Method for evaluating fresh feeling and / or palatability

Abstract

The present invention provides a method for evaluating a fresh feeling and / or palatability, and the like. An evaluation method according to the present invention is a method for evaluating a fresh feeling and / or palatability, the method comprising: adding or exposing a substance to be evaluated to at least one olfactory receptor polypeptide among an olfactory receptor OR6B1 and polypeptides having an amino acid sequence that shares an identity of at least 90% with the amino acid sequence of the OR6B1, and measuring the response of the olfactory receptor polypeptide; and evaluating the fresh feeling and / or palatability of the substance to be evaluated, on the basis of the measured response.

Description

Method for evaluating freshness and / or palatability

[0001] The present invention relates to a method for evaluating freshness and / or palatability.

[0002] In recent years, there has been a growing preference for a fresh sensation, and the number of products that claim to have a "fresh" sensation is on the rise. The appeal range is extremely broad, and it is not limited to juices and chuhai drinks that boast the sensation of freshly squeezed fruit, but also dairy products such as fresh cheese and fresh butter, and even baked bread and confectionery such as "fresh" bread and "fresh" cookies.

[0003] In a broad sense, freshness can be considered as a sensation associated with freshly made food, and for example, the fragrant, freshly baked feeling of bread, the sweet, freshly cooked feeling of rice, and the mellow, freshly brewed feeling of coffee can all be considered as a type of freshness. In other words, the freshness of food does not necessarily depend on whether it is unheated, but can be considered as a sensation that evokes the freshness brought about by freshly made food, and plays a very important role in food preference.

[0004] Substances associated with imparting or enhancing a fresh feeling include acetaldehyde, butyraldehyde, and isobutyraldehyde. According to the Food Safety Commission website, these aldehydes are easily produced by fermentation or heating and are found in a variety of foods, including fruits, vegetables, beans, alcoholic beverages, tea leaves, breads, baked goods, soft drinks, meats, and dairy products. Acetaldehyde, in particular, is a highly volatile compound that volatilizes along with other fruit aroma components, enhancing the perception of fresh fruit aroma (Patent Document 1). It is also believed to contribute to the freshness and fermented feel of fermented foods, such as bread, butter, and cheese (Non-Patent Document 1, Patent Document 2). In addition to aldehydes, a variety of compounds, including ketone compounds such as 2,3-pentanedione and diacetyl, alcohol compounds such as 3,6-nonadienol, and spice extracts such as fenugreek, are also believed to be associated with imparting or enhancing a freshness and fermented feeling (Patent Documents 3 and 4).

[0005] Thus, although many materials related to imparting or enhancing a fresh feeling have been reported, in the example of the fresh feeling of fruit as described in Patent Document 4, the current situation is that "although many materials that reproduce the fruit aroma itself have been found, no material that accurately expresses the fresh feeling portion of fruit has been found." This is thought to be mainly due to the lack of biological indices for objectively evaluating fresh feeling, and freshness evaluation is based solely on ambiguous subjective sensory evaluation.

[0006] In recent years, comprehensive screening of the responses of various odorants to the approximately 400 types of olfactory receptors present in humans has led to a rough understanding of which scent notes respond to which olfactory receptors (Non-Patent Document 2, Patent Document 5). In particular, odorants with musk scents are known to respond very specifically to the olfactory receptor OR5AN1, and the stronger the musk scent, the stronger the response to OR5AN1. Therefore, OR5AN1 response can be used as an objective indicator of the odor intensity of musk scents (Non-Patent Document 3).

[0007] It has also been found that the response of some olfactory receptors induces emotions and behaviors such as likes and dislikes, or attraction and avoidance (Non-Patent Document 4). Genome-Wide Association Studies (GWAS) conducted on human populations of hundreds of thousands of people have indicated an association between olfactory receptor genes and specific eating habits and food preferences (Non-Patent Documents 5 and 6). Therefore, it is believed that some olfactory receptors are involved in the formation of eating habits and food preferences by eliciting specific emotions or sensations.

[0008] To date, no olfactory receptor has been shown to have a clear "causal relationship" with the perception of freshness. Patent Document 5 discloses OR1D2, OR10A2, and OR9A4 as olfactory receptors associated with fresh aromas; however, the document only posits a "correlation" association, and does not exclude spurious correlations or confounding factors. For example, OR1D2, which is associated with fresh aromas in Patent Document 5, responds to a wide range of compounds, such as lactone compounds and citrus compounds contained in fruits (Non-Patent Document 6). As a result, OR1D2 is confounded with various aroma descriptions associated with fruits, such as sweet, fresh, fruity, apricot, pineapple, peach, orangeflower, and sulfurous, making it unclear whether it is truly associated with fresh aromas. Patent Document 5 also discloses that bready aromas are associated with OR6B1.

[0009] Patent Document 6 discloses that acetaldehyde, propanal, butanal, and pentanal, which are considered to be malodorous substances, respond to OR6B1. However, because acetaldehyde odor is not suppressed by an OR6B1 response inhibitor (antagonist), it has been suggested that OR6B1 may not actually be involved in the recognition of aldehyde odor (Non-Patent Document 7), and the function of OR6B1 remains unknown. Patent Document 7 also discloses that aldehydes such as isobutyraldehyde respond to OR6B2. Non-Patent Document 5 describes a strong correlation between the genomic region containing the OR6B1 gene and fruit intake, but does not mention at all why this genomic region is associated with fruit intake, and the causal relationship is unknown.

