CN115840026B - Use of olfactory receptors for the recognition of 4-methoxybenzaldehyde and method for detecting 4-methoxybenzaldehyde - Google Patents
Use of olfactory receptors for the recognition of 4-methoxybenzaldehyde and method for detecting 4-methoxybenzaldehyde Download PDFInfo
- Publication number
- CN115840026B CN115840026B CN202310140517.XA CN202310140517A CN115840026B CN 115840026 B CN115840026 B CN 115840026B CN 202310140517 A CN202310140517 A CN 202310140517A CN 115840026 B CN115840026 B CN 115840026B
- Authority
- CN
- China
- Prior art keywords
- olfactory receptor
- methoxybenzaldehyde
- response value
- tested
- or2j2
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for detecting 4-methoxybenzaldehyde. The method comprises the following steps: 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-methoxybenzaldehyde or not based on the response value; wherein the olfactory receptor comprises OR2J2 and/OR 2W1. The 4-methoxybenzaldehyde in the method can activate the olfactory receptor, so that a sample to be detected is contacted with the olfactory receptor, if the sample to be detected contains 4-methoxybenzaldehyde, the olfactory receptor can be activated, a response value after the olfactory receptor is activated is obtained, and whether the sample to be detected contains 4-methoxybenzaldehyde can be determined according to the response value.
Description
Technical Field
The invention relates to the technical field of chemical detection, in particular to application of an olfactory receptor in identifying 4-methoxybenzaldehyde and a method for detecting 4-methoxybenzaldehyde, and more particularly relates to application of the olfactory receptor in identifying 4-methoxybenzaldehyde, application of 4-methoxybenzaldehyde in activating the olfactory receptor, a method for detecting 4-methoxybenzaldehyde, a method for detecting food raw materials and a method for evaluating quality of a spice product.
Background
4-methoxybenzaldehyde (CAS number 123-11-5), commonly known as anisaldehyde (p-anisaldehyde), has a molecular structure comprising a benzene ring, a methoxy group and an aldehyde group, and has a fragrance similar to that of strong pimpinella anisum and hawthorn. Natural products are found in fennel, star anise, vanilla beans, acacia, and the like.
The 4-methoxybenzaldehyde mainly has the following applications: 1) The method is mainly used for preparing daily use and food essence such as vanilla, spice, apricot, cream, fennel, caramel, cherry, chocolate, walnut, raspberry, strawberry, peppermint and the like; 2) 4-methoxybenzaldehyde is an excellent brightening agent for temporarily allowing edible flavors to be used as a non-cyanide galvanization DE additive in GB2760-86 in China, can improve anodic polarization in a wider current range, obtains a bright coating and creates favorable conditions for environmental protection; 3) Are intermediates of antihistaminic drugs in the pharmaceutical industry for the manufacture of the antimicrobial drug amoxicillin; 4) The compound with nitrogen functional groups is tracked as a developer.
Humans have excellent olfactory perceptibility and can sensitively and rapidly recognize and distinguish odors in daily foods, mainly because odorants activate human Olfactory Receptors (ORs) to trigger nerve impulses, and transmit information about odors to the brain. About 400 olfactory receptors are currently known in humans, but no report has been made on the detection of olfactory receptors for 4-methoxybenzaldehyde.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides the application of the OR2J2 and/OR OR2W1 olfactory receptor in recognizing 4-methoxybenzaldehyde, the olfactory receptor can rapidly recognize the 4-methoxybenzaldehyde, can detect the 4-methoxybenzaldehyde in a perfume product, and can be used for evaluating the quality of the perfume product.
In a first aspect of the invention, the invention proposes the use of an olfactory receptor comprising OR2J2 and/OR OR2W1 for the recognition of 4-methoxybenzaldehyde. The inventor finds through experiments that after the OR2J2 and the OR2W1 are stimulated by 4-methoxybenzaldehyde respectively, the OR2J2 and the OR2W1 can be activated, so that the 4-methoxybenzaldehyde can be effectively identified by utilizing the olfactory receptor, and a foundation is laid for the identification of the 4-methoxybenzaldehyde and the detection of a sample containing the 4-methoxybenzaldehyde.
