Method for researching mutual antagonism among aromas based on olfaction receptor

Abstract

The invention discloses a method for researching mutual antagonism among aromas based on an olfactory receptor. The method comprises the following steps: constructing an olfactory receptor in-vitro heterologous expression cell model and constructing an olfactory receptor expression element, expressing and measuring an olfactory receptor; pre-preparing an aroma compound binary mixture; and finally, determining the activation effect of the binary aroma system on the olfactory receptor, and comparing the activation effects of the single substance and the binary system on the receptor so as to research the interaction effect of the aroma compounds at the cell-receptor level. The method is a brand-new method for researching mutual antagonism among aromas based on the olfactory receptor, the defect that the interaction of flavor substances can be researched only through human body sensory experiments in the prior art is overcome, and a more scientific and objective flavor experiment research scheme is provided by the method. The displayed result is visual and reliable, and the applicability is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of food flavor, and particularly relates to a method for studying the antagonistic interaction between aromas based on olfactory receptors. Background Art

[0002] Through advanced cell biology methods, the molecular mechanisms of human olfactory perception and odor interaction can be elucidated at the receptor level. For example, researchers found that OR2T11 is a small molecule thiol-specific receptor, revealing the key role of metal ions in mediating odor receptor interactions. Based on this discovery, subsequent studies demonstrated the efficacy of deodorants in inhibiting malodorous sulfides, where β-ionone became an effective antagonist by reducing the level of OR2T11 under the action of sulfides.

[0003] The systematic characterization of the olfactory receptor activation pattern has greatly promoted the in-depth understanding of food flavor by humans. Notably, researchers have discovered the interactions between specific receptors and odorants, including the activation of OR5M3 and OR8D1 by the structural homologs furanone and sotolon, respectively, and the selective response of OR8H1 to 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN), a characteristic flavor compound in Riesling wine. In addition, the development of olfactory receptor heterologous expression systems has enabled people to study the interactions between flavor compounds and receptors at the molecular level, providing important insights into chemosensory mechanisms. The construction of mammalian model cells and related studies on olfactory receptor orphan have provided a good basis and reference for our study of the binary interaction of aroma substances.

[0004] Based on this, the present invention provides a method for studying the synergistic interaction between two different types of aroma substances based on an olfactory receptor heterologous expression model, especially for studying the interaction between 2,3,5-trimethylpyrazine (TMP) and L-menthol (L-MT). On the one hand, it makes up for the deficiency of the research technology for studying the interaction between aroma substances from the cell-receptor level. On the other hand, it fills the gap in the research on the interaction relationship of the TMP and L-MT binary aroma system in the prior art. Summary of the Invention

[0005] The object of the present invention is to propose a method for studying the antagonistic effect between two different types of aroma substances based on an olfactory receptor heterologous expression model. This method adopts a new binary ligand preparation process, pre-prepares aroma compounds to treat cells, and uses a multifunctional microplate reader to detect the chemiluminescence changes caused by the fluctuation of cAMP production in cells, and then analyzes the interaction between different aroma substances at the receptor level. The determination method is scientific, reasonable, highly sensitive, simple to operate, fast and efficient in detection, and consumes a small amount of samples, making up for the problem of the lack of research methods for the interaction of binary aroma systems at the cell-receptor level in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a method for studying the antagonistic effect between two different types of aroma substances based on an olfactory receptor heterologous expression model, including the following steps:

[0008] S1: Olfactory receptor heterologous expression:

[0009] S101: Construction of olfactory receptor expression element: Connect the target gene, namely the olfactory receptor expression fragment, and the detection tag sequence together on the expression vector;

[0010] S102: Cell culture: Transfect the olfactory receptor expression fragment in S101 into human non-olfactory model cells, and culture the cells under appropriate conditions;

[0011] S103: Detection of olfactory receptor heterologous expression: The detection tag and the target gene are co-expressed, and the expression of the detection tag is detected;

[0012] S2: Preparation of odorants: Pre-mix the odorants to obtain a binary system odorant, and dilute it to the required concentration with a solvent gradient and set a series of concentration gradients;

[0013] S3: Activation of the model cells expressing the receptor with odorants: The cells after heterologous expression of the receptor need to be incubated with 88% HBSS, 10% FBS, and 2% pGlosensor-22F (Promega Corporation, USA). After incubation, add the pre-prepared odorants to the cells to obtain the final operating concentration and activate the olfactory receptor in the cells;

[0014] S4: Determination of chemiluminescence by microplate reader: Perform chemiluminescence detection on the cells activated in S3, and measure the luminescence values of the cells at different time points within a certain time by setting a kinetic cycle;

[0015] S5: Chemiluminescence data analysis: Statistically analyze the data obtained in S4 to obtain a response-concentration curve and a response-time curve, and characterize and study the interaction effect of the binary odor system through the chemiluminescence changes when the cells are stimulated by single odorants and binary system odorants.

