Method for enhancing flavor sensitivity

Abstract

To enhance flavor sensitivity.SOLUTION: A method for improving flavor sensitivity of a subject, the method comprising reducing amounts of Methanethiol (MTH) and Dimethyl sulfide (DMS) in an oral cavity of the subject, where the flavor sensitivity is flavor sensitivity to a flavor derived from at least one selected from the group consisting of Dipropyl disulfide (DPDS), Methional (MTNA), Dimethyl disulfide (DMDS) and 2-Methyl-3-furanthiol (MFT).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for increasing flavor sensitivity. [Background technology]

[0002] Smells play an important role in enriching our daily lives. For example, smells add a rich flavor to food, and the scents added to everyday products provide various pleasant sensations, such as a sense of cleanliness. On the other hand, unpleasant smells are also important. Smells also play a major role in quickly alerting us to any deterioration in health or hygiene. The importance of these smells can be seen in the fact that the taste of food is impaired when a cold or other respiratory illness occurs, and that elderly people with a weakened sense of smell are at risk because they are unable to detect odors added to gas.

[0003] In mammals such as humans, odors are perceived by olfactory receptors in olfactory nerve cells located in the olfactory epithelium, which extends to the deepest part of the nasal cavity. Odorants taken into the nasal cavity act on and activate olfactory receptors, which then transmit signals from the olfactory nerve cells to the central nervous system, resulting in the perception of odor. It has been reported that humans have 396 genes encoding olfactory receptors. The quality of the odor we perceive from a particular odorant is thought to be determined by the combination of approximately 400 olfactory receptors activated by that odorant. That is, individual olfactory receptors perceive multiple structurally similar odorants with different affinities, while individual odorants are perceived by multiple olfactory receptors. Furthermore, it has been reported that odorants that activate one olfactory receptor inhibit the activation of another olfactory receptor. The combination of these multiple olfactory receptor responses results in the recognition of individual odors.

[0004] For example, hydrogen sulfide (H2S), methanethiol (MTH), and dimethyl sulfide (DMS) are known to be typical components of bad breath, an example of malodor. H2S has been reported to activate human olfactory receptors OR2T11, OR2T1, and OR2T6, MTH to activate OR4S2, OR2T11, and OR2T1 (Patent Document 1, Non-Patent Document 1), and DMS to activate OR4S2 (Non-Patent Document 2). Furthermore, among the key food odorants (KFOs) that contribute to the flavor of food (Non-Patent Document 3), for example, it is known that dimethyl disulfide (DMDS) activates OR4S2 and OR2T11, dimethyl trisulfide (DMTS) activates OR4S2, 2-furfurylthiol (2FT) activates OR4S2 and OR2T11, and 4-methyl-4-sulfanylpentan-2-one (MSP) activates OR2T11 (Patent Documents 2 to 5, Non-Patent Document 2).

[0005] Continuing to smell a certain odor reduces sensitivity to that odor. This phenomenon is called odor adaptation. Furthermore, continuing to smell a certain odor can reduce sensitivity to another odor. This phenomenon is called odor cross-adaptation. Patent Document 6 discloses that the activity of olfactory receptors is involved in odor cross-adaptation, and that cross-adaptation is observed between odorants that are perceived by the same olfactory receptor. In other words, when an olfactory receptor is exposed to a certain odorant and its responsiveness is reduced, its responsiveness to another odorant is also reduced, which is thought to be odor cross-adaptation. The applicant has previously found that body odor affects olfactory sensitivity to other substances that activate the same olfactory receptor via the olfactory receptor that responds to it (Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2020-10629 [Patent Document 2] Japanese Patent Application Publication No. 2017-006122 [Patent Document 3] Patent Publication No. 2021-099326 [Patent Document 4] Japanese Patent Application Publication No. 2024-17361 [Patent Document 5] Japanese Patent Application Publication No. 2022-187349 [Patent Document 6] International Publication No. 2016 / 194788 [Non-patent literature]

[0007] [Non-Patent Document 1] Fukutani, Y. et al. Curr. Biol. 33, 2235-2245.e4 (2023) [Non-patent document 2] Ijichi, C. et al. Chem. Senses 44, 465-481 (2019) [Non-patent document 3] Dunkel, A. et al. Angew. Chemie - Int. Ed. 53, 7124-7143 (2014) Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to a method for improving an individual's olfactory sensitivity to specific odorants that contribute to the flavor of food, and further to a method for improving flavor sensitivity to the flavor derived from the specific odorant. [Means for solving the problem]

[0009] The inventors conducted a cross-adaptation experiment using key food odorants (KFOs) known to activate olfactory receptors activated by MTH and DMS, the main substances that cause bad breath, as well as newly discovered KFOs that activate these olfactory receptors, as candidate odorants that undergo cross-adaptation through bad breath adaptation. As a result, they found that only certain odorants undergo cross-adaptation through bad breath adaptation.

[0010] That is, the present invention provides the following 1) to 4). 1) A method for improving the flavor sensitivity of a subject, comprising reducing the amount of methanethiol (MTH) and dimethyl sulfide (DMS) in the oral cavity of the subject, wherein the flavor sensitivity is to at least one flavor derived from a substance selected from the group consisting of dipropyl disulfide (DPDS), methional (MTNA), dimethyl disulfide (DMDS), and 2-methyl-3-furanthiol (MFT). 2) A method for improving the olfactory sensitivity of a subject, comprising reducing the amount of MTH and DMS in the subject's oral cavity, wherein the olfactory sensitivity is to at least one selected from the group consisting of DPDS, MTNA, DMDS, and MFT. 3) A method for selecting a flavor sensitivity enhancer, comprising identifying a substance that removes MTH and DMS, wherein the flavor sensitivity is to a flavor derived from at least one selected from the group consisting of DPDS, MTNA, DMDS, and MFT. 4) A method for selecting an olfactory sensitivity enhancer, comprising identifying a substance that removes MTH and DMS, wherein the olfactory sensitivity is olfactory sensitivity to at least one selected from the group consisting of DPDS, MTNA, DMDS, and MFT. [Effects of the Invention]