In a second aspect of the invention, the invention proposes the use of 4-methoxybenzaldehyde for activating an olfactory receptor comprising OR2J2 and/OR OR2W1. The inventor finds through experiments that after the OR2J2 and the OR2W1 are stimulated by 4-methoxybenzaldehyde respectively, the OR2J2 and the OR2W1 can be activated, so that the 4-methoxybenzaldehyde can be effectively identified by utilizing the olfactory receptor, and a foundation is laid for the identification of the 4-methoxybenzaldehyde and the detection of a sample containing the 4-methoxybenzaldehyde.
In a third aspect of the present invention, the present invention provides a method for detecting 4-methoxybenzaldehyde. 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-methoxybenzaldehyde or not based on the response value; wherein the olfactory receptor comprises OR2J2 and/OR 2W1. From the foregoing, it is known that 4-methoxybenzaldehyde can activate the above-mentioned olfactory receptor, and thus, a sample to be tested is contacted with the above-mentioned olfactory receptor, if 4-methoxybenzaldehyde is contained in the sample to be tested, the above-mentioned olfactory receptor can be activated, and a response value after the olfactory receptor is activated is obtained, and according to the response value, whether 4-methoxybenzaldehyde is contained in the sample to be tested can be determined.
In a fourth aspect of the invention, the invention provides a method of detecting food ingredients. 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 the raw materials of the sample to be tested based on the response value; wherein the olfactory receptor comprises OR2J2 and/OR 2W1. From the foregoing, it is known that 4-methoxybenzaldehyde can activate the above-mentioned olfactory receptor, and thus, the sample to be tested is contacted with the above-mentioned olfactory receptor, if 4-methoxybenzaldehyde is contained in the sample to be tested, the above-mentioned olfactory receptor can be activated, and the response value after the olfactory receptor is activated is obtained, and it can be determined that the raw material containing 4-methoxybenzaldehyde is present in the sample to be tested, and if the olfactory receptor has no response value, the raw material containing 4-methoxybenzaldehyde is not present in the sample to be tested.
In a fifth aspect of the invention, the invention provides a method of assessing the quality of a fragrance product. According to an embodiment of the invention, the method comprises: contacting a perfume product to be tested with an olfactory receptor, and determining a response value of the olfactory receptor after contact; determining the quality of the perfume product to be tested based on the response value; wherein the olfactory receptor comprises OR2J2 and/OR 2W1. From the foregoing, it can be seen that 4-methoxybenzaldehyde can activate the above olfactory receptor, and thus, the perfume product to be tested is contacted with the above olfactory receptor, if the perfume product to be tested contains 4-methoxybenzaldehyde, the above olfactory receptor can be activated, and a response value after the olfactory receptor is activated is obtained, and the quality of the perfume product to be tested can be determined according to the magnitude of the response value.
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-methoxybenzaldehyde to stimulate 398 olfactory receptors (hOR) in a human being in example 1 of the present invention;
FIG. 2 is a graph showing the dose dependence of OR2J2 and OR2W1 in response to 4-methoxybenzaldehyde in example 1 of the present invention;
FIG. 3 is a graph showing the dose dependence of OR2J2 response to the extract of Hui brand Anise and Foeniculum vulgare in example 2 of the present invention;
FIG. 4 is a graph showing the dose-dependent response of OR2J2 to illicium verum oil produced by Guangxi farmers and to illicium verum essential oil of the plant Yisheng brand in example 3 of the present invention;
FIG. 5 shows the response of OR2J2 to tandem brand Fennel gas in example 4 of the present invention.
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 present invention provides a use of an olfactory receptor for recognizing 4-methoxybenzaldehyde, a use of 4-methoxybenzaldehyde for activating an olfactory receptor, a method for detecting 4-methoxybenzaldehyde, a method for detecting a food material and a method for evaluating the quality of a flavor product, which will be described in detail below, respectively.
Use of the same
In a first aspect of the invention, the invention proposes the use of an olfactory receptor comprising OR2J2 and/OR OR2W1 for the recognition of 4-methoxybenzaldehyde. The inventor finds through experiments that after the OR2J2 and the OR2W1 are stimulated by 4-methoxybenzaldehyde respectively, the OR2J2 and the OR2W1 can be activated, so that the 4-methoxybenzaldehyde can be effectively identified by utilizing the olfactory receptor, and a foundation is laid for the identification of the 4-methoxybenzaldehyde and the detection of a sample containing the 4-methoxybenzaldehyde. The inventor further discovers that the recognition of 4-methoxybenzaldehyde gas in the same space can be realized by adopting the olfactory receptor, and the recognition possibility of bionic olfactory recognition is provided.