[0010] JP 2015-27311 A JP 2014-8049 A JP 2019-106518 A JP 2016-198025 A WO2023 / 013792A1 JP 2023-117130 A JP 2023-84478 A

[0011] Toyo Food Research Institute Research Report, 30, 79-85, (2014) Flavor and Fragrance Journal, 2015, 30, 342-361 The Journal of Neuroscience, April 20, 2016, 36(16):4482-44 Nature Communications 10:209 (2019) Nature Communications 11:1467 (2020) Nature Communications 13:2743 (2022) ECRO XXIV DIJON: “OR1D2 IS A BROADLY TUNED HUMAN OLFACTORY RECEPTOR”. September 19, 2014, Posters The 95th Annual Meeting of the Japanese Biochemical Society. [1P-370] Identification of key amino acid residues responsible for differences in functional expression between aldehyde-responsive olfactory receptors

[0012] Although freshness is a very important sensation in food preference, it is a subjective and ambiguous sensation, and its biological substance has been unclear. Furthermore, although compounds that impart freshness are known, it has not been elucidated until now how freshness is biologically perceived or how it is related to the formation of eating habits and food preferences. Under these circumstances, it is desired to clarify the biological substance of freshness and to evaluate freshness qualitatively and / or quantitatively.

[0013] The present invention has been made in consideration of the above circumstances, and provides the following method for evaluating freshness and / or palatability.

[0014] [1] A method for evaluating freshness and / or palatability, comprising adding or exposing a substance to be evaluated to at least one olfactory receptor polypeptide selected from olfactory receptor OR6B1 and polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence of OR6B1, measuring the response of the olfactory receptor polypeptide, and evaluating the freshness and / or palatability of the substance to be evaluated based on the measured response.

[0015] [2] A method for screening for a substance that imparts, enhances, or suppresses freshness and / or palatability, wherein the screening for the imparting substance comprises adding or exposing a test substance to at least one olfactory receptor polypeptide selected from olfactory receptor OR6B1 and polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence of OR6B1, measuring the response of the olfactory receptor polypeptide, and identifying the test substance as a substance that imparts freshness and / or palatability based on the measured response; and the screening for the enhancing substance comprises adding or exposing a test substance together with an OR6B1-responsive substance to at least one olfactory receptor polypeptide selected from olfactory receptor OR6B1 and polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence of OR6B1, measuring the response of the olfactory receptor polypeptide, and identifying the test substance as a substance that enhances freshness and / or palatability based on the measured response; or The screening for the inhibitory substance comprises adding or exposing a test substance together with an OR6B1-responsive substance to at least one olfactory receptor polypeptide selected from olfactory receptor OR6B1 and polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence of OR6B1, measuring the response of the olfactory receptor polypeptide, and identifying the test substance as a substance that inhibits freshness and / or palatability based on the measured response.

[0016] [3] The method according to [1] or [2] above, wherein the freshness is the freshness of fruit or the freshness of processed foods. [4] The method according to [1] or [2] above, wherein the response of the olfactory receptor polypeptide is measured on cells that endogenously express the olfactory receptor polypeptide, or on cells in which the olfactory receptor polypeptide is artificially expressed by genetic manipulation. [5] The method according to [1] or [2] above, wherein the response of the olfactory receptor polypeptide is measured by a reporter assay or calcium imaging. [6] An agent for imparting or enhancing freshness and / or palatability, comprising a substance that imparts or enhances freshness and / or palatability, identified by the method of [2] above. [7] The agent for imparting or enhancing freshness and / or palatability according to [6] above, wherein the substance that imparts or enhances freshness and / or palatability is methylisoeugenol.

[0017] The present invention provides a method for qualitatively and quantitatively evaluating freshness and / or palatability, which have previously been subjective and vague sensations, using an OR6B1 response. It also provides a screening method for substances that impart, enhance, or suppress freshness and / or palatability, and substances identified by the method can be used to control freshness and improve food preference, for example.

[0018] a) A diagram showing the vapor-phase response of olfactory receptor OR6B1 to various fruits. b) A diagram showing the results of vapor-phase screening of all 454 types of human olfactory receptors for pink grapefruit. A diagram showing the results of screening flavor ingredients that respond to the olfactory receptor OR6B1. A diagram showing the vapor-phase response of the olfactory receptor OR6B1 to freshly baked bread, butter, and cream cheese. A diagram showing the results of liquid-phase screening of all 454 types of human olfactory receptors for various freshness-imparting materials. A diagram showing the results of measuring the concentration-dependent inhibition of the olfactory receptor OR6B1 response of various freshness-imparting materials. A diagram showing the results of measuring the enhancement of the olfactory receptor OR6B1 response over time by an olfactory receptor OR6B1 response enhancer. A diagram showing the results of measuring the inhibition of the olfactory receptor OR6B1 response over time by an olfactory receptor OR6B1 response inhibitor. A diagram showing the differences in olfactory receptor OR6B1 response strength to various orange flavors. A diagram showing the results of a sensory evaluation test of the freshness of various orange flavors.

[0019] Overview of the present invention The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, modifications can be made as appropriate without departing from the spirit of the present invention. This specification encompasses the entire specification of Japanese Patent Application No. 2024-029824 (filed February 29, 2024), which is the basis for claiming priority to this application. In addition, all publications cited in this specification, such as prior art documents, published patent applications, patent publications, and other patent documents, are incorporated herein by reference.

