as causes of "dusty odors." Pyridines and pyrazines are considered to be the main causative substances of "dusty odors," but it has not been determined that all "dusty odors" are pyridines or pyrazines, and not all pyrazines have a "dusty odor." According to Non-Patent Document 2, the odor of pyrazine compounds changes sharply with slight differences in the side chain structure, from herb-like, burnt, nut-like, chocolate-like, earthy, or grassy odors, and pyrazines containing a methoxy group in particular are known to have a very grassy (bell pepper-like), dusty, earthy odor, i.e., a "dusty odor."

[0005] The generation of such "dust odor" reduces the commercial value of food and causes discomfort in living spaces. Therefore, the search for fragrance materials that reduce the "dust odor" is extremely important for improving the quality of life in both aspects of food, clothing, and shelter. The search for such malodor-reducing or deodorizing substances typically involves identifying the malodor-causing substance, followed by a sensory evaluation by an odor evaluation expert, who then identifies a vast number of candidate malodor-suppressing substances one by one. This conventional method suffers from the problem of low throughput. Recently established molecular biology techniques search for olfactory receptors that respond to the identified malodor-causing substance, and then simultaneously screen in vitro for candidate malodor-suppressing substances that act as antagonists that inhibit the olfactory receptor response. This allows for the rapid identification of malodor-suppressing materials, using olfactory receptor antagonists against specific malodor-causing substances as malodor blockers. However, when screening for malodor suppressants that suppress specific olfactory receptor responses, the primary challenge is to identify the substance that causes the malodor, since it is generally necessary to identify the olfactory receptor that responds to the malodor before the substance that causes the malodor can be identified.

[0006] There are approximately 400 types of human olfactory receptors, and a single odorant often responds to several types of olfactory receptors. These receptors are identified by a combinatorial code, which combines the combined responses of narrowly-tuned olfactory receptors, which recognize specific odor notes and structures, and broadly-tuned olfactory receptors, which recognize a wide range of odorants (Non-Patent Document 3). Therefore, in the search for malodor-suppressing materials, it is important to identify the olfactory receptors that are not broadly-tuned but are truly involved in the recognition of the malodor, among the several olfactory receptors that respond to malodor-causing substances. For example, Non-Patent Document 4 reports that, among the several olfactory receptors that respond to androstenone, the olfactory receptor OR7D4 is a truly important olfactory receptor for the recognition of androstenone odor.

[0007] Furthermore, it has been reported that narrowly-tuned olfactory receptors often selectively respond to compounds with a certain degree of similar structure, although the strength of the response may vary, and that there are cases in which the same olfactory receptor has the odor specificity to respond to compounds of different structures as long as they have the same odor note. For example, Non-Patent Document 5 shows that the narrowly-tuned olfactory receptor OR5AN1, which has extremely high odor specificity for musk scents, responds to the macrocyclic musk group and the nitromusk group, which are compound groups with different structures, and that if the main odor note is the same, they are recognized by the same olfactory receptor.

[0008] To date, no olfactory receptor has been identified that is clearly linked to the "dusty odor." However, Non-Patent Document 6 shows that 2-isopropyl-3-methoxypyrazine, 2-methoxy-3-(1-methylpropyl)pyrazine, and 2-isobutyl-3-methoxypyrazine respond to the olfactory receptor OR2AG1, and that these substances have the odor characteristics of the so-called "dusty odor," which include green, musty, bell pepper, and galbanum-like odors. However, Non-Patent Document 6 did not conduct in vitro receptor inhibition experiments or sensory evaluations to determine whether the "dusty odor" is reduced by odorants (antagonists) that block the OR2AG1 response. Therefore, the possibility that the "dusty odor" is perceived via olfactory receptors other than OR2AG1 cannot be ruled out, and it remains uncertain whether OR2AG1 is linked to the aroma notes of the "dusty odor."

[0009] Furthermore, diosmin and 2-methylisoborneol are important malodorous substances that share similar odor characteristics to the "dusty odor" and are often described as moldy, earthy, or muddy. These odors are often expressed as two-word contractions, such as "musty" or "moldy"-"earthy." However, these odors are more accurately described as moldy based on their dominant odor characteristics, and are distinct from the "dusty odor." These two substances are known to specifically respond to the olfactory receptors OR11A1 and OR2M3, respectively (Patent Document 2). Furthermore, pyrazines with side chain lengths of C1 to C2 are thought to respond to OR5K1 (Non-Patent Document 6, Patent Document 3), but only some of these OR5K1-responsive pyrazines with side chain lengths of C1 to C2 possess the odor characteristics of the "dusty odor."

[0010] JP 2009-131228 A JP 2014-235098 A JP 2015-512032 A

[0011] Journal of Chromatography A, 1428 (2016) 72-85. Shigeharu Inoue, Microorganisms and Aromas: The Power of Aroma in the Microworld, p.60-61 (2002). Journal of Neuroscience 22 June 2011, 31 (25) 9179-9191. Nature. 2007 Sep 27;449(7161):468-72. The Journal of Neuroscience, April 20, 2016, 36(16):4482-44. ECRO XXV Istanbul: “Functional characterization of human olfactory receptors responding to pyrazine odorants”. September 1, 2015, Posters. Nature Communications 10:209 (2019). J Vis Exp. 2019 Apr 23; (146): 10.3791 / 59446.