It should be noted that, if the olfactory receptor is activated after the 4-methoxybenzaldehyde stimulates the olfactory receptor, the olfactory receptor is "4-methoxybenzaldehyde can be identified by the olfactory receptor"; if the olfactory receptor is not activated after the 4-methoxybenzaldehyde stimulates the olfactory receptor, the olfactory receptor is "the olfactory receptor can not recognize the 4-methoxybenzaldehyde".
In a preferred embodiment of the invention, the olfactory receptor is OR2J2.
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-methoxybenzaldehyde 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-methoxybenzaldehyde can be determined by detecting the change of the concentration of cAMP.
In a second aspect of the invention, the invention proposes the use of 4-methoxybenzaldehyde for activating an olfactory receptor comprising OR2J2 and/OR OR2W1. The inventor finds through experiments that after the OR2J2 and the OR2W1 are stimulated by 4-methoxybenzaldehyde respectively, the OR2J2 and the OR2W1 can be activated, so that the 4-methoxybenzaldehyde can be effectively identified by utilizing the olfactory receptor, and a foundation is laid for the identification of the 4-methoxybenzaldehyde and the detection of a sample containing the 4-methoxybenzaldehyde.
In a preferred embodiment of the invention, the olfactory receptor is OR2J2.
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-methoxybenzaldehyde 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-methoxybenzaldehyde can be determined by detecting the change of the concentration of cAMP.
Method
In a third aspect of the present invention, the present invention provides a method for detecting 4-methoxybenzaldehyde. 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-methoxybenzaldehyde or not based on the response value; wherein the olfactory receptor comprises OR2J2 and/OR 2W1. From the foregoing, it is known that 4-methoxybenzaldehyde can activate the above-mentioned olfactory receptor, and thus, a sample to be tested is contacted with the above-mentioned olfactory receptor, if 4-methoxybenzaldehyde is contained in the sample to be tested, the above-mentioned olfactory receptor can be activated, and a response value after the olfactory receptor is activated is obtained, and according to the response value, whether 4-methoxybenzaldehyde is contained in the sample to be tested can be determined.
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-methoxybenzaldehyde) 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 invention, the olfactory receptor is OR2J2.
According to an embodiment of the invention, the olfactory receptor presence response value is an indication that 4-methoxybenzaldehyde 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-methoxybenzaldehyde.
It should be noted that the "indication without 4-methoxybenzaldehyde" means that 4-methoxybenzaldehyde is completely absent in the sample to be measured; or a small amount of 4-methoxybenzaldehyde exists in the sample to be detected, but cannot be detected.
According to an embodiment of the present invention, the olfactory receptor is selected from OR2J2, and the olfactory receptor presence response value is an indication of the presence of 4-methoxybenzaldehyde OR an indication of the presence of not less than 30 μm of 4-methoxybenzaldehyde in the sample to be tested; alternatively, the olfactory receptor absence response value is an indication of the absence of 4-methoxybenzaldehyde or an indication of the presence of less than 30 μm of 4-methoxybenzaldehyde in the sample to be tested.
According to an embodiment of the present invention, the olfactory receptor is selected from OR2W1, and the olfactory receptor presence response value is an indication of the presence of 4-methoxybenzaldehyde in the sample to be tested OR an indication of the presence of 4-methoxybenzaldehyde not lower than 100 μm; alternatively, the olfactory receptor absence response value is an indication that the test sample does not contain 4-methoxybenzaldehyde or contains less than 100 μm 4-methoxybenzaldehyde.
According to an embodiment of the invention, the method further comprises: and determining the content of 4-methoxybenzaldehyde 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 predetermined amount of 4-methoxybenzaldehyde and the olfactory receptor. Thus, the content of 4-methoxybenzaldehyde in the sample to be detected 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 is such that when 4-methoxybenzaldehyde or a sample containing 4-methoxybenzaldehyde is used to stimulate cells containing an olfactory receptor, if the olfactory receptor is activated, the intracellular cAMP concentration increases, cAMP binds to the promoter region of CRE-luciferase and causes the luciferase to be transcribed and translated, thus by detecting the activity of the luciferase, the response of the olfactory receptor can be characterized and whether the olfactory receptor recognizes 4-methoxybenzaldehyde 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, the GloSensor cAMP detection kit provides a substrate of the luciferase variant, the method can measure the change of the cAMP concentration in real time, and the content of 4-methoxybenzaldehyde in a sample to be detected can be detected rapidly and sensitively.
