Use of olfactory receptors in the recognition of 4-ethylguaiacol and methods for detecting 4-ethylguaiacol

Abstract

This invention proposes the use of olfactory receptors in the identification of 4-ethylguaiacol, wherein the olfactory receptors include at least one selected from the following: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2, and OR10H3. In this invention, after stimulating the above-mentioned olfactory receptors with 4-ethylguaiacol, all of the above-mentioned olfactory receptors are activated. Therefore, 4-ethylguaiacol can be effectively identified using these olfactory receptors, laying the foundation for the identification of 4-ethylguaiacol and the detection of samples containing 4-ethylguaiacol.

Description

[0001] Case information

[0002] This application is a divisional application of the patent application filed on February 8, 2023, with China National Intellectual Property Administration, patent application number 202310141341.X, entitled "Use of olfactory receptors in recognizing 4-ethylguaiacol and method for detecting 4-ethylguaiacol". Technical Field

[0003] This invention relates to the field of chemical detection technology. Specifically, this invention relates to the use of olfactory receptors in recognizing 4-ethylguaiacol and a method for detecting 4-ethylguaiacol. More specifically, this invention relates to the use of olfactory receptors in recognizing 4-ethylguaiacol, the use of 4-ethylguaiacol in activating olfactory receptors, a method for detecting 4-ethylguaiacol, a method for evaluating the quality of soy sauce products, and a method for judging the processing technology of soy sauce products. Background Technology

[0004] 4-Ethylguaiacol, also known as 4-ethyl-2-methoxyphenol, or simply 4-EG (4-ethylguaiacol, CAS No. 2785-89-9), is a colorless to pale yellow liquid with a slight phenolic odor. It is usually described as a substance with a soy sauce aroma, a smoky aroma, and a clove aroma. It is a natural aroma compound.

[0005] The content of 4-ethylguaiacol is closely related to the quality of soy sauce. It is not only a major aroma component of soy sauce but also plays a role in moderating its saltiness; 1–5 mg / L of 4-ethylguaiacol can significantly improve the flavor quality of soy sauce. Furthermore, 4-ethylguaiacol is a key aroma component that distinguishes soy sauce produced using high-salt liquid-state fermentation processes from those produced using low-salt solid-state fermentation processes. In recent years, there have been examples of increasing the 4-ethylguaiacol content in soy sauce fermentation processes by using mixed cultures of different strains or by adding pure cultures of strains during fermentation.

[0006] Mammals possess an excellent sense of smell, enabling them to quickly and sensitively identify and distinguish odors in food. This is primarily because odorants activate olfactory receptors (ORs), triggering nerve impulses that transmit odor information to the brain. However, there are currently no reports on the detection of olfactory receptors for 4-ethylguaiacol. Summary of the Invention

[0007] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides a method for detecting 4-ethylguaiacol using olfactory receptors. This method can rapidly identify 4-ethylguaiacol and can be used to assist in evaluating the quality and flavor of soy sauce.

[0008] In one aspect of the invention, the present invention provides the use of olfactory receptors in the identification of 4-ethylguaiacol, said olfactory receptors comprising at least one selected from the following: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2, and OR10H3. The inventors have experimentally discovered that stimulating the aforementioned olfactory receptors with 4-ethylguaiacol activates all of them. Therefore, 4-ethylguaiacol can be effectively identified using these olfactory receptors, laying the foundation for the identification of 4-ethylguaiacol and the detection of samples containing 4-ethylguaiacol.

[0009] It should be noted that if the olfactory receptor is activated after 4-ethylguaiacol stimulates it, it is called "olfactory receptor can recognize 4-ethylguaiacol"; if the olfactory receptor is not activated after 4-ethylguaiacol stimulates it, it is called "olfactory receptor cannot recognize 4-ethylguaiacol".

[0010] In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and / or MOR179-7.

[0011] According to an embodiment of the present invention, the recognition is manifested through changes in the activity of olfactory receptors.

[0012] According to embodiments of the present invention, the activity change includes at least one of the following signal changes: luciferase, secretory alkaline phosphatase, fluorescent protein, fluorescent probe, cAMP, IP3, calcium ions, current, and pH.

[0013] For example, the recognition is manifested through an increase in cAMP downstream of the olfactory receptor. The inventors discovered through experiments that when 4-ethylguaiacol stimulates cells expressing olfactory receptors, the intracellular cAMP concentration increases after the olfactory receptors are activated. By detecting changes in cAMP concentration, it can be determined whether the olfactory receptors can recognize 4-ethylguaiacol.

[0014] In another aspect of the invention, the use of 4-ethylguaiacol in activating olfactory receptors, said olfactory receptors comprising at least one of the following: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2, and OR10H3. The inventors have experimentally discovered that stimulating the aforementioned olfactory receptors with 4-ethylguaiacol activates them, thereby enabling the effective identification of 4-ethylguaiacol and laying the foundation for the identification of 4-ethylguaiacol and the detection of samples containing 4-ethylguaiacol.

[0015] In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and / or MOR179-7.

[0016] According to an embodiment of the present invention, the activation is manifested through changes in the activity of olfactory receptors.

[0017] According to embodiments of the present invention, the activity change includes at least one of the following signal changes: luciferase, secretory alkaline phosphatase, fluorescent protein, fluorescent probe, cAMP, IP3, calcium ions, current, and pH.

[0018] For example, the recognition is manifested through an increase in cAMP downstream of the olfactory receptor. The inventors discovered through experiments that when 4-ethylguaiacol stimulates cells expressing olfactory receptors, the intracellular cAMP concentration increases after the olfactory receptors are activated. By detecting changes in cAMP concentration, it can be determined whether the olfactory receptors can recognize 4-ethylguaiacol.

[0019] In another aspect, the present invention provides a method for detecting 4-ethylguaiacol. According to an embodiment of the invention, the method includes: contacting a test sample with an olfactory receptor and determining the response value of the olfactory receptor after contact; determining, based on the response value, whether the test sample contains 4-ethylguaiacol; wherein the olfactory receptor includes at least one selected from the following: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2, and OR10H3. As is known, 4-ethylguaiacol can activate the aforementioned olfactory receptors. Therefore, when the test sample is contacted with the aforementioned olfactory receptors, if the test sample contains 4-ethylguaiacol, the olfactory receptors can be activated, and a response value after activation of the olfactory receptors is obtained. Based on the response value, it can be determined whether the test sample contains 4-ethylguaiacol.

[0020] It should be noted that the term "contact" in this article should be interpreted broadly, encompassing both direct and indirect contact, without any specific limitations.

[0021] For example, the test sample in liquid state (either liquid itself or prepared into liquid using a solvent) is mixed with the olfactory receptor and then brought into contact (i.e., the test sample and the olfactory receptor are in direct contact); alternatively, the test sample and the olfactory receptor can be placed in the same space, and the odor molecules (4-ethylguaiacol) released by the test sample itself come into contact with the olfactory receptor (i.e., the test sample and the olfactory receptor are indirectly contacted).

[0022] In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and / or MOR179-7.

[0023] According to an embodiment of the present invention, the presence response value of the olfactory receptor is an indication that the sample to be tested contains 4-ethylguaiacol; or, the absence response value of the olfactory receptor is an indication that the sample to be tested does not contain 4-ethylguaiacol.

[0024] It should be noted that "indication of absence of 4-ethylguaiacol" means that 4-ethylguaiacol is completely absent in the sample to be tested; or that a small amount of 4-ethylguaiacol is present in the sample to be tested, but cannot be detected.

[0025] In one embodiment of the present invention, the olfactory receptor is selected from MOR170-15, and the presence response value of the olfactory receptor is an indication that the test sample contains 4-ethylguaiacol or contains not less than 3 μM of 4-ethylguaiacol; or, the absence response value of the olfactory receptor is an indication that the test sample does not contain 4-ethylguaiacol or contains less than 3 μM of 4-ethylguaiacol.