[0012] Although "dust odor" is a significant odor present in food and the living environment, only a portion of the causative substances is known, and its chemical identity remains unclear. Furthermore, although olfactory receptors that respond to some compounds with the aroma characteristics of "dust odor" have been disclosed, it is unclear whether they are truly narrowly-tuned olfactory receptors associated with the perception of "dust odor." Under these circumstances, it is desirable to clarify the chemical identity of "dust odor," identify olfactory receptors that specifically respond to "dust odor," and provide methods for efficiently screening candidate substances for "dust odor" suppression materials.

[0013] The present invention has been made in consideration of the above circumstances, and provides the following method for screening candidate substances for malodor-suppressing materials.

[0014] [1] A method for screening malodor-suppressing materials, comprising: allowing a test substance and a malodor-causing substance to act on at least one olfactory receptor polypeptide selected from the group consisting of olfactory receptor OR2AG2 and polypeptides that contain an amino acid sequence that is 80% or more identical to the amino acid sequence of the olfactory receptor and that are responsive to malodor-causing substances; measuring the response of the olfactory receptor polypeptide to the malodor-causing substance; and identifying a test substance that suppresses the response of the olfactory receptor polypeptide based on the measured response as a candidate substance for a malodor-suppressing material. [2] The method according to [1], wherein the malodor is a malodor that can be described as a "dusty smell," and has at least one odor characteristic selected from the group consisting of dusty, earthy, powdery, medicinal, and moldy, and includes in its main aroma note at least one expression selected from the group consisting of "dusty," "earthy," and "bell pepper-like or vegetable-like" as a descriptor for expressing the odor characteristic, or a similar expression thereto, and is a smell that may be unpleasant to humans. [3] The method according to [2], wherein the malodor is a smell that can be described as a "dusty smell," and has at least one expression selected from the group consisting of "dusty," "bell pepper-like," and "vegetable-like" as a descriptor for expressing the odor characteristic, in its main aroma note. [4] The method according to [3], wherein the malodor is an odor containing, as a main aroma note, at least one description selected from the group consisting of "dusty" and "bell pepper" as a descriptor expressing the aroma characteristics. [5] The method according to any one of [1] to [4], wherein the malodor is an odor responsive to the olfactory receptor OR2AG2. [6] The malodor-causing substance is represented by the following formula (1) or (2): [In formula (1), R １ represents a hydrogen atom, an unbranched alkyl group having 1 to 3 carbon atoms, an alkyl group having 4 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms; R２ and R ３ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms, and in formula (2), R ４ and R ５ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms.] [7] The method according to any one of [1] to [6], wherein the response of the olfactory receptor polypeptide to the malodor-causing substance is measured on a cell that endogenously expresses the olfactory receptor, or on a cell in which the olfactory receptor has been artificially expressed by genetic engineering. [8] The method according to any one of [1] to [7], wherein the response of the olfactory receptor polypeptide is measured by a reporter assay or calcium imaging.

[0015] By using the method of the present invention, it is possible to screen for candidate substances for "dust odor" suppression materials that can inhibit the binding of the causative substances of "dust odor," a serious malodor in food and living spaces, to the olfactory receptor OR2AG2. Furthermore, according to one embodiment of the present invention, it has been identified that the chemical entity of the malodor expressed sensorially, abstractly, or subjectively as a "dust odor" is a malodor responsive to the olfactory receptor OR2AG2. Therefore, even if the causative substance of a "dust odor" is unknown, it is possible to utilize the fact that the causative substance always contained in a mixture containing the "dust odor" binds to the olfactory receptor OR2AG2, and to screen for candidate substances for suppressing the "dust odor" without identifying the causative substance of the "dust odor."

[0016]

[0033] Figure 1 shows the results of olfactory receptor screening for 2-isobutyl-3-methoxypyrazine. The responses of the olfactory receptor OR2AG2 to 1,367 odorants were screened, and the figures show the percentage of those that responded with a) a fold increase value of 3 or more to OR2AG2, and b) a fold increase value of 50 or more to OR2AG2, and the proportion of their main aroma notes.

[0034] Figure 1 shows the results of measuring the concentration-dependent response of the olfactory receptor OR2AG2 to a) 3-ethyl-2-pentylpyridine (3E2P-pyridine), b) 2-methoxy-3-isopropylpyrazine (2M3IP-pyrazine), c) 2-isobutyl-3-methoxypyrazine (2IB3M-pyrazine), and d) 2-sec-butyl-3-methoxypyrazine (2sB3M-pyrazine).