In a fourth aspect of the invention, the invention provides a method of detecting food ingredients. 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 the raw materials of the sample to be tested based on the response value; wherein the olfactory receptor comprises OR2J2 and/OR 2W1. From the foregoing, it is known that 4-methoxybenzaldehyde can activate the above-mentioned olfactory receptor, and thus, the sample to be tested is contacted with the above-mentioned olfactory receptor, if 4-methoxybenzaldehyde is contained in the sample to be tested, the above-mentioned olfactory receptor can be activated, and the response value after the olfactory receptor is activated is obtained, and it can be determined that the raw material containing 4-methoxybenzaldehyde is present in the sample to be tested, and if the olfactory receptor has no response value, the raw material containing 4-methoxybenzaldehyde is not present in the sample to be tested.
In a preferred embodiment of the invention, the olfactory receptor is OR2J2.
According to an embodiment of the present invention, the olfactory receptor presence response value is an indication that the sample to be tested contains at least one of star anise, fennel, vanilla bean, dill, corn and acacia; alternatively, the absence of a response value for the olfactory receptor is an indication that the sample to be tested does not contain star anise, fennel, vanilla bean, dill, corn and acacia.
According to embodiments of the present invention, the perfume raw material in the sample to be tested has a mass-to-volume ratio of not less than 0.01g/ml, for example not less than 0.02g/ml, 0.03 g/ml, 0.04g/ml, 0.05 g/ml, 0.06g/ml, 0.07 g/ml, 0.08g/ml, etc.
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.
According to an embodiment of the present 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 activity change is byAt least one of the detection methods determines: 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 is such that when 4-methoxybenzaldehyde or a sample containing 4-methoxybenzaldehyde is used to stimulate cells containing an olfactory receptor, if the olfactory receptor is activated, the intracellular cAMP concentration increases, cAMP binds to the promoter region of CRE-luciferase and causes the luciferase to be transcribed and translated, thus by detecting the activity of the luciferase, the response of the olfactory receptor can be characterized and whether the olfactory receptor recognizes 4-methoxybenzaldehyde 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, the GloSensor cAMP detection kit provides a substrate of the luciferase variant, the method can measure the change of the cAMP concentration in real time, and the content of 4-methoxybenzaldehyde in a sample to be detected can be detected rapidly and sensitively.
In a fifth aspect of the invention, the invention provides a method of assessing the quality of a fragrance product. According to an embodiment of the invention, the method comprises: contacting a perfume product to be tested with an olfactory receptor, and determining a response value of the olfactory receptor after contact; determining the quality of the perfume product to be tested based on the response value; wherein the olfactory receptor comprises OR2J2 and/OR 2W1. From the foregoing, it can be seen that 4-methoxybenzaldehyde can activate the above olfactory receptor, and thus, the perfume product to be tested is contacted with the above olfactory receptor, if the perfume product to be tested contains 4-methoxybenzaldehyde, the above olfactory receptor can be activated, and a response value after the olfactory receptor is activated is obtained, and the quality of the perfume product to be tested can be determined according to the magnitude of the response value. The perfume product to be tested can be a perfume final product or an intermediate product in the production of the perfume product, and when the perfume product is the perfume final product, the quality of the perfume final product can be controlled according to the quality of the perfume product to be tested, and when the perfume product is the intermediate product, the perfume product can be used for knowing the production and the preparation of the perfume product.
As used herein, the term "flavor product" refers to any product comprising botanical flavors (star anise, fennel, vanilla bean, corn, and acacia), such as flavor powders, flavor oils (e.g., flavor essential oils or edible flavor oils), and the like, without limitation.
Illustratively, when the flavor product is a flavor essential oil, including, but not limited to, an essential oil product containing at least one of fennel oil, star anise oil, acacia oil, and corn oil.
According to an embodiment of the present invention, the perfume product to be tested is subjected to a dilution treatment in advance before the contacting.
Illustratively, the flavor product is a flavor powder, and the diluted mass to volume ratio of the flavor product is not less than 0.01g/ml, such as not less than 0.02g/ml, 0.03 g/ml, 0.04g/ml, 0.05 g/ml, 0.06g/ml, 0.07 g/ml, 0.08g/ml, and the like.
