CN114720645B - Use of olfactory receptor for identifying 3-methylthiopropanal and method for detecting 3-methylthiopropanal - Google Patents
Use of olfactory receptor for identifying 3-methylthiopropanal and method for detecting 3-methylthiopropanal Download PDFInfo
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Abstract
The invention relates to the use of olfactory receptors for identifying 3-methylthiopropanal and to a method for detecting 3-methylthiopropanal. Use of an olfactory receptor comprising at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1; the method comprises the following steps: contacting a sample to be tested with olfactory receptors, determining a response value of the olfactory receptors, and determining whether the sample to be tested contains 3-methylthiopropanal or not based on the response value, wherein the olfactory receptors comprise at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The olfactory receptor of the invention can quickly identify the 3-methylthiopropanal, and can detect the 3-methylthiopropanal in products such as drinks, fruits or fruit products and the like so as to determine the freshness of the products or the types of fruits contained in the products.
Description
Technical Field
The invention relates to the technical field of chemical detection, in particular to application of an olfactory receptor in identifying 3-methylthiopropanal and a method for detecting 3-methylthiopropanal, and more particularly relates to application of the olfactory receptor in identifying 3-methylthiopropanal, application of 3-methylthiopropanal in activating the olfactory receptor, a method for detecting 3-methylthiopropanal, a method for detecting freshness of drinks, fruits or fruit products, a method for detecting types of fruits contained in food, a method for distinguishing beer or red wine and a method for monitoring whether the fruit products are subjected to high-temperature disinfection treatment or not.
Background
Olfactory Receptors (ORs), also known as odorant receptors, are chemoreceptors expressed in the cell membrane of olfactory neurons (OSNs). Olfactory receptors activated by odorants trigger nerve impulses, which convey information about the odor to the brain. These receptors are G protein-coupled receptors (GPCRs) located on the cell surface membrane at the dendritic tip of olfactory neurons. Olfactory receptors form a multigene family, with about 400 genes in humans and 1200 genes in mice.
3-methylthiopropanal (CAS number 3268-49-3), commonly known as yolk aldehyde, is a colorless or yellowish liquid whose molecular structure contains both a methylthio group and an aldehyde group, and exhibits the odor characteristics of baked potatoes, onions or potato chips. 3-methylthiopropanal is widely found in fruits, fruit preparations and alcoholic products. However, no report has been made on the detection of olfactory receptors for 3-methylthiopropanal.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides the application of the olfactory receptor in identifying 3-methylthiopropanal, the application of 3-methylthiopropanal in activating the olfactory receptor, a method for detecting 3-methylthiopropanal, a method for detecting the freshness of wine, fruits or fruit products, a method for detecting the types of fruits contained in food, a method for distinguishing beer or red wine and a method for monitoring whether the fruit products are subjected to high-temperature disinfection treatment.
The present invention has been completed based on the following findings of the inventors:
the 3-methylthiopropanal is widely used in fruits, fruit products, oat flour, sake, wine, beer, white spirit and other foods. Research shows that the durian, mango and other fruits are rich in 3-methylthio propionaldehyde, and the-methylthio propionaldehyde can be detected in the Beijing-flavored roast duck. However, 3-methylthiopropanal is difficult to detect in freshly squeezed pineapple juice, and is one of the most odorous substances in commercial pineapple juice that has been pasteurized. In addition, the fresh wine (new wine) does not contain 3-methylthiopropanal, but the wine after being stored for a while (old wine) contains 3-methylthiopropanal, and the content is higher the longer the storage time is.
Based on the above, the inventors have found through a large number of experiments that the adoption of four olfactory receptors, MOR244-3, MOR256-17, MOR180-1 and OR2C1 can detect 3-methylthiopropanal, and have the advantages of high detection sensitivity and the like. Specifically, the inventors selected a subset of olfactory receptors from the human and mouse olfactory receptor repertoires and then stimulated cells expressing different olfactory receptors with different concentrations of 3-methylthiopropanal. As a result, it was found that the mouse olfactory receptors MOR244-3, MOR256-17 and MOR180-1, and the human olfactory receptor OR2C1, which MOR244-3 and MOR256-17 were significantly responsive to 30 μ M3-methylthiopropanal, could be activated to various degrees. In addition, the inventor also adopts the cells respectively containing MOR244-3 and MOR256-17 to detect the 3-methylthiopropanal in different fruits, fruit products and wines, wherein the detection results of the fresh orange juice, the fresh grapefruit juice and the fresh mango juice are consistent with the results reported in the research. In addition, the inventor also detects the pineapple juice which is not subjected to pasteurization treatment and the pineapple juice which is subjected to pasteurization treatment by using cells respectively containing MOR244-3 and MOR256-17, and finds that compared with fresh pineapple juice, the 3-methylthiopropanal of the pineapple juice which is subjected to pasteurization treatment is remarkably improved, and the detection result is consistent with the result reported in the research. Therefore, olfactory receptors can be used to detect the high temperature sterilization of fruit products (e.g., pineapple juice).