[0023] Figure 1 shows the concentration-dependent suppression of OR2AG2 responses by olfactory receptor OR2AG2 antagonists against a) 3-ethyl-2-pentylpyridine, b) 2-methoxy-3-isopropylpyrazine, c) 2-isobutyl-3-methoxypyrazine, and d) 2-sec-butyl-3-methoxypyrazine. Figure 2 shows the results of a sensory test demonstrating that olfactory receptor OR2AG2 antagonists against a) 3-ethyl-2-pentylpyridine (3E2P-pyridine), b) 2-methoxy-3-isopropylpyrazine (2M3IP-pyrazine), c) 2-isobutyl-3-methoxypyrazine (2IB3M-pyrazine), and d) 2-sec-butyl-3-methoxypyrazine (2sB3M-pyrazine) suppress the "dust odor." Figure 3 shows the concentration-dependent response of olfactory receptor OR2AG2 to an extract containing a "dust odor" of unknown components. 1 shows the results of measuring the concentration-dependent suppression of OR2AG2 responses by an antagonist of olfactory receptor OR2AG2 against an extract having a "dusty odor" of unknown components. 2 shows the results of a sensory test demonstrating that an antagonist of olfactory receptor OR2AG2 against an extract having a "dusty odor" of unknown components suppresses the "dusty odor."

[0017] Overview of the Invention The present invention identifies and utilizes the olfactory receptor OR2AG2, which responds specifically to substances that cause the malodor described as a "dusty odor." This provides a method for efficiently screening candidate substances for materials that suppress the "dusty odor," as well as a "dusty odor" suppression composition that reduces the unpleasantness of the "dusty odor."

[0018] To elucidate the chemical nature of the malodor sensorially, abstractly, or subjectively described as "dusty odor," the present inventors screened 396 human olfactory receptors for 2-isobutyl-3-methoxypyrazine, a representative substance of "dusty odor," and identified that 2-isobutyl-3-methoxypyrazine, a representative substance of "dusty odor," characteristically responds strongly to the olfactory receptor OR2AG2 (Figure 1). Furthermore, the present inventors screened the responses of the olfactory receptor OR2AG2 to 1,367 odorants with various structures and aroma notes, and summarized the relationship between the OR2AG2 response and aroma notes. As a result, we obtained the following novel findings: (i) many of the odorants that respond to OR2AG2 have the characteristics of a "dust odor," (ii) all of the odorants that respond particularly strongly are compounds with strong "dust odor" characteristics, and in particular, (iii) a group of compounds represented by the following formula (1) or (2) shows a strong response to OR2AG2 (Figures 2 and 3). [In formula (1), R １ represents a hydrogen atom, an unbranched alkyl group having 1 to 3 carbon atoms, an alkyl group having 4 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms; R ２ and R ３ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms, and in formula (2), R ４ and R ５ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms.

[0019] Examples of the alkyl group having 1 to 6 carbon atoms, which may be branched, include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, and 2-ethylbutyl. Of these, methyl, ethyl, isopropyl, isobutyl, sec-butyl, and n-pentyl are preferred. Examples of the unbranched alkyl group having 1 to 3 carbon atoms include methyl, ethyl, and propyl. Examples of the alkyl group having 4 to 6 carbon atoms, which may be branched, include n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, and 2-ethylbutyl. Examples of the alkoxy group having 1 to 3 carbon atoms include methoxy, ethoxy, propoxy, and isopropoxy.

[0020] Furthermore, the inventors discovered that antagonists that block the response of the olfactory receptor OR2AG2 have the effect of suppressing "dust odors" (Figures 4 and 5), indicating that the olfactory receptor OR2AG2 is a narrowly-tuned olfactory receptor strongly linked to the recognition of "dust odors." These findings identified the chemical entity of the malodor previously described as "dust odors" sensorily, abstractly, or subjectively as a malodor responsive to the olfactory receptor OR2AG2. Further investigations by the inventors confirmed the response of the olfactory receptor OR2AG2 not only to the causative substance of "dust odors" but also to "dust odors" with unknown components, and discovered that by searching for olfactory receptor OR2AG2 antagonists and utilizing their masking effects, it is possible to evaluate and select malodor-suppressing materials (Figures 6, 7, and 8). The present invention was discovered based on these novel findings.

[0021] The screening method for candidate substances for malodor-suppressing materials according to the present invention (hereinafter referred to as the screening method of the present invention) will be specifically described below. As described above, the screening method of the present invention is a method for screening for malodor-suppressing materials, and comprises: exposing a test substance and a malodor to at least one olfactory receptor polypeptide selected from the group consisting of olfactory receptor OR2AG2 and polypeptides that contain an amino acid sequence having 80% or more identity to the amino acid sequence of the olfactory receptor and that are responsive to malodor-causing substances; measuring the response of the olfactory receptor polypeptide to the malodor; and identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response as a candidate substance for a malodor-suppressing material. In the present invention, "malodor" refers to a malodor for which candidate substances for suppression materials can be screened using the above method, and more preferably refers to a malodor that is described as a "dusty odor."

[0022] The present invention can be implemented, for example, according to the following embodiments.