Illustratively, the fragrance product is a fragrance essential oil, the diluted volume ratio of the fragrance product being not less than 0.0004, e.g. not less than 0.0004, 0.0005, 0.0008, 0.0010, 0.0012, 0.0016, 0.0020, 0.0025, 0.0030, 0.0032, etc.
According to an embodiment of the present invention, the perfume product to be tested contains at least one of star anise, fennel, vanilla bean, dill, corn and acacia.
In the present invention, "fennel" and "fennel" are synonymous.
In a preferred embodiment of the invention, the olfactory receptor is OR2J2.
According to an embodiment of the invention, the response value being higher than a predetermined threshold value is an indication of the qualification of the perfume product to be tested.
It should be noted that the "predetermined threshold" may be obtained by detecting a large number of perfume products (for example, 20, 50, 100, 150, 200, and more) using the olfactory receptor of the present invention, and counting the response values obtained based on the large number of perfume 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.
In some alternative embodiments of the invention, the method may be used to evaluate the quality of a flavour product containing a predetermined starting material, for example, star anise, fennel, vanilla bean, dill, maize and acacia.
For example, the predetermined threshold may be 50 to 60 μg/1.2ml, or the molar concentration may be 300 to 400 μM, when used to evaluate the quality of a fennel-containing flavor product.
According to the embodiment of the invention, a plurality of perfume products to be tested are respectively contacted with an olfactory receptor, and the response value of the olfactory receptor after the contact is determined; and determining the quality of the perfume product to be tested based on the response value, wherein the response value is higher and is an indication that the quality of the perfume product to be tested is higher.
For example, as shown in fig. 4, when two types of perfume products to be tested exist, the quality of the perfume product to be tested (such as star anise oil produced by peasant in guangxi province) with high response value is better than the quality of the perfume product to be tested (such as star anise oil produced by plant Yisheng brand).
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 is such that when 4-methoxybenzaldehyde or a sample containing 4-methoxybenzaldehyde is used to stimulate cells containing an olfactory receptor, if the olfactory receptor is activated, the intracellular cAMP concentration increases, cAMP binds to the promoter region of CRE-luciferase and causes the luciferase to be transcribed and translated, thus by detecting the activity of the luciferase, the response of the olfactory receptor can be characterized and whether the olfactory receptor recognizes 4-methoxybenzaldehyde 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, the GloSensor cAMP detection kit provides a substrate of the luciferase variant, the method can measure the change of the cAMP concentration in real time, and the content of 4-methoxybenzaldehyde in a sample to be detected can be detected rapidly and sensitively.
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 398 olfactory receptors from a human olfactory receptor library, then prepared a gene construct containing olfactory receptors, golf, CRE-Luciferase, and pRL-SV40, and transfected the gene construct into HEK293T cells using a transfection reagent Lipofectamine2000 (Invitrogen). After 24 hours of incubation, 4-methoxybenzaldehyde was diluted to a concentration of 300. Mu.M with medium, cells were stimulated with diluted 4-methoxybenzaldehyde, and incubated for 2-4 hours. If the olfactory receptor is activated, the intracellular cAMP concentration is increased, cAMP binds to the promoter region of CRE-luciferase and promotes transcription and translation of luciferase, and the response of the olfactory receptor can be characterized by detecting the activity of luciferase, so that 2 human olfactory receptors OR2J2 (Genbank Gene ID:26707,protein ID:NP_112167.2) and OR2W1 (Genbank Gene ID:26692,protein ID:NP_112165.1) which produce responses are finally obtained, and the results are shown in FIG. 1.
The inventors also examined the dose curves of the above 2 olfactory receptors in response to 4-methoxybenzaldehyde at different concentrations by stimulating cells containing the olfactory receptors OR2J2 and OR2W1 respectively with 4-methoxybenzaldehyde, and examined the results as shown in FIG. 2, wherein the horizontal axis represents the logarithm of the 4-methoxybenzaldehyde concentration (M) and the vertical axis represents the fold change in response to the blank (without 4-methoxybenzaldehyde). As a result, it was found that the response of OR2J2 to 4-methoxybenzaldehyde was strongest in the above 2 olfactory receptors, and that the response fold changes of OR2J2 and OR2W1 were about 20-fold and 12-fold, respectively, with sensitivities of 30. Mu.M and 100. Mu.M, respectively.
