CN116200456A - Method for detecting food deterioration by using olfactory receptor - Google Patents
Method for detecting food deterioration by using olfactory receptor Download PDFInfo
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- CN116200456A CN116200456A CN202310467723.1A CN202310467723A CN116200456A CN 116200456 A CN116200456 A CN 116200456A CN 202310467723 A CN202310467723 A CN 202310467723A CN 116200456 A CN116200456 A CN 116200456A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention relates to a method for detecting food deterioration by using an olfactory receptor. The olfactory receptor comprises at least one selected from the group consisting of: OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f; the compound comprises at least one selected from the group consisting of: amine compounds, hydrogen sulfide, hexanal, octanol and dimethyl sulfide; the method comprises the following steps: contacting an olfactory receptor with a meat sample to be tested, and determining a response value of the olfactory receptor; and determining whether the meat sample to be detected is deteriorated based on the response value. The selected olfactory receptor can finish the rapid detection of the food sample within 15 minutes, and has the advantages of simple operation, high flux, high sensitivity and the like.
Description
Technical Field
The invention relates to the technical field of chemical detection, in particular to a method for detecting food deterioration by using an olfactory receptor and application thereof, and more particularly relates to application of the olfactory receptor in identifying a compound, a method for detecting the compound, a method for evaluating meat deterioration and a method for detecting meat food storage time.
Background
Olfactory Receptors (ORs), also known as odorants, are chemoreceptors expressed in the cell membrane of olfactory neurons (OSNs). Olfactory receptors form a polygene family, with about 400 genes in humans and about 1200 genes in mice. 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 tip of the olfactory neuron dendrites.
In the sensory analysis methods currently established internationally, professional evaluator can evaluate whether food quality meets the corresponding standard by examining the smell of food through nose. However, human olfaction has remained deficient in odor assessment: firstly, the reflection of the smell is quite subjective, the reliability of the result can be influenced by a plurality of factors, and the result cannot be quantified; secondly, some detection objects have toxic and side effects on human bodies; third, high throughput detection cannot be achieved. In addition, other conventional food safety detection and analysis methods have limitations in terms of time, cost, throughput, and the like. The compounds generated by the olfactory receptor in the meat deterioration detection process have higher safety, can realize high-flux detection, and have shorter detection time and lower cost.
Thus, there is a need in the art to develop a method that can assess food quality using olfactory receptors.
Disclosure of Invention
The present invention aims to solve at least to some extent one of the technical problems existing in the prior art.
To this end, the invention provides the use of an Olfactory Receptor (OR) in the recognition of a compound. The olfactory receptor of the invention can safely and effectively detect the compounds generated in the food spoilage process, and can be used for evaluating the meat spoilage.
The present invention has been completed based on the following findings by the inventors:
the different processes of food spoilage can produce a variety of characteristic volatile compounds. For example, meat products spoil to octanol (octanol), trimethylamine (trimethylamine), dimethyl sulfide (dimethyl sulfide), dimethyl disulfide(dimethyl disulfide) hydrogen sulfide (H) 2 S, hydrogen sulfide) and the like; deterioration of beef also produces hexanal (hexanal). In addition, the deterioration of meat foods is accompanied by the degradation of amino acids, and besides sulfides, various amine substances are generated, and both substances have strong pungent odor.
Different volatile compounds are generated in the deterioration process of different meat samples, the types and the contents of the volatile compounds in the samples of the same meat sample at different storage times are different, and the response mode generated by the combination of the volatile compounds stimulated by the olfactory receptor generated by food can reflect the state of the food. Because of the huge number of olfactory receptors of animals, the compounds can be detected with pertinence and more high efficiency, and the method is safer and more convenient in practical application.
Based on this, in a first aspect of the invention, the invention proposes the use of an olfactory receptor in the recognition of a compound, said olfactory receptor comprising at least one selected from the group consisting of: OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f; the compound comprises at least one selected from the group consisting of: amine compounds, hydrogen sulfide, hexanal, octanol and dimethyl sulfide.