[0020] The present invention provides a method for qualitatively and quantitatively evaluating freshness and / or palatability by using the response of the olfactory receptor OR6B1 as an index. It also provides a method for screening substances that impart, enhance, or suppress freshness and / or palatability by using the response of the olfactory receptor OR6B1 as an index, and substances identified by this method can be used to, for example, control freshness and improve food preference (i.e., regulate freshness and / or palatability).

[0021] While examining data from a genome-wide association study (GWAS) targeting a human population of several hundred thousand people, the inventors noticed that a genomic region (QTL: Quantitative Trait Locus) containing OR6B1 was strongly associated with fresh fruit consumption, but not with dried fruit consumption. This led them to speculate that OR6B1 may be involved in the recognition of freshness (Table 1). OR6B1 responds to various fruit aromas (Fig. 1a), indicating that actual fresh fruit contains OR6B1-responsive components. Furthermore, OR6B1 responded most strongly to fruit aromas among all human olfactory receptors (Fig. 1b), demonstrating that OR6B1 is a receptor with a strong influence on human olfactory perception.

[0022] Screening for substances that respond to OR6B1 revealed that OR6B1 responded not only to substances that impart a sense of freshness, such as aldehydes commonly present in fruit, but also to substances that impart a sense of freshness to bread and butter (Figure 2, Table 2). Further examination of the genome-wide association analysis revealed that the top three dietary habits significantly associated with OR6B1 were the consumption of fruit, bread, and butter (Table 3), which was completely consistent with the OR6B1 response screening. This demonstrated a causal relationship in which a "response" to OR6B1 "confers" a sense of freshness, leading to the formation of preferences for fruit, bread, and butter.

[0023] Furthermore, because the freshness-imparting substances responded only to OR6B1, the possibility that they imparted the freshness via an olfactory receptor that responded in common to other than OR6B1 could be ruled out (Figure 4). Furthermore, the OR6B1 response of the freshness-imparting substances was concentration-dependent (Figure 5), which appears to support the finding that the higher the content of freshness-imparting materials such as acetaldehyde, the stronger the "fresh fruit aroma" perceived by the drinker (Patent Document 1).

[0024] Furthermore, the present inventors conducted screening of OR6B1 response enhancers and OR6B1 response suppressors and identified OR6B1 response regulators, such as methylisoeugenol and cinnamic aldehyde (Figures 6 and 7).

[0025] The present inventors further investigated the relationship between the intensity of the OR6B1 response and the perception of freshness. In vitro and sensory evaluation tests demonstrated that the stronger the OR6B1 response, the stronger the perceived freshness (Figures 8 and 9: Comparison of Flavors 1 to 3). Furthermore, flavors containing or exposed to cinnamic aldehyde, an OR6B1 response inhibitor, clearly impaired the perceived freshness (Figures 8 and 9: Comparison of Flavors 2 and 4). Thus, the suppression of freshness by an OR6B1 response inhibitor demonstrated for the first time that freshness is recognized and / or regulated via the OR6B1 response. These findings demonstrate that freshness is a sensation evoked via the OR6B1 response, and that the freshness or freshness of processed foods, such as bread and butter, that require heating are biologically equivalent to the freshness of fruit in a broader sense. The present invention was discovered based on these novel findings.

[0026] The method for evaluating freshness and / or palatability according to the present invention (hereinafter referred to as the "evaluation method of the present invention") is specifically described below. The evaluation method of the present invention is a method for evaluating freshness and / or palatability using, as an index, the response of olfactory receptor OR6B1 or at least one olfactory receptor polypeptide among polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence of OR6B1 (hereinafter also referred to as "olfactory receptor OR6B1, etc." in this specification) to a substance to be evaluated (which may include, but is not limited to, various compounds, compositions, mixtures, etc., such as foods themselves and flavor compositions).

[0027] In detail, as described above, the evaluation method of the present invention is a method for evaluating freshness and / or palatability, which includes adding or exposing a substance to be evaluated to an olfactory receptor such as OR6B1, measuring the response of the olfactory receptor polypeptide, and evaluating the freshness and / or palatability of the substance to be evaluated based on the measured response.

[0028] In the present invention, the "freshness" refers to a sensation that can be evaluated by the above-mentioned method, and more preferably refers to the freshness of fruit and the freshness of processed foods. Furthermore, in the present invention, the "freshness" of processed foods refers to a sensation evoked by components that volatilize from foods within a few minutes after the completion of cooking involving fermentation or heat treatment, such as the fresh and refreshing sensation evoked by the aroma of freshly baked bread, the aroma of freshly made cream cheese, or the aroma of freshly brewed coffee. The evaluation method of the present invention can be carried out, for example, according to the following embodiment.

[0029] That is, one embodiment of the present invention is a method for evaluating freshness and / or palatability by measuring the response of olfactory receptor OR6B1 or the like to a test sample (food, fragrance composition, etc.) as a substance to be evaluated, the method comprising: (i) a step of contacting olfactory receptor OR6B1 or the like with the aroma of the test sample, which is the substance to be evaluated, and measuring the basal activity response of the olfactory receptor before contact with the aroma of the test sample (T0) and the response of the olfactory receptor to the aroma of the test sample at any time after contact with the aroma of the test sample (Tx); and (ii) a step of comparing the basal activity response of the olfactory receptor before contact with the aroma of the test sample (T0) measured in step (i) with the response of the olfactory receptor to the aroma of the test sample at any time after contact with the aroma of the test sample (Tx), and calculating the change in responsiveness.