[0023] In one embodiment, the present invention provides a method for screening test substances for candidate substances for suppressing a "dust odor" using an olfactory receptor that is responsive to a "dust odor"-causing substance, comprising: (i) a step of interacting a "dust odor" with an olfactory receptor selected from the group consisting of olfactory receptor OR2AG2 and proteins (polypeptides) that contain an amino acid sequence that is 80% or more identical to the amino acid sequence of the olfactory receptor and that is responsive to a "dust odor"-causing substance, and measuring the basal activity response (T0) of the olfactory receptor before the interaction with the "dust odor"-causing substance and the response (Tx) of the olfactory receptor to the "dust odor" at any time after the interaction with the "dust odor"-causing substance; and (ii) The method is characterized by comprising the steps of: (i) comparing the basal activity response (T0) of the olfactory receptors before the application of the "dust odor"-causing substance, which is the measurement result in step (i), with the response (Tx) of the olfactory receptors to the "dust odor" at any time after the application of the "dust odor"-causing substance, and calculating and determining the change in responsiveness; (iii) applying a test substance to the "dust odor"-causing substance in step (i) and calculating and determining the change in responsiveness in the same manner as in step (ii); and (iv) selecting the test substance whose value in step (iii) is reduced compared to step (ii) as a candidate substance for a "dust odor" suppressing material.

[0024] The screening method of the present invention selects candidate substances for suppressing malodors or "dusty odors" from among test substances, using as an index the responsiveness of the test substance to an olfactory receptor selected from the group consisting of olfactory receptor OR2AG2 and proteins (polypeptides) that contain an amino acid sequence that is 80% or more identical to the amino acid sequence of the olfactory receptor and that are responsive to substances that cause malodors or "dusty odors."

[0025] It is generally known that a single odorant activates multiple olfactory receptors, resulting in emotions and behaviors such as likes and dislikes, attraction and avoidance. Non-Patent Document 7 indicates that each olfactory receptor is responsible for the "quality" of an odor. After measuring OR2AG2 responses to 1,367 odorants with various structures and aroma notes and carefully examining the relationship between OR2AG2 responses and aroma notes, it was found that substances responding to OR2AG2 possess at least some odor components of a "dusty odor." Furthermore, an OR2AG2 antagonist compound reduced the unpleasantness of the "dusty odor." This indicates that OR2AG2 is a narrowly-tuned olfactory receptor associated with the "quality" of unpleasant odors ("dusty odors") that induce emotions such as dislike and avoidance. Therefore, by evaluating the responsiveness of a test substance to the olfactory receptor OR2AG2, it is possible to select from the test substances a candidate substance that can prevent the "dust odor" from interacting with the olfactory receptor OR2AG2. Note that, in this specification, the term "test substance" refers to, but is not limited to, the subject of investigation into the malodor-suppressing effect, and refers to a compound, composition, or mixture. Also, in this specification, the term "malodor-suppressing material" refers to, but is not limited to, a compound, composition, or mixture that can suppress malodor. Each step of the screening method of the present invention (the aforementioned steps (i) to (iv)) will be explained below.

[0026] <Step (i)> In step (i), an olfactory receptor selected from the group consisting of olfactory receptor OR2AG2 and proteins (polypeptides) that contain an amino acid sequence that is 80% or more identical to the amino acid sequence of the olfactory receptor and that is responsive to a substance that causes a "dust odor" is reacted with the substance, and the basal activity response (T0) of the olfactory receptor before the substance that causes the "dust odor" is reacted with the olfactory receptor and the response (Tx) of the olfactory receptor to the "dust odor" at an arbitrary time after the action of the substance that causes the "dust odor" are measured. In order to appropriately compare the measurement results for the basal activity response (T0) and the response (Tx), it is preferable that the measurement conditions be the same, except for the presence or absence of the action of the substance that causes the "dust odor."

[0027] The olfactory receptor used is an olfactory receptor selected from the group consisting of OR2AG2 and proteins (polypeptides) that contain an amino acid sequence that is 80% or more identical to the amino acid sequence of OR2AG2 and that respond to substances that cause "dust odors." OR2AG2 is registered in GenBank as NM_001004490 and is a protein consisting of the amino acid sequence (SEQ ID NO: 2) encoded by DNA from positions 893 to 1843 of the base sequence shown in SEQ ID NO: 1. Because the olfactory receptor OR2AG2 selectively responds to substances that cause "dust odors," screening methods using OR2AG2 are expected to contribute to the development of materials that suppress "dust odors."

[0028] The olfactory receptor may be selected from the group consisting of proteins that contain an amino acid sequence that is 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more identical to the amino acid sequence of OR2AG2 and that exhibits responsiveness to substances that cause "dust odors." Herein, the sequence identity of amino acid sequences is calculated using the BLAST search algorithm (publicly available from NCBI). Responsiveness to substances that cause "dust odors" refers to a change in the response value when comparing the basal activity response (T0) of the olfactory receptor before exposure to the substance that causes "dust odors" with the response (Tx) of the olfactory receptor to the "dust odor" at any time after exposure to the substance that causes "dust odors." One type of olfactory receptor may be used, or two or more types may be used in combination.