According to the embodiment of the invention, the olfactory receptor can be used as a chemical sensor, can accurately detect compounds, can be used for detecting and diagnosing the possibly existing compounds, and can greatly save cost and time in practical detection application.
It should be noted that, if the above-mentioned compounds stimulate the olfactory receptor, the olfactory receptor may be activated, i.e. "the olfactory receptor may recognize the above-mentioned compounds"; if the above compound stimulates the olfactory receptor, the olfactory receptor is not activated, i.e., the olfactory receptor is "unrecognizable to the above compound".
According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following technical features:
according to an embodiment of the present invention, at least one of OR2W1, MOR203-1 and MOR261-1 is used to recognize octanol (octanol).
According to an embodiment of the present invention, at least one of OR2W1 and MOR256-17 is used to identify hexanal (hexanal).
According to an embodiment of the present invention, the MOR244-3 is used to identify dimethyl sulfide (dimethyl sulfide).
According to an embodiment of the present invention, the MOR256-17 is used to identify hydrogen sulfide (H 2 S）。
According to an embodiment of the present invention, the amine compound includes at least one selected from the group consisting of tyramine (tyramine), 2-methylbutylamine (2-methylputylamine), isobutylamine (isobutylamine), butylamine (butylamine), benzylamine (benzamine), 2-phenylethylamine (2-phenylethanolamine), dimethyl-N, N-phenylethylamine (dimethyl-2-phenylethanamine), cyclohexylamine (Cyclohexylamine), trimethylamine (N, N-dimethylamine), N-methylpiperidine (N-methylpiperidine), N-dimethylcyclohexylamine (N, N-dimethylhexylamine), and N, N-dimethyln-octylamine (N, N-dimethyloctylamine).
According to an embodiment of the invention, the hTAAR1 is used for identifying at least one of tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenethylamine and dimethyl-N, N-phenethylamine.
According to an embodiment of the invention, the mTAAR3 is used for recognizing at least one of 2-methylbutylamine, isobutylamine and cyclohexylamine.
According to an embodiment of the invention, the mTAAR4 is used to recognize dimethyl-N, N-phenethylamine.
According to an embodiment of the invention, the mTAAR5 is used for recognizing trimethylamine.
According to an embodiment of the invention, the mTAAR7f is used for identifying at least one of N-methylpiperidine, N-dimethylcyclohexylamine and N, N-dimethyln-octylamine.
According to an embodiment of the invention, the mTAAR1 is used for identifying butylamine, isobutylamine.
According to an embodiment of the invention, the recognition is manifested by a change in the activity of the olfactory receptor.
Illustratively, the inventors can be achieved by transfecting olfactory receptors into HEK293T cells and stimulating the cells with compounds at different concentration gradients and incubating for 3-4 hours. If the olfactory receptor is activated, the intracellular cAMP concentration will increase, cAMP will bind to the promoter region of CRE-luciferase and promote transcription and translation of luciferase, so that the activity of luciferase can characterize the response 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.
It should be noted that the luciferase and secreted alkaline phosphatase signals are commonly used to detect cellular metabolic activity and the presence of a marker substance in the organism; the fluorescent protein and fluorescent probe signals can be used to detect local chemical activity of intracellular molecules, and the fluorescent-labeled cellular proteins can be used to track cell life cycle. The fluorescent probe can measure pH, ion concentration and the like; cAMP and IP3 are signaling molecules that cells control metabolic and functional turnover during intracellular signaling. Calcium ions are also active signaling molecules involved in many physiological and biochemical processes within cells, and determining both levels within a cell can capture information about the cell's process of signaling. The current and pH signals can be used to detect the state of ion channels on cell membranes and differences in intracellular and extracellular pH. If metabolic and functional changes occur in the cell, resulting in changes in the state of ion channels and transport bodies on the membrane, changes in surface potential or pH can be measured to reflect changes in the activity of the cell.
In a second aspect of the invention, the invention provides a method of detecting a compound. 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;
determining whether the sample to be tested contains a compound or not based on the response value;
wherein the olfactory receptor comprises at least one selected from the group consisting of: OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f.