Example 2: detection of Anise and Foeniculum vulgare products by OR2J2 olfactory receptors
In this example, the food flavoring of star anise and fennel was examined by cells expressing OR2J2 using the method of example 1. The star anise and the fennel are tandem brands, the star anise and the fennel are solid, and the inventor extracts aroma substances by water, specifically: respectively taking 20g of star anise and fennel, adding into 250ml of distilled water, boiling for 20min, collecting the residual liquid, supplementing to 24ml, and diluting, wherein the specific dilution is shown in the abscissa of FIG. 3; the diluted star anise and fennel were then tested using the method of example 1. The results of the test are shown in FIG. 3, wherein the horizontal axis represents the mass-to-volume ratio (in g/ml) and the vertical axis represents the response change times relative to the blank (without adding star anise and fennel).
The result shows that the content of 4-methoxybenzaldehyde in the fennel of the tandem brand is obviously higher than that of the fennel of the tandem brand, and when the content of the fennel and the fennel is 0.08g/ml, the response multiple of the fennel can reach about 7 times, and the response multiple of the fennel can reach about 32 times. Thus, 4-methoxybenzaldehyde in the flavoring can be sensitively detected by using the OR2J2 olfactory receptor.
Example 3: detection of OR2J2 olfactory receptor on different brands of star anise essential oil
In this example, 2 types of star anise essential oils were detected by cells expressing OR2J2 using the method of example 1. The 2 kinds of star anise essential oil are respectively star anise oil produced by Guangxi farmers and star anise essential oil of plant Yisheng brand. The inventor firstly dilutes the 2 essential oils to 1/4 by ethanol respectively, and then dilutes the 2 essential oils again by a CD293 culture medium, wherein the specific dilution is shown in the abscissa of FIG. 4; the diluted 2 types of star anise essential oils were then tested using the method of example 1. The results of the test are shown in FIG. 4, wherein the horizontal axis represents the volume ratio and the vertical axis represents the response change times relative to the blank (without addition of star anise essential oil).
As can be seen from fig. 4, when the ratio of the star anise oil is 0.0032, that is, when the star anise oil is diluted 320 times, the response multiple of the square-western farmer self-produced star anise oil can reach about 8 times, the response multiple of the plant Yisheng brand star anise essential oil reaches about 4 times, the square-western farmer self-produced star anise oil has a higher content of 4-methoxybenzaldehyde than the plant Yisheng brand star anise essential oil, which indicates that the quality of the square-western farmer self-produced star anise oil is better, and the extraction process of the square-western farmer self-produced star anise oil is more helpful for preserving key aroma components in food. Thus, the use of the OR2J2 olfactory receptor can be used to assess the quality of commercially available essential oils.
Example 4: detection of Fennel released gas by OR2J2 olfactory receptor
In this example, the method of example 1 was used to detect the release of gas from fennel by cells expressing OR2J2. The method comprises the following steps: weighing 20g of fennel, placing the fennel in a closed aluminum box, allowing the fennel to naturally release fragrance for 12 hours, and then placing cells expressing the OR2J2 olfactory receptor into the fennel to stimulate for 4 hours; cells that were not exposed to the fragrance of fennel were set as a blank. The results of the detection are shown in fig. 5, and the results show that the response times of the cells expressing the OR2J2 olfactory receptor to the gas in the aluminum box are about 3 times that of the blank group, and the bionic olfactory recognition of the gas by using the biological olfactory receptor cells is possible.
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, including OR2J2 and/OR 2W1, for the recognition of 4-methoxybenzaldehyde;
wherein the amino acid sequence of OR2J2 is the protein ID of Genbank: an amino acid sequence shown in NP 112167.2, wherein the amino acid sequence of OR2W1 is protein ID of Genbank: NP-112165.1.
Use of 4-methoxybenzaldehyde for activating an olfactory receptor, comprising OR2J2 and/OR 2W1;
wherein the amino acid sequence of OR2J2 is the protein ID of Genbank: an amino acid sequence shown in NP 112167.2, wherein the amino acid sequence of OR2W1 is protein ID of Genbank: NP-112165.1.