[0029] In the present invention, the term "dusty odor" refers to an odor that is dusty and, in some cases, has at least one odor characteristic selected from the group consisting of "earthy," "grassy," "powdery," "medicinal," and "moldy." The odor characteristic is expressed by a descriptor selected from the group consisting of "dusty," "earthy," and "grassy (bell pepper-like or vegetable-like)," or a similar descriptor, and the odor may be unpleasant to humans. The term "similar descriptor" is not particularly limited and may be recognized by those skilled in the art as a descriptor expressing the same odor characteristic. For example, similar descriptors to "earthy" include "galbanum" and "smoky." Furthermore, for example, similar expressions to "bell pepper-like or vegetable-like" include "vegetable smell" and "bell pepper-like." According to this definition, for example, diosmin and 2-methylisoborneol, whose main aroma notes are expressed by the descriptor "musty" or "moldy," are not recognized as "dusty odors." Examples of substances that cause malodors, particularly substances that cause "dusty odors," include compounds represented by the following formula (1) or (2): [In formula (1), R １ represents a hydrogen atom, an unbranched alkyl group having 1 to 3 carbon atoms, an alkyl group having 4 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms; R ２ and R ３ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms, and in formula (2), R ４ and R ５ and each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms.] These pyridines or pyrazines may contain one or more of them. Specific examples of the functional group in the formula are as described above.

[0030] In the present invention, the "dusty odor" may be a "dusty odor" consisting of a single substance that causes the "dusty odor," or a complex odor containing the "dusty odor," such as an extract of a fraction containing the "dusty odor." As long as a "dusty odor" is present, the substance that causes the "dusty odor" must be present; the substance that causes the "dusty odor" does not need to be specifically identified. Furthermore, in the present invention, interacting an olfactory receptor with a substance that causes the "dusty odor" includes not only contacting or adding a solution of a single substance that causes the "dusty odor," or a solution of a complex odor containing the "dusty odor," such as an extract of a fraction that has the "dusty odor," to olfactory receptor-expressing cells, but also includes gas-phase exposure. Methods for obtaining a response by exposing olfactory receptor-expressing cells to an odor are known, for example, in Non-Patent Document 8.

[0031] In the present invention, the method for interacting an olfactory receptor with a substance that causes a "dust odor" and measuring the response of the olfactory receptor to the substance that causes a "dust odor" is not particularly limited. For example, the substance that causes a "dust odor" may be interacted with cells isolated from a living organism that expresses an olfactory receptor, and the response of the olfactory receptor may be measured, or the substance that causes a "dust odor" may be interacted with an olfactory receptor on cells in which the olfactory receptor has been artificially expressed by genetic manipulation, and the response of the olfactory receptor may be measured. The time for interacting the olfactory receptor with the substance that causes a "dust odor" cannot be generalized because it depends on the concentration of the substance that causes a "dust odor," but is 0 to 4 hours in the luciferase assay method of the present invention and a few seconds to a few minutes in the calcium imaging method.

[0032] Cells in which olfactory receptors are artificially expressed through genetic manipulation can be produced by transforming cells with a vector incorporating a gene encoding the olfactory receptor.

[0033] In a preferred embodiment of the present invention, the N-terminal 20 amino acid residues of bovine rhodopsin may be incorporated together with the olfactory receptor. Incorporation of the N-terminal 20 amino acid residues of bovine rhodopsin can promote the expression of the olfactory receptor on the cell membrane. Bovine rhodopsin is registered on the NCBI (GenBank) website under "Accession number: NM_001014890.2" or "Accession number: NP_001014890.1." Bovine rhodopsin is a protein (polypeptide) consisting of an amino acid sequence (SEQ ID NO: 4) encoded by DNA from the 1st to 1047th positions of the nucleotide sequence shown in SEQ ID NO: 3. Alternatively, instead of bovine rhodopsin, a protein (polypeptide) may be used that contains an amino acid sequence that is 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more identical to the amino acid sequence shown in SEQ ID NO: 4 and that can promote the expression of the olfactory receptor on the cell membrane. It should be noted that amino acid residues of other proteins (polypeptides) may be used, not limited to bovine rhodopsin, as long as they can promote the expression of olfactory receptors on the cell membrane.

[0034] The method for measuring the response of an 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, it activates intracellular G protein, which then activates adenylate cyclase, converting ATP to cyclic AMP (cAMP), thereby increasing the amount of cAMP in the cell. Therefore, the response of an olfactory receptor can be measured by measuring the amount of cAMP. Methods for measuring the amount of cAMP include ELISA and reporter assays. Among these, it is preferable to measure the response of an olfactory receptor using a reporter assay using a luminescent substance such as luciferase.

[0035] <Step (ii)> In step (ii), the basal activity response (T0) of the olfactory receptor before the exposure of the "dusty odor"-causing substance, as measured in step (i), is compared with the olfactory receptor response (Tx) to the "dusty odor" at an arbitrary time after the exposure of the "dusty odor"-causing substance, and a change in responsiveness is calculated. According to one embodiment of the present invention, the change in responsiveness may be evaluated using as an index the fold increase value, which is obtained by dividing the measurement result of the olfactory receptor response (Tx) to the "dusty odor"-causing substance at an arbitrary time after the exposure of the "dusty odor"-causing substance in step (i) by the measurement result of the basal activity response (T0) of the olfactory receptor before the exposure of the "dusty odor"-causing substance. For example, when measuring the response of an olfactory receptor by a reporter assay method using a luminescent substance such as luciferase, evaluation can be performed using a "dusty odor" that exhibits a fold increase value of preferably 2 or more, more preferably 4 or more, even more preferably 5 or more, or alternatively 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 45 or more, or 50 or more.