[0026] In one embodiment of the present invention, the olfactory receptor is selected from MOR179-7, and the presence response value of the olfactory receptor is an indication that the test sample contains 4-ethylguaiacol or contains not less than 10 μM of 4-ethylguaiacol; or, the absence response value of the olfactory receptor is an indication that the test sample does not contain 4-ethylguaiacol or contains less than 10 μM of 4-ethylguaiacol.

[0027] According to an embodiment of the present invention, the method further includes: determining the content of 4-ethylguaiacol in the sample to be tested based on a standard curve, wherein the standard curve is a curve corresponding to a predetermined amount of 4-ethylguaiacol and the olfactory receptor response value. Thus, the content of 4-ethylguaiacol in the sample to be tested can be detected.

[0028] According to an embodiment of the present invention, the olfactory receptor is provided by a cell expressing the olfactory receptor or a transgenic cell.

[0029] According to embodiments of the present invention, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.

[0030] In some alternative embodiments of the present invention, the eukaryotic cells include, but are not limited to, cells selected from cell groups isolated from the olfactory substrate, HEK293 cells, CHO cells, Xenopus oocytes, HeLa cells, COS cells, and yeast cells.

[0031] According to an embodiment of the present invention, the prokaryotic cells are selected from bacteria.

[0032] According to an embodiment of the present invention, the response value is obtained by detecting changes in the activity of the olfactory receptor.

[0033] According to embodiments of the present invention, the change in activity is determined by at least one of the following detection methods: luciferase detection, secretory alkaline phosphatase detection, fluorescent protein detection, fluorescent probe detection, Ca2+ concentration detection, current detection, isotope labeling, antibody detection, and pH detection.

[0034] For example, the detection method of luciferase involves stimulating cells containing olfactory receptors with 4-ethylguaiacol or a sample containing 4-ethylguaiacol. If the olfactory receptors are activated, the intracellular cAMP concentration increases. cAMP binds to the promoter region of CRE-luciferase and promotes the transcription and translation of luciferase. Therefore, by detecting the activity of luciferase, the response of olfactory receptors can be characterized, and it can be determined whether olfactory receptors can recognize 4-ethylguaiacol.

[0035] According to an embodiment of the present invention, the response value is obtained by detecting changes in cAMP concentration in the cells.

[0036] For example, changes in cAMP concentration in the cells were obtained using the GloSensor™ cAMP assay kit. The GloSensor-20F cAMP gene construct pre-expresses a luciferase variant. Increased cAMP concentration causes a conformational change in the luciferase variant, causing the luciferase to transition from an inactive to an active state. The GloSensor™ cAMP assay kit provides the substrate for the luciferase variant. This method can measure changes in cAMP concentration in real time and can rapidly and sensitively detect the content of 4-ethylguaiacol in the sample.

[0037] In another aspect, the present invention provides a method for evaluating the quality of soy sauce products. According to an embodiment of the invention, the method includes: contacting the soy sauce product to be tested with olfactory receptors, determining the response value of the olfactory receptors after contact; and determining the quality of the soy sauce product to be tested based on the response value; wherein the olfactory receptors include at least one selected from the following: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2, and OR10H3. As is known, 4-ethylguaiacol can activate the above-mentioned olfactory receptors. Therefore, when the soy sauce product to be tested is contacted with the above-mentioned olfactory receptors, if the soy sauce product to be tested contains 4-ethylguaiacol, the above-mentioned olfactory receptors can be activated, and the response value after activation of the olfactory receptors is obtained. The quality of the soy sauce product to be tested can be determined according to the magnitude of the response value.

[0038] According to an embodiment of the present invention, before the contact, the soy sauce product to be tested is diluted in advance, and the dilution ratio is 10 to 1000 times, for example, 10 to 500 times, 10 to 400 times, 10 to 300 times, 10 to 200 times, 10 to 100 times, 10 to 90 times, 20 to 80 times, 30 to 70 times, etc.

[0039] In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and / or MOR179-7.

[0040] According to an embodiment of the present invention, the response value being higher than a first predetermined threshold is an indication that the quality of the soy sauce product under test is qualified.

[0041] In this paper, the "predetermined threshold" is obtained by statistically analyzing the response values ​​obtained from a large number of soy sauce products (e.g., 20, 50, 100, 150, 200 or more) using the olfactory receptor of this invention. The "predetermined threshold" can be a directly obtained response value, or a corresponding multiple relative to the control group, and its specific type is not limited.

[0042] It should be noted that, as those skilled in the art will know, the production of 4-ethylguaiacol during soy sauce fermentation significantly improves the quality of soy sauce. When a soy sauce product contains approximately 1-2 mg / L of 4-ethylguaiacol, its quality can be significantly altered. Generally, the concentration of 4-ethylguaiacol in soy sauce products should not exceed 5 mg / L. Based on this, the first predetermined threshold is not lower than 6.5 μM. Of course, this threshold can be further adjusted according to actual conditions. The setting range of the first predetermined threshold is 6.5 μM-33 μM, for example, it can be any value among 6.57 μM-32.89 μM, 13.14 μM-32.89 μM, and 6.57 μM-13.14 μM.

[0043] For example, the first predetermined threshold is approximately 6.57 μM or 13.14 μM.

[0044] According to an embodiment of the present invention, the quality of the soy sauce product to be tested is proportional to the response value.

[0045] In some optional embodiments of the present invention, multiple soy sauce products to be tested are respectively brought into contact with olfactory receptors to determine the response value of the olfactory receptors after contact; based on the response value, the quality of the soy sauce products to be tested is determined, wherein a higher response value is an indication of higher quality of the soy sauce products to be tested.

[0046] For example, such as Figure 4As shown, the olfactory receptors of this invention were used to test the quality of Haoji Organic Fresh Soy Sauce, Lee Kum Kee Premium Soy Sauce, and Golden Lion Soy Sauce. The response value of Organic Fresh Soy Sauce was > the response value of Lee Kum Kee Premium Soy Sauce > the response value of Golden Lion Soy Sauce. Therefore, Haoji Organic Fresh Soy Sauce has the best quality, followed by Lee Kum Kee Premium Soy Sauce, and lastly Golden Lion Soy Sauce.

[0047] According to an embodiment of the present invention, the olfactory receptor is provided by a cell expressing the olfactory receptor or a transgenic cell.

[0048] According to embodiments of the present invention, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.

[0049] In some alternative embodiments of the present invention, the eukaryotic cells include, but are not limited to, cells selected from cell groups isolated from the olfactory substrate, HEK293 cells, CHO cells, Xenopus oocytes, HeLa cells, COS cells, and yeast cells.

[0050] According to an embodiment of the present invention, the prokaryotic cells are selected from bacteria.

[0051] According to an embodiment of the present invention, the response value is obtained by detecting changes in the activity of the olfactory receptor.

[0052] According to embodiments of the present invention, the change in activity is determined by at least one of the following detection methods: luciferase detection, secretory alkaline phosphatase detection, fluorescent protein detection, fluorescent probe detection, Ca2+ concentration detection, current detection, isotope labeling, antibody detection, and pH detection.

[0053] For example, the detection method of luciferase involves stimulating cells containing olfactory receptors with 4-ethylguaiacol or a sample containing 4-ethylguaiacol. If the olfactory receptors are activated, the intracellular cAMP concentration increases. cAMP binds to the promoter region of CRE-luciferase and promotes the transcription and translation of luciferase. Therefore, by detecting the activity of luciferase, the response of olfactory receptors can be characterized, and it can be determined whether olfactory receptors can recognize 4-ethylguaiacol.

[0054] According to an embodiment of the present invention, the response value is obtained by detecting changes in cAMP concentration in the cells.

[0055] For example, changes in cAMP concentration in the cells were obtained using the GloSensor™ cAMP assay kit. The GloSensor-20F cAMP gene construct pre-expresses a luciferase variant. Increased cAMP concentration causes a conformational change in the luciferase variant, causing the luciferase to transition from an inactive to an active state. The GloSensor™ cAMP assay kit provides the substrate for the luciferase variant. This method can measure changes in cAMP concentration in real time and can rapidly and sensitively detect the content of 4-ethylguaiacol in the sample.

[0056] In another aspect, the present invention provides a method for determining the processing technology of soy sauce products. According to an embodiment of the present invention, the method includes: contacting the soy sauce product to be tested with olfactory receptors, and determining the response value of the olfactory receptors after contact; determining the processing technology of the soy sauce product to be tested based on the response value; wherein the olfactory receptors include at least one selected from the following: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2, and OR10H3. As is known, 4-ethylguaiacol can activate the above-mentioned olfactory receptors. Therefore, by contacting the soy sauce product to be tested with the above-mentioned olfactory receptors, if the soy sauce product to be tested contains 4-ethylguaiacol, the above-mentioned olfactory receptors can be activated, and the response value after activation of the olfactory receptors is obtained. The content of 4-ethylguaiacol can be determined based on the magnitude of the response value, thereby determining the processing technology of the soy sauce product.

[0057] According to embodiments of the present invention, the processing technology includes a high-salt dilute-state fermentation process or a low-salt solid-state fermentation process. Those skilled in the art will know that 4-ethylguaiacol is a key aroma component distinguishing soy sauce produced using high-salt dilute-state fermentation and low-salt solid-state fermentation processes. Therefore, using the method of the present invention, the soy sauce product to be tested is brought into contact with the aforementioned olfactory receptors. If the soy sauce product contains 4-ethylguaiacol, the aforementioned olfactory receptors can be activated, and the response value after activation is obtained. Based on the magnitude of the response value, the content of 4-ethylguaiacol can be determined, thereby judging the processing technology of the soy sauce product.

[0058] According to an embodiment of the present invention, before the contact, the soy sauce product to be tested is diluted by a dilution factor of 10 to 1000 times, for example, 10 to 500 times, 10 to 400 times, 10 to 300 times, 10 to 200 times, 10 to 100 times, 10 to 90 times, 20 to 80 times, 30 to 70 times, etc.

[0059] In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and / or MOR179-7.

[0060] According to an embodiment of the present invention, the response value being higher than a second predetermined threshold is an indication that the soy sauce product to be tested was obtained through a high-salt dilute-state fermentation process.

[0061] According to an embodiment of the present invention, the second predetermined threshold is not less than 1 mg / L.

[0062] For example, the second predetermined threshold is approximately 6.5 μM.

[0063] According to an embodiment of the present invention, the olfactory receptor is provided by a cell expressing the olfactory receptor or a transgenic cell.

[0064] According to embodiments of the present invention, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.

[0065] In some alternative embodiments of the present invention, the eukaryotic cells include, but are not limited to, cells selected from cell groups isolated from the olfactory substrate, HEK293 cells, CHO cells, Xenopus oocytes, HeLa cells, COS cells, and yeast cells.

[0066] According to an embodiment of the present invention, the prokaryotic cells are selected from bacteria.

[0067] According to an embodiment of the present invention, the response value is obtained by detecting changes in the activity of the olfactory receptor.

[0068] According to embodiments of the present invention, the change in activity is determined by at least one of the following detection methods: luciferase detection, secretory alkaline phosphatase detection, fluorescent protein detection, fluorescent probe detection, Ca2+ concentration detection, current detection, isotope labeling, antibody detection, and pH detection.

[0069] For example, the detection method of luciferase involves stimulating cells containing olfactory receptors with 4-ethylguaiacol or a sample containing 4-ethylguaiacol. If the olfactory receptors are activated, the intracellular cAMP concentration increases. cAMP binds to the promoter region of CRE-luciferase and promotes the transcription and translation of luciferase. Therefore, by detecting the activity of luciferase, the response of the olfactory receptors can be characterized, and it can be determined whether the olfactory receptors can recognize 4-ethylguaiacol.

[0070] According to an embodiment of the present invention, the response value is obtained by detecting changes in cAMP concentration in the cells.

[0071] For example, changes in cAMP concentration in the cells were obtained using the GloSensor™ cAMP assay kit. The GloSensor-20F cAMP gene construct pre-expresses a luciferase variant. Increased cAMP concentration causes a conformational change in the luciferase variant, causing the luciferase to transition from an inactive to an active state. The GloSensor™ cAMP assay kit provides the substrate for the luciferase variant. This method can measure changes in cAMP concentration in real time and can rapidly and sensitively detect the content of 4-ethylguaiacol in the sample.

[0072] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0073] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0074] Figure 1 The response spectra of 4-ethylguaiacol to 400 olfactory receptors (ORs) in humans and mice in Example 1 of the present invention are shown.

[0075] Figure 2 The dose-dependent curves of MOR170-15, MOR179-7 and MOR139-3 in response to 4-ethylguaiacol in Example 1 of the present invention are shown.

[0076] Figure 3 The response of MOR170-15 and MOR179-7 to Lee Kum Kee's premium flavor in Embodiment 2 of the present invention;

[0077] Figure 4 This describes the response of MOR170-15 and MOR179-7 to Lee Kum Kee Premium Soy Sauce, Hao Kee Organic Fresh Soy Sauce, and Golden Lion Soy Sauce in Example 3 of the present invention. Detailed Implementation

[0078] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0079] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0080] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0081] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0082] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0083] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0084] In this document, the term "about" is used to provide textual support for precise numerical values ​​preceding it, as well as numerical values ​​that are close to or approximate to a specific stated numerical value. In determining whether a numerical value is close to or approximates a specifically stated numerical value, the unstated numerical value that is close to or approximates can be a numerical value that, in the context in which it is provided, provides a substantial equivalent to the specifically stated numerical value. The term "about" or "approximately" refers to an acceptable error for a particular value as determined by those skilled in the art, which depends in part on how the value is measured or determined. In some embodiments, the term "about" or "approximately" refers to within 1, 2, 3, or 4 standard deviations. In some embodiments, the term "about" or "approximately" refers to within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

[0085] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0086] Example 1: Screening of olfactory receptors

[0087] This embodiment utilizes the dual-luciferase method (Dual-Luciferase). TM The Luciferase Assay System (Promega) was used to determine the activity of olfactory receptors. The inventors selected all olfactory receptors from the human olfactory receptor library and a partial mouse olfactory receptor library, totaling 400 receptors. Gene constructs containing olfactory receptors, Golf, CRE-Luciferase, and pRL-SV40 were then prepared and transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). After culturing for 24 hours, 4-ethylguaiacol was diluted to 300 μM in culture medium, and the cells were stimulated with the diluted 4-ethylguaiacol and incubated for 2-4 hours. When olfactory receptors are activated, intracellular cAMP concentration increases. cAMP binds to the promoter region of CRE-luciferase, promoting the transcription and translation of luciferase. By detecting luciferase activity, the response of olfactory receptors can be characterized. Nine responsive olfactory receptors were ultimately obtained, including four mouse olfactory receptors (MOR170-15 (Genbank Gene ID: 404313), MOR179-7 (Genbank Gene ID: 258362), MOR125-5_p (Genbank Gene ID: 404316), and MOR106-4 (Genbank Gene ID: 258661, protein ID: NP_666878.2)) and four human olfactory receptors (OR2W1 (Genbank Gene ID: 26692), OR2J2 (Genbank Gene ID: 26707), and OR9Q2 (Genbank Gene ID: 26707). Gene ID: 219957) and OR10H3 (Genbank Gene ID: 26532) were obtained, and the results were as follows: Figure 1 As shown. The inventors selected the two with the strongest responses for subsequent experiments, namely MOR170-15 and MOR179-7.

[0088] Previous studies have reported that 4-ethylguaiacol can activate the olfactory receptor MOR139-3. Based on this, the inventors stimulated cells containing olfactory receptors MOR139-3, MOR170-15, and MOR179-7 with different concentrations of 4-ethylguaiacol and observed the dose-response curves of the four olfactory receptors at different concentrations. The results are as follows: Figure 2 As shown, the horizontal axis represents the logarithm of the 4-ethylguaiacol concentration (M), and the vertical axis represents the fold change in response relative to the blank control (without 4-ethylguaiacol).

[0089] The results showed that MOR139-3 had the weakest response to 4-ethylguaiacol, only about 3 times at 300 μM. In contrast, at the same concentration, MOR170-15 (discovered in this invention) showed a response fold of about 24 times to 4-ethylguaiacol, and MOR179-7 showed a response fold of about 15 times. Clearly, compared to MOR139-3, the newly discovered MOR170-15 and MOR179-7 showed significantly higher response folds. Specifically, the sensitivity of 4-ethylguaiacol was 3 μM, and the sensitivity of MOR179-7 was 10 μM for initiation of the reaction. Both olfactory receptors were more sensitive to low concentrations of 4-ethylguaiacol.

[0090] Example 2: Detection of different olfactory receptors on soy sauce products

[0091] In this embodiment, the method of Example 1 was used to detect Lee Kum Kee's premium soy sauce product using cells expressing MOR170-15 and MOR179-7, respectively. The inventors diluted the soy sauce 50-fold using CD293 medium, and then used the method of Example 1 to detect the diluted soy sauce using cells expressing MOR170-15 and MOR179-7, respectively. The detection results are shown below. Figure 3 The horizontal axis represents the types of olfactory receptors, and the vertical axis represents the fold change in response relative to the blank control (no soy sauce added).

[0092] The results showed that both MOR170-15 and MOR179-7 olfactory receptors could detect activation signals. Among them, MOR170-15 had the highest response, about 9 times, while MOR179-7 had a response of about 3 times.

[0093] Example 3: Detection of different olfactory receptors on soy sauce produced by different processing techniques

[0094] In this embodiment, the method of Example 1 was used to detect soy sauce produced by different processing techniques by using cells expressing MOR170-15 and MOR179-7 respectively. Among them, the high-salt liquid fermented soy sauce was Lee Kum Kee Premium Soy Sauce (ordinary soy sauce) and Hao Kee Organic Fresh Soy Sauce (organic soy sauce); the low-salt liquid fermented soy sauce was Golden Lion Soybean Soy Sauce.

[0095] The inventors used CD293 medium to dilute the two types of soy sauce 50-fold, and then used the method in Example 1 to test the diluted soy sauce with cells expressing MOR170-15 and MOR179-7, respectively. The test results are shown below. Figure 3The horizontal axis represents the types of olfactory receptors and soy sauce brands, while the vertical axis represents the fold change in response relative to the blank control (no soy sauce added). The black bars represent Lee Kum Kee Premium Soy Sauce, the dark gray bars represent Hao Ji Organic Fresh Soy Sauce, and the light gray bars represent Golden Lion Soy Sauce.

[0096] The results showed that both olfactory receptors could detect 4-ethylguaiacol in soy sauce; both olfactory receptors also showed that the content of 4-ethylguaiacol in high-salt diluted soy sauce was higher than that in low-salt diluted soy sauce, exhibiting a consistent trend. Therefore, this further demonstrates that the processing technology of soy sauce can be determined using these two olfactory receptors.

[0097] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0098] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of olfactory receptors in the recognition of 4-ethylguaiacol, said olfactory receptors comprising at least one of the following: MOR179-7, MOR125-5_p and MOR106-4; in, The MOR179-7 has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 258362; The MOR125-5_p has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 404316; The MOR106-4 has an amino acid sequence as shown in the Genbank protein ID: NP_666878.

2.

2. Use of 4-ethylguaiacol in activating olfactory receptors, said olfactory receptors comprising at least one of the following: MOR179-7, MOR125-5_p and MOR106-4; in, The MOR179-7 has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 258362; The MOR125-5_p has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 404316; The MOR106-4 has an amino acid sequence as shown in the Genbank protein ID: NP_666878.

2.

3. The use according to claim 1 or 2, characterized in that, The olfactory receptors include those selected from MOR179-7; The recognition or activation is manifested through changes in the activity of olfactory receptors; The activity change includes at least one of the following signal changes: Luciferase, secretory alkaline phosphatase, fluorescent protein, fluorescent probe, cAMP, IP3, calcium ions, current, and pH.

4. A method for detecting 4-ethylguaiacol, characterized in that, include: The sample to be tested is brought into contact with an olfactory receptor, and the response value of the olfactory receptor after contact is determined. Based on the response value, determine whether the sample to be tested contains 4-ethylguaiacol; The olfactory receptors include at least one of the following: MOR179-7, MOR125-5_p and MOR106-4; The MOR179-7 has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 258362; The MOR125-5_p has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 404316; The MOR106-4 has an amino acid sequence as shown in the Genbank protein ID: NP_666878.

2.

5. The method according to claim 4, characterized in that, The presence of the olfactory receptor response value indicates that the sample to be tested contains 4-ethylguaiacol; or, the absence of the olfactory receptor response value indicates that the sample to be tested does not contain 4-ethylguaiacol. The method further includes: Based on the standard curve, the content of 4-ethylguaiacol in the sample to be tested was determined. The standard curve is the curve corresponding to a predetermined amount of 4-ethylguaiacol and the olfactory receptor response value.

6. A method for evaluating the quality of soy sauce products, characterized in that, include: The soy sauce product to be tested is brought into contact with olfactory receptors, and the response value of the olfactory receptors after contact is determined. Based on the response value, the quality of the soy sauce product to be tested is determined; The olfactory receptors include at least one of the following: MOR179-7, MOR125-5_p and MOR106-4; The MOR179-7 has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 258362; The MOR125-5_p has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 404316; The MOR106-4 has an amino acid sequence as shown in the Genbank protein ID: NP_666878.

2.

7. The method according to claim 6, characterized in that, The response value being higher than the first predetermined threshold is an indication that the quality of the soy sauce product under test is qualified. Multiple soy sauce products to be tested were brought into contact with olfactory receptors to determine the response value of the olfactory receptors after contact. Based on the response value, the quality of the soy sauce product to be tested is determined. A higher response value indicates a higher quality soy sauce product being tested.

8. A method for determining the processing technology of soy sauce products, characterized in that, include: The soy sauce product to be tested is brought into contact with olfactory receptors, and the response value of the olfactory receptors after contact is determined. Based on the response value, the processing technology of the soy sauce product to be tested is determined; The olfactory receptors include at least one of the following: MOR179-7, MOR125-5_p and MOR106-4; The MOR179-7 has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 258362; The MOR125-5_p has an amino acid sequence encoded by a nucleotide sequence as shown in Genbank's Gene ID: 404316; The MOR106-4 has an amino acid sequence as shown in the Genbank protein ID: NP_666878.

2.

9. The method according to claim 8, characterized in that, The processing technology includes high-salt dilute-state fermentation or low-salt solid-state fermentation.

10. The method according to any one of claims 4 to 9, characterized in that, The olfactory receptors include those selected from MOR179-7; The olfactory receptor is provided by cells expressing the olfactory receptor or by transgenic cells; The response value was obtained by detecting changes in cAMP concentration in the cells; The cells or transgenic cells are eukaryotic cells or prokaryotic cells; The response value is obtained by detecting changes in the activity of the olfactory receptors; The change in activity is determined by at least one of the following detection methods: Luciferase assay, secretory alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, Ca 2+ Concentration detection method, current detection method, isotope labeling method, antibody detection method and pH detection method.

Images