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-methoxybenzaldehyde, 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-methoxybenzaldehyde or not based on the response value;
wherein the olfactory receptor comprises OR2J2 and/OR 2W1;
wherein the amino acid sequence of OR2J2 is the protein ID of Genbank: an amino acid sequence shown in NP 112167.2, wherein the amino acid sequence of OR2W1 is protein ID of Genbank: NP-112165.1.
5. The method of claim 4, wherein the olfactory receptor presence response value is an indication that the test sample contains 4-methoxybenzaldehyde; or, the absence of a response value for the olfactory receptor is an indication that the sample to be tested does not contain 4-methoxybenzaldehyde;
the method further comprises:
and determining the content of 4-methoxybenzaldehyde 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 predetermined amount of 4-methoxybenzaldehyde and the olfactory receptor.
6. A method of detecting a food material, comprising:
contacting a sample to be tested with an olfactory receptor, wherein 4-methoxybenzaldehyde can activate the olfactory receptor, and determining the response value of the olfactory receptor after contact;
determining whether the sample to be detected contains raw materials of 4-methoxybenzaldehyde or not based on the response value;
wherein the olfactory receptor is OR2J2, and the amino acid sequence of OR2J2 is the protein ID of Genbank: NP-112167.2.
7. The method of claim 6, wherein the olfactory receptor presence response value is indicative of the presence of at least one of star anise, fennel, vanilla bean, dill, corn, and acacia in the test sample; or alternatively, the process may be performed,
the absence of a response value for the olfactory receptor is an indication that the sample to be tested does not contain star anise, fennel, vanilla bean, dill, corn and acacia.
8. A method of evaluating the quality of a fragrance product, comprising:
contacting a perfume product to be tested with an olfactory receptor, and determining a response value of the olfactory receptor after contact;
determining the quality of the perfume product to be tested based on the response value;
if the perfume product to be tested contains 4-methoxybenzaldehyde, the olfactory receptor can be activated, a response value of the activated olfactory receptor is obtained, and the quality of the perfume product to be tested is determined according to the response value;
the olfactory receptor is OR2J2, and the amino acid sequence of OR2J2 is Genbank protein ID: NP-112167.2.
9. The method of claim 8, wherein the flavor product to be tested comprises at least one of star anise, fennel, vanilla bean, corn, and acacia;
the response value is higher than a preset threshold value and is an indication of qualified quality of the perfume product to be detected;
contacting a plurality of perfume products to be tested with an olfactory receptor respectively, and determining the response value of the olfactory receptor after contact;
determining the quality of the perfume product to be tested based on the response value,
wherein the higher response value is an indication of higher quality of the perfume product to be tested.
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310140517.XA CN115840026B (en) | 2023-02-13 | 2023-02-13 | Use of olfactory receptors for the recognition of 4-methoxybenzaldehyde and method for detecting 4-methoxybenzaldehyde |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310140517.XA CN115840026B (en) | 2023-02-13 | 2023-02-13 | Use of olfactory receptors for the recognition of 4-methoxybenzaldehyde and method for detecting 4-methoxybenzaldehyde |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115840026A CN115840026A (en) | 2023-03-24 |
| CN115840026B true CN115840026B (en) | 2023-05-23 |
Family
ID=85579974
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310140517.XA Active CN115840026B (en) | 2023-02-13 | 2023-02-13 | Use of olfactory receptors for the recognition of 4-methoxybenzaldehyde and method for detecting 4-methoxybenzaldehyde |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115840026B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726551A (en) * | 2013-12-15 | 2015-06-24 | 西姆莱斯股份公司 | Method For Evaluating The Scent Performance Of Perfumes And Perfume Mixtures |
| CN109071626A (en) * | 2016-02-24 | 2018-12-21 | 阿罗姆斯公司 | For detecting smell, fragrance and the biosensor of taste |
| CN110214273A (en) * | 2016-09-30 | 2019-09-06 | 马斯公司 | Use the screening technique and the noval chemical compound identified in this way of olfactory receptor |