In a first aspect of the invention, the invention proposes the use of an olfactory receptor for the recognition of 3-methylthiopropanal. According to an embodiment of the invention, the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor experimentally found that after stimulating MOR244-3, MOR256-17, MOR180-1 and OR2C1 with 3-methylthiopropanal, MOR244-3, MOR256-17, MOR180-1 and OR2C1 can be activated, so that the olfactory receptor can be used for effectively identifying 3-methylthiopropanal, and a foundation is laid for identifying 3-methylthiopropanal and detecting a sample containing 3-methylthiopropanal.
In a second aspect of the invention, the invention proposes the use of 3-methylthiopropanal for activating olfactory receptors comprising at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor experimentally found that after stimulating MOR244-3, MOR256-17, MOR180-1 and OR2C1 with 3-methylthiopropanal, MOR244-3, MOR256-17, MOR180-1 and OR2C1 can be activated, so that the olfactory receptor can be used for effectively identifying 3-methylthiopropanal, and a foundation is laid for identifying 3-methylthiopropanal and detecting a sample containing 3-methylthiopropanal.
In a third aspect of the invention, the invention provides a method for detecting 3-methylthiopropanal. According to an embodiment of the invention, the method comprises: contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor; determining whether the sample to be tested contains 3-methylthiopropanal or not based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor finds through experiments that the 3-methylthiopropanal can activate the olfactory receptor, if the sample to be detected contains the 3-methylthiopropanal, the sample to be detected is contacted with the olfactory receptor, the olfactory receptor is activated, a response value after the olfactory receptor is activated is obtained, and whether the sample to be detected contains the 3-methylthiopropanal or not can be determined according to the response value.
In a fourth aspect of the invention, a method of detecting the freshness of a brew, fruit or fruit preparation is presented. According to an embodiment of the invention, the method comprises: contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor; determining the freshness degree of the sample to be detected based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor finds through experiments that the 3-methylthiopropanal can activate the olfactory receptor, if the sample to be detected contains the 3-methylthiopropanal, the sample to be detected is contacted with the olfactory receptor, the olfactory receptor is activated, the activated response value can be obtained, the condition that the sample to be detected contains the 3-methylthiopropanal can be determined according to the response value, and the freshness degree of the sample to be detected can be determined according to the condition that different samples to be detected contain the 3-methylthiopropanal.
In a fifth aspect of the invention, a method of detecting the type of fruit contained in a food product is presented. According to an embodiment of the invention, the method comprises: contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor; determining the fruit type contained in the sample to be tested based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1; the sample to be tested only contains one fruit. The inventor finds through experiments that different fruit-containing foods (such as fruit pulp obtained by grinding, fresh fruit juice, beverage containing fruit juice and the like) contain different concentrations of 3-methylthiopropanal, a sample to be tested is contacted with the olfactory receptor, the olfactory receptor is activated, a response value after activation can be obtained, and the condition that the sample to be tested contains the 3-methylthiopropanal can be determined according to the response value, so that the type of the fruit in the foods can be determined.
In a sixth aspect of the invention, a method of differentiating between beer and red wine is presented. According to an embodiment of the invention, the method comprises: contacting a plurality of products to be detected with olfactory receptors, and respectively determining response values of the olfactory receptors corresponding to the products to be detected, wherein the products to be detected are beer and/or red wine; determining the type of the product to be detected based on the response values of the olfactory receptors corresponding to the products to be detected; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor finds through experiments that the content of the 3-methylthiopropanal in the beer is higher than that of the red wine, the 3-methylthiopropanal in the sample to be detected can activate the olfactory receptor by contacting the sample to be detected with the olfactory receptor to obtain a corresponding response value, the condition that the sample to be detected contains the 3-methylthiopropanal can be determined according to the response value, and whether various samples to be detected are type products or the types of various samples to be detected can be determined by the condition that the samples to be detected contain the 3-methylthiopropanal.
In a seventh aspect of the invention, a method of monitoring whether a fruit preparation has been subjected to a pasteurization treatment is presented. According to an embodiment of the invention, the method comprises: contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor; determining whether the sample to be detected is subjected to high-temperature disinfection treatment or not based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor tests that 3-methylthiopropanal in some fruit products (such as pineapple products) is obviously increased after pasteurization treatment, the fruit products are contacted with the olfactory receptors, if the olfactory receptors are activated, corresponding response values can be obtained, and whether the fruit products are subjected to high-temperature sterilization treatment or not can be judged by comparing the response values with the response values of the fruit products which are not subjected to high-temperature sterilization treatment or not, so that the high-temperature sterilization treatment link of the fruit products can be monitored, and the processing condition of the fruit products can be ensured.
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.