[0011] The present invention provides a method for improving an individual's olfactory sensitivity to specific odorants and flavor sensitivity to the flavors derived from the specific odorants by controlling the amounts of MTH and DMS in the oral cavity. The present invention also provides various means and substances that can control the amounts of MTH and DMS in the oral cavity and thereby improve an individual's olfactory sensitivity to specific odorants and flavor sensitivity to the flavors derived from the specific odorants. [Brief explanation of the drawings]

[0012] [Figure 1] Responsiveness of OR4S2 and OR2T11 (primate consensus; cOR2T11) to sulfur-containing KFOs (sulfur-containing KFOs). A) Structural formulas of the tested sulfur-containing KFOs and their abbreviations used herein. B) Responsiveness of OR4S2 and cOR2T11 (primate consensus) to the test fragrances. The upper row shows 10 μM stimulation conditions, and the lower row shows 100 μM stimulation conditions. In both cases, 300 μM copper chloride was added to the stimulation solution. Plots show data from each experimental run. Bars represent average values. n=1-3. ND indicates that the test could not be performed due to cytotoxicity (Not determined; ND). C) Enlarged view of B. [Figure 2]Search for sulfur-containing KFOs that cause cross-adaptation with MTH and DMS. A) List of test products used in this study. Odor characters are listed from the Good Scent Company database. The Agonists column indicates whether each test product is recognized by OR4S2 or OR2T11 (primate consensus). Open circles indicate that an effect on OR4S2 or OR2T11 has been reported in the literature, while closed circles indicate that a response was observed in Example 1 of the present specification. B) Structural formulas of the evaluation samples. C) Experimental paradigm for this study. The presentation order of the evaluation and adaptation fragrances was randomized for each participant. D) Changes in the sensory intensity of the evaluation fragrances following adaptation to the main halitosis-causing substances. Columns indicate the results before (before adaptation) and after (after adaptation). The vertical axis represents the sensory intensity value perceived when smelling the intensity evaluation substances. Gray lines represent the sensory intensity data for each participant. The black plot indicates the median, and the upper and lower ends of the whiskers indicate the 75th and 25th percentiles, respectively. [Figure 3] Verification experiment for Example 2. A) Experimental paradigm for this test. The order in which the sensory evaluation fragrances and the adaptation fragrances were presented was randomized for each participant. B) to D) Changes in the sensory intensity of the sensory evaluation fragrances following the adaptation procedure for the test sample. The columns show the results for Before: before the adaptation procedure, and After: after the adaptation procedure. The vertical axis shows the sensory intensity value felt when smelling the intensity evaluation substance. The gray line shows the intensity data for each participant. The black plot indicates the median, and the top and bottom of the whiskers indicate the 75th and 25th percentiles, respectively. *; p<0.05 with Bonferroni correction. DETAILED DESCRIPTION OF THE INVENTION

[0013] As used herein, "odor cross-adaptation" in relation to a target odor refers to a phenomenon in which olfactory sensitivity to a target odor is reduced or altered by prior exposure to the odor of a substance other than the causative substance of the target odor and becoming accustomed to that odor. Patent Document 6 clarifies that "odor cross-adaptation" is a phenomenon based on olfactory receptor agonism. That is, the olfactory receptor for the causative substance of the target odor responds to a causative substance of a different odor before responding to the causative substance of the target odor, and then its responsiveness decreases, resulting in only a weak response even when later exposed to the causative substance of the target odor, resulting in a reduction or alteration of the intensity of the target odor perceived by the individual.

[0014] The names of olfactory receptors used herein follow the names registered in Genbank ( / www.ncbi.nlm.nih.gov / genbank / ).

[0015] As used herein, "Key Food Odorants (KFOs)" refers to substances present in a food at levels above the olfactory detection threshold, which contribute to the flavor of the food. Here, flavor refers to smell and taste. When consuming food, smell and taste are perceived together, and smell plays an important role in characterizing the flavor.

[0016] As used herein, "improved olfactory sensitivity" refers to an increase in the sensory intensity of a target odor, a suppression of a decrease in the sensory intensity of a target odor, or a decrease in the detection threshold for the target odor. If the target odor is an odor that contributes to flavor, improved olfactory sensitivity can be rephrased as "improved flavor sensitivity," and improved flavor sensitivity refers to an increase in the sensory intensity of the target flavor, a suppression of a decrease in the sensory intensity of the target flavor, or a decrease in the detection threshold for the target flavor.

[0017] As used herein, "bad breath" refers to an odor that is constantly present in an individual's oral cavity and emanates from the oral cavity or respiratory system, and includes both pathological bad breath and physiological bad breath.

[0018] The odor to which humans are most frequently exposed may be their own body odor (e.g., bad breath). Body odor should continue to affect a person's sense of smell, causing odor adaptation. In fact, it is empirically well-known that humans have difficulty detecting their own body odor. Furthermore, when adaptation to body odor occurs, the aforementioned cross-adaptation occurs simultaneously, and it is presumed that not only sensitivity to the body odor in question but also olfactory sensitivity to substances other than body odor decreases. The present applicant has previously found that body odor, via the olfactory receptors that respond to it, affects olfactory sensitivity to odorants other than body odor that activate the same olfactory receptors (Patent Document 2).

[0019] When these other odorants are KFOs that contribute to the flavor of food, adaptation to body odor may affect sensitivity to the food's flavor, preventing the enjoyment of the food's original flavor and potentially reducing its palatability. Therefore, the inventors conducted further research and, as shown in the Examples below, conducted a cross-adaptation experiment using KFOs known to activate olfactory receptors activated by methanethiol (MTH) and dimethyl sulfide (DMS), the major odorants responsible for bad breath, as well as newly discovered KFOs that activate these olfactory receptors, as candidate odorants for cross-adaptation through bad breath adaptation. The cross-adaptation experiment consisted of (1) a step of presenting the odorant to the subject and having the subject evaluate the odor intensity of the odorant, (2) a step of adapting the subject to a bad breath model (a mixture of MTH and DMS), and (3) a step of presenting the odorant to the subject again and having the subject evaluate the odor intensity of the odorant. In this experiment, the subject in step (1) before adaptation step (2) can be considered a model of a state not adapted to bad breath, and the subject in step (3) after adaptation step (2) can be considered a model of a state after adaptation to bad breath. The odor intensity evaluations in steps (1) and (3) were compared, and odorants whose odor intensity decreased in step (3) could be correctly detected as odorants undergoing cross-adaptation through adaptation step (2). Experimental results showed that continued sniffing of a mixture of MTH and DMS, the major causative substances of bad breath, resulted in a decrease in odor intensity perception for certain KFOs. However, in the above experimental system, there were also KFOs for which no decrease in odor intensity perception was observed. Although both KFOs that showed a decrease in odor intensity perception and KFOs that did not show a decrease in odor intensity perception activated the same olfactory receptors, the responsiveness of the olfactory receptors to these KFOs in the model of a state after adaptation to bad breath was unexpectedly not uniform, suggesting selectivity in the KFOs that undergo cross-adaptation through adaptation to MTH and DMS.

[0020] The above results indicate that when olfactory neurons are exposed to MTH and DMS, the main substances that cause bad breath, their olfactory sensitivity to specific KFOs decreases due to adaptation and cross-adaptation to the odors caused by MTH and DMS. In other words, once a person becomes accustomed to their own bad breath containing MTH and DMS, it becomes difficult to detect the food aromas contributed by these specific KFOs, and their flavor sensitivity to the flavors derived from these specific KFOs decreases.

[0021] Furthermore, the above results show that, conversely, temporary oral halitosis care that reduces the amounts of MTH and DMS in the oral cavity improves olfactory sensitivity to the odors of the specific KFOs, and thus improves flavor sensitivity to the flavors derived from the specific KFOs. For example, when oral care is performed while one has bad breath immediately after waking up, the odor containing the odors resulting from MTH and DMS is reduced (the amounts of MTH and DMS in the oral cavity are reduced), eliminating cross-adaptation, which is thought to result in a stronger perception of the odors of the specific KFOs and the flavors of foods containing the specific KFOs. Therefore, according to the present invention, by reducing the amounts of MTH and DMS in the oral cavity, it is possible to improve olfactory sensitivity to the specific KFOs and improve flavor sensitivity to the flavors derived from the specific KFOs. Such a technique for improving olfactory and flavor sensitivity would be suitable for those who require improved olfactory and flavor sensitivity.

[0022] Furthermore, the above results indicate that a substance or means contributing to bad breath care (e.g., a cleanser or body deodorant) is effective in improving, even if only temporarily, the olfactory sensitivity to the odor of the specific KFOs and the flavor sensitivity to the flavor derived from the specific KFOs. By administering the substance or means contributing to bad breath care to an individual, the olfactory sensitivity to the odor of the specific KFOs and the flavor sensitivity to the flavor derived from the specific KFOs can be improved.

[0023] Thus, in one aspect, the present invention provides a method for improving the olfactory sensitivity of a subject, comprising reducing the amount of MTH and DMS in the subject's oral cavity, wherein the olfactory sensitivity is to at least one selected from the group consisting of dipropyl disulfide (DPDS), methional (MTNA), dimethyl disulfide (DMDS), 2-methyl-3-furanthiol (MFT), and 2-furfurylthiol (2FT).

[0024] In another aspect, the present invention provides a method for improving the flavor sensitivity of a subject, comprising reducing the amount of MTH and DMS in the oral cavity of the subject, wherein the flavor sensitivity is flavor sensitivity to at least one flavor derived from DPDS, MTNA, DMDS, MFT, and 2FT.

[0025] MTH and DMS are the main substances that cause bad breath, and since MTH and DMS are usually present in the oral cavity of individuals with bad breath, reducing the amounts of MTH and DMS in the oral cavity is synonymous with reducing bad breath. Means for reducing a subject's bad breath include washing away bad breath-causing substances from the oral cavity using water or oral care products such as toothpaste, mouthwash, oral tablets, and oral sprays, adsorbing or decomposing bad breath-causing substances, killing bad breath-causing bacteria, suppressing the metabolism or inhibiting enzymes of bad breath-causing bacteria, suppressing the formation or decomposing of biofilms containing bad breath-causing bacteria, and cleaning or sterilizing dentures.

[0026] By reducing the amounts of MTH and DMS in the oral cavity, the olfactory sensitivity of the subject to the above odorants, which undergo cross-adaptation due to adaptation to MTH and DMS, which are the causative substances of bad breath, and the flavor sensitivity of the flavors derived from these odorants are improved. More specifically, in the present invention, by reducing the amounts of MTH and DMS in the oral cavity, the olfactory sensitivity of the subject to the above odorants among odorants that activate olfactory receptors that respond to MTH and / or DMS, which are the causative substances of bad breath, and the flavor sensitivity of the flavors derived from these odorants are improved. In the present invention, the odorant targeted for improving olfactory sensitivity and flavor sensitivity is at least one selected from the group consisting of DPDS, MTNA, DMDS, MFT, and 2FT. From the viewpoint of improving sensitivity, it is preferably at least one selected from the group consisting of DPDS, MTNA, DMDS, and MFT, more preferably at least one selected from the group consisting of DPDS, MTNA, and MFT, and even more preferably at least one selected from the group consisting of DPDS and MTNA. Here, the olfactory receptors for MTH are OR4S2 and OR2T11, and the olfactory receptor for DMS is OR4S2. The olfactory receptors for DPDS are OR4S2 and OR2T11, the olfactory receptor for MTNA is OR2T11, the olfactory receptors for DMDS are OR4S2 and OR2T11, the olfactory receptors for MFT are OR4S2 and OR2T11, and the olfactory receptors for 2FT are OR4S2 and OR2T11. The fact that DPDS, MTNA, and MFT activate the above olfactory receptors was newly discovered by the present inventors.

[0027] DPDS contributes to the flavor of fresh onion, MTNA to the flavor of soy sauce or potato, DMDS to the fresh flavor of citrus fruits, MFT to the flavor of roasted meat, and 2FT to the flavor of coffee. Therefore, the flavor sensitivity to which is improved by the method of improving flavor sensitivity of the present invention can be at least one selected from the group consisting of fresh onion flavor mainly composed of DPDS, soy sauce flavor and potato flavor mainly composed of MTNA, fresh citrus flavor mainly composed of DMDS, roasted meat flavor mainly composed of MFT, and coffee flavor mainly composed of 2FT.From the viewpoint of improving sensitivity, the flavor sensitivity can be preferably at least one selected from the group consisting of fresh onion flavor mainly composed of DPDS, soy sauce flavor and potato flavor mainly composed of MTNA, fresh citrus flavor mainly composed of DMDS, and roasted meat flavor mainly composed of MFT, more preferably at least one selected from the group consisting of fresh onion flavor mainly composed of DPDS, soy sauce flavor and potato flavor mainly composed of MTNA, and roasted meat flavor mainly composed of MFT, and even more preferably at least one selected from the group consisting of fresh onion flavor mainly composed of DPDS, and soy sauce flavor and potato flavor mainly composed of MTNA. For example, foods that contain DPDS as the main component and have the flavor of fresh onion include beef, leeks, onions, pork, apple brandy, grape brandy, asafoetida oil, peanuts, durian, capers, and cabbage. Foods that contain MTNA as the main component and have the flavor of soy sauce or potato include red wine, asparagus, apple brandy, morels, potato mushrooms, roasted sesame seeds, whiskey, shrimp, Emmental cheese, oats, krill, and osipek cheese. Orange juice, cacao, oysters, cashew apples, bonito flakes, pumpkin, Camembert cheese, fried duck skin, duck liver, curry leaves, mushrooms, beef stew with vegetables, guava, cupuacu, green asparagus, Gruyere cheese, grape brandy, grapefruit juice, capers, yeast extract, Gouda cheese, coffee, cocoa, lamb, wheat dough, salmon, salami, crayfish, sherry, turkey, potatoes, soy sauce, white wine, Swiss cheese,Soda bread, taco shells, onions, tamarind, cod, various cheeses, cheddar cheese, chicken soup, chocolate chip cookies, molasses, corn, toast, doughnuts, tomatoes, tomato sauce, tomato paste, truffles, rapeseed, fresh apples, sake, roasted chicken skin, garlic, parsnips, baguettes, parsley, honey, passion fruit, vanilla, paprika, Parmesan cheese, breadcrumbs, bread and leaven, breadfruit, hamburger steaks, pumpkin seed oil, bread dough, peanuts, beer, pistachio nuts, oyster mushrooms, feijoa, pork, These include pork stewed with vegetables, grapes, French fries, hazelnuts, spinach, whey protein, scallops, hops, popcorn, potato chips, white asparagus, white mushrooms, macadamia nuts, trout, mustard, miso (soybean, rice, fish), melon, rice cakes, pear brandy, rye flour, rye, rum, licorice, apples, apple juice, cider, lobster, wine, crab, citrus fruits, beef, milk and dairy products, fish, chicken, black tea, yeast, sugar, soy sauce, Chinese liquor (Baijiu), beans, malt, eggs, and lamb. Foods with the fresh flavour of citrus fruits include asafoetida oil, apple brandy, argan oil, squid, strawberries, potato mushrooms, whiskey, shrimp, peas, oats, krill, oysters, blackcurrants, cashew apples, crab, cauliflower and broccoli, citrus fruits, mushrooms, cabbage, beef, guava and feijoa, crispbread, grape brandy, watercress, capers, coffee, cocoa, lamb, rice, cherries, cowpeas, sapodilla, crayfish, sherry, potatoes, soy sauce, Swiss cheese, sukiyaki, Spanish cheese, sausages. Double bread, radish, yam, soybeans, various cheeses, cheddar cheese, dates, tequila, chili peppers, corn, tomatoes, trashi, chicken, truffles, rapeseed, sake, garlic, green onions, pineapple, parsley, honey, passion fruit, papaya, clams, peanuts, beer, loquat, pork, hazelnuts, pecans, whey protein, scallops, hops, popcorn, potato chips, macadamia nuts, mustard, mango, miso (soybeans, rice, fish), melon, peaches, rye, green algae, rooibos tea, rutabaga, wine, Brussels sprouts,Examples of foods with a roasted meat flavor that contain MFT as the main component include roasted sesame seeds, cashew apples, fried duck skin, citrus fruits, beef, yeast extract, coffee, lamb, salami, white sesame seeds, cheddar cheese, chicken, truffles, roasted chicken skin, vanilla, beer, lamb, braised pork, whey protein, wine, milk and dairy products, fish, yeast, and pork. Foods that have a coffee flavor include roasted sesame seeds, shrimp, duck liver, beef, green asparagus, coffee, lamb, sesame oil, salami, white sesame, chicken, corn, chicken stock, roasted chicken skin, chicken, pumpkin seed oil, peanuts, beef, beer, lamb, hazelnuts, popcorn, white asparagus, mustard, lobster, wine, yeast, yeast extract, rapeseed, wheat, Chinese liquor (baijiu), and pork.

[0028] In the present invention, the improvement in olfactory sensitivity to the above-mentioned odorants and the improvement in flavor sensitivity to the flavors derived from the above-mentioned odorants in a subject are achieved within a relatively short time after the treatment for reducing the amounts of MTH and DMS in the oral cavity is performed. Therefore, the improvement in olfactory sensitivity and flavor sensitivity according to the present invention can be an immediate improvement. In the present invention, the "immediate" improvement in olfactory sensitivity and flavor sensitivity preferably refers to an improvement in olfactory sensitivity and flavor sensitivity occurring within 1 hour, more preferably within 10 minutes, after the treatment for reducing the amounts of MTH and DMS in the oral cavity.

[0029] In another aspect, the present invention provides a means for improving a subject's olfactory sensitivity, wherein the olfactory sensitivity is to at least one selected from the group consisting of DPDS, MTNA, DMDS, MFT, and 2FT. In yet another aspect, the present invention provides a means for improving a subject's flavor sensitivity, wherein the flavor sensitivity is to a flavor derived from at least one selected from the group consisting of DPDS, MTNA, DMDS, MFT, and 2FT. The odorants targeted for improving olfactory sensitivity and flavor sensitivity in the present invention are the same as those in the above-described methods for improving olfactory sensitivity and flavor sensitivity. Preferably, the means for improving olfactory sensitivity and flavor sensitivity may be a means for removing halitosis-causing substances, including MTH and DMS, from the oral cavity. The means for removing halitosis-causing substances removes the subject's halitosis, including MTH and DMS, thereby improving the subject's olfactory sensitivity to the odorants and flavor sensitivity to flavors derived from the odorants.

[0030] In one embodiment, the present invention provides an olfactory sensitivity enhancer for the above odorants, which contains as an active ingredient a substance for removing halitosis-causing substances, including MTH and DMS, from the oral cavity. In another embodiment, the present invention provides a flavor sensitivity enhancer for flavors derived from the above odorants, which contains as an active ingredient a substance for removing halitosis-causing substances, including MTH and DMS, from the oral cavity. Preferably, the enhancer is an olfactory sensitivity enhancer for the above odorants among odorants that activate olfactory receptors for MTH and / or DMS, which are halitosis-causing substances, and an enhancer for flavor sensitivity for flavors derived from the above odorants. Preferably, the olfactory sensitivity enhancer and flavor sensitivity enhancer can be immediate olfactory sensitivity enhancers and flavor sensitivity enhancers, respectively. Preferably, the active ingredient is a substance that cleans, adsorbs, inhibits the generation, inhibits the volatilization, or decomposes halitosis-causing substances, including MTH and DMS.

[0031] Substances for removing bad breath-causing substances, including MTH and DMS, from the oral cavity include oral care products such as toothpaste, mouthwash, oral tablets, and oral sprays, as well as denture cleaners. These oral care products may optionally contain, but are not limited to, the following ingredients: bases or humectants such as water, sorbitol, propylene glycol, glycerin, and ethanol; fluorides (e.g., sodium fluoride and sodium monofluorophosphate); bactericides such as benzethonium chloride, benzalkonium chloride, cetylpyridinium chloride, isopropylmethylphenol, chlorhexidine hydrochloride, and triclosan; copper, zinc, or aluminum salts such as copper gluconate, copper citrate, copper sulfate, copper chloride, copper chlorophyllin sodium, zinc gluconate, zinc citrate, zinc sulfate, zinc chloride, zinc oxide, aluminum hydroxide, and aluminum lactate; and anhydrous silicic acid. Thickeners such as thickening silica (such as thickening silica), carboxymethyl cellulose, carrageenan, xanthan gum, and polyacrylic acid; abrasives such as anhydrous silicic acid (such as abrasive silica) and calcium carbonate; active agents such as sodium lauryl sulfate, sodium lauroyl methyl taurate, sodium lauroyl glutamate, sodium myristoyl glutamate, coconut oil fatty acid amidopropyl betaine, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan stearate, glycerin fatty acid esters, sucrose fatty acid esters, and polyoxyethylene stearyl ether; fragrances or sweeteners such as menthol and sodium saccharin; preservatives such as parabens; cyclodextrin; and zeolite.

[0032] Furthermore, the bad breath prevention ingredients that can be used in oral care products can themselves be used in the present invention as a means for removing bad breath-causing substances used in the olfactory sensitivity enhancing method and the flavor sensitivity enhancing method, or as active ingredients of the olfactory sensitivity enhancer and the flavor sensitivity enhancer. Examples of bad breath prevention ingredients that can be used in the oral care product include bactericides such as benzethonium chloride, benzalkonium chloride, cetylpyridinium chloride, isopropylmethylphenol, chlorhexidine hydrochloride, and triclosan; copper, zinc, or aluminum salts such as copper gluconate, copper citrate, copper sulfate, copper chloride, copper chlorophyllin sodium, zinc gluconate, zinc citrate, zinc sulfate, zinc chloride, zinc oxide, aluminum hydroxide, and aluminum lactate; surfactants such as sodium lauryl sulfate, sodium lauroyl methyl taurate, sodium lauroyl glutamate, sodium myristoyl glutamate, coconut oil fatty acid amidopropyl betaine, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan stearate, glycerin fatty acid esters, sucrose fatty acid esters, and polyoxyethylene stearyl ether; cyclodextrin; zeolite, etc. These bad breath prevention ingredients can be used alone or in appropriate combination as the active ingredient of the olfactory sensitivity enhancer and flavor sensitivity enhancer of the present invention.

[0033] In yet another aspect, the present invention provides a method for selecting an olfactory sensitivity enhancer, wherein the olfactory sensitivity is olfactory sensitivity to at least one selected from the group consisting of DPDS, MTNA, DMDS, MFT, and 2FT. In yet another aspect, the present invention provides a method for selecting a flavor sensitivity enhancer, wherein the flavor sensitivity is flavor sensitivity to a flavor derived from at least one selected from the group consisting of DPDS, MTNA, DMDS, MFT, and 2FT. The odorants targeted for olfactory sensitivity enhancement and flavor sensitivity enhancement in the present invention are the same as those in the olfactory sensitivity enhancement method and flavor sensitivity enhancement method described above. As described above, removing halitosis-causing substances, including MTH and DMS, from a subject can improve the subject's olfactory sensitivity to the above odorants and flavor sensitivity to flavors derived from the above odorants. Therefore, substances that remove halitosis-causing substances, including MTH and DMS, can be identified as olfactory sensitivity enhancers and flavor sensitivity enhancers. Preferably, the olfactory sensitivity enhancers and flavor sensitivity enhancers can be instantaneous olfactory sensitivity enhancers and flavor sensitivity enhancers.

[0034] In one embodiment, the method for selecting an olfactory sensitivity enhancer and a flavor sensitivity enhancer of the present invention involves examining the ability of a test substance to remove targeted halitosis-causing substances, preferably MTH and DMS. For example, this method examines the ability of the test substance to cleanse, adsorb, inhibit the generation of, inhibit volatilization, or decompose the halitosis-causing substances. Test substances having such an effect can be identified as olfactory sensitivity enhancers and flavor sensitivity enhancers. A substance with a stronger effect in removing halitosis-causing substances may function as a more powerful olfactory sensitivity enhancer and flavor sensitivity enhancer. The type of test substance is not particularly limited as long as it is a substance desired for use as an olfactory sensitivity enhancer and flavor sensitivity enhancer. For example, the test substance may be a substance expected to cleanse, adsorb, inhibit the generation of, inhibit volatilization, or decompose halitosis-causing substances. The test substance may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, or may be a compound, composition, or mixture. The test substance may also be a substance that has been previously evaluated or identified for its olfactory sensitivity enhancing effect, or its effect of cleaning, adsorbing, inhibiting the generation, inhibiting the volatilization or decomposing substances that cause bad breath. In another embodiment, the method for selecting an olfactory sensitivity enhancer and a flavor sensitivity enhancer of the present invention comprises sensory evaluation of the olfactory sensitivity enhancing effect and / or flavor sensitivity enhancing effect of a substance identified as having the effect of removing substances that cause bad breath, preferably sensory evaluation of the olfactory sensitivity enhancing effect from the viewpoint of ease of evaluation. For example, after a substance that removes substances that cause bad breath is identified from among the test substances as described above, the olfactory sensitivity enhancing effect and / or flavor sensitivity enhancing effect of the substance, preferably the olfactory sensitivity enhancing effect, may be sensory evaluated. Alternatively, a substance that has already been identified as removing substances that cause bad breath may be sensory evaluated for its olfactory sensitivity enhancing effect and / or flavor sensitivity enhancing effect, preferably the olfactory sensitivity enhancing effect. [Example]

[0035] The present invention will be described in more detail below with reference to examples. The odor substances used in the following examples are shown in Table 1.

[0036] [Table 1]

[0037] Reference Example 1 Preparation of olfactory receptor-expressing cells 1) Cloning of the human olfactory receptor gene OR4S2 The OR4S2 gene (SEQ ID NO: 1) was cloned by PCR using human genomic DNA female (G1521: Promega) as a template based on the sequence information registered in GenBank. The PCR-amplified gene was inserted into the pENTR vector (Invitrogen) according to the manufacturer's instructions, and then recombined with the NotI and AscI sites in the pENTR vector downstream of the Flag-Rho tag sequence in the pME18S vector.

[0038] 2) Cloning of olfactory receptor genes For OR2T11, a primate consensus was used in which amino acids highly common between homologous genes of primate olfactory receptors and the human olfactory receptor OR2T11 were introduced into the human olfactory receptor (Patent Publication No. 2023-002482). To determine the primate consensus for OR2T11, we performed a BLAST search using the amino acid sequence of human OR2T11 (NP_001001964.1) as the query sequence and the target organism name "primate." Phylogenetic tree analysis was performed on the homologous genes obtained. A clade was selected that included human OR2T11 and OR2T11 from other species, while containing as few genes as possible other than OR2T11. Of the 16 genes in that clade, 15 genes, excluding human OR2T11, were identified as primate orthologs. Next, we performed alignment analysis and identified consensus amino acids for the amino acid sequences of the 16 genes, including human OR2T11, as described below. Alignment analysis of the identified gene clusters was performed using ClustalW, and further adjustments were made to optimize the alignment based on amino acids or amino acid motifs highly conserved among olfactory receptors. Ballesteros-Weinstein residue numbering was assigned based on the results of an alignment of all mouse olfactory receptors shown in the literature (Ikegami K et al. PNAS 117:2957-2967 (2020)). Based on the alignment results, consensus olfactory receptors were designed using Jalview. In the alignment, if there was one amino acid residue at a position corresponding to each amino acid position in the original human olfactory receptor amino acid sequence that was different from the amino acid residue in the reference amino acid sequence and occurred with a frequency of 50% or higher, the amino acid residue in the reference amino acid sequence was modified to that amino acid residue. Furthermore, even if there was one amino acid residue at a position corresponding to each amino acid position in the original reference human olfactory receptor amino acid sequence that was different from the amino acid residue in the reference amino acid sequence and had an occurrence frequency of 50%, if the occurrence frequency of the amino acid residue in the reference amino acid sequence was also 50%, the amino acid residue in the reference amino acid sequence was not modified.On the other hand, if there was an amino acid with an occurrence frequency of 60% or more at a position corresponding to a deletion position in the original reference human olfactory receptor amino acid sequence, the reference amino acid sequence was modified to insert the most conserved amino acid at the deletion position. The topology of the olfactory receptor was confirmed using the Transmembrane Hidden Markov Model (TMHMM). The DNA sequence (SEQ ID NO: 2) encoding the designed olfactory receptor polypeptide (OR2T11 consensus (SEQ ID NO: 3)) was obtained by DNA synthesis after optimizing the base sequence codons corresponding to the amino acid sequence for expression in human cultured cells. EcoRI and XhoI sites were added to both ends of this base sequence, and it was recombined into the EcoRI and XhoI sites created downstream of the Flag-Rho tag sequence on the pME18S vector.

[0039] 3) Construction of pME18S-human RTP1S vector The gene encoding human RTP1S, which translocates olfactory receptor proteins produced in cultured cells to the cell membrane surface, was inserted into the EcoRI and XhoI sites of another pME18S vector.

[0040] 4) Preparation of olfactory receptor-expressing cells HEK293 cells expressing the above olfactory receptors were prepared. The reaction solution with the composition shown in Table 2 was prepared and left to stand in a clean bench for 15 minutes, after which it was added to each well of a 96-well plate (BD). Next, 100 μL of HEK293 cells suspended in DMEM (Nacalai) were added to each well at a concentration of 2 × 10 5 cells / cm 2 The cells were seeded at 100°C and cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. As a control, cells that do not express olfactory receptors (mock) were transfected with an empty vector that did not incorporate the olfactory receptor gene.

[0041] [Table 2]

[0042] Reference Example 2 Reporter gene assay (Dual-Glo™ luciferase assay system) Olfactory receptors expressed in HEK293 cells increase intracellular cAMP levels by coupling with endogenous Gαs and activating adenylate cyclase. In this study, odor responses were measured using a luciferase reporter gene assay, which monitors the increase in intracellular cAMP levels as luminescence from the firefly luciferase gene (luc2P-CRE-Hygro). A Renilla luciferase gene fused downstream of the CMV promoter (pRL-CMV) was also co-transfected and used as an internal control to correct for errors in gene transfection efficiency and cell number. Luciferase activity was measured using the Dual-Glo™ luciferase assay system (Promega) according to the product's instructions. For various stimulation conditions, the luminescence value derived from firefly luciferase was divided by the luminescence value derived from Renilla luciferase to calculate Fluc / Rluc. The Fluc / Rluc induced by odorant stimulation was divided by the Fluc / Rluc in cells not stimulated by the odorant, and the fold increase was calculated as an index of response strength.

[0043] Example 1: Search for key food odorants (KFOs) that activate halitosis receptors Hydrogen sulfide (hereinafter referred to as H2S), methanethiol (hereinafter referred to as MTH), and dimethyl sulfide (hereinafter referred to as DMS) are known as typical components of bad breath. Patent Document 1 and Non-Patent Document 1 report that OR2T11, OR2T1, and OR2T6 are human olfactory receptors activated by H2S, and OR4S2, OR2T11, and OR2T1 are human olfactory receptors activated by MTH. Non-Patent Document 2 also reports that OR4S2 is an olfactory receptor activated by DMS. If we could select KFOs that activate the above-mentioned odor receptors, we could efficiently identify KFOs that undergo cross-adaptation through adaptation to bad breath. Here, we selected KFOs that activate OR2T11 and OR4S2. First, we selected KFOs for olfactory receptor response measurements. Of the 226 KFOs in 227 foods listed in Dunkel et al.'s meta-analysis, 34 KFOs containing sulfur atoms in their molecular structures, i.e., volatile sulfur compounds (VSCs), were selected (Non-Patent Document 3). Of these substances, five flavors—MTH, DMS, dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and 2-furfurylthiol (2FT)—are known to be agonists of OR4S2 and / or OR2T11 or OR4S2 in Patent Documents 1-4 and Non-Patent Documents 1 and 2. Therefore, we selected them as candidate flavors that undergo cross-adaptation through bad breath. Next, to explore other candidate odorants, we examined the olfactory receptor activity of 10 KFOs (Fig. 1A). Six odorants, 4-Methyl-4-sulfanylpentan-2-one (MSP), 4-Methoxy-2-methylbutane-2-thiol (MMBT), Methional (MTNA), 2-Methyl-3-furanthiol (MFT), Dipropyl disulfide (DPDS), and Dipropyl trisulfide (DPTS), activated OR2T11 (primate consensus) (Fig. 1B). In addition, Methionol (MTNO), MFT, and DPDS activated OR4S2 (Fig. 1B). Among these KFOs that activate at least one of OR2T11 and OR4S2, five KFOs (MSP, MFT, DPDS, MTNO, and MTNA) were selected as candidate flavors that undergo cross-adaptation due to oral odor. In this way, MTH, DMS, DMDS, DMTS, 2FT, MTNA, MTNO, MFT, MSP, and DPDS were selected as candidate KFOs likely to have their sensory intensity reduced by halitosis adaptation. Furthermore, as shown below, KFOs for which olfactory sensitivity decreased before and after the intervention of adapting to halitosis MTH and DMS were identified.

[0044] Example 2 Identification of substances that cause halitosis and reduce olfactory sensitivity 1) Method We investigated whether sniffing a mixture of MTH and DMS (sample name: Oral malodor; OMA) for two minutes would reduce the perceived intensity of the test product, KFOs (Figure 2A-C). The method is described below. Eighteen participants participated in the study. The odor sample was prepared by adding 35 mL of diluted odor solution to a 50 mL standard bottle. Participants were asked to take a quick sniff of the bottle's opening. The perceived odor intensity was then recorded on a 10 cm VAS. The adaptation procedure consisted of sniffing the presented sample for two minutes. After two minutes, the same odor sample was presented again, and participants were asked to rate the odor intensity using the same method as before the adaptation procedure. Muscone (MSC), which is thought to activate a different olfactory receptor than OR2T11 and OR4S2, was used as a negative control for the adaptation substance. A list of the test products used in this experiment is shown in Figures 2A and B. This experiment was conducted in a standard laboratory between 9:00 AM and 5:00 PM. Generally, physiological bad breath shows diurnal variations, peaking immediately after waking up, and is subsequently reduced by saliva secretion, eating, and oral care. This experiment was conducted during the day, and no oral care or dietary restrictions were imposed. In other words, the period before the intervention of continuously sniffing OMA can be considered a "model of a state where subjects are not adapted to bad breath," and the period after the intervention can be considered a "model of a state where subjects have adapted to bad breath."

[0045] 2) Results First, we confirmed the validity of the experimental system. In the positive control condition (adaptation odor → intensity evaluation odor (the symbols before and after the symbol → indicate the adaptation odor and intensity evaluation odor, respectively): MSC → MSC, OMA (MTH + DMS) → MTH, OMA → DMS), in which self-adaptation is expected to reduce the perceived intensity of the sensory evaluation substances, we observed a statistically significant decrease or a tendency toward a decrease in the intensity after the adaptation procedure (Figure 2D). This indicates that the 2-minute adaptation procedure, in which odorants were continuously smelled, induced self-adaptation as designed. On the other hand, in the OMA → MSC, MSC → MTH, and MSC → DMS conditions, in which the adaptation odor and intensity evaluation odor were combined to activate different sets of olfactory receptors, no statistical difference was observed before and after adaptation (Figure 2D). This result suggests that the intervention of continuously smelling both adaptation odors for 2 minutes does not necessarily reduce the perceived odor intensity for all sensory evaluation substances. In other words, it was confirmed that this cross-adaptation test system can correctly detect odorants that cross-adapt through OMA adaptation. Using this experimental system, we searched for sulfur-containing KFOs that cross-adapted with OMA adaptation. Results indicated a tendency for the median sensory intensity of 2FT, MTNA, and MFT to decrease (p<0.1 by Wilcoxon signed-rank test without multiple testing correction) (Fig. 2D). In particular, the sensory intensity of MTH, DMS, DMDS, and DPDS decreased statistically significantly with OMA adaptation (p<0.05 by Wilcoxon signed-rank test with Bonferroni correction) (Fig. 2D). On the other hand, despite activating odor receptors, no decrease in sensory intensity was observed with OMA adaptation for DMTS, MTNO, and MSP (Fig. 2D). This may be due to the presence of other olfactory receptors important for odor intensity perception, in addition to the OMA-activated olfactory receptors. In summary, these findings suggest that continued exposure to a mixture of the major oral malodors MTH and DMS leads to cross-adaptation to multiple KFOs, including DMDS, 2FT, MTNA, MFT, and DPDS. These aroma compounds contribute to the freshness of citrus fruits (DMDS), coffee (2FT), soy sauce and potatoes (MTNA), roasted meat (MFT), and fresh onion (DPDS) (Synthetic Flavorings Editorial Committee. Revised and Expanded Edition: Synthetic Flavorings: Chemistry and Product Knowledge. The Chemical Daily; Schieberle, P. Food Chem. 55, 145-152 (1996); Czerny, M., Mayer, F. & Grosch, W. J. Agric. Food Chem. 47, 695-699 (1999); Kaneko, H. J. Brewing Association 110, 20-25 (2015); Semmelroch, P. & Grosch, W. J. Agric. Food Chem. 44, 537-543 (1996)). This suggests that once people become accustomed to their own bad breath, they become less able to detect the aroma of foods containing these aroma compounds. Conversely, for example, if you have bad breath immediately after waking up, reducing bad breath through oral care may cancel cross-adaptation, resulting in a stronger perception of the food flavors.

[0046] Example 3 Verification of Example 2 In Example 2, the selected KFOs DMDS, 2FT, MTNA, MFT, and DPDS demonstrated the possibility of cross-adaptation with malodor-causing substances (MTH + DMS). However, because the results of Example 2 were exploratory, there was a possibility that false positives were included. Therefore, verification experiments were conducted on these KFOs, 2FT, MTNA, and DPDS, under various control conditions, such as adaptation to odorless samples and self-adaptation conditions.

[0047] 1) Method The odor sample was prepared by adding 35 mL of diluted odor solution to a 50 mL standard bottle. During the test, participants were asked to take a light sniff of the bottle opening. Afterwards, they were asked to record the perceived odor intensity on a 10 cm VAS. The adaptation procedure involved sniffing the presented sample for two minutes. After two minutes, the same odor sample was presented again, and participants were asked to evaluate the odor intensity in the same way as before the adaptation procedure.

[0048] 2) Results The results are shown in Figure 3. For 2FT, DPDS, and MTNA, no decrease in sensory intensity was observed in the negative control adaptation condition (None) or the MSC adaptation condition. A statistically significant decrease in sensory intensity was observed in all self-adaptation conditions (Wilcoxon signed rank test, p < 0.0083 with Bonferroni correction). This indicates that this test system can accurately capture the decrease in sensory intensity due to adaptation. Furthermore, a statistically significant decrease in sensory intensity was observed for 2FT, DPDS, and MTNA in the OMA adaptation condition (Figures 3B, C, and D). Furthermore, while a statistically significant decrease in 2FT sensory intensity was not observed in the DMS adaptation condition, a statistically significant decrease in 2FT sensory intensity was observed in the MTH adaptation condition (Figure 3B). These results suggest that at least MTH contributes to the cross-adaptation effect of OMA on 2FT. For DPDS and MTNA, no statistically significant decrease in sensory intensity was observed in either the DMS or MTH conditions (Figures 3C and D). These results suggest that both DMS and MTH are required for the decrease in the sensory intensity of DPDS and MTNA due to adaptation to OMA. There may be other olfactory receptors besides OR4S2 and OR2T11 that play a major role in the perception of the sensory intensity of each substance, DPDS and MTNA, and these olfactory receptors may be more strongly activated by the mixture of DMS and MTH (OMA) than by DMS or MTH alone, and thus undergo sufficient adaptation. The above results demonstrate that the OMA found in Example 2 certainly induces cross-adaptation to the KFOs.

Claims

1. A method for improving flavor sensitivity in a subject, comprising reducing the amounts of methanethiol (MTH) and dimethyl sulfide (DMS) in the oral cavity of the subject, wherein the flavor sensitivity is flavor sensitivity to at least one flavor derived from at least one selected from the group consisting of dipropyl disulfide (DPDS), methional (MTNA), dimethyl disulfide (DMDS), and 2-methyl-3-furanthiol (MFT).

2. 2. The method according to claim 1, wherein the flavor sensitivity is a flavor sensitivity to at least one flavor derived from DPDS, MTNA, and MFT.

3. The method of claim 1 , comprising reducing bad breath in the subject.

4. The method of claim 1 , further comprising rinsing the subject's mouth.

5. 2. The method of claim 1, wherein the flavor sensitivity is to at least one flavor selected from the group consisting of fresh onion flavor, soy sauce flavor, potato flavor, fresh citrus flavor, and roasted meat flavor.

6. A method for improving the olfactory sensitivity of a subject, comprising reducing the amount of MTH and DMS in the subject's oral cavity, wherein the olfactory sensitivity is olfactory sensitivity to at least one selected from the group consisting of DPDS, MTNA, DMDS, and MFT.

7. The method according to claim 6, wherein the olfactory sensitivity is olfactory sensitivity to at least one selected from the group consisting of DPDS, MTNA, and MFT.

8. 7. The method of claim 6, comprising reducing bad breath in the subject.

9. The method of claim 6, further comprising rinsing the subject's oral cavity.

10. A method for selecting a flavor sensitivity enhancer, comprising identifying a substance that removes MTH and DMS, wherein the flavor sensitivity is flavor sensitivity to at least one flavor derived from DPDS, MTNA, DMDS, and MFT.

11. The method according to claim 10, wherein the flavor sensitivity is a flavor sensitivity to a flavor derived from at least one selected from the group consisting of DPDS, MTNA, and MFT.

12. The method according to claim 10, further comprising subjecting the identified substance that removes MTH and DMS to a sensory evaluation for its effect of improving olfactory sensitivity.

13. A method for selecting an olfactory sensitivity enhancer, comprising identifying a substance that removes MTH and DMS, wherein the olfactory sensitivity is olfactory sensitivity to at least one selected from the group consisting of DPDS, MTNA, DMDS, and MFT.

14. The method according to claim 13, wherein the olfactory sensitivity is olfactory sensitivity to at least one selected from the group consisting of DPDS, MTNA, and MFT.

15. The method according to claim 13, further comprising subjecting the identified substance that removes MTH and DMS to a sensory evaluation for its effect of improving olfactory sensitivity.

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