| CN114720645A (en) * | 2022-05-18 | 2022-07-08 | 汉王科技股份有限公司 | Use of olfactory receptor for recognizing 3-methylthiopropanal and method for detecting 3-methylthiopropanal |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2558577B1 (en) * | 2010-04-16 | 2018-12-12 | Nuevolution A/S | Bi-functional complexes and methods for making and using such complexes |
| JP2021521248A (en) * | 2018-04-16 | 2021-08-26 | ラ ホヤ インスティテュート フォア アラージー アンド イムノロジー | Modulation of immune response by targeting olfactory receptor activity |
| US20200399558A1 (en) * | 2019-06-21 | 2020-12-24 | The Regents Of The University Of California | Methods for identifying, compounds identified and compositions thereof |
-
2023
- 2023-02-13 CN CN202310140517.XA patent/CN115840026B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726551A (en) * | 2013-12-15 | 2015-06-24 | 西姆莱斯股份公司 | Method For Evaluating The Scent Performance Of Perfumes And Perfume Mixtures |
| CN109071626A (en) * | 2016-02-24 | 2018-12-21 | 阿罗姆斯公司 | For detecting smell, fragrance and the biosensor of taste |
| CN110214273A (en) * | 2016-09-30 | 2019-09-06 | 马斯公司 | Use the screening technique and the noval chemical compound identified in this way of olfactory receptor |
| CN114720645A (en) * | 2022-05-18 | 2022-07-08 | 汉王科技股份有限公司 | Use of olfactory receptor for recognizing 3-methylthiopropanal and method for detecting 3-methylthiopropanal |
Non-Patent Citations (1)
| Title |
|---|
| Ectopic Odorant Receptor Responding to Flavor Compounds: Versatile Role in Health and Disease;Tao TONG 等;Pharmaceutics;第13卷;1314:1-36 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115840026A (en) | 2023-03-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lendor et al. | Solid phase microextraction-based miniaturized probe and protocol for extraction of neurotransmitters from brains in vivo | |
| Mozzi et al. | Metabolomics as a tool for the comprehensive understanding of fermented and functional foods with lactic acid bacteria | |
| US9249453B2 (en) | Method for identifying a malodor inhibitor | |
| Vidal et al. | Multiclass analysis of antibiotic residues in honey by ultraperformance liquid chromatography− tandem mass spectrometry | |
| Taylor‐Clark et al. | Ozone activates airway nerves via the selective stimulation of TRPA1 ion channels | |
| Angleson et al. | Monitoring secretion in real time: capacitance, amperometry and fluorescence compared | |
| Price et al. | Nesfatin‐1 influences the excitability of paraventricular nucleus neurones | |
| CN115902128B (en) | Use of olfactory receptors for the recognition of 4-ethylguaiacol and method for detecting 4-ethylguaiacol | |
| Lee et al. | The identification and quantification of biogenic amines in Korean turbid rice wine, Makgeolli by HPLC with mass spectrometry detection | |
| CN114720645B (en) | Use of olfactory receptor for identifying 3-methylthiopropanal and method for detecting 3-methylthiopropanal | |
| Arce et al. | Ion mobility spectrometry a versatile analytical tool for metabolomics applications in food science | |
| Pleadin et al. | Rapid immunoassay method for the determination of clenbuterol and salbutamol in blood | |
| Pioggia et al. | A composite sensor array impedentiometric electronic tongue: Part II. Discrimination of basic tastes | |
| Haag et al. | Key food furanones furaneol and sotolone specifically activate distinct odorant receptors | |
| Mulinacci et al. | Evaluation of lignans and free and linked hydroxy‐tyrosol and tyrosol in extra virgin olive oil after hydrolysis processes | |
| CN115840026B (en) | Use of olfactory receptors for the recognition of 4-methoxybenzaldehyde and method for detecting 4-methoxybenzaldehyde | |
| Fulop et al. | Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells | |
| Wickham et al. | Advances in studying phasic dopamine signaling in brain reward mechanisms | |
| EP3944855A2 (en) | Method for screening of unpleasant odor masking agents | |
| Ackroff et al. | Flavor preferences conditioned by oral monosodium glutamate in mice | |
| Guliy et al. | Bacteria-based electro-optical platform for ampicillin detection in aquatic solutions | |
| Sakata et al. | Coordinated regulation of TORC2 signaling by MCC/eisosome‐associated proteins, Pil1 and tetraspan membrane proteins during the stress response | |
| CN116200456B (en) | Method for detecting food deterioration by using olfactory receptor | |
| EFSA Panel on Contaminants in the Food Chain (CONTAM) | Scientific Opinion on marine biotoxins in shellfish–Cyclic imines (spirolides, gymnodimines, pinnatoxins and pteriatoxins) | |
| Burseg et al. | Application of the olfactoscan method to study the ability of saturated aldehydes in masking the odor of methional |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |