CN115902128B - Use of olfactory receptors for the recognition of 4-ethylguaiacol and method for detecting 4-ethylguaiacol - Google Patents
Use of olfactory receptors for the recognition of 4-ethylguaiacol and method for detecting 4-ethylguaiacol Download PDFInfo
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Abstract
The invention provides an application of an olfactory receptor in identifying 4-ethyl guaiacol and a method for detecting the 4-ethyl guaiacol. The method comprises the following steps: contacting a sample to be tested with an olfactory receptor, determining a response value of the olfactory receptor after contact, and determining whether 4-ethyl guaiacol is contained in the sample to be tested based on the response value; in the use in the identification of 4-ethylguaiacol and the above method, the olfactory receptor comprises at least one selected from the group consisting of: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2 and OR10H3. In the invention, after the olfactory receptors are stimulated by the 4-ethyl guaiacol respectively, the olfactory receptors can be activated, so that the 4-ethyl guaiacol can be effectively identified by using the olfactory receptors, and a foundation is laid for detecting samples containing the 4-ethyl guaiacol.
Description
Technical Field
The invention relates to the technical field of chemical detection, in particular to application of an olfactory receptor in identifying 4-ethyl guaiacol and a method for detecting the 4-ethyl guaiacol, and more particularly relates to application of the olfactory receptor in identifying the 4-ethyl guaiacol, application of the 4-ethyl guaiacol in activating the olfactory receptor, a method for detecting the 4-ethyl guaiacol, a method for evaluating the quality of a soy product and a method for judging the processing technology of the soy product.
Background
4-ethyl guaiacol, also known as 4-ethyl-2-methoxyphenol, 4-EG (4-ethylguaiacol, CAS number 2785-89-9), is a colorless to pale yellow liquid, and the smell of miniphenol, a substance commonly described as a sauce flavor, smoke flavor, and butane flavor, is a natural aroma compound.
The content of the 4-ethyl guaiacol is closely related to the quality of the soy sauce, and the 4-ethyl guaiacol not only is a main aroma component of the soy sauce, but also has the effect of relieving salty taste, and the flavor quality of the soy sauce can be obviously improved by 1-5 mg/L of the 4-ethyl guaiacol. In addition, 4-ethyl guaiacol is a key aroma component for distinguishing between a high-salt dilute fermentation process and a low-salt solid fermentation process of soy sauce. In recent years, there have been examples of increasing the content of 4-ethyl guaiacol in soy sauce fermentation engineering by using a mixed culture of different strains or by adding a pure culture of the strains during fermentation.
Mammals have excellent olfactory perceptibility and can sensitively and rapidly recognize and distinguish odors in foods, mainly because odorants activate human Olfactory Receptors (ORs) to trigger nerve impulses, and transmit information about odors to the brain. However, no report has been made on detection of olfactory receptors for 4-ethyl guaiacol.
Disclosure of Invention
The present invention aims to solve at least to some extent one of the technical problems existing in the prior art. Therefore, the invention provides a method for detecting 4-ethyl guaiacol by adopting an olfactory receptor, and the method can rapidly identify the 4-ethyl guaiacol and can be used for assisting in evaluating the quality and the flavor of soy sauce.
In one aspect, the invention provides the use of an olfactory receptor in the recognition of 4-ethyl guaiacol, said olfactory receptor comprising at least one selected from the group consisting of: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2 and OR10H3. The inventor finds through experiments that after the olfactory receptors are stimulated by the 4-ethyl guaiacol respectively, the olfactory receptors can be activated, so that the 4-ethyl guaiacol can be effectively identified by using the olfactory receptors, and a foundation is laid for the identification of the 4-ethyl guaiacol and the detection of a sample containing the 4-ethyl guaiacol.
It should be noted that, if 4-ethyl guaiacol stimulates the olfactory receptor, the olfactory receptor is activated, namely "4-ethyl guaiacol can be recognized by the olfactory receptor"; if the olfactory receptor is not activated after the 4-ethyl guaiacol stimulates the olfactory receptor, the olfactory receptor is "the olfactory receptor cannot recognize the 4-ethyl guaiacol".
In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and/or MOR179-7.
According to an embodiment of the invention, the recognition is manifested by a change in the activity of the olfactory receptor.
According to an embodiment of the invention, the change in activity comprises at least one of the following signal changes: luciferase, secreted alkaline phosphatase, fluorescent protein, fluorescent probe, cAMP, IP3, calcium ion, current and pH.
Illustratively, the recognition is manifested by an increase in cAMP downstream of the olfactory receptor. The inventor finds through experiments that 4-ethyl guaiacol stimulates cells expressing an olfactory receptor, after the olfactory receptor is activated, the concentration of cAMP in the cells is increased, and whether the olfactory receptor can recognize 4-ethyl guaiacol can be determined by detecting the change of the concentration of cAMP.
In another aspect of the invention, the invention proposes the use of 4-ethyl guaiacol for activating an olfactory receptor comprising at least one selected from the group consisting of: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2 and OR10H3. The inventor finds through experiments that after the olfactory receptors are stimulated by the 4-ethyl guaiacol respectively, the olfactory receptors can be activated, so that the 4-ethyl guaiacol can be effectively identified by using the olfactory receptors, and a foundation is laid for the identification of the 4-ethyl guaiacol and the detection of a sample containing the 4-ethyl guaiacol.
In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and/or MOR179-7.
According to an embodiment of the invention, the activation is manifested by a change in the activity of the olfactory receptor.
According to an embodiment of the invention, the change in activity comprises at least one of the following signal changes: luciferase, secreted alkaline phosphatase, fluorescent protein, fluorescent probe, cAMP, IP3, calcium ion, current and pH.
Illustratively, the activation is manifested by an increase in cAMP downstream of the olfactory receptor. The inventor finds through experiments that 4-ethyl guaiacol stimulates cells expressing an olfactory receptor, after the olfactory receptor is activated, the concentration of cAMP in the cells is increased, and whether the olfactory receptor can activate 4-ethyl guaiacol can be determined by detecting the change of the concentration of cAMP.
In yet another aspect of the invention, the invention provides a method of detecting 4-ethyl guaiacol. According to an embodiment of the invention, the method comprises: contacting a sample to be tested with an olfactory receptor, and determining a response value of the olfactory receptor after contact; determining whether the sample to be detected contains 4-ethyl guaiacol or not based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2 and OR10H3. From the foregoing, it is apparent that 4-ethylguaiacol activates the above-mentioned olfactory receptor, and thus, a sample to be tested is brought into contact with the above-mentioned olfactory receptor, and if 4-ethylguaiacol is contained in the sample to be tested, the above-mentioned olfactory receptor can be activated, and a response value after activation of the olfactory receptor is obtained, and whether 4-ethylguaiacol is contained in the sample to be tested can be determined based on the response value.
It should be noted that "contact" in this specification is to be understood in a broad sense, and may be a direct contact or an indirect contact, and is not particularly limited.
Illustratively, the sample to be tested (either liquid per se or prepared as a liquid using a solvent) is contacted with the olfactory receptor after being mixed in a liquid state (i.e., the sample to be tested is in direct contact with the olfactory receptor); or directly placing the sample to be tested and the olfactory receptor in the same space, wherein the odor molecules (4-ethyl guaiacol) released by the sample to be tested are contacted with the olfactory receptor (namely, the sample to be tested and the olfactory receptor are indirectly contacted).
In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and/or MOR179-7.
According to an embodiment of the invention, the olfactory receptor presence response value is an indication that 4-ethyl guaiacol is contained in the sample to be tested; alternatively, the absence of a response value for the olfactory receptor is an indication that the test sample does not contain 4-ethylguaiacol.
The "indication of no 4-ethyl guaiacol" means that 4-ethyl guaiacol is completely absent from the sample to be tested; or that a small amount of 4-ethyl guaiacol is present in the sample to be tested but cannot be detected.
In one embodiment of the invention, the olfactory receptor is selected from MOR170-15, and the olfactory receptor presence response value is an indication of the presence of 4-ethyl guaiacol in the test sample or an indication of the presence of not less than 3. Mu.M of 4-ethyl guaiacol; alternatively, the olfactory receptor absence response value is an indication that the test sample does not contain 4-ethylguaiacol or contains less than 3 μm of 4-ethylguaiacol.
In one embodiment of the invention, the olfactory receptor is selected from MOR179-7, and the olfactory receptor presence response value is an indication of the presence of 4-ethyl guaiacol in the test sample or an indication of the presence of not less than 10. Mu.M 4-ethyl guaiacol; alternatively, the olfactory receptor absence response value is an indication that the test sample does not contain 4-ethylguaiacol or contains less than 10 μm of 4-ethylguaiacol.
According to an embodiment of the invention, the method further comprises: and determining the content of the 4-ethyl guaiacol in the sample to be detected based on a standard curve, wherein the standard curve is a curve corresponding to the response value of the preset amount of the 4-ethyl guaiacol and the olfactory receptor. Thus, the content of 4-ethyl guaiacol in the sample to be tested can be detected.
According to an embodiment of the invention, the olfactory receptor is provided by a cell or transgenic cell expressing the olfactory receptor.
According to an embodiment of the invention, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.
In some alternative embodiments of the invention, the eukaryotic cells include, but are not limited to, cells selected from the group of cells isolated from the olfactory substrate, HEK293 cells, CHO cells, xenopus oocytes, hela cells, COS cells, yeast cells, and the like.
According to an embodiment of the invention, the prokaryotic cell is selected from bacteria.
According to an embodiment of the invention, the response value is obtained by detecting a change in the activity of the olfactory receptor.
According to an embodiment of the invention, 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, amperometric detection, isotopic labeling, antibody detection and pH detection.
Illustratively, the luciferase assay detects that when 4-ethylguaiacol or a sample containing 4-ethylguaiacol is used to stimulate cells containing an olfactory receptor, if the olfactory receptor is activated, the intracellular cAMP concentration increases, cAMP binds to the CRE-luciferase promoter region and causes the luciferase to be transcribed and translated, thus, by detecting the luciferase activity, the response of the olfactory receptor can be characterized and whether the olfactory receptor recognizes 4-ethylguaiacol can be determined.
According to an embodiment of the invention, the response value is obtained by detecting a change in the concentration of cAMP in the cell.
Illustratively, the change in cAMP concentration in the cell is obtained by using a GloSensorTMcAMP detection kit. The GloSensor-20F cAMP gene construct can pre-express a luciferase variant, the increase of the cAMP concentration can cause the conformational change of the luciferase variant, so that the luciferase is converted from an inactive state to an active state, a substrate of the luciferase variant is provided by a GloSensor cAMP detection kit, the change of the cAMP concentration can be measured in real time, and the content of 4-ethyl guaiacol in a sample to be detected can be rapidly and sensitively detected.
In yet another aspect of the present invention, the present invention provides a method of evaluating the quality of soy sauce products. According to an embodiment of the invention, the method comprises: contacting a soy sauce product to be tested with an olfactory receptor, and determining a response value of the olfactory receptor after contact; determining the quality of the soy sauce product to be tested based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2 and OR10H3. From the previous results, the 4-ethyl guaiacol can activate the olfactory receptor, so that the soy product to be tested is contacted with the olfactory receptor, if the soy product to be tested contains the 4-ethyl guaiacol, the olfactory receptor can be activated, a response value after the olfactory receptor is activated is obtained, and the quality of the soy product to be tested can be determined according to the magnitude of the response value.
According to the embodiment of the invention, the soy sauce product to be tested is subjected to dilution treatment in advance before the contact, wherein the dilution factor is 10-1000 times, for example 10-500 times, 10-400 times, 10-300 times, 10-200 times, 10-100 times, 10-90 times, 20-80 times, 30-70 times and the like.
In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and/or MOR179-7.
According to the embodiment of the invention, the response value is higher than the first preset threshold value and is an indication that the soy sauce product to be tested is qualified.
In this context, a "predetermined threshold" may be obtained by detecting a large number of soy products (e.g., 20, 50, 100, 150, 200, and more) using the olfactory receptor of the present invention, and based on the response values obtained for the large number of soy products. The "predetermined threshold" may be a response value obtained directly, may be a corresponding multiple with respect to the control group, and the specific type is not limited.
It should be noted that, as those skilled in the art can know, the quality of soy sauce is significantly improved if 4-ethyl guaiacol is produced during fermentation of soy sauce, and the quality of soy sauce can be significantly changed when the soy sauce product contains about 1-2mg/L of 4-ethyl guaiacol, and the concentration of 4-ethyl guaiacol in the soy sauce product cannot exceed 5mg/L. Based on this, the first predetermined threshold value is not lower than 6.5. Mu.M, but of course, the threshold value can be continuously adjusted according to the actual situation, and the setting range of the first predetermined threshold value is 6.5. Mu.M-33. Mu.M, for example, any point value of 6.57. Mu.M-32.89. Mu.M, 13.14. Mu.M-32.89. Mu.M, and 6.57. Mu.M-13.14. Mu.M.
Illustratively, the first predetermined threshold is about 6.57 μΜ or 13.14 μΜ.
According to the embodiment of the invention, the quality of the soy sauce product to be tested is proportional to the response value.
In some alternative embodiments of the present invention, a plurality of soy products to be tested are contacted with an olfactory receptor, respectively, and the response value of the olfactory receptor after the contact is determined; and determining the quality of the soy sauce product to be tested based on the response value, wherein the response value is higher and is an indication that the quality of the soy sauce product to be tested is higher.
Illustratively, as shown in FIG. 4, the quality of organic fresh soy sauce, plum-brocade-flavor extremely fresh and golden lion soybean soy sauce is detected by using the olfactory receptor of the present invention, the response value of the organic fresh soy sauce is higher than that of the plum-golden lion-flavored extremely-fresh soy sauce, so that the organic fresh soy sauce is better in quality, the plum-golden-flavored extremely-fresh soy sauce is obtained, and the golden lion-flavored extremely-fresh soy sauce is obtained.
According to an embodiment of the invention, the olfactory receptor is provided by a cell or transgenic cell expressing the olfactory receptor.
According to an embodiment of the invention, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.
In some alternative embodiments of the invention, the eukaryotic cells include, but are not limited to, cells selected from the group of cells isolated from the olfactory substrate, HEK293 cells, CHO cells, xenopus oocytes, hela cells, COS cells, yeast cells, and the like.
According to an embodiment of the invention, the prokaryotic cell is selected from bacteria.
According to an embodiment of the invention, the response value is obtained by detecting a change in the activity of the olfactory receptor.
According to an embodiment of the invention, 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, amperometric detection, isotopic labeling, antibody detection and pH detection.
Illustratively, the luciferase assay detects that when 4-ethylguaiacol or a sample containing 4-ethylguaiacol is used to stimulate cells containing an olfactory receptor, if the olfactory receptor is activated, the intracellular cAMP concentration increases, cAMP binds to the CRE-luciferase promoter region and causes the luciferase to be transcribed and translated, thus, by detecting the luciferase activity, the response of the olfactory receptor can be characterized and whether the olfactory receptor recognizes 4-ethylguaiacol can be determined.
According to an embodiment of the invention, the response value is obtained by detecting a change in the concentration of cAMP in the cell.
Illustratively, the change in cAMP concentration in the cell is obtained by using a GloSensorTMcAMP detection kit. The GloSensor-20F cAMP gene construct can pre-express a luciferase variant, the increase of the cAMP concentration can cause the conformational change of the luciferase variant, so that the luciferase is converted from an inactive state to an active state, a substrate of the luciferase variant is provided by a GloSensor cAMP detection kit, the change of the cAMP concentration can be measured in real time, and the content of 4-ethyl guaiacol in a sample to be detected can be rapidly and sensitively detected.
In yet another aspect of the present invention, a method for determining a soy sauce product processing technique is provided. According to an embodiment of the invention, the method comprises: contacting a soy sauce product to be tested with an olfactory receptor, and determining a response value of the olfactory receptor after contact; determining the processing technology of the soy sauce product to be tested based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR170-15, MOR179-7, MOR125-5_p, MOR106-4, OR2W1, OR2J2, OR9Q2 and OR10H3. From the previous results, the 4-ethyl guaiacol can activate the olfactory receptor, so that the soy product to be tested is contacted with the olfactory receptor, if the soy product to be tested contains the 4-ethyl guaiacol, the olfactory receptor can be activated, a response value of the activated olfactory receptor is obtained, the content of the 4-ethyl guaiacol can be determined according to the size of the response value, and the processing technology of the soy product is further judged.
According to an embodiment of the invention, the processing process comprises a high salt dilute fermentation process or a low salt solid fermentation process. As known by those skilled in the art, 4-ethyl guaiacol is also a key aroma component for distinguishing the high-salt dilute state fermentation process and the low-salt solid state fermentation process of soy, therefore, by adopting the method of the invention, the soy product to be tested is contacted with the olfactory receptor, if the soy product to be tested contains 4-ethyl guaiacol, the olfactory receptor can be activated, a response value after the olfactory receptor is activated is obtained, the content of 4-ethyl guaiacol can be determined according to the size of the response value, and the processing process of the soy product is further judged.
According to the embodiment of the invention, the soy sauce product to be tested is subjected to dilution treatment in advance before the contact, wherein the dilution factor is 10-1000 times, for example, 10-500 times, 10-400 times, 10-300 times, 10-200 times, 10-100 times, 10-90 times, 20-80 times, 30-70 times and the like.
In a preferred embodiment of the present invention, the olfactory receptor is MOR170-15 and/or MOR179-7.
According to an embodiment of the invention, the response value being higher than a second predetermined threshold value is an indication that the soy sauce product to be tested is obtained by a high salt dilute fermentation process.
According to an embodiment of the invention, the second predetermined threshold is not lower than 1mg/L.
The second predetermined threshold is, for example, about 6.5 μm.
According to an embodiment of the invention, the olfactory receptor is provided by a cell or transgenic cell expressing the olfactory receptor.
According to an embodiment of the invention, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.
In some alternative embodiments of the invention, the eukaryotic cells include, but are not limited to, cells selected from the group of cells isolated from the olfactory substrate, HEK293 cells, CHO cells, xenopus oocytes, hela cells, COS cells, yeast cells, and the like.
According to an embodiment of the invention, the prokaryotic cell is selected from bacteria.
According to an embodiment of the invention, the response value is obtained by detecting a change in the activity of the olfactory receptor.
According to an embodiment of the invention, 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, amperometric detection, isotopic labeling, antibody detection and pH detection.
Illustratively, the luciferase assay detects that when 4-ethylguaiacol or a sample containing 4-ethylguaiacol is used to stimulate cells containing an olfactory receptor, if the olfactory receptor is activated, the intracellular cAMP concentration increases, cAMP binds to the CRE-luciferase promoter region and causes the luciferase to be transcribed and translated, and thus, by detecting the luciferase activity, the response of the olfactory receptor can be characterized, i.e., whether the olfactory receptor recognizes 4-ethylguaiacol can be determined.
According to an embodiment of the invention, the response value is obtained by detecting a change in the concentration of cAMP in the cell.
Illustratively, the change in cAMP concentration in the cell is obtained by using a GloSensorTMcAMP detection kit. The GloSensor-20F cAMP gene construct can pre-express a luciferase variant, the increase of the cAMP concentration can cause the conformational change of the luciferase variant, so that the luciferase is converted from an inactive state to an active state, a substrate of the luciferase variant is provided by a GloSensor cAMP detection kit, the change of the cAMP concentration can be measured in real time, and the content of 4-ethyl guaiacol in a sample to be detected can be rapidly and sensitively detected.
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.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a graph showing the response of 4-ethyl guaiacol to human and murine 400 Olfactory Receptors (OR) in example 1 of the present invention;
FIG. 2 is a graph showing the dose-dependent curves of MOR170-15, MOR179-7 and MOR139-3 in response to 4-ethyl guaiacol in example 1 of the present invention;
FIG. 3 is a graph showing the very fresh response of MOR170-15 and MOR179-7 to the taste of the brocade in example 2 of the present invention;
FIG. 4 shows the response of MOR170-15 and MOR179-7 to the taste of the plum blossom in example 3 of the present invention to the organic light soy sauce and the golden lion soy sauce.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
The term "about" is used herein to provide literal support for the exact numerical value preceding it, as well as numerical values that are close or approximate preceded by the term. In determining whether a numerical value is close or approximates a specifically stated numerical value, the close or approximated non-stated numerical value may be a numerical value that, in the context in which it is provided, provides substantial equivalence of the specifically stated numerical value. The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain 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.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: screening of olfactory receptors
The activity of the olfactory receptor was determined in this example using the double luciferase assay (Dual-Glo ™ Luciferase Assay System, promega). The inventors selected a total olfactory receptor library from the human olfactory receptor library and a partial olfactory receptor library of mice for a total of 400 olfactory receptors, then prepared a gene construct containing olfactory receptors, golf, CRE-Luciferase and pRL-SV40, and transfected the gene construct into HEK293T cells using the transfection reagent Lipofectamine2000 (Invitrogen). After 24 hours of incubation, 4-ethyl guaiacol was diluted to a concentration of 300. Mu.M with the medium, cells were stimulated with the diluted 4-ethyl guaiacol, and incubated for 2-4 hours. If the olfactory receptor is activated, the intracellular cAMP concentration increases, cAMP binds to the promoter region of CRE-luciferase and causes transcription and translation of luciferase, and the response of the olfactory receptor can be characterized by detecting the activity of luciferase, and 9 responding olfactory receptors are finally obtained, including 4 mouse olfactory receptors (MOR 170-15 (Gen. ID: 404313), MOR179-7 (Gen. ID: 258362), MOR125-5_p (Gen. Gene ID: 404316), MOR106-4 (Gen. Gene ID:258661,protein ID:NP_666878.2), and 4 human olfactory receptors (OR 2W1 (Gen. Gene ID: 26692), OR2J2 (Gen. ID: 26707), OR9Q2 (Gen. Gene ID: 219957) and OR10H3 (Gen. Gene. ID: 2626)), as shown in FIG. 1. The inventors selected the 2 most responsive follow-up experiments, MOR170-15 and MOR179-7, respectively.
Previous studies reported that 4-ethylguaiacol activated olfactory receptor MOR139-3, based on which the inventors stimulated cells containing the olfactory receptors MOR139-3, MOR170-15 and MOR179-7, respectively, with different concentrations of 4-ethylguaiacol, and observed the dose curves of the 4 olfactory receptors in response to 4-ethylguaiacol at different concentrations, the results are shown in FIG. 2, wherein the horizontal axis represents the logarithm of 4-ethylguaiacol concentration (M) and the vertical axis represents the fold change in response relative to the blank (without 4-ethylguaiacol).
As a result, it was found that MOR139-3 responded to 4-ethyl guaiacol only about 3-fold at 300. Mu.M, whereas at the same concentration, the response times of MOR170-15 to 4-ethyl guaiacol in the present invention could be about 24-fold, and the response times of MOR179-7 to 4-ethyl guaiacol could be about 15-fold, and it was apparent that the response times of MOR170-15 and MOR179-7 newly found in the present invention were higher than those of MOR 139-3. Of these, the sensitivity of 4-ethyl guaiacol was 3. Mu.M, and the sensitivity of MOR179-7 was 10. Mu.M, the two olfactory receptors that initiated the reaction were more sensitive to low concentrations of 4-ethyl guaiacol.
Example 2: detection of soy products by different olfactory receptors
In this example, the detection of a plum-flavored extremely fresh soy sauce product was performed by using the method of example 1, using cells expressing MOR170-15 and MOR179-7, respectively. The inventors performed 50-fold dilution of soy sauce using CD293 medium, and then examined the diluted soy sauce with cells expressing MOR170-15 and MOR179-7, respectively, using the method of example 1. The results of the assay are shown in FIG. 3, in which the horizontal axis indicates olfactory receptor type and the vertical axis indicates fold change in response relative to the blank (without soy addition).
The results indicate that activation signals are detected for both MOR170-15 and MOR179-7 olfactory receptors, with the highest response of MOR170-15 being about 9-fold and MOR179-7 being about 3-fold.
Example 3: soy sauce produced by different processing techniques is detected by different olfactory receptors
In this example, the method of example 1 was used to detect soy sauce produced by different processing techniques by cells expressing MOR170-15 and MOR179-7, respectively, wherein the high salt diluted fermented soy sauce is plum-flavored extremely fresh (ordinary soy sauce) and organic-flavored fresh soy sauce (organic soy sauce); the low-salt dilute state fermented soy sauce is golden lion soybean soy sauce.
The inventors performed 50-fold dilutions of each of the 2 soy sauces using CD293 medium, and then examined the diluted soy sauces using the method of example 1 for cells expressing MOR170-15 and MOR179-7, respectively. The results of the test are shown in FIG. 4, wherein the horizontal axis shows olfactory receptor type and soy brand, and the vertical axis shows the response change times relative to the blank (without soy added), wherein the black bar graph shows the organic fresh soy, the dark gray bar graph shows the plum-brocade flavor, and the light gray bar graph shows the golden lion soybean soy.
The results show that 4-ethyl guaiacol in soy sauce can be detected by all the 2 olfactory receptors; the content of 4-ethyl guaiacol in the high-salt dilute soy sauce is higher than that of the low-salt dilute soy sauce, and the smell receptors can detect that the content of 4-ethyl guaiacol in the high-salt dilute soy sauce is higher than that of the low-salt dilute soy sauce, so that the content of 4-ethyl guaiacol in the high-salt dilute soy sauce is consistent. Therefore, it was further demonstrated that the processing technology of soy sauce can be judged by using the above 2 olfactory receptors.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. Use of an olfactory receptor, MOR170-15, for the recognition of 4-ethylguaiacol;
wherein the amino acid sequence of MOR170-15 is as Genbank's Gene ID: 404313.
Use of 4-ethylguaiacol for activating an olfactory receptor, said olfactory receptor being MOR170-15;
wherein the amino acid sequence of MOR170-15 is as Genbank's Gene ID: 404313.
3. The use according to claim 1 or2, wherein the recognition or activation is manifested by a change in the activity of an olfactory receptor;
the change in activity includes at least one of the following signal changes:
luciferase, secreted alkaline phosphatase, fluorescent protein, fluorescent probe, cAMP, IP3, calcium ion, current and pH.
4. A method for detecting 4-ethyl guaiacol, comprising:
contacting a sample to be tested with an olfactory receptor, and determining a response value of the olfactory receptor after contact;
determining whether the sample to be detected contains 4-ethyl guaiacol or not based on the response value;
wherein the olfactory receptor is MOR170-15, and the amino acid sequence of MOR170-15 is as GenE ID of Genbank: 404313.
5. The method of claim 4, wherein the olfactory receptor presence response value is an indication that the test sample contains 4-ethyl guaiacol; alternatively, the absence of a response value for the olfactory receptor is an indication that the test sample does not contain 4-ethylguaiacol;
the method further comprises:
and determining the content of the 4-ethyl guaiacol in the sample to be detected based on a standard curve, wherein the standard curve is a corresponding curve of the response value of the preset amount of the 4-ethyl guaiacol and the olfactory receptor.
6. A method of evaluating the quality of a soy sauce product, comprising:
contacting a soy sauce product to be tested with an olfactory receptor, and determining a response value of the olfactory receptor after contact;
determining the quality of the soy sauce product to be tested based on the response value;
wherein the olfactory receptor is MOR170-15, and the amino acid sequence of MOR170-15 is as GenE ID of Genbank: 404313.
7. The method of claim 6, wherein the response value being higher than a first predetermined threshold value is an indication that the soy sauce product to be tested is of acceptable quality;
contacting a plurality of soy products to be tested with an olfactory receptor, and determining the response value of the olfactory receptor after contact;
determining the quality of the soy sauce product to be tested based on the response value,
the response value is higher, and is an indication that the quality of the soy sauce product to be tested is higher.
8. A method for judging a processing process of a soy sauce product, comprising:
contacting a soy sauce product to be tested with an olfactory receptor, and determining a response value of the olfactory receptor after contact;
determining the processing technology of the soy sauce product to be tested based on the response value;
wherein the olfactory receptor is MOR170-15, and the amino acid sequence of MOR170-15 is as GenE ID of Genbank: 404313.
9. The method of claim 8, wherein the processing process comprises a high salt dilute fermentation process or a low salt solid fermentation process.
10. The method according to any one of claims 4 to 9, wherein the olfactory receptor is provided by a cell expressing the olfactory receptor or a transgenic cell;
the response value is obtained by detecting a change in the concentration of cAMP in the cell;
the cell or transgenic cell is eukaryotic or prokaryotic;
the response value is obtained by detecting a change in the activity of the olfactory receptor;
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, amperometric detection, isotopic labeling, antibody detection and pH detection.
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