Wherein the compound comprises at least one selected from the group consisting of: amine compounds, octanol, dimethyl sulfide, hydrogen sulfide and hexanal.
The inventors found that the presence or absence of specific compounds (amine compounds, octanol, dimethyl sulfide, hydrogen sulfide, and hexanal) in a sample to be tested can be detected by using the above olfactory receptor. Olfactory receptors are more rapid, sensitive, intuitive than traditional compound analysis methods (e.g., thermal, spectroscopic, chromatographic, etc.), and have unique advantages over organic compounds that cannot be detected by traditional chemical methods (e.g., hydrogen sulfide, hexanal, octanol, dimethyl sulfide, and tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, and dimethyl-N, N-phenylethylamine, etc.). Thus, the method of the present invention can provide a more efficient, reliable, and economical method of analyzing compounds (e.g., qualitative analysis, etc.).
According to an embodiment of the present invention, the method for detecting a compound may further include at least one of the following technical features:
according to an embodiment of the invention, the olfactory receptor presence response value is an indication that the test sample contains a compound; alternatively, the absence of a response value for the olfactory receptor is an indication that the test sample does not contain a compound. I.e., the reaction results of the olfactory receptor, can be used to determine whether a test sample contains a particular compound.
The "indication without compound" means that the compound is not present in the sample to be tested; or that a small amount of the aforementioned compound is present in the sample to be tested, but cannot be detected.
According to an embodiment of the invention, the method further comprises: and determining the content of the compound in the sample to be detected based on a standard curve, wherein the standard curve is a curve corresponding to the predetermined quantitative compound and the response value. Namely, the content of the compound in the sample to be measured is determined by establishing a curve (i.e., a standard curve) of the correspondence between the concentration of the compound and the response value. Specifically, the response value of the sample to be measured is measured, and then the content of the compound in the sample to be measured is calculated according to the compound concentration corresponding to the corresponding response value in the standard curve.
In a third aspect of the invention, the invention provides a method of assessing meat spoilage. According to an embodiment of the invention, the method comprises: contacting an olfactory receptor with a meat sample to be tested, and determining a response value of the olfactory receptor; determining whether the meat sample to be tested is deteriorated based on the response value;
wherein the spoiled meat sample to be tested comprises at least one of the following compounds: amine compounds, octanol, dimethyl sulfide, hydrogen sulfide and hexanal;
the olfactory receptor comprises at least one selected from the group consisting of: OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f.
According to the method provided by the embodiment of the invention, the response value of the olfactory receptor can be measured by contacting the olfactory receptor with the meat sample to be measured, and whether the meat sample to be measured is degenerated can be determined based on the response value. The detection method can finish the rapid detection of the food sample within 15 minutes, and can be popularized and applied as a food safety monitoring technology with simple operation, high flux and sensitivity.
The response value of the olfactory receptor refers to the degree of response caused by sensing the smell of the meat sample.
According to an embodiment of the present invention, the meat sample to be tested includes at least one selected from chicken, pork, fish, shrimp, and beef.
According to an embodiment of the present invention, the amine compound includes at least one selected from the group consisting of tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenylethylamine, dimethyl-N, N-phenylethylamine, cyclohexylamine, N-methylpiperidine, trimethylamine, N-dimethylcyclohexylamine and N, N-dimethyl-N-octylamine.
According to an embodiment of the present invention, at least one of OR2W1, MOR203-1 and MOR261-1 is used to identify octanol.
According to an embodiment of the present invention, at least one of OR2W1 and MOR256-17 is used to identify hexanal.
According to an embodiment of the present invention, the MOR244-3 is used to identify dimethyl sulfide.
According to an embodiment of the present invention, the MOR256-17 is used to identify hydrogen sulfide.
According to an embodiment of the invention, the hTAAR1 is used for identifying at least one of tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenethylamine and dimethyl-N, N-phenethylamine.
According to an embodiment of the invention, the mTAAR3 is used for recognizing at least one of 2-methylbutylamine, isobutylamine and cyclohexylamine.
According to an embodiment of the invention, the mTAAR4 is used to recognize dimethyl-N, N-phenethylamine.
According to an embodiment of the invention, the mTAAR5 is used for recognizing trimethylamine.
According to an embodiment of the invention, the mTAAR7f is used for identifying at least one of N-methylpiperidine, N-dimethylcyclohexylamine and N, N-dimethyln-octylamine.
According to an embodiment of the invention, the mTAAR1 is used for identifying butylamine, isobutylamine.
According to an embodiment of the invention, the meat sample to be tested is fish meat, and the olfactory receptor comprises at least one of OR2W1, MOR261-1, MOR256-17, hTAAR1, mTAAR3, mTAAR7f.
According to an embodiment of the invention, the meat sample to be tested is shrimp, and the olfactory receptor comprises at least one of OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR4, mTAAR 5.
According to an embodiment of the invention, the meat sample to be tested is beef, and the olfactory receptor comprises at least one of OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR4, mTAAR 5.
According to an embodiment of the invention, the meat sample to be tested is chicken, and the olfactory receptor comprises at least one of OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f.
According to an embodiment of the present invention, the meat sample to be tested is pork and the olfactory receptor comprises at least one of MOR244-3, hTAAR1, mTAAR4, mTAAR 5.
In a fourth aspect of the present invention, a method of detecting the time of storage of a meat product is presented. According to an embodiment of the invention, the method comprises: contacting a meat sample to be tested with an olfactory receptor, and determining a response value of the olfactory receptor; and determining the storage time of the meat sample to be tested based on the response value. The smell of the meat sample to be detected is detected by the method to judge whether the meat sample is overdue or not.
Wherein the compounds in the meat sample to be tested are different with the change of the storage time, and the meat sample to be tested comprises at least one of the following compounds:
amine compounds, octanol, dimethyl sulfide, hydrogen sulfide and hexanal.
The olfactory receptor comprises at least one selected from the group consisting of:
OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f.
According to an embodiment of the present invention, before determining the storage time of the meat sample to be tested, further comprising: preparing standard response value curves of the meat sample to be tested at different storage times; and comparing the response value with the standard response value curve to determine the storage time of the meat sample to be tested.
Illustratively, the method establishes a curve (i.e., a standard curve) of a predetermined correspondence between the storage time and the response value to determine the storage time of the meat sample. Specifically, the response value in the sample to be measured is measured, and then the storage time of the meat sample to be measured is obtained according to the storage time of the meat sample corresponding to the corresponding response value in the standard curve. In this application, the inventors take fish as an example, and verify the storage time using the olfactory receptor of the present invention. Likewise, the methods described herein are applicable to other meats as well.
According to an embodiment of the present invention, the sample to be tested includes at least one selected from chicken, pork, fish, shrimp and beef.
According to an embodiment of the present invention, the amine compound includes at least one selected from the group consisting of tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenylethylamine, dimethyl-N, N-phenylethylamine, cyclohexylamine, N-methylpiperidine, trimethylamine, N-dimethylcyclohexylamine and N, N-dimethyl-N-octylamine.
According to an embodiment of the present invention, at least one of OR2W1, MOR203-1 and MOR261-1 is used to identify octanol.
According to an embodiment of the present invention, at least one of OR2W1 and MOR256-17 is used to identify hexanal.
According to an embodiment of the present invention, the MOR244-3 is used to identify dimethyl sulfide.
According to an embodiment of the present invention, the MOR256-17 is used to identify hydrogen sulfide.
According to an embodiment of the invention, the hTAAR1 is used for identifying at least one of tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenethylamine and dimethyl-N, N-phenethylamine.
According to an embodiment of the invention, the mTAAR3 is used for recognizing at least one of 2-methylbutylamine, isobutylamine and cyclohexylamine.
According to an embodiment of the invention, the mTAAR4 is used to recognize dimethyl-N, N-phenethylamine.
According to an embodiment of the invention, the mTAAR5 is used for recognizing trimethylamine.
According to an embodiment of the invention, the mTAAR7f is used for identifying at least one of N-methylpiperidine, N-dimethylcyclohexylamine and N, N-dimethyln-octylamine.
According to an embodiment of the invention, the mTAAR1 is used for identifying butylamine, isobutylamine.
According to an embodiment of the invention, the meat sample to be tested is fish meat, and the olfactory receptor comprises at least one of OR2W1, MOR261-1, MOR256-17, hTAAR1, mTAAR3, mTAAR7f.
According to an embodiment of the invention, the meat sample to be tested is shrimp, and the olfactory receptor comprises at least one of OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR4, mTAAR 5.
According to an embodiment of the invention, the meat sample to be tested is beef, and the olfactory receptor comprises at least one of OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR4, mTAAR 5.
According to an embodiment of the invention, the meat sample to be tested is chicken, and the olfactory receptor comprises at least one of OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f.
According to an embodiment of the present invention, the meat sample to be tested is pork and the olfactory receptor comprises at least one of MOR244-3, hTAAR1, mTAAR4, mTAAR 5.
According to an embodiment of the invention, the olfactory receptor is provided by a cell or transgenic cell expressing the olfactory receptor. The cells expressing the olfactory receptor include eukaryotic cells and prokaryotic cells. The eukaryotic cells include HEK293T cells, yeast cells, HEK293 cells, CHO cells, xenopus oocytes, heLa cells and cells selected from the group of cells isolated from the olfactory substrate. According to an embodiment of the invention, the cells expressing the olfactory receptor are HEK293 cells.
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, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, calcium concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay. In the examples of the present invention, a luciferase assay is used, whereby a change in cell activity is measured by adding a luciferin substrate to a cell system expressing a luciferase, which is capable of hydrolyzing the luciferin substrate.
According to an embodiment of the invention, the response value is obtained by detecting a change in cAMP concentration in the cellsAnd (5) obtaining the product. According to an embodiment of the present invention, the change in cAMP concentration in the cells is achieved by using GloSensor TM cAMP detection kit. The GloSensor-20F cAMP gene construct can pre-express a luciferase variant, the increase of 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, and finally, the response condition of an olfactory receptor can be characterized through the activity of the luciferase.
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 olfactory receptors to volatile compounds characteristic of deterioration of various foods in example 1 of the present invention;
FIG. 2 is a graph showing the response of trace amine receptors (TAARs) to various amines in example 1 of the present invention;
FIG. 3 is a plot of the response of 2 primary Olfactory Receptors (OR) to fish at various storage times in example 2 of the present invention;
FIG. 4 is a plot of the response of 2 TAARs to fish at different storage times for example 2 of this invention;
FIG. 5 is a plot of the response of 3 OR versus 4 TAARs for fish at different storage times in example 2 of this invention;
FIG. 6 is a plot of the response of 5 OR versus 6 TAARs for shrimp at various storage times in example 2 of this invention;
FIG. 7 is a graph showing the response of 5 OR versus 6 TAARs for beef at various storage times in example 2 of this invention;
FIG. 8 is a graph showing the response of 5 OR versus 6 TAARs for chicken at various storage times in example 2 of this invention;
FIG. 9 is a plot of the response of 5 OR versus 6 TAARs in example 2 of this invention to pork at various storage times.
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.
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.
In this application, the protein IDs of the olfactory receptors OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f are shown as NCBI protein_id= "ALI87719.1", NCBI protein_id= "AAL60888.1", NCBI protein_id= "AAL60763.1", NCBI protein_id= "AAL61239.1", NCBI protein_id= "AAL60958.1", NCBI protein_id= "NP 612200.1", NCBI protein_id= "NP 444435.1", NCBI protein_id= "NP_001008429.1", NCBI protein_KB_ 70138.1", protein/protein_tQ5 QD, uniprotein_SwtSwt Q5QD08.1, respectively.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: screening of olfactory receptors
The activity of the olfactory receptor was determined in this example using the double luciferase assay (Dual-Glo ™ Luciferase Assay System, promega). The inventors selected a part of olfactory receptors from a human and mouse 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, the cell culture medium was aspirated, replaced with 25. Mu.L of compound dilutions of different concentration gradients (0, 0.3. Mu.M, 1. Mu.M, 3. Mu.M, 10. Mu.M, 30. Mu.M, 100. Mu.M, 300. Mu.M, 1000. Mu.M) diluted with CD293 medium, the cells were stimulated (FIG. 1), and incubated for 3-4 hours. If the olfactory receptor is activated, the intracellular cAMP concentration is increased, cAMP is combined with a CRE-luciferase promoter region, transcription and translation of luciferase are promoted, and the response condition of the olfactory receptor can be characterized by detecting the activity of the luciferase.
As shown in FIG. 1, the olfactory receptors OR2W1, MOR203-1 and MOR261-1 can be used to recognize octanol; OR2W1 and MOR256-17 can be used to identify hexanal; MOR244-3 is used to identify dimethyl sulfide; MOR244-3 and MOR256-17 were used to identify methylthiopropanal; MOR256-17 is used to identify hydrogen sulfide; mTAAR5 is used to recognize trimethylamine. Wherein the horizontal axis represents the logarithm of the compound concentration, and the vertical axis represents the fold change in the luminous intensity (ratio of the luminous intensity of the compound-stimulated group to the luminous intensity of the non-compound-stimulated group).
In addition, the inventors verified the pairing relationship between a plurality of amine substances (tyramine), 2-methylbutylamine (2-methylethylamine), isobutylamine (isobutylamine), butylamine (butyl amine), benzylamine (benzoylamine), 2-phenylethylamine (2-phenylethylamine), dimethyl-N, N-phenylethylamine (dimethyl-2-phenylethylamine), cyclohexylamine (Cyclohexylamine), N-methylpiperidine (N-methylpiperidine), N-dimethylcyclohexylamine (N, N-dimethyloctylamine)) and TAAR in this example. The cell construction process is the same as above, and the trace amine receptor is stimulated by adopting a single stimulation mode.
The results are shown in FIG. 2, which shows that hTAAR1 can be used to recognize tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenethylamine, and dimethyl-N, N-phenethylamine; mTAAR3 can be used to recognize 2-methylbutylamine, isobutylamine, and cyclohexylamine; mTAAR4 is used to recognize dimethyl-N, N-phenethylamine; mTAAR7f was used to recognize N-methylpiperidine, N-dimethylcyclohexylamine and N, N-dimethyl-N-octylamine.
Example 2: olfactory receptors for food storage time detection
To achieve rapid detection of food samples, the inventors first tried to detect the stimulation of olfactory receptors by food samples using a real-time fluorescence detection technique (GloSensorTM cAMP Assay). Gene constructs containing olfactory receptor, golf and pGlosensor-20F cAMP were prepared and transfected into HEK293T cells using the transfection reagent Lipofectamine2000 (Invitrogen) and after 24 hours incubation, the olfactory molecules in the test sample activated the olfactory receptor, which resulted in an increase in intracellular second messenger cAMP. The pGlosensor-20F cAMP gene construct used in GloSensorTM cAMP Assay can rapidly determine the change in cAMP concentration in real time by expressing a variant of luciferase in advance, and increasing cAMP concentration causes conformational change of the variant of luciferase, so that luciferase is converted from inactive state to active state, catalyzes substrate, and generates luminescence signal.
The preparation steps of the sample to be tested are as follows:
weighing food sample (fish, shrimp, beef, chicken, pork) 5 g, cutting with aseptic instrument in ultra clean bench, placing into 50 mL aseptic centrifuge tube, adding 30 mL PBS solution, tightening the cap, loosening two circles, and storing at 25deg.C under 50% relative humidity for 8 days. Samples were taken every 24 th h, 2 mL each time, filtered through a 0.22 μm filter, frozen, and food samples were labeled 0, 1, 2, 3, 4, 5, 6, 7 days (day 0 of sample preparation).
Analysis of results:
FIG. 3 shows the response curves of two major olfactory receptors OR (MOR 203-1, MOR 244-3) to fish at different storage times.
On the left is the real-time response curve of olfactory receptors versus fish at day 0 and day 2, with the horizontal axis being the time of detection (minutes) and the vertical axis being the relative luminescence intensity (luminescence signal readings at each time point minus baseline). The results show that fish meat stored for a period of 2 days can significantly activate MOR203-1 and MOR244-3, reaching a plateau within 15 minutes.
The right side is the detection result of olfactory receptor on continuous 8-day fish samples, the horizontal axis is (days), and the vertical axis is the luminescence intensity difference (the maximum value of luminescence signal of real-time response curve per day minus the baseline). The results showed that the difference in luminous intensity showed an increasing trend with increasing storage time, reaching substantially the peak at day 4.
FIG. 4 shows the response curves of two trace amine receptors mTAAR4 and mTAAR5 to different shelf-time fish. Similar to the response of the two primary olfactory receptors OR to fish as shown in fig. 1. Notably, TAAR responds more strongly to the same fish sample, with a value of 3-4 times OR relative to the luminous intensity and the difference in luminous intensity.
FIG. 5 shows the response curves of the other 3 OR's (OR 2W1, MOR261-1, MOR 256-17) and 4 TAARs (hTAAR 1, mTAAR3, mTAAR7 f) to fish at different storage times. Wherein, OR2W1, MOR261-1, MOR256-17, hTAAR1, mTAAR3 and mTAAR7f can be used for identifying the compound generated in the fish meat spoilage process, and the increase of the storage time of OR2W1, MOR261-1, MOR256-17 and hTAAR1 can still continuously monitor the compound change.
FIGS. 6, 7, 8 and 9 are response curves for 5 major olfactory receptors (OR 2W1, MOR203-1, MOR261-1, MOR256-17, MOR 244-3) and 6 trace amine receptors (hTAAR 1, mTAAR3, mTAAR4, mTAAR5, mTAAR7 f) for four food samples, respectively, shrimp, beef, chicken, pork.
Among them, FIG. 6 shows that OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR4 and mTAAR5 are superior in identifying compounds generated during shrimp deterioration.
FIG. 7 shows that OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR4 and mTAAR5 are better for identifying compounds generated in the beef deterioration process.
FIG. 8 shows that OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f are useful for identifying compounds produced during chicken spoilage.
FIG. 9 shows that MOR244-3, hTAAR1, mTAAR4, mTAAR5 are useful for identifying compounds produced during spoilage of pork.
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 (11)
1. Use of an olfactory receptor in the recognition of a compound, said olfactory receptor comprising at least one selected from the group consisting of:
OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f;
the compound comprises at least one selected from the group consisting of:
amine compounds, hydrogen sulfide, hexanal, octanol and dimethyl sulfide.
2. The use according to claim 1, wherein at least one of OR2W1, MOR203-1 and MOR261-1 is used to identify octanol;
at least one of said OR2W1 and MOR256-17 is used to identify hexanal;
the MOR244-3 is used for identifying dimethyl sulfide;
the MOR256-17 is used to identify hydrogen sulfide.
3. Use according to claim 1, characterized in that the amine compound comprises at least one selected from tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenethylamine, dimethyl-N, N-phenethylamine, cyclohexylamine, trimethylamine, N-methylpiperidine, N-dimethylcyclohexylamine and N, N-dimethyl-N-octylamine;
the hTAAR1 is used to identify at least one of tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenethylamine, and dimethyl-N, N-phenethylamine;
the mTAAR3 is used for identifying at least one of 2-methylbutylamine, isobutylamine and cyclohexylamine;
the mTAAR4 is used for identifying dimethyl-N, N-phenethylamine;
the mTAAR5 is used for recognizing trimethylamine;
the mTAAR7f is used for identifying at least one of N-methylpiperidine, N-dimethylcyclohexylamine and N, N-dimethyln-octylamine;
the mTAAR1 is used for identifying butylamine and isobutylamine.
4. The use according to claim 1, wherein said recognition 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.
5. A method of detecting a compound, comprising:
contacting a sample to be tested with an olfactory receptor, and determining a response value of the olfactory receptor;
determining whether the sample to be tested contains a compound or not based on the response value;
wherein the olfactory receptor comprises at least one selected from the group consisting of:
OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f;
the compound comprises at least one selected from the group consisting of:
amine compounds, octanol, dimethyl sulfide, hydrogen sulfide and hexanal.
6. The method of claim 5, wherein the olfactory receptor presence response value is an indication that the test sample contains a compound; alternatively, the olfactory receptor absence response value is an indication that the test sample does not contain a compound;
the method further comprises:
and determining the content of the compound in the sample to be detected based on a standard curve, wherein the standard curve is a curve corresponding to the predetermined quantitative compound and the response value.
7. A method of assessing spoilage of meat comprising:
contacting an olfactory receptor with a meat sample to be tested, and determining a response value of the olfactory receptor;
determining whether the meat sample to be tested is deteriorated based on the response value;
wherein the spoiled meat sample to be tested comprises at least one of the following compounds:
amine compounds, octanol, dimethyl sulfide, hydrogen sulfide and hexanal;
the olfactory receptor comprises at least one selected from the group consisting of:
OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f.
8. A method of detecting the time of storage of a meat product comprising:
contacting a meat sample to be tested with an olfactory receptor, and determining a response value of the olfactory receptor;
determining the storage time of the meat sample to be tested based on the response value;
wherein the compounds in the meat sample to be tested are different with the change of the storage time, and the meat sample to be tested comprises at least one of the following compounds:
amine compounds, octanol, dimethyl sulfide, hydrogen sulfide and hexanal;
the olfactory receptor comprises at least one selected from the group consisting of:
OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f;
before determining the storage time of the meat sample to be tested, further comprising:
preparing standard response value curves of the meat sample to be tested at different storage times;
and comparing the response value with the standard response value curve to determine the storage time of the meat sample to be tested.
9. The method of claim 7 or 8, wherein the meat sample to be tested comprises at least one selected from the group consisting of chicken, pork, fish, shrimp, and beef;
the amine compound comprises at least one selected from tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenethylamine, dimethyl-N, N-phenethylamine, cyclohexylamine, N-methylpiperidine, trimethylamine, N-dimethylcyclohexylamine and N, N-dimethyl-N-octylamine;
at least one of said OR2W1, MOR203-1 and MOR261-1 is used to identify octanol;
at least one of said OR2W1 and MOR256-17 is used to identify hexanal;
the MOR244-3 is used for identifying dimethyl sulfide;
the MOR256-17 is used for identifying hydrogen sulfide;
the hTAAR1 is used to identify at least one of tyramine, 2-methylbutylamine, isobutylamine, butylamine, benzylamine, 2-phenethylamine, and dimethyl-N, N-phenethylamine;
the mTAAR3 is used for identifying at least one of 2-methylbutylamine, isobutylamine and cyclohexylamine;
the mTAAR4 is used for identifying dimethyl-N, N-phenethylamine;
the mTAAR5 is used for recognizing trimethylamine;
the mTAAR7f is used for identifying at least one of N-methylpiperidine, N-dimethylcyclohexylamine and N, N-dimethyln-octylamine;
the mTAAR1 is used for identifying butylamine and isobutylamine;
the meat sample to be detected is fish meat, and the olfactory receptor comprises at least one of OR2W1, MOR261-1, MOR256-17, hTAAR1, mTAAR3 and mTAAR7f;
the meat sample to be detected is a shrimp, and the olfactory receptor comprises at least one of OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR4 and mTAAR 5;
the meat sample to be detected is beef, and the olfactory receptor comprises at least one of OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR4 and mTAAR 5;
the meat sample to be tested is chicken, and the olfactory receptor comprises at least one of OR2W1, MOR203-1, MOR261-1, MOR256-17, MOR244-3, hTAAR1, mTAAR3, mTAAR4, mTAAR5 and mTAAR7f;
the meat sample to be tested is pork, and the olfactory receptor comprises at least one of MOR244-3, hTAAR1, mTAAR4 and mTAAR 5.
10. The method of any one of claims 5-8, wherein the olfactory receptor is provided by a cell or transgenic cell expressing the olfactory receptor;
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, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, calcium concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay.
11. The method of claim 10, wherein the response value is obtained by detecting a change in cAMP concentration in the cell.
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