[0030] In one embodiment, the change in responsiveness may be evaluated using as an index the fold increase value, which is calculated by dividing the olfactory receptor response (Tx) to the odor of the test sample at any time after the test sample's exposure to the odor in step (i) by the measurement result of the basal activity response (T0) of the olfactory receptor before the test sample's exposure to the odor. For example, when the olfactory receptor response is measured by a reporter assay using a luminescent substance such as luciferase, a fold increase value of preferably 1 or more, more preferably 1.2 or more, and even more preferably 2 or more can be considered to indicate a response. A larger fold increase value indicates a stronger response, indicating a stronger sense of freshness and a test sample with higher palatability.

[0031] The screening method for substances that impart freshness and / or palatability according to the present invention is described in detail below. As described above, the screening method comprises adding or exposing a test substance to an olfactory receptor such as OR6B1, measuring the response of the olfactory receptor polypeptide, and identifying the test substance as a substance that imparts freshness and / or palatability based on the measured response.

[0032] In this screening method, the term "test substance" refers to, but is not limited to, a compound, composition, mixture, or test specimen (such as a food product itself or a flavoring composition) that has the effect of eliciting a response from olfactory receptor OR6B1 or the like.

[0033] Furthermore, one embodiment of the screening method includes the following steps: (i) contacting a test substance with an olfactory receptor OR6B1 or the like, and measuring the basal activity response of the olfactory receptor before contact with the test substance (T0) and the response of the olfactory receptor to the test substance at any time after contact with the test substance (Tx); and (ii) comparing the basal activity response of the olfactory receptor before contact with the test substance (T0) measured in step (i) with the response of the olfactory receptor to the odor of the test sample at any time after contact with the test substance (Tx), and calculating the change in responsiveness.

[0034] In one embodiment, the change in responsiveness may be evaluated using as an index the fold increase value obtained by dividing the olfactory receptor response (Tx) to the aroma of the test sample at any time after contact of the test sample with the aroma in step (i) by the measurement result of the basal activity response (T0) of the olfactory receptor before contact with the aroma of the test sample. For example, when the olfactory receptor response is measured by a reporter assay using a luminescent substance such as luciferase, a fold increase value of preferably 1 or more, more preferably 1.2 or more, and even more preferably 2 or more can be determined to be an OR6B1-responsive substance that is responsive, and an OR6B1-responsive substance with a higher fold increase value can be selected as a substance that imparts freshness and / or palatability.

[0035] The screening method for substances that enhance or suppress freshness and / or palatability according to the present invention is specifically described below. Specifically, as described above, the screening method comprises adding or exposing a test substance together with an OR6B1-responsive substance to an olfactory receptor such as OR6B1, measuring the response of the olfactory receptor polypeptide, and identifying the test substance as a substance that enhances or suppresses freshness and / or palatability based on the measured response.

[0036] In this screening method, the term "test substance" refers to, but is not limited to, a compound, composition, mixture, or test specimen (such as a food product itself or a flavoring composition) that has the effect of enhancing or suppressing the response of olfactory receptor OR6B1 or the like.

[0037] Furthermore, one embodiment of the screening method includes: (i) measuring the basal activity response (T0) of the olfactory receptor before contacting the olfactory receptor OR6B1 or the like with an OR6B1-responsive substance and the response (Tx) of the olfactory receptor to a test substance at any time after contact with the OR6B1-responsive substance; (ii) comparing the basal activity response (T0) of the olfactory receptor before contact with the OR6B1-responsive substance measured in step (i) with the response (Tx) of the olfactory receptor to the OR6B1-responsive substance at any time after contact with the OR6B1-responsive substance, and calculating a change in responsiveness; (iii) mixing the test substance with the OR6B1-responsive substance in step (i), and calculating a change in responsiveness in the same manner as in step (ii); and (iv) and selecting a test substance for which the value in step (iii) is increased or decreased compared to step (ii) as a substance that enhances or suppresses freshness and palatability.

[0038] Here, in one embodiment, the change in responsiveness may be evaluated using as an index the fold increase value obtained by dividing the measurement result at Tx by the measurement result at T0 in steps (i) and (iii). For example, when the response of an olfactory receptor is measured by a reporter assay method using a luminescent substance such as luciferase, when the maximum fold increase value in step (i) is normalized to 1, if the relative value of the maximum fold increase value in step (iii) is 1 or more, more preferably 1.2 or more, and even more preferably 2 or more, the substance can be determined to be an OR6B1 response enhancer. If the relative value is 1 or less, more preferably 0.8 or less, and even more preferably 0.5 or less, the substance can be determined to be an OR6B1 response suppressor, and each of these substances can be selected as a substance that enhances or suppresses freshness and / or palatability.

[0039] Each step (steps (i) to (iv)) in each of the above-mentioned screening methods will now be described in more detail.

[0040] <Step (i)> In step (i), the olfactory receptor OR6B1 or the like is contacted with a test substance, and the basal activity response of the olfactory receptor before contact with the test substance (T0) and the response of the olfactory receptor to the odor of the test sample at any time after contact with the odor of the test sample (Tx) are measured.

[0041] The nucleotide sequence and amino acid sequence of olfactory receptor OR6B1 are registered on the NCBI (GenBank) website (http: / / www.ncbi.nlm.nih.gov / ) as "Accession number: NM_001005281.1" and "Accession number: NP_001005281.1," and olfactory receptor OR6B1 is a protein (polypeptide) consisting of the amino acid sequence (SEQ ID NO: 2) encoded by the DNA from the 1st to 936th positions of the nucleotide sequence shown in SEQ ID NO: 1.