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The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a graph of the dose of 4 olfactory receptors responding to 3-methylthiopropanal in example 1 of the present invention;
FIG. 2 is a graph showing the relative response intensity of MOR244-3 and MOR256-17 to 3-methylthiopropanal in fresh orange juice and fresh mango juice, in example 3 of the present invention;
FIG. 3 is a graph of the relative response intensity of MOR244-3 and MOR256-17 to 3-methylthiopropanal in fresh orange, grapefruit and mango juice, in example 4 of the present invention;
FIG. 4 is a graph showing the relative response intensity of MOR244-3 and MOR256-17 to 3-methylthiopropanal in fresh orange juice and 3 orange juice drinks in accordance with example 5 of the present invention;
FIG. 5 is a graph of the relative response intensity of MOR244-3 and MOR256-17 to 3-methylthiopropanal in fresh mango juice and in concentrated reduced mango juice according to example 6 of the present invention;
FIG. 6 is a graph of the relative response strength of MOR244-3 and MOR256-17 of example 7 of the present invention to 3-methylthiopropanal in freshly squeezed pineapple juice and pasteurized processed pineapple juice;
FIG. 7 is a graph showing the relative response intensity of MOR244-3 and MOR256-17 to 3-methylthiopropanal in draft beer, dark beer and red wine in example 8 of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to implicitly indicate the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, 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 "comprise" are open-ended expressions that include the elements indicated in the present invention, but do not exclude other elements.
As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs, and instances where it does not.
The invention provides a use of an olfactory receptor for identifying 3-methylthiopropanal, a use of 3-methylthiopropanal for activating the olfactory receptor, a method for detecting 3-methylthiopropanal, a method for detecting the freshness of a drink, a fruit or a fruit preparation, a method for detecting the kind of fruit contained in a food, a method for distinguishing a beer or a red wine, and a method for monitoring whether the fruit preparation is subjected to a high-temperature sterilization treatment, which will be described in detail below, respectively.
Use of
In a first aspect of the invention, the invention proposes the use of an olfactory receptor for the recognition of 3-methylthiopropanal. According to an embodiment of the invention, the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor experimentally found that when 3-methylthiopropanal is used to stimulate MOR244-3, MOR256-17, MOR180-1 and OR2C1 respectively, all the four olfactory receptors can be activated, so that the olfactory receptors can be used to effectively identify 3-methylthiopropanal, and a foundation is laid for the detection of a sample containing 3-methylthiopropanal.
It should be noted that, if the olfactory receptor is stimulated by 3-methylthiopropanal, the olfactory receptor is activated, i.e., "the olfactory receptor can recognize 3-methylthiopropanal"; if the olfactory receptor is stimulated by the 3-methylthiopropanal, the olfactory receptor is not activated, namely, the olfactory receptor can not recognize the 3-methylthiopropanal.
According to an embodiment of the invention, said recognition or activation is represented by a change in the activity of olfactory receptors.
According to an embodiment of the invention, the activity change comprises at least one of the following signal changes: cAMP, IP3, calcium ion, current, and pH.
According to an embodiment of the invention, said recognition is manifested by an increase in cAMP downstream of the olfactory receptor. The inventor finds out through experiments that 3-methylthiopropanal stimulates cells expressing olfactory receptors, the cAMP concentration in the cells is increased after the olfactory receptors are activated, and whether the olfactory receptors can recognize the 3-methylthiopropanal can be determined by detecting the change of the cAMP concentration.
In a second aspect of the invention, the invention proposes the use of 3-methylthiopropanal for activating olfactory receptors comprising at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor finds that after 3-methylthiopropanal is used for stimulating MOR244-3, MOR256-17, MOR180-1 and OR2C1, all the four olfactory receptors can be activated, so that the olfactory receptors can be used for effectively identifying 3-methylthiopropanal, and the method lays a foundation for identifying 3-methylthiopropanal and detecting a sample containing 3-methylthiopropanal.
According to an embodiment of the invention, said activation is manifested by an increase in cAMP downstream of the olfactory receptor. The inventor finds out through experiments that 3-methylthiopropanal stimulates cells expressing the plurality of olfactory receptors, if the olfactory receptors are activated, the cAMP concentration in the cells is increased, and finally the 3-methylthiopropanal is determined to be capable of activating the four olfactory receptors by detecting the change of the cAMP concentration.
Method
In a third aspect of the invention, the invention provides a method for detecting 3-methylthiopropanal. According to an embodiment of the invention, the method comprises: contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor; determining whether the sample to be detected contains 3-methylthiopropanal or not based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor finds through experiments that the 3-methylthiopropanal can activate the olfactory receptor, if the sample to be detected contains the 3-methylthiopropanal, the sample to be detected is contacted with the olfactory receptor, the olfactory receptor can be activated, a response value after activation is obtained, and whether the sample to be detected contains the 3-methylthiopropanal or not can be determined according to the response value.
According to an embodiment of the invention, said olfactory receptor present response value is an indication that 3-methylthiopropanal is contained in said sample to be tested; alternatively, the olfactory receptor non-presence response value is indicative that 3-methylthiopropanal is not present in the test sample.
Note that the "indication that 3-methylthiopropanal is not contained" means that 3-methylthiopropanal is not present in the sample to be tested at all; or a small amount of 3-methylthiopropanal is present in the sample to be tested but cannot be detected.
According to an embodiment of the invention, said olfactory receptor is selected from MOR244-3 and/or MOR256-17, said olfactory receptor presence response value is an indication that said sample to be tested contains 3-methylthiopropanal or an indication that it contains not less than 30 μ M of 3-methylthiopropanal; alternatively, the olfactory receptor non-presence response value is an indication that no 3-methylthiopropanal is present in the sample to be tested or an indication that less than 30 μ M of 3-methylthiopropanal is present.
According to an embodiment of the invention, said olfactory receptor is selected from MOR180-1 and/OR OR2C1, said olfactory receptor presence response value being an indication that said sample to be tested contains 3-methylthiopropanal OR an indication that it contains not less than 100 μ M of 3-methylthiopropanal; alternatively, the olfactory receptor non-presence response value is an indication that no 3-methylthiopropanal is present in the sample to be tested or an indication that less than 100 μ M of 3-methylthiopropanal is present.
According to an embodiment of the invention, the method further comprises: and determining the content of the 3-methylthiopropanal in the sample to be detected based on a standard curve, wherein the standard curve is a curve corresponding to a predetermined amount of the 3-methylthiopropanal and the olfactory receptor response value. Therefore, the content of the 3-methylthio propionaldehyde in the sample to be detected can be detected.
In a fourth aspect of the invention, a method of detecting the freshness of a brew, fruit or fruit preparation is presented. According to an embodiment of the invention, the method comprises: contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor; determining the freshness degree of the sample to be detected based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor finds through experiments that the 3-methylthiopropanal can activate the olfactory receptor, if the sample to be detected contains the 3-methylthiopropanal, the sample to be detected is contacted with the olfactory receptor, the olfactory receptor can be activated to obtain a response value after activation, the condition that the sample to be detected contains the 3-methylthiopropanal can be determined according to the response value, and the freshness degree of the sample to be detected can be determined according to the condition that different samples to be detected contain the 3-methylthiopropanal.
According to an embodiment of the invention, the method further comprises: contacting a control sample with an olfactory receptor, and determining a response value of the olfactory receptor, wherein the control sample is fresh wine, fruit or a fruit preparation; the absence of a significant difference in the response value of the test sample from the response value of the control sample is an indication that the test sample is fresh; alternatively, a significant difference in the response value of the test sample and the response value of the control sample is indicative that the test sample is stale. The inventor compares the response value (detection group) of the 3-methylmercapto propionaldehyde in the wine, the fruit or the fruit product to be detected with the response value (control group) of the 3-methylmercapto propionaldehyde in the known fresh wine, the fruit or the fruit product, and can quickly detect the freshness of the sample to be detected by analyzing the difference of the response values of the detection group and the control group.
In a fifth aspect of the invention, a method of detecting the type of fruit contained in a food product is presented. According to an embodiment of the invention, the method comprises: contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor; determining the fruit type contained in the sample to be tested based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1; the sample to be tested only contains one fruit. The inventor finds through experiments that different fruit-containing food (such as fruit pulp obtained by grinding, freshly squeezed fruit juice, beverage containing fruit juice and the like) contains different concentrations of 3-methylthiopropanal, the olfactory receptor is activated by contacting a sample to be tested with the olfactory receptor, a response value after activation can be obtained, and the condition that the sample to be tested contains the 3-methylthiopropanal can be determined according to the response value, so that the type of the fruit in the food can be determined.
According to an embodiment of the invention, the method further comprises: and comparing the response value with response values of various known fruits to determine the fruit type contained in the sample to be tested. The inventor compares the response value of the sample to be detected with the response values of various known fruits, and finds the fruit corresponding to the response value closest to the response value of the sample to be detected, namely the fruit type contained in the sample to be detected.
According to an embodiment of the invention, the food product is selected from freshly extracted juice.
According to an embodiment of the invention, the plurality of known fruits includes oranges, grapefruit, mango, and pineapple.
In a sixth aspect of the invention, a method of differentiating between beer or wine is presented. According to an embodiment of the invention, the method comprises: contacting a plurality of products to be detected with olfactory receptors, and respectively determining response values of the olfactory receptors corresponding to the products to be detected, wherein the products to be detected are beer and/or red wine; determining the type of the product to be detected based on the response values of the olfactory receptors corresponding to the products to be detected; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor finds through experiments that the content of the 3-methylthiopropanal in the beer is higher than that of the red wine, the 3-methylthiopropanal in the sample to be detected can activate the olfactory receptor by contacting the sample to be detected with the olfactory receptor to obtain a corresponding response value, the condition that the sample to be detected contains the 3-methylthiopropanal can be determined according to the response value, and whether various samples to be detected are products or not can be determined or further the types of various samples to be detected can be determined by the condition that the samples to be detected contain the 3-methylthiopropanal.
According to the embodiment of the invention, the absence of significant difference in the response values of the olfactory receptors corresponding to the plurality of products to be tested is an indication that the plurality of products to be tested are beer or red wine. Therefore, whether multiple samples to be detected are the same wine or not can be detected quickly.
According to the embodiment of the invention, the fact that the response values of the olfactory receptors corresponding to the products to be tested are not lower than a first preset value indicates that the products to be tested are beer; or, the fact that the response values of the olfactory receptors corresponding to the products to be detected are lower than a first preset value indicates that the products to be detected are red wine. Therefore, the sample to be detected can be further rapidly distinguished to be beer or red wine.
It should be noted that the first predetermined value refers to an experimentally obtained response value for distinguishing beer from red wine, and can be determined according to different detection methods, for example, by detecting a change in cAMP concentration, the first predetermined value can be set to 500.
According to the embodiment of the invention, the response values of the olfactory receptors corresponding to the products to be tested have significant difference, and the significant difference is an indication that the products to be tested are beer and red wine. Therefore, beer or red wine in the sample to be detected can be distinguished quickly.
According to the embodiment of the invention, the response values of the olfactory receptors corresponding to the products to be detected have significant difference, the fact that the response value of the olfactory receptors is not lower than the first preset value indicates that the corresponding products to be detected are beer, and the fact that the response value of the olfactory receptors is lower than the first preset value indicates that the corresponding products to be detected are red wine. Therefore, beer or red wine in the sample to be detected can be further and quickly distinguished.
In a seventh aspect of the invention, a method of monitoring whether a fruit preparation has been subjected to a pasteurization treatment is presented. According to an embodiment of the invention, the method comprises: contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor; determining whether the sample to be detected is subjected to high-temperature disinfection treatment or not based on the response value; wherein the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR256-17, MOR180-1 and OR2C 1. The inventor tests that 3-methylthiopropanal in some fruit products (such as pineapple products) is obviously increased after high-temperature sterilization treatment, the fruit products are contacted with the olfactory receptors, if the olfactory receptors are activated, corresponding response values can be obtained, and whether the fruit products are subjected to high-temperature sterilization treatment or not can be judged by comparing the response values with the response values of the fruit products which are not subjected to high-temperature sterilization treatment or not, so that the processing condition of the fruit products can be ensured.
According to the embodiment of the invention, the fact that the response values of the olfactory receptors corresponding to the products to be tested are not lower than a second preset value is an indication that the products to be tested are subjected to high-temperature disinfection treatment; or, the fact that the response value of the olfactory receptor corresponding to the products to be tested is lower than a second preset value is an indication that the products to be tested are not subjected to high-temperature disinfection treatment. Therefore, whether the high-temperature disinfection treatment is carried out on the fruit product can be rapidly monitored, so that the processing link of the fruit product is ensured, and the quality safety of the fruit product is improved.
The second predetermined value is a response value obtained through experiments for distinguishing a fruit preparation subjected to a high-temperature sterilization treatment from a fruit preparation not subjected to a high-temperature sterilization treatment, and may be determined according to different detection methods, for example, by detecting a change in cAMP concentration, and the first predetermined value may be set to 200.
According to an embodiment of the invention, the product to be tested is a pineapple product.
According to an embodiment of the invention, the pineapple product comprises pineapple juice or pineapple can.
According to an embodiment of the invention, the high temperature sterilization process is selected from a pasteurisation process.
According to the embodiment of the invention, the pasteurization treatment is carried out at 70-100 ℃ for 20-60 min.
According to an embodiment of the present invention, the methods of the third, fourth, fifth, sixth and seventh aspects may further include at least one of the following technical features:
according to an embodiment of the invention, the olfactory receptor comprises at least one selected from the group consisting of: MOR244-3, MOR180-1 and OR2C 1. Experiments show that the olfactory receptor can specifically recognize 3-methylthiopropanal.
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 invention, the eukaryotic cell comprises at least one of a cell selected from the group of cells isolated from the olfactory substrate, a HEK293 cell, a CHO cell, a xenopus oocyte, a Hela cell, a COS cell and a yeast cell.
According to an embodiment of the invention, the prokaryotic cell is selected from bacteria.
According to an embodiment of the invention, said response value is obtained by detecting a change in activity of said olfactory receptor.
According to an embodiment of the invention, said activity change is determined by at least one of the following detection methods: luciferase assay, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, Ca 2+ Concentration detection method, current detection method, isotope labeling method, antibody detection method, and pH detection method。
Illustratively, the luciferase assay is performed by stimulating olfactory receptor-containing cells with 3-methylthiopropanal or a sample containing 3-methylthiopropanal, wherein when olfactory receptors are activated, intracellular cAMP concentration increases, cAMP binds to the CRE-luciferase promoter region and promotes transcription and translation of luciferase, and thus, by detecting luciferase activity, it is possible to characterize the olfactory receptor response and determine whether olfactory receptor can recognize 3-methylthiopropanal.
According to an embodiment of the invention, said response value is obtained by detecting a change in cAMP concentration in said cell.
According to an embodiment of the invention, the change of cAMP concentration in said cells is performed by using GloSensor TM And obtaining the cAMP detection kit. The GloSensor-20F cAMP gene construct can pre-express a luciferase variant, and the increase of cAMP concentration can cause the conformation change of the luciferase variant, so that the luciferase is converted from an inactive state to an active state, GloSensor TM The cAMP detection kit provides a substrate of a luciferase variant, and the method can be used for measuring the change of the concentration of cAMP in real time and rapidly and sensitively detecting the content of 3-methylthiopropanal in a sample to be detected.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: screening of olfactory receptors
In this example, the olfactory receptor activity is measured by the Dual-Luciferase method (Dual-Glo-Luciferase Assay System, Promega). The inventors selected a partial olfactory receptor from the pool of human and mouse 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 culture, 3-methylthiopropanal was diluted with the medium to a concentration of 500. mu.M, the cells were stimulated with the diluted 3-methylthiopropanal, and incubated for 2-4 hours. If the olfactory receptor is activated, the cAMP concentration in cells is increased, the cAMP can be combined with a promoter region of CRE-luciferase and promote the transcription and translation of luciferase, the response condition of the olfactory receptor can be characterized by detecting the activity of the luciferase, and finally 3 mouse olfactory receptors MOR244-3, MOR256-17 and MOR180-1 which generate responses and a human olfactory receptor OR2C1 which generates responses are obtained.
The inventors also determined that different concentrations of 3-methylthiopropanal stimulate cells containing the above 4 olfactory receptors, respectively, and observed dose curves of the 4 olfactory receptors in response to 3-methylthiopropanal at different concentrations, with the results shown in FIG. 1, where the horizontal axis is the logarithm of the 3-methylthiopropanal concentration (M) and the vertical axis is the fold change in response relative to the blank control (without 3-methylthiopropanal). As a result, MOR244-3 and MOR256-17 among the above four olfactory receptors were found to respond most strongly to 3-methylthiopropanal with a fold change of about 25-fold or 10-fold, respectively, and the sensitivities were all 30. mu.M.
Wherein, the amino acid sequence of MOR244-3 is shown as follows:
MGALNQTRVTEFIFLGLTDNWVLEILFFVPFTVTYMLTLLGNFLIVVTIVFTPRLHNPMYFFLSNLSFIDICHSSVTVPKMLEGLLLERKTISFDNCIAQLFFLHLFACSEIFLLTIMAYDRYVAICIPLHYSNVMNMKVCVQLVFALWLGGTIHSLVQTFLTIRLPYCGPNIIDSYFCDVPPVIKLACTDTYLTGILIVSNSGTISLVCFLALVTSYTVILFSLRKKSAEGRRKALSTCSAHFMVVTLFFGPCIFLYTRPDSSFSIDKVVSVFYTVVTPLLNPLIYTLRNEEVKTAMKHLRQRRICS（SEQ ID NO:1）。
the amino acid sequence of MOR256-17 is shown below:
MEVDSNSSSGSFILMGVSDHPHLEIIFFAVILASYLLTLVGNLTIILLSRLDARLHTPMYFFLSNLSSLDLAFTTSSVPQMLKNLWGPDKTISYGGCVTQLYVFLWLGATECILLVVMAFDRYVAVCRPLHYMTVMNPRLCWGLAAISWLGGLGNSVIQSTFTLQLPFCGHRKVDNFLCEVPAMIKLACGDTSLNEAVLNGVCTFFTVVPVSVILVSYCFIAQAVMKIRSVEGRRKAFNTCVSHLVVVFLFYGSAIYGYLLPAKSSNQSQGKFISLFYSVVTPMVNPLIYTLRNKEVKGALGRLLGKGRGAS（SEQ ID NO:2）。
the amino acid sequence of MOR180-1 is as follows:
MDKENHSVVTEFVFMGITQDPQLQIIFFVVFLLVYLVNVIGNVGMIILIITDSQLHTPMYFFLCNLSFVDLGYSSAIAPRMLADFLTKHKVISFSSCATQFAFFVGFVDAECYVLAAMAYDRFVAICRPLHYSTLMSKKVCLVLMLGSYFAGLVSLVAHTSLTFSLSYCGSNIINHFFCEIPPLLALSCSDTYISEILLFSLCGFIEFSTILIIFISYAFILIAIIRIRSAEGRLKAFSTCGSHLTGVTLFYGTVMFMYLRPTSSYSLDQDKWASVFYTIIIPMLNPLIYSLRNKDVKAAFKKLIGKKPQ（SEQ ID NO:3）。
the amino acid sequence of OR2C1 is shown below:
MDGVNDSSLQGFVLMGISDHPQLEMIFFIAILFSYLLTLLGNSTIILLSRLEARLHTPMYFFLSNLSSLDLAFATSSVPQMLINLWGPGKTISYGGCITQLYVFLWLGATECILLVVMAFDRYVAVCRPLRYTAIMNPQLCWLLAVIACLGGLGNSVIQSTFTLQLPLCGHRRVEGFLCEVPAMIKLACGDTSLNQAVLNGVCTFFTAVPLSIIVISYCLIAQAVLKIRSAEGRRKAFNTCLSHLLVVFLFYGSASYGYLLPAKNSKQDQGKFISLFYSLVTPMVNPLIYTLRNMEVKGALRRLLGKGREVG（SEQ ID NO:4）。
example 2: response of four olfactory receptors to different compounds
In this example, the method of example 1 was used to determine the response of MOR244-3, MOR256-17, MOR180-1 and OR2C1 to 3 structural analogs of 3-methylthiopropanal (also known as "odorant molecules"), wherein the structural analogs of 3-methylthiopropanal (methional) were 3-methylthiopropanol (methionol), n-valeraldehyde (pentanal) and pentanol (pentanol), and the specific test results are shown in Table 1. Wherein, the hydroxyl in the 3-methylthio propanol replaces the aldehyde group of the 3-methylthio propionaldehyde; the carbon atom in n-valeraldehyde replaces the sulfur atom of the 3-methylthiopropanal; the hydroxyl and carbon atoms in amyl alcohol replaced the aldehyde and sulfur atoms of 3-methylthiopropanal simultaneously, "+ + + +" indicates a response with a change of > 10, "+" indicates a response with a change of 5< X <10(X is the detected response with a change of ≦ 5, "-" indicates no response. The results in table 1 show that replacement of the aldehyde and/or methylthio groups significantly reduces or eliminates the activation effect on 4 olfactory receptors.
Table 1: results of the response of the four olfactory receptors to different compounds
| MOR256-17 | MOR244-3 | MOR180-1 | OR2C1 | |
| methional | ++ | +++ | + | + |
| methionol | + | - | - | - |
| pentanal | - | - | - | - |
| pentanol | + | - | - | - |
Example 3: MOR244-3 and MOR256-17 detection of 3-methylthiopropanal in different freshly squeezed fruit juices
The detection method in this example is described in example 1, except that in this example, a luciferase variant was used in place of the original dual luciferase, and cells expressing olfactory receptors of MOR244-3 and MOR256-17, respectively, were used to detect the presence of 3-methylthiopropanal in fresh orange juice and fresh mango juice, the specific results of which are shown in FIG. 2. The results show a peak around 15 minutes (min) with strong signals for both olfactory receptors.
pGloSensor-20F cAMP (Promega) constructs are co-transfected into cells, which pre-express a luciferase variant. After olfactory receptor is activated, an increase in cAMP concentration can cause a conformational change in the luciferase variant, which allows the luciferase to be converted from an inactive state to an active state using a GloSensor TM The cAMP detection kit can rapidly detect the change of the concentration of cAMP in real time. Thus, binding of GloSensor using cells expressing olfactory receptors would be useful TM The cAMP assay allows for rapid and sensitive detection of the presence of 3-methylthiopropanal in food (e.g., freshly squeezed juice).
Example 4: MOR244-3 and MOR256-17 detection of 3-methylthiopropanal in different freshly squeezed fruit juices
The detection method in this example is shown in example 3, and the results of comparison of the content of 3-methylthiopropanal in three fresh fruit juices, orange, grapefruit and mango, using cells expressing MOR244-3 and MOR256-17 olfactory receptors, respectively, are shown in FIG. 3. The results show that the content of the 3-methylmercapto propionaldehyde in the three fresh squeezed fruit juices is mango > orange > grapefruit, and the results of the research results reported are consistent, and the results of the two olfactory receptors show a consistent trend.
Example 5: MOR244-3 and MOR256-17 detection of 3-methylthiopropanal in four orange-flavored drinks
The detection method in this example is shown in example 3, and the content of 3-methylthiopropanal in four orange beverages, namely freshly squeezed orange juice, pooled and reduced orange juice, fruit grain orange and orange meiniada fizzwater, was compared using cells expressing MOR244-3 and MOR256-17 olfactory receptors, respectively, and the results are shown in fig. 4. The results show that the content of 3-methylthiopropanal in the fresh orange juice is equivalent to that in the concentrated reduced orange juice from the pooled sources, the grain size is inferior to that in the orange minora soda, 3-methylthiopropanal is hardly contained in the orange, and the results of the two olfactory receptors show a consistent trend.
Example 6: MOR244-3 and MOR256-17 detection of 3-methylthiopropanal in 2 mango drinks
The detection method in this example is shown in FIG. 5, in which the content of 3-methylthiopropanal in freshly squeezed mango juice and in concentrated reduced mango juice was compared using cells expressing MOR244-3 and MOR256-17 olfactory receptors, respectively, in example 3. The results show that the content of the fresh mango juice is higher, and the results of two olfactory receptors show a consistent trend.
Example 7: MOR244-3 and MOR256-17 monitoring of pineapple juice sanitization process
The detection method in this example is shown in example 3, and the content of 3-methylthiopropanal in two pineapple juices, i.e., freshly squeezed pineapple juice and processed pineapple juice obtained by pasteurizing (30 min at 80 ℃) the freshly squeezed pineapple juice, was compared using cells expressing olfactory receptors MOR244-3 and MOR256-17, respectively, and the results are shown in FIG. 6. The result shows that the content of the 3-methylmercapto propionaldehyde in the freshly squeezed pineapple juice is increased, the results of two olfactory receptors show a consistent trend and accord with the reported research result.
Example 8: MOR244-3 and MOR256-17 detection of 3-methylthiopropanal in 3 alcoholic products
The assay in this example, see example 3, compares the content of 3-methylthiopropanal in draft beer, black beer and red wine using cells expressing MOR244-3 and MOR256-17 olfactory receptors, respectively, and the results are shown in FIG. 7. The results show that the content of 3-methylthiopropanal in beer (draught beer and black beer) is high, while that in red wine is low, and the results for the two olfactory receptors show a consistent trend.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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 invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer 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, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Hanwang science and technology, Inc
<120> use of olfactory receptor for identifying 3-methylthiopropanal and method for detecting 3-methylthiopropanal
<130> BI3220666
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<170> PatentIn version 3.5
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Claims (10)
1. Use of an olfactory receptor, MOR244-3, wherein the amino acid sequence of MOR244-3 is as set forth in SEQ ID NO 1, for the recognition of 3-methylthiopropanal.
Use of 3-methylthiopropanal in activating an olfactory receptor, said olfactory receptor being MOR244-3, said MOR244-3 having an amino acid sequence as set forth in SEQ ID NO: 1.
3. Use according to claim 1 or2, characterized in that said recognition or activation is represented by a change in the activity of olfactory receptors;
the activity change comprises at least one of the following signal changes:
cAMP, IP3, calcium ion, current, and pH.
4. A method for detecting 3-methylthiopropanal, comprising:
contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor;
determining whether the sample to be tested contains 3-methylthiopropanal or not based on the response value;
wherein, the olfactory receptor is MOR244-3, and the amino acid sequence of the MOR244-3 is shown as SEQ ID NO. 1.
5. The method of claim 4, wherein the olfactory receptor present response value is an indication that 3-methylthiopropanal is contained in the test sample; alternatively, the olfactory receptor non-presence response value is an indication that 3-methylthiopropanal is not present in the test sample;
the method further comprises:
and determining the content of the 3-methylthio propionaldehyde in the sample to be tested based on a standard curve, wherein the standard curve is a corresponding curve of a preset amount of the 3-methylthio propionaldehyde and the response value of the olfactory receptor.
6. A method of detecting the freshness of a brew, fruit or fruit preparation, comprising:
contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor;
determining the freshness degree of the sample to be detected based on the response value;
wherein, the olfactory receptor is MOR244-3, and the amino acid sequence of the MOR244-3 is shown as SEQ ID NO. 1.
7. A method for detecting a fruit type contained in a food, comprising:
contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor;
determining the fruit type contained in the sample to be tested based on the response value;
wherein, the olfactory receptor is MOR244-3, and the amino acid sequence of the MOR244-3 is shown as SEQ ID NO. 1;
the sample to be tested only contains one fruit.
8. A method of differentiating between beer and red wine comprising:
contacting a plurality of products to be detected with olfactory receptors, and respectively determining response values of the olfactory receptors corresponding to the products to be detected, wherein the products to be detected are beer and/or red wine;
determining the type of the product to be detected based on the response values of the olfactory receptors corresponding to the products to be detected;
wherein, the olfactory receptor is MOR244-3, and the amino acid sequence of the MOR244-3 is shown as SEQ ID NO. 1.
9. A method of monitoring whether a fruit preparation is subjected to a high temperature sterilization process, comprising:
contacting a sample to be detected with an olfactory receptor, and determining a response value of the olfactory receptor;
determining whether the sample to be detected is subjected to high-temperature disinfection treatment or not based on the response value;
wherein, the olfactory receptor is MOR244-3, and the amino acid sequence of the MOR244-3 is shown as SEQ ID NO. 1.
10. The method of any one of claims 4 to 9, wherein the olfactory receptor is provided by a cell or transgenic cell expressing the olfactory receptor; alternatively, the response value is obtained by detecting a change in cAMP concentration in the cell;
the cell or the transgenic cell is a eukaryotic cell or a prokaryotic cell; said response value is obtained by detecting a change in activity of said olfactory receptor;
the change in activity is determined by at least one of the following detection methods:
luciferase assay, secreted 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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| US20050176009A1 (en) * | 2002-04-23 | 2005-08-11 | Doron Lancet | Polymorphic olfactory receptor genes and arrays, kits and methods utilizing information derived therefrom for genetic typing of individuals |
| EP2565271A1 (en) * | 2011-09-02 | 2013-03-06 | Philip Morris Products S.A. | Threonine synthase from Nicotiana tabacum and methods and uses thereof |
| ES2655840T3 (en) * | 2013-05-31 | 2018-02-21 | Chemcom S.A. | Olfactory receptors involved in the perception of sweat carboxylic acids and their use |
| JP6371336B2 (en) * | 2015-06-17 | 2018-08-08 | 花王株式会社 | Odor suppressor for polysulfide compounds |
| US10209239B1 (en) * | 2017-08-16 | 2019-02-19 | Aromyx Corporation | Method of making an aromagraph comprising ectopic olfactory receptors |
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