[0036] <Step (iii)> In step (iii), a test substance is allowed to act on the substance causing the "dust odor" in step (i), and the change in responsiveness is calculated in the same manner as in step (ii). In order to properly compare the measurement results for the basal activity response (T0) and the measurement results for the response (Tx), it is preferable that the measurement conditions are the same, except for the presence or absence of the action of the substance causing the "dust odor" and the test substance.

[0037] <Step (iv)> In step (iv), a test substance that exhibits a reduced value in step (iii) compared to step (ii) is selected as a candidate substance for a material that suppresses "dust odor." In the present invention, when a reduction in the change value of responsiveness is observed by comparing the measurement results of the change values ​​in steps (ii) and (iii), the test substance used in step (iii) can be evaluated as a candidate substance for a material that suppresses "dust odor."

[0038] In this manner, candidate substances for suppressing malodors, particularly those described as "dusty odors," can be screened from among the test substances. According to the present invention, candidate substances for suppressing "dusty odors" can be selected from a large number of test substances without problems such as olfactory fatigue and individual differences that arise from sensory evaluations conducted using the human sense of smell. The selected substances can be used as candidate substances for "dusty odor" suppression. Novel compounds with optimal odors can be developed by modifying the selected substances as needed. Furthermore, it is possible to develop fragrance materials that can suppress "dusty odors" and have optimal odors by blending the selected substances with other fragrance materials. The screening method of the present invention can contribute to the development of new fragrance materials for suppressing "dusty odors."

[0039] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0040] Example 1 Identification of an Olfactory Receptor Responding to 2-Isobutyl-3-Methoxypyrazine, a Representative Substance of "Dusty Odor" (1-1) Cloning of Olfactory Receptor Gene The human olfactory receptor gene was obtained by cloning by PCR from Human Genomic DNA: Female (Promega) based on the base sequence information (see SEQ ID NO: 1) registered on the NCBI (GenBank) website mentioned above. DNA encoding the N-terminal 20 amino acid residues of bovine rhodopsin (DNA consisting of bases 1 to 60 of the base sequence of SEQ ID NO: 3) was inserted into the pME18S vector, and the obtained human olfactory receptor gene was inserted downstream of this to obtain a human olfactory receptor gene expression vector.

[0041] (1-2) Expression of Olfactory Receptor Genes in HEK293T Cells 0.05 μg of human olfactory receptor gene expression vector, 0.01 μg of RTP1S vector, 0.01 μg of firefly luciferase vector pGL4.29 (Promega) containing a cAMP response element promoter, and 0.005 μg of Renilla luciferase vector pGL4.74 (Promega) containing a thymidine kinase promoter were dissolved in 10 μL of Opti-MEM I (Gibco) to prepare a gene solution (for one well). HEK293T cells were seeded in 96-well plates (Biocoat, Corning) at a cell number that would reach confluence after 24 hours, each in an amount of 100 μL. The gene solution was added to each well by lipofection according to the instructions for Lipofectamine 3000, thereby transfecting the cells. The cells were then cultured at 37°C in a 5% carbon dioxide atmosphere for 24 hours.

[0042] (1-3) Luciferase Reporter Gene Assay After removing the culture medium, 60 μL of a sample aroma compound prepared in CD293 (Gibco) medium (supplemented with 20 μM L-glutamine) at a concentration suitable for measurement was added to each well. After 3 hours of stimulation, luciferase activity was measured according to the instructions for the Dual-Luciferase Reporter Assay System (Promega). The response intensity of the olfactory receptor was measured using the fold increase value, which was calculated by dividing the luciferase activity generated by stimulation with an aroma compound by the luciferase activity generated in a test system without the aroma compound.

[0043] (1-4) Olfactory Receptor Screening with 2-Isobutyl-3-Methoxypyrazine Based on the above (1-1) to (1-3), HEK293 cells expressing each of the 396 human olfactory receptors were prepared, and the response of each of the 396 olfactory receptors was calculated as a fold increase value by adding 1 mM 2-isobutyl-3-methoxypyrazine. The results are shown in Figure 1.

[0044] Example 2: Proof that the olfactory receptor OR2AG2 is an odor note-specific receptor for substances that cause "dusty odors" (2-1) Screening of odorants that respond to OR2AG2 A human olfactory receptor OR2AG2 gene expression vector was cloned, and the olfactory receptor OR2AG2 gene was expressed in HEK293T cells. A total of 1,367 odorants were added to the cells at the maximum optimal concentration within the final concentration range of 1 μM to 1 mM that did not cause cell damage, and a luciferase reporter gene assay was performed. All 1,367 odorants used were assigned odor note information by an odor expert. For odorants that showed a response value above a predetermined level in the luciferase reporter gene assay, the main odor note in the odor note information was extracted, and the proportion of each odor note was determined. The results are shown in Figure 2.

[0045] (2-2) Confirmation that "dusty odor" compounds respond to OR2AG2 in a concentration-dependent manner. The human olfactory receptor OR2AG2 gene expression vector was cloned, and the olfactory receptor OR2AG2 gene was expressed in HEK293T cells. Representative odorants with a "dusty odor," which are OR2AG2-responsive compounds shown in Figure 2, were added to the cells to final concentrations of 3 μM, 10 μM, 30 μM, 100 μM, 300 μM, and 700 μM, and a luciferase reporter gene assay was performed. The results are shown in Figure 3.

[0046] (2-3) Identification of OR2AG2 Antagonists for "Dusty Odor" Compounds A human olfactory receptor OR2AG2 gene expression vector was cloned, and the olfactory receptor OR2AG2 gene was expressed in HEK293T cells. Antagonist screening was performed using a luciferase reporter gene assay to measure the effect of test substances on OR2AG2 response inhibition. In the luciferase reporter gene assay, approximately 500 test substances were mixed with each "dusty odor" compound sample. The fold increase value in the test without the test substance was set to 1, and the fold increase value in the test with each test substance mixed was calculated as the normalized response. The concentrations of each "dusty odor" compound used as a test sample were a) 300 μM for 3-ethyl-2-pentylpyridine, b) 500 μM for 2-methoxy-3-isopropylpyrazine, c) 500 μM for 2-isobutyl-3-methoxypyrazine, and d) 500 μM for 2-sec-butyl-3-methoxypyrazine. The concentration of each test substance was appropriately adjusted to a final concentration range of 1 μM to 1 mM so that no cell damage occurred. Test substances with a normalized response of less than 1 are considered antagonists that suppress the OR2AG2 response, and test substances with smaller normalized response values ​​are considered to be strong antagonists. As examples of antagonists for the "dust odor" compounds a) to d), a) 2,4-decadienal, b) trans-2-nonenal, c) 2,4-undecadienal, and d) 2,4-decadienal were identified. The effect of these antagonists on the concentration-dependent inhibition of the OR2AG2 response of each test substance was measured using a luciferase reporter gene assay. The test concentrations were a) 3-ethyl-2-pentylpyridine (300 μM), b) 2-methoxy-3-isopropylpyrazine (500 μM), c) 2-isobutyl-3-methoxypyrazine (500 μM), and d) 2-sec-butyl-3-methoxypyrazine (500 μM), and the antagonist concentrations for each test substance were 0 μM, 10 μM, 30 μM, 100 μM, and 300 μM. The results are shown in Figure 4.

[0047] (2-4) Evaluation of the Ability of OR2AG2 Antagonists to Mask Dust Odors The ability of OR2AG2 antagonists to suppress the dust odor of each sample was confirmed by sensory evaluation. 10 μL of a 100 mM aqueous solution of each sample (a) 3-ethyl-2-pentylpyridine, b) 2-methoxy-3-isopropylpyrazine, c) 2-isobutyl-3-methoxypyrazine, or d) 2-sec-butyl-3-methoxypyrazine was dropped onto Muette paper, and simultaneously, 10 μL of sterile water (control) or 10 μL of 200 μM OR2AG2 antagonists for each sample (a) 2,4-decadienal, b) trans-2-nonenal, c) 2,4-undecadienal, or d) 2,4-decadienal was dropped onto the paper. The sensory evaluation test was conducted by a panel of five or more people (Sample A: 7 people, Samples B-D: 5 people). The odor intensity when distilled water was dropped onto the "dust odor" compound was rated as 3 to 5, and the intensity of the "dust odor" when the test substance was dropped onto it was rated as 0 (no unpleasant odor) to 5 (very strong unpleasant odor). The average values ​​were calculated from the obtained values. The results are shown in Figure 5.

[0048] Example 3 Application to the Search for Masking Agents Using the OR2AG2 Response of an Unknown "Dusty Odor" (3-1) Confirmation of the OR2AG2 Response of an Unknown "Dusty Odor" To extract the "dusty odor" that occurs when aspartame is combined with a certain fruit flavor, as described in Patent Document 1, a well-known "dusty odor" whose causative substance is unknown, aspartame, trans-2-hexenal, and hexanal were mixed in a 1:1:1 (mass ratio) mixture, and the reaction mixture was gently heated at 60°C for one week and extracted with hexane. This extract was fractionated by medium-pressure HPLC using a silica gel column with hexane and ethyl acetate, then by medium-pressure HPLC using an ODS column with water and acetonitrile, and further fractionated by high-pressure HPLC using an ODS column with water and acetonitrile, to obtain an odor solution containing the "dusty odor." This "dust odor" solution with unknown components was added to HEK293T cells expressing the olfactory receptor OR2AG2 gene at final concentrations of 0.05%, 0.1%, and 0.2% by mass, and a luciferase reporter gene assay was performed. The results are shown in Figure 6.

[0049] (3-2) Identification of OR2AG2 Antagonists for Unknown "Dusty Odors" As in (2-3) of Example 2, HEK293T cells expressing the olfactory receptor OR2AG2 gene were used to screen for antagonists using a luciferase reporter gene assay to determine the inhibitory effect of test substances on the OR2AG2 response to an odor solution of unknown components containing the sample "dusty odor." Approximately 500 test substances were mixed at final concentrations of 1 μM to 1 mM with 0.1% by mass of the sample odor solution of unknown components containing the "dusty odor." Normalized responses were calculated, and OR2AG2 antagonists for the "dusty odor" of unknown components that yielded normalized responses less than 1 were identified. The effect of trans-2-nonenal, an example of an antagonist, on suppressing the OR2AG2 response to a "dust odor" solution of unknown components was measured by luciferase reporter gene assay. The final concentration of the sample was 0.1% by mass, and the concentrations of trans-2-nonenal relative to the sample were 0 μM, 1 μM, 2 μM, and 4 μM. The results are shown in Figure 7.

[0050] (3-3) Evaluation of the Masking Ability of an OR2AG2 Antagonist for a "Dust Odor" of Unknown Components Ten μL of a "dust odor" solution of unknown components was dropped onto Muette paper, and simultaneously, 10 μL of sterile water (control) or 10 μL of 200 μM trans-2-nonenal was dropped. The sensory evaluation test was conducted by five panelists. The odor intensity when distilled water was dropped into the "dust odor" solution was rated as 3 to 5, and the intensity of the "dust odor" when trans-2-nonenal was dropped was rated as 0 (no unpleasant odor perceived) to 5 (very strong unpleasant odor perceived). The average values ​​were calculated from the obtained values. The results are shown in Figure 8.

[0051] Results of the Example: The responses of 396 olfactory receptors to 2-isobutyl-3-methoxypyrazine (1 mM), a representative substance of "dust odor," were measured. The olfactory receptor OR2AG2 showed an extremely strong response (Figure 1). The response of OR2AG2 to 1,367 odorants with diverse structures and aroma notes was specific to "dust odors" (Figure 2). In particular, a group of compounds represented by formula (1a) or (1b) responded to OR2AG2, and the OR2AG2 response to various "dust odor" compounds was concentration-dependent (Figure 3). The OR2AG2 response to various "dust odor" compounds was concentration-dependently inhibited by an OR2AG2 antagonist (Figure 4). In humans, the OR2AG2 antagonist significantly reduced the perception of the "dust odor" of various "dust odor" compounds (Figure 5). This finding demonstrates that the "dust odor" is recognized via OR2AG2. OR2AG2 is a novel "dust odor" receptor whose biological function has not been identified until now. Similarly, even mixed extracts containing unknown "dust odor" components showed a concentration-dependent response to OR2AG2 if they contained a "dust odor" (Figure 6). The OR2AG2 response to the "dust odor" with unknown components was concentration-dependently inhibited by an OR2AG2 antagonist (Figure 7), significantly reducing the perception of the "dust odor" (Figure 8). This suggests that even if the causative substance has not yet been identified, it is possible to screen candidate substances for suppressing "dust odor" by utilizing the OR2AG2 response.

[0052] By using the screening method of the present invention, it is possible to select candidate substances for malodor-suppressing materials, particularly for "dust odor" suppressing materials, from a large number of test substances. The screening method of the present invention is expected to contribute to the development of malodor-suppressing materials, particularly for "dust odor" suppressing materials.

Claims

1. A method for screening malodor-suppressing materials, comprising: applying a test substance and a malodor-causing substance to at least one olfactory receptor polypeptide selected from the group consisting of olfactory receptor OR2AG2 and polypeptides that contain an amino acid sequence that is 80% or more identical to the amino acid sequence of the olfactory receptor and that are responsive to malodor-causing substances; measuring the response of the olfactory receptor polypeptide to the malodor-causing substance; and identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response as a candidate for a malodor-suppressing material.

2. The method according to claim 1, wherein the malodor is a malodor that can be described as a "dusty smell", has at least one odor characteristic selected from the group consisting of dusty, earthy, powdery, medicinal and moldy, and contains at least one main odor note selected from the group consisting of "dusty", "earthy" and "bell pepper" or "vegetable" as a descriptor expressing the odor characteristic, or a similar expression thereto, and is an odor that can be unpleasant to humans in some cases.

3. The method according to claim 1, wherein the malodor is an olfactory receptor OR2AG2-responsive malodor.

4. The odor-causing substance is represented by the following formula (1) or (2): [In formula (1), R １ represents a hydrogen atom, an unbranched alkyl group having 1 to 3 carbon atoms, an alkyl group which may be branched having 4 to 6 carbon atoms, or an alkoxy group having 1 to 3 carbon atoms; R ２ and R ３ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms; ４ and R ５ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms which may be branched, or an alkoxy group having 1 to 3 carbon atoms.

5. The method according to claim 1, wherein the response of the olfactory receptor polypeptide to the malodor-causing substance is measured on cells that endogenously express the olfactory receptor or on cells in which the olfactory receptor is artificially expressed by genetic manipulation.

6. The method according to claim 1, wherein the response of the olfactory receptor polypeptide is measured by a reporter assay or calcium imaging.

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