[0042] The olfactory receptor OR6B1 increases the intake of fresh foods through the recognition of freshness. Therefore, measuring OR6B1 responses as indicators and screening for substances that induce, enhance, or suppress OR6B1 responses can contribute to the evaluation and regulation of freshness and palatability.

[0043] The olfactory receptor polypeptide can be not only the olfactory receptor OR6B1, but also proteins (polypeptides) that contain an amino acid sequence that is 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identical to the amino acid sequence of the olfactory receptor OR6B1, either separately from or together with the olfactory receptor OR6B1. However, proteins (polypeptides) that contain an amino acid sequence that is 80% or more, preferably 85% or more identical may also be used. Note that, in this specification, sequence identity of amino acid sequences is calculated using the BLAST search algorithm (publicly available from NCBI).

[0044] In the present invention, the method for contacting the olfactory receptor OR6B1 or the like with a test substance and measuring the response of the olfactory receptor is not particularly limited. For example, the test substance may be contacted with cells isolated from an organism expressing the olfactory receptor and the response of the olfactory receptor may be measured, or the test substance may be contacted with 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 which the olfactory receptor is contacted with the test substance depends on the concentration of the test substance and the measurement method, and therefore cannot be generalized; however, in the luciferase assay method of the present invention, the time is 0 to 60 minutes, preferably 0 to 30 minutes, and in the calcium imaging method, it is several seconds to several minutes.

[0045] Cells in which the olfactory receptor OR6B1 or the like is artificially expressed by genetic manipulation can be produced by transforming cells with a vector incorporating a gene encoding the olfactory receptor.

[0046] 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 OR6B1, etc. By incorporating the N-terminal 20 amino acid residues of bovine rhodopsin, it is possible to promote the expression of the olfactory receptor on the cell membrane.

[0047] The nucleotide sequence and amino acid sequence of bovine rhodopsin have been registered on the NCBI (GenBank) website under "Accession number: NM_001014890.2" and "Accession number: NP_001014890.1." Bovine rhodopsin is a protein (polypeptide) consisting of the amino acid sequence (SEQ ID NO: 4) encoded by the DNA from the 1st to 1047th positions of the nucleotide sequence shown in SEQ ID NO: 3.

[0048] Alternatively, instead of or together with bovine rhodopsin, a protein (polypeptide) may be used which contains an amino acid sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more identity to the amino acid sequence shown in SEQ ID NO: 4 and which can promote cell membrane expression of olfactory receptors.

[0049] In addition, amino acid residues of other proteins (polypeptides) may be used, not limited to bovine rhodopsin, as long as they can promote cell membrane expression of olfactory receptor OR6B1 or the like.

[0050] The method for measuring the response of olfactory receptor OR6B1 and the like 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 the 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 the olfactory receptor using a reporter assay using a luminescent substance such as luciferase. In addition, any substance involved in olfactory receptor signal transduction can be measured because its amount increases or decreases depending on the response.

[0051] <Step (ii)> In step (ii), the basal activity response (T0) of the olfactory receptor OR6B1 etc. before contact with the test substance, which is the measurement result in step (i), is compared with the response (Tx) of the olfactory receptor to the test substance at any time after contact with the test substance, and the change in responsiveness is calculated.

[0052] According to one embodiment of the present invention, the change in responsiveness may be evaluated using as an index the fold increase value obtained by dividing the olfactory receptor response (Tx) to the test substance at any time after contact with the test substance in step (i) by the measurement result of the basal activity response (T0) of the olfactory receptor before contact with the test substance. For example, when the olfactory receptor response is measured by a reporter assay method using a luminescent substance such as luciferase, a fold increase value of preferably 1 or more, more preferably 1.2 or more, and even more preferably 2 or more can be determined to be a response to OR6B1, and the substance can be evaluated as an OR6B1-responsive substance, and therefore as a substance that imparts a freshness sensation or a palatability-imparting substance.

[0053] <Step (iii)> In step (iii), when the test substance in steps (i) and (ii) is a specific OR6B1-responsive substance, the specific OR6B1-responsive substance and the test substance are mixed, and the change in responsiveness is calculated in the same manner as in step (ii).

[0054] <Step (iv)> In step (iv), when the test substances in steps (i) and (ii) are specific OR6B1-responsive substances, a test substance that increases or decreases the change value when the specific OR6B1-responsive substance determined in step (iii) is mixed with the test substance, compared to the change value determined in step (ii) with the specific OR6B1-responsive substance alone, is selected as a substance that enhances or suppresses freshness and / or palatability.

[0055] According to one embodiment of the present invention, the change in responsiveness may be evaluated using as an index the fold increase value obtained by dividing the olfactory receptor response (Tx) to the test substance at any time after contact with the test substance in steps (i) and (iii) by the measurement result of the basal activity response (T0) of the olfactory receptor before contact with the test substance. For example, when the olfactory receptor response is measured by a reporter assay using a luminescent substance such as luciferase, when the maximum fold increase value in step (i) is normalized to 1, if the relative value of the maximum fold increase value in step (iii) is 1 or more, more preferably 1.2 or more, and even more preferably 2 or more, the substance can be determined to be an OR6B1 response enhancer. If the relative value is 1 or less, more preferably 0.8 or less, and even more preferably 0.5 or less, the substance can be determined to be an OR6B1 response suppressor, and each of these substances is selected as a substance that enhances or suppresses freshness and palatability.

[0056] The specific explanations regarding the steps (i) and (ii) above can also be appropriately adopted in the evaluation method of the present invention.

[0057] In the present invention, by calculating the change in responsiveness of various test substances as the measurement result in step (ii), the strength of the OR6B1 response can be evaluated as an index of the strength of freshness or the level of preference, and if an increase in the change in responsiveness is observed, the test substance can be evaluated as an OR6B1-responsive substance, and therefore as a substance that imparts freshness or preference. Furthermore, in the present invention, when the test substance in steps (i) and (ii) is a specific OR6B1-responsive substance, if an increase or decrease in the change in responsiveness is observed by comparing the measurement results in steps (ii) and (iii), the test substance used in step (iii) can be evaluated as a substance that enhances or suppresses freshness and preference.

[0058] As described above, the freshness and palatability of test substances can be qualitatively and quantitatively evaluated as an OR6B1 response. Furthermore, substances that impart, enhance, or suppress the OR6B1 response can be selected from the test substances, and the selected substances can be added to foods as substances that impart, enhance, or suppress freshness and / or palatability, and used to regulate freshness and / or palatability. Based on the selected substances, the responsiveness to OR6B1 can be modified as needed to develop novel compounds with optimal odors. Furthermore, the selected substances can be blended with other flavor materials, enabling the development of flavor materials that boast optimal odors and the ability to improve freshness and / or palatability. By using the evaluation method and screening methods of the present invention, new flavor materials with improved freshness and / or palatability can be developed and evaluated.

[0059] The present invention also provides a freshness and / or palatability imparting agent, enhancer, or suppressor, which comprises a substance that imparts, enhances, or suppresses the above-mentioned freshness and / or palatability, obtained by each screening method of the present invention. Examples of such imparting agents or enhancers include, but are not limited to, methylisoeugenol, and examples of such suppressors include, but are not limited to, cinnamic aldehyde. By using such imparting agents, enhancers, or suppressors, it is possible to adjust the freshness and / or palatability (impart, enhance, or suppress) of desired foods, flavoring compositions, and the like.

[0060] 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.

[0061] [Example 1] Extraction and estimation of genes related to freshness (1-1) Table 1 shows the lead SNP of the genomic region (QTL) most significantly associated with the "population that eats fresh fruit every day," extracted from the background data analyzed in the aforementioned Non-Patent Document 5, its p-value, and the name of the nearest olfactory receptor gene. For comparison, information on the "population that eats dried fruit every day" for the same lead SNP is also provided. In addition, the table shows the lead SNP of the genomic region associated with the consumption of various fruits, extracted from the background data analyzed in the aforementioned Non-Patent Document 6, its p-value, and the name of the nearest olfactory receptor gene.

[0062]

[0063] Example 2: Proof that a "response" to OR6B1 "confers" a fresh feeling (2-1) Cloning of Olfactory Receptor Genes Each human olfactory receptor gene was obtained by PCR cloning from Human Genomic DNA: Female (Promega) based on the nucleotide sequence information registered on the NCBI (GenBank) website. The OR6B1 gene used was the sequence of SEQ ID NO: 1 (NM_001005281.1). DNA encoding the N-terminal 20 amino acid residues of bovine rhodopsin (DNA consisting of bases 1 to 60 of the nucleotide sequence of SEQ ID NO: 3) was inserted into the pME18S vector, and the obtained human olfactory receptor gene was inserted downstream of that to obtain a human olfactory receptor gene expression vector.

[0064] (2-2) Luciferase Assay 0.05 μg of human olfactory receptor gene expression vector, 0.01 μg of RTP1S vector, and 0.01 μg of firefly luciferase vector pGloSensor™-22F cAMP Plasmid (Promega) containing a CMV promoter were dissolved in 10 μL of Opti-MEM I (Gibco) to prepare a gene solution (for one well). HEK293T cells were seeded in 100 μL aliquots into a 96-well plate (Biocoat, Corning) at a cell number that would reach subconfluence after 24 hours, and the gene solution was added or exposed to each well by lipofection according to the Lipofectamine 3000 instructions, thereby transfecting the cells. The cells were then cultured at 37°C in a 5% carbon dioxide atmosphere for 24 hours. After removing the culture medium, the plates were washed once with HEPES buffer, and 30 μL (for gas-phase measurements) or 100 μL (for liquid-phase measurements) of GloSensor™ cAMP Reagent (Promega) adjusted to a predetermined concentration with FBS-containing HEPES buffer was added to or exposed to each well. The top of the plate was immediately sealed with a seal and allowed to stand at room temperature for 2 hours. The response value (T0) of each well immediately before odor exposure was measured using a plate reader, and the response value (Tx) was measured every minute starting immediately after odor stimulation to measure luminescence values.

[0065] (2-3) Measurement of the Gas-Phase Response of Fresh Fruit Olfactory Receptors In luciferase assays, the analyte was spread on the underside of the plate lid beforehand, and the plate was then covered with the analyte, saturating each well with the aroma of the analyte. Ten minutes after the sealed exposure (T10), the response value of each well was measured again using a plate reader. The olfactory receptor response strength was evaluated by the fold increase (T10 / T0) for the screening of all 454 human olfactory receptors against pink grapefruit (Figure 1a), and the response of various fruits to the human olfactory receptor OR6B1 was evaluated by the ΔF / F0 value (T10-T0) / T0 (Figure 1b).

[0066] (2-4) Screening of Flavor Materials Responding to OR6B1 A luciferase assay was performed using a human olfactory receptor OR6B1 gene expression vector. Each well was exposed to 10 μL of a flavor material library solution prepared at 10 × 30 ppm in HEPES buffer. The time course of the luciferase activity response was measured before addition (T0) and every minute thereafter (T1, T2, ..., T20) to obtain the response value of the human olfactory receptor OR6B1 to each flavor material. The olfactory receptor response intensity was calculated by first dividing the maximum luciferase activity response value generated in each well after stimulation with each flavor material by the luciferase activity before stimulation with each flavor material to calculate the FIsample(MAX) value. This value was then divided by the average FIcontrol(MAX) value of the solvent-only control placed on each plate to evaluate the normalized response value. The response results for all 746 types of flavor raw materials screened are shown in Figure 2, and the top raw materials that provided responses above a certain level are extracted and shown in Table 2 with their aroma information attached.

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[0068] (2-5) Extraction of dietary habits suspected to be influenced by the genomic region containing OR6B1 Table 3 shows dietary habits, lead SNPs, and p-values ​​that are significantly associated with the genomic region containing the OR6B1 gene, which were extracted from the background data analyzed in Non-Patent Documents 5 and 6.

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[0070] (2-6) Measurement of OR6B1 Gas-Phase Responses to Freshly Baked Bread, Butter, and Cheese In luciferase assays, the target substance was spread on the underside of the plate lid beforehand, and the plate was then covered with the lid, saturating each well with the target substance's aroma and sealing it. Ten minutes after the sealed exposure (T10), the response value of each well was measured again using a plate reader, and the response of various fruits to the human olfactory receptor OR6B1 was evaluated using the ΔF / F0 value, expressed as (T10 - T0) / T0. The results are shown in Figure 3.

[0071] Example 3: Proof that the olfactory receptor OR6B1 is the only receptor imparting freshness perception (3-1) Olfactory receptor screening of freshness perception materials A total of 454 types of human olfactory receptor genes were cloned into expression vectors, and luciferase assays were performed using HEK293T cells expressing each olfactory receptor gene. Representative samples of freshness perception materials, which are OR6B1-responsive compounds shown in Figure 2, were diluted with 300 μM CuCl to make 10x1 mM or 10x3 mM. ２ The human olfactory receptors were prepared in a HEPES buffer containing 10 μL of the solution, and each well was exposed to the solution. The time-dependent changes in luciferase activity were measured every minute starting immediately after the addition, thereby obtaining the time-dependent response of each human olfactory receptor. The response strength of the olfactory receptors was evaluated using the maximum fold increase value, which was calculated by dividing the luciferase activity induced by the aroma compound stimulation by the luciferase activity before the aroma compound stimulation. The results are shown in Figure 4.

[0072] (3-2) Measurement of concentration-dependent OR6B1 response of freshness-imparting material A human olfactory receptor OR6B1 gene expression vector was cloned, and a luciferase assay was performed using HEK293T cells expressing the olfactory receptor OR6B1 gene. Representative samples of the freshness-imparting material, which is an OR6B1-responsive compound shown in Figure 2, were added or exposed to final concentrations of 1 mM, 700 μM, 300 μM, 100 μM, or 0 μM, or 700 μM, 300 μM, 70 μM, 30 μM, 0 μM, or 100 ppm, 70 ppm, 30 ppm, 10 ppm, or 0 ppm. The time-dependent change in luciferase activity was measured every minute starting immediately after addition to obtain the time-dependent response of the olfactory receptor OR6B1. The response strength of the olfactory receptor OR6B1 was evaluated by calculating the fold increase value by dividing the luciferase activity at each time point induced by aroma compound stimulation by the luciferase activity before aroma compound stimulation, and then subtracting the fold increase value at each time point for the 0 μM well to obtain the Δfold increase value. The results are shown in Figure 5.

[0073] (3-3) Screening of Flavor Materials that Enhance OR6B1 Responses Using HEK293T cells expressing the olfactory receptor OR6B1 gene, flavor materials that enhance OR6B1 responses in the presence of acetaldehyde were screened by luciferase assay. 827 test substances were added to or exposed to 275 μM acetaldehyde to a final concentration of 30 ppm. The time course of luciferase activity was measured every minute starting immediately after addition to obtain the time course response of each human olfactory receptor. The maximum fold increase value in a control test in which 275 μM acetaldehyde alone was added or exposed to no test substance was defined as 1, and the relative fold increase value in a test in which each test substance was mixed was calculated as the fold increase enhancement value (FIE value). Flavor materials identified in Example 2 (2-4) that have an FIE value of 1.5 or higher and do not elicit an OR6B1 response by themselves were used as OR6B1 response enhancers. As an example, the time-dependent response enhancement results for methyl isoeugenol are shown in Figure 6.

[0074] (3-4) Screening of Flavor Ingredients Suppressing OR6B1 Responses Using HEK293T cells expressing the olfactory receptor OR6B1 gene, screening was performed to measure the effect of test substances on OR6B1 response suppression by luciferase assay. 827 test substances were added to or exposed to 600 μM acetaldehyde at a final concentration of 30 ppm to each well. The time-dependent changes in luciferase activity were measured every minute starting immediately after addition to obtain the time-dependent responses of each human olfactory receptor. The relative suppression value (RS value) was calculated by taking the maximum fold increase value in a control test in which only 600 μM acetaldehyde was added or exposed to the test substance and no test substance was added, and then calculating the relative fold increase value in the test in which each test substance was added. Flavor materials with an RS value of 0.5 or less were used as OR6B1 response inhibitors, and the time-dependent response inhibition results of cinnamic aldehyde as an example are shown in Figure 7.

[0075] (3-5) Evaluation of OR6B1 response to orange flavor

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[0077] Using HEK293T cells expressing the olfactory receptor OR6B1 gene, the OR6B1 response to orange flavors 1 to 4 prepared according to the formulations shown in Table 4 above was measured by luciferase assay. Each of orange flavors 1 to 4 was added to or exposed to each well to a final concentration of 0.1%, and the time-dependent change in luciferase activity was measured every minute starting immediately after addition to calculate the time-dependent response (fold increase value) of each human olfactory receptor. The results are shown in Figure 8.

[0078] (3-6) Sensory Evaluation Test of Orange Flavor Freshness Sensory evaluation was conducted to clarify the relationship between OR6B1 response intensity and sensation. Water containing 0.1% of the above orange flavors 1 to 4 was consumed, and a sensory evaluation test was conducted with nine general panelists to evaluate the orange freshness felt immediately after drinking. The intensity of the freshness was rated on a 10-point scale from 0 (not felt) to 9 (very strong feeling). The results are shown in Figure 9.

[0079] Results of the Example: The genomic region containing OR6B1 was strongly associated with the consumption of fresh fruit, but not with the consumption of dried fruit. This suggests that OR6B1 is strongly associated with the perception of fruit freshness (Table 1). Various fruit aromas respond to OR6B1 (Fig. 1a), indicating that actual fresh fruit contains OR6B1-responsive components. OR6B1 is the receptor with the strongest response among all human olfactory receptors (Fig. 1b), demonstrating that OR6B1 is the olfactory receptor with the strongest influence on human fruit aroma perception.

[0080] Screening for substances that respond to OR6B1 revealed that OR6B1 responded not only to substances that confer a sense of freshness, such as aldehydes commonly present in fruit, but also to substances that confer a sense of freshness in bread and butter (Figure 2, Table 2). These results were in perfect agreement with population genome analysis data showing that the top dietary habits significantly associated with OR6B1 were the consumption of fruit, bread, and butter (Table 3). This demonstrated a causal relationship between the "response" to OR6B1, which "confers" a sense of freshness, and the formation of preferences for fruit, bread, and butter.

[0081] Because the freshness-imparting substances shared only the OR6B1 response, it is unlikely that they imparted freshness via an olfactory receptor that shared a common response to other olfactory receptors (Figure 4). The OR6B1 response of the freshness-imparting substances was concentration-dependent (Figure 5). Screening of OR6B1 response enhancers and OR6B1 response inhibitors identified OR6B1 response modulators, such as methylisoeugenol and cinnamic aldehyde (Figures 6 and 7). Using these, flavors that enhanced the OR6B1 response increased freshness in sensory evaluation tests, while flavors that suppressed the OR6B1 response suppressed freshness (Figures 8 and 9). Thus, the suppression of freshness by OR6B1 response inhibitors demonstrated for the first time that freshness is perceived via the OR6B1 response. This finding also clarified that the freshness perceived in processed foods, such as bread and butter, which require heating, is a broadly defined freshness that is biologically equivalent to the freshness of fruit.

[0082] The present invention provides a method for qualitatively and quantitatively evaluating freshness and / or palatability, which have previously been subjective and vague sensations, using an OR6B1 response. It also provides a screening method for substances that impart, enhance, or suppress freshness and / or palatability. Substances identified by this method can be used to control freshness and improve food preference, for example, and are expected to contribute to the development of technologies for improving food preference.

Claims

1. A method for evaluating freshness and / or palatability, comprising adding or exposing a substance to be evaluated to at least one olfactory receptor polypeptide selected from the olfactory receptor OR6B1 and polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence of said OR6B1, measuring the response of said olfactory receptor polypeptide, and evaluating the freshness and / or palatability of said substance to be evaluated based on the measured response.

2. A method for screening for substances that impart, enhance, or suppress freshness and / or palatability, wherein the screening for the imparting substance comprises adding or exposing a test substance to at least one olfactory receptor polypeptide selected from olfactory receptor OR6B1 and polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence of OR6B1, measuring the response of the olfactory receptor polypeptide, and identifying the test substance as a substance that imparts freshness and / or palatability based on the measured response; and the screening for the enhancing substance comprises adding or exposing a test substance together with an OR6B1-responsive substance to at least one olfactory receptor polypeptide selected from olfactory receptor OR6B1 and polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence of OR6B1, measuring the response of the olfactory receptor polypeptide, and identifying the test substance as a substance that enhances freshness and / or palatability based on the measured response; or The screening for the inhibitory substance comprises adding or exposing a test substance together with an OR6B1-responsive substance to at least one olfactory receptor polypeptide selected from olfactory receptor OR6B1 and polypeptides having an amino acid sequence that is at least 90% identical to the amino acid sequence of OR6B1, measuring the response of the olfactory receptor polypeptide, and identifying the test substance as a substance that inhibits freshness and / or palatability based on the measured response.

3. The method according to claim 1 or 2, wherein the freshness is the freshness of fruit or the freshness of processed food.

4. The method of claim 1 or 2, wherein the response of the olfactory receptor polypeptide is measured on cells that endogenously express the olfactory receptor polypeptide or on cells in which the olfactory receptor polypeptide is artificially expressed by genetic manipulation.

5. The method according to claim 1 or 2, wherein the response of the olfactory receptor polypeptide is measured by a reporter assay or calcium imaging.

6. An agent for imparting or enhancing freshness and / or palatability, comprising a substance for imparting or enhancing freshness and / or palatability, identified by the method of claim 2.

7. The agent for imparting or enhancing freshness and / or palatability according to claim 6, wherein the substance for imparting or enhancing freshness and / or palatability is methylisoeugenol.

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