[0016] In some embodiments, the target genes in S1 are OR5K1 (GenBank: KP290526.1) and OR2W1 (GenBank: KP290589.1), the expression vector is pGL4.29, and the detection tag is the green fluorescent protein GFP-tag.

[0017] In some embodiments, in S1, the expression of olfactory receptor proteins is observed using an upright and inverted integrated microscope.

[0018] In some embodiments, the olfactory receptors include, but are not limited to, the OR5K1 olfactory receptor and the OR2W1 olfactory receptor.

[0019] In some embodiments, in S1, the target gene encoding the olfactory receptor is amplified from human genomic DNA by polymerase chain reaction (PCR) using specific primers and ligated into the vector pGL4.29 using T4-DNA ligase.

[0020] In some embodiments, in S1, the cell lines include, but are not limited to, HEK293 and Hana3A; further preferably, the cell line is Hana3A, and the culture conditions are 100% humidity, 37 °C, and 5% CO2.

[0021] In some embodiments, the cells in S1 should be inoculated into a specific white 96-well plate (Thermo Scientific Nunc F96 MicroWell white, #13710).

[0022] In some embodiments, the odorants in S2 are prepared from odor substances and the ligand solvent DMSO (ST038, Beyotime, Shanghai).

[0023] In some embodiments, the binary system odorants in S2 belong to a premix, and the two odor substance mixtures are first mixed before stimulating the cells.

[0024] In some embodiments, the odor substances include, but are not limited to, 2,3,5-trimethylpyrazine that specifically responds to the OR5K1 olfactory receptor and L-menthol that responds to the OR2W1 olfactory receptor.

[0025] In some embodiments, the preparation method of the binary system odorants is specifically as follows: two different odorants are mixed at a concentration ratio of 1:1 and set at different concentration gradients in the range of 0.1 - 1000 μM.

[0026] In some embodiments, all odorants in S2 and S3 are diluted from pure reagents with dimethyl sulfoxide (DMSO). First, prepare a 2M stock solution of each odorant, and then dilute it successively to prepare an operating solution with a suitable concentration. The odorants required for the odor interaction experiment are prepared in advance. The stock solutions are pre-mixed in the required proportions and then diluted into the operating solution. 1 μL of the odorant is added to 99 μL of cell culture medium. In other words, ultimately, the ligand stimulating the cells is the operating solution with a concentration of 1 / 100.

[0027] In some embodiments, in S3, when adding the ligand to the cells cultured in a 96-well plate, add an equal volume (1 μL) of the odorant quickly at one time with a multi-channel pipette. Further preferably, use a low-binding 10 μL pipette tip; accurate pipetting is required, and the cells cannot be touched during the sample addition process.

[0028] In some embodiments, in S4, chemiluminescence is the detection index for the change in olfactory receptor activity, which is achieved by measuring the amount of cAMP by the pGlosensor method.

[0029] In some embodiments, in S4, the incubation time is 2 h before measuring with an enzyme-linked immunosorbent assay (ELISA) reader; when testing on the machine, the measurement interval for each well is 30 s, and the total measurement time is 10 min.

[0030] And / or, the optimal temperature range for incubation is 20°C - 25°C, and the optimal temperature for measurement with an ELISA reader is 25°C.

[0031] The positive and progressive effects of the present invention are as follows:

[0032] Compared with the existing detection methods, the method for studying the mutual antagonism between aromas based on olfactory receptors of the present invention makes up for the deficiencies in the research technology of the interaction between aroma substances from the cell-receptor level. This method is simple and fast, the results are intuitive and reliable, and it has a wide range of applicability, helping people better understand the interaction between olfactory receptors and odorants, building a mature research system for exploring the interaction between olfactory receptors and different aroma substances, and providing an objective verification method.

[0033] During the research process, the inventors also found that after 2,3,5-trimethylpyrazine (TMP) and L-menthol (L-MT) are mixed, they can mutually inhibit the activation levels of OR5K1 and OR2W1, realizing the research on the antagonistic effect of TMP and L-MT aroma substances at the cell-receptor level. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the response-concentration curve of OR5K1 to TMP and L-MT in Example 1 of the present invention;

[0035] Figure 2Schematic diagram of the change of the response-concentration curve on the OR5K1 receptor after equal-proportion mixing of TMP and L-MT in Example 1 of the present invention;

[0036] Figure 3 Schematic diagram of the change of the response-time curve on the OR5K1 receptor after equal-proportion mixing of TMP and L-MT in Example 1 of the present invention;

[0037] Figure 4 Schematic diagram of the response-concentration curve of OR2W1 to TMP and L-MT in Example 2 of the present invention;

[0038] Figure 5 Schematic diagram of the change of the response-concentration curve on the OR2W1 receptor after equal-proportion mixing of TMP and L-MT in Example 2 of the present invention;

[0039] Figure 6 Schematic diagram of the change of the response-time curve on the OR2W1 receptor after equal-proportion mixing of TMP and L-MT in Example 2 of the present invention. Detailed implementation manners

[0040] The present invention will be specifically described below in conjunction with the embodiments. The technical solutions of the present invention will be clearly and completely described to facilitate the understanding of those skilled in the art. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. At the same time, for the raw materials not detailed below, they are all commercially available products; the process steps or preparation methods not detailed are all process steps or preparation methods known to those skilled in the art.

[0041] The core instruments used in the following embodiments of the present invention include:

[0042] Microplate reader, an instrument of model Spark produced by TECAN, Switzerland.

[0043] Example 1 Research on the interaction between 2,3,5-trimethylpyrazine and L-menthol based on the OR5K1 olfactory receptor

[0044] 1. Olfactory receptor expression

[0045] 1) Cell culture

[0046] ① First, culture the cells in a 10 cm dish until 100% adherent.

[0047] ② The 96-well plate can be incubated with 0.1 mg / mL poly-D-lysine (or poly-L-lysine) overnight in advance.

[0048] ③ Passage into 96-well plates: Wash the cells with 3 ml of PBS, add 3 ml of PBS and 0.5 ml of trypsin, incubate for 2 min, transfer to a 15-ml centrifuge tube, centrifuge at 800 rpm for 4 min. Resuspend the cells in 2 ml of complete medium and divide them equally into 4 15-ml centrifuge tubes. Make up the complete medium to 10 ml in each centrifuge tube, and transfer the cell suspension to 4 96-well plates for culturing for one day.

[0049] To prevent bacterial and mycoplasma contamination, levofloxacin at a concentration of 1 / 1000 can be added, and double antibiotics at a concentration of 1 / 500 can be supplemented at each passage.

[0050] 2) Construction of OR5K1 expression element

[0051] The target gene OR5K1 encoding the olfactory receptor was amplified from human genomic DNA by polymerase chain reaction (PCR) using specific primers, and then ligated into the vector pGL4.29 using T4-DNA ligase. The sequence information of each gene can be found in GenBank (http: / / www.ncbi.nlm.nih.gov / genbank / ). Plasmid sequencing was performed using Oxford Nanopore technology.

[0052] 3) Cell transfection

[0053] Cell transfection was carried out using Lipofectamine TM 3000 transfection method.

[0054] Cells can be used for experiments when they have covered 70% or more of the bottom of the culture container. The following transfection protocol is for a whole 96-well plate.

[0055] ① Preparation of transfection solutions A and B: Solution A is 500 μl of Opti-MEM + 30 μl of liposome, and solution B is 500 μl of Opti-MEM + 18 μg of DNA (6000 ng of OR5K1 + 6000 ng of 22F + 3000 ng of mRTPs + 3000 ng of Ga) + 30 μl of P3000.

[0056] ② Mix solutions A and B, gently vortex and incubate for 15 - 20 min.

[0057] ③ Prepare a 15-ml centrifuge tube, add 10 ml of Opti-MEM to the tube, add the mixed solution obtained in step 2 → vortex and pour it into a pipette trough.

[0058] ④ Pour out the original medium in the 96-well plate → use a multi-channel pipette to add the liquid in the pipette trough in step 3 to the 96-well plate, 100 μl per well.

[0059] ⑤Change the medium / supplement the medium with a multi-channel pipette and a pipette trough 6 - 8 h after transfection.

[0060] ⑥Observe the receptor expression under an upright and inverted microscope. Observe the cell morphology and quantity under bright field and observe the GFP expression under the green fluorescence channel.

[0061] 2. Ligand preparation

[0062] The three required aroma substances are 2,3,5 - trimethylpyrazine (TMP) and L - menthol (L - MT). All odorants are diluted with dimethyl sulfoxide (DMSO) from pure reagents. We first prepare a 2 M stock solution of each odorant, and then dilute it successively to prepare a working solution with an appropriate concentration. The odorants required for the odor interaction experiment are prepared in advance. We pre - mix the stock solutions in the required ratio and then dilute them into the working solution. Add 1 μL of the odorant to 99 μL of cell culture medium. In other words, the final ligand stimulating the cells is the working solution with a concentration of 1 / 100. Finally, the solutions we obtain are 0.1 μM L - MT + 0.1 μM TMP, 0.5 μM L - MT + 0.5 μM TMP,......, 1000 μM L - MT + 1000 μM TMP.

[0063] 3. Use a TECAN Spark microplate reader to measure the activation of olfactory receptors.

[0064] Cells transfected for 18 - 24 h can be used to detect specific response signals.

[0065] ①Prepare the incubation solution: Prepare 88% HBSS, 10% FBS, and 2% 22F reagent according to the kit requirements. That is, 8800 μL HBSS, 1000 μL FBS, and 200 μL 22F reagent. Vortex and mix well.

[0066] ②Pour out the medium in the 96 - well plate, and add the incubation solution obtained in step 1 with a multi - channel pipette. Incubate in the dark for 2 h.

[0067] ③Perform the detection on the instrument: The detection interval for each well is 30 s, and the total detection time is 10 min.

[0068] 4. Data processing

[0069] ①Baseline determination: Conduct no less than 3 rounds of detection when no odor stimulation is added, and take the average value of the last 3 detection results as the baseline (Base);

[0070] ②Response intensity determination: The formula for calculating the response intensity is as follows:

[0071]

[0072] In the formula:

[0073] Lum: The response intensity of cells after stimulation with odor compounds, expressed as a multiple;

[0074] x: The reading of the experimental group wells measured by the instrument at time t;

[0075] x0: The reading of the CON group wells measured by the instrument at time t;

[0076] ③ Use Origin2019 data processing software to plot the response-time curve with a dot-line graph for the response values within 10 min of each treatment group. Calculate the response values of each group at each concentration, fit them with the Logestic function to obtain the response-concentration curve. After subtracting the control group from all data, perform maximum normalization and express it as the mean ± SD (n = 4). RLU = relative light unit.

[0077] Example 2 Study on the interaction between 2,3,5-trimethylpyrazine and L-menthol based on the OR2W1 olfactory receptor

[0078] 1. Olfactory receptor expression

[0079] 1) Cell culture

[0080] ① First, culture the cells in a 10 cm dish until 100% adherent.

[0081] ② The 96-well plate can be pre-incubated with 0.1 mg / mL poly-D-lysine (or poly-L-lysine) overnight.

[0082] ③ Passage the cells into the 96-well plate: Wash the cells with 3 ml PBS, add 3 ml PBS and 0.5 ml trypsin, incubate for 2 min, transfer to a 15 ml centrifuge tube, centrifuge at 800 rpm for 4 min, resuspend the cells with 2 ml of complete medium and divide them equally into 4 15 ml centrifuge tubes. Make up the complete medium to 10 ml in each centrifuge tube, transfer the cell suspension to 4 96-well plates and culture for one day.

[0083] To prevent bacterial and mycoplasma contamination, 1 / 1000 levofloxacin agent can be added, and 1 / 500 double antibody can be supplemented during each passage.

[0084] 2) Construction of OR2W1 expression element

[0085] Through polymerase chain reaction (PCR), use specific primers to amplify the target gene OR2W1 encoding the olfactory receptor from human genomic DNA, and then use T4-DNA ligase to ligate it into the vector pGL4.29. The sequence information of each gene can be found in GenBank (http: / / www.ncbi.nlm.nih.gov / genbank / ). Plasmid sequencing uses Oxford Nanopore technology.

[0086] 3) Cell transfection

[0087] Cell transfection was performed using Lipofectamine TM 3000 transfection method.

[0088] The cells can be used for experiments when they have covered 70% or more of the bottom of the culture vessel. The following transfection protocol is for a whole 96-well plate.

[0089] ① Preparation of transfection solutions A and B: Solution A is 500 μl of Opti-MEM + 30 μl of liposome, and solution B is 500 μl of Opti-MEM + 18 μg of DNA (6000 ng of OR2W1 + 6000 ng of 22F + 3000 ng of mRTPs + 3000 ng of Ga) + 30 μl of P3000.

[0090] ② Mix solutions A and B, gently vortex and incubate for 15 - 20 min.

[0091] ③ Prepare a 15-ml centrifuge tube, add 10 ml of Opti-MEM to the tube, add the mixed solution obtained in step 2 → vortex and pour it into a pipette trough.

[0092] ④ Pour out the original medium in the 96-well plate → use a multichannel pipette to add the liquid in the pipette trough in step 3 to the 96-well plate, 100 μl per well.

[0093] ⑤ At 6 - 8 h after transfection, change the medium / supplement the medium using a multichannel pipette and a pipette trough.

[0094] ⑥ Observe the receptor expression under an upright and inverted microscope. Observe the cell morphology and number under bright field and the GFP expression under the green fluorescence channel.

[0095] 2. Ligand preparation

[0096] The three required aroma substances are 2,3,5-trimethylpyrazine (TMP) and L-menthol (L-MT). All odorants are diluted with dimethyl sulfoxide (DMSO) from pure reagents. First, prepare a 2 M stock solution of each odorant, and then dilute them successively to prepare the working solutions with appropriate concentrations. The odorants required for the odor interaction experiment are prepared in advance. The stock solutions are pre-mixed in the required proportions and then diluted into the working solutions. 1 μl of odorant is added to 99 μl of cell culture medium. In other words, the final ligand stimulating the cells is the working solution at a concentration of 1 / 100. Finally, the obtained solutions are 0.1 μM L-MT + 0.1 μM TMP, 0.5 μM L-MT + 0.5 μM TMP,..., 1000 μM L-MT + 1000 μM TMP.

[0097] 3. Detection of olfactory receptor activation using a TECAN Spark microplate reader.

[0098] Cells transfected for 18 - 24 h can be used for detecting specific response signals.

[0099] ① Prepare the incubation solution: Prepare 88% HBSS, 10% FBS, and 2% 22F reagent according to the kit requirements. That is, 8800 ul HBSS, 1000 ul FBS, and 200 ul 22F reagent. Vortex to mix evenly.

[0100] ② Pour out the culture medium in the 96 - well plate, and add the incubation solution obtained in step 1 using a multi - channel pipette. Incubate in the dark for 2 h.

[0101] ③ Perform detection on the instrument: The measurement interval for each well is 30 s, and the total measurement time is 10 min.

[0102] 4. Data processing

[0103] ① Baseline determination: Conduct at least 3 rounds of detections when no odor stimulation is added, and take the average of the last 3 detection results as the baseline (Base);

[0104] ② Response intensity determination: The response intensity calculation formula is as follows:

[0105]

[0106] In the formula:

[0107] Lum: The response intensity of cells after odor compound stimulation, expressed as a multiple;

[0108] x: The reading of the experimental group well measured by the instrument at time t;

[0109] x0: The reading of the CON group well measured by the instrument at time t;

[0110] ③ Use the Origin2019 data processing software to plot the response - time curve with the response values of each treatment group within 10 min using a dot - line graph. Calculate the response values of each group at each concentration, fit them with the Logestic function to obtain the response - concentration curve. After subtracting the control group from all data, perform maximum normalization and express as mean ± SD (n = 4). RLU = relative light unit.

[0111] In the examples of the present invention, the EC values of the specific receptors OR5K1 for TMP and OR2W1 for L - MT were measured. 50 value:

[0112] Table 1

[0113] receptor ligand <![CDATA[EC 50 value]]> OR5K1 TMP 27.67 ± 2.02 μM OR5K1 L-MT NA OR2W1 TMP NA OR2W1 L-MT 22.69 ± 3.85 μM

[0114] Figure 1 / 4 is a schematic diagram of the response-concentration curves of OR5K1 / OR2W1 to TMP and L-MT in Example 1 / 2 of the present invention;

[0115] Figure 2 / 3 is a schematic diagram of the changes in the response-concentration / response-time curves on the OR5K1 receptor after equimolar mixing of TMP and L-MT in Example 1 of the present invention;

[0116] Figure 5 / 6 is a schematic diagram of the changes in the response-concentration / response-time curves on the OR2W1 receptor after equimolar mixing of TMP and L-MT in Example 2 of the present invention.

[0117] Figure 1 The results showed that the response of OR5K1 to TMP was concentration-dependent, and the response value gradually increased in the range of TMP concentration from 0.1 to 1000 uM. According to the fitted curve, the EC50 value of TMP was 27.67 ± 2.02 μmol / L, while FA and L-MT did not generate response signals on OR5K1 with the increase of concentration, and their concentration-response relationships could not prove significant responses.

[0118] Figure 2 and Figure 3 The results showed that compared with the group treated with TMP alone, the chemiluminescence level of cells was significantly reduced when the same concentration of L-MT was mixed with TMP, and this inhibition existed at each concentration. This confirmed that L-MT could inhibit the activation of TMP on OR5K1 at the same concentration. It is worth noting that when the concentration of L-MT was greater than 100 uM, a more severe reversal effect would occur because high-concentration L-MT, as a GPCR antagonist, played a dominant inhibitory role. Therefore, even when the concentration of TMP was as high as 500 uM or 1000 uM, the response signal obtained by the mixture stimulation no longer increased, and the binary ligand system no longer had concentration dependence. In order to better obtain the response-concentration "S"-shaped curve, the inventors did not consider fitting the data points greater than 100 uM to a function.

[0119] Figure 4 The results showed that OR2W1 had concentration dependence on L-MT only in the range of 1-100 uM, and the EC50 value was 22.69 ± 3.85 μmol / L. This may be because high-concentration L-MT would damage the cell membrane and have an adverse effect on the activity of cells or G protein-coupled receptors.

[0120] Figure 5 and Figure 6The results showed that in the presence of the same concentration of TMP, the activation effect of L-MT on OR2W1 was lower than that of pure L-MT, and the response of the binary mixed ligand within the range of 0.1 - 100 uM was concentration-dependent, similar to the results shown on OR5K1. This indicates that the conclusion that the two odorants inhibit each other can be drawn from the perspective of both the receptors of TMP and L-MT. The difference is that at the same concentration, the inhibitory effect of TMP on L-MT on OR2W1 is weaker than the mutual inhibition between the two on OR5K1.

[0121] Finally, it should also be noted that in the present invention, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not explicitly listed, or further includes elements inherent to such process, method, article or device.

[0122] Although the present disclosure has been disclosed above through the description of specific embodiments of the present disclosure, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present disclosure within the spirit and scope of the appended claims. These modifications, improvements or equivalents should also be considered to be included within the scope claimed by the present disclosure.

Claims

1. A method for studying the mutual antagonism between aromas based on olfactory receptors, characterized in that It includes the following steps: S1: Heterologous expression of olfactory receptors: S101: Construction of olfactory receptor expression element: Connect the target gene, i.e., the olfactory receptor expression fragment, and the detection tag sequence together on the expression vector. S102: Cell culture: Transfect the olfactory receptor expression fragment in S101 into human non-olfactory model cells and culture the cells under appropriate conditions. S103: Detection of heterologous expression of olfactory receptors: The detection tag and the target gene are co-expressed, and the expression of the detection tag is detected. S2: Preparation of odorants: Pre-mix the odorants to obtain a binary system odorant, and dilute it to the required concentration with a solvent gradient and set a series of concentration gradients. S3: Activating the model cells expressing the receptor with odorants: The cells after heterologous expression of the receptor need to be incubated with 88% HBSS, 10% FBS, and 2% pGlosensor-22F. After incubation, add the pre-prepared odorant to the cells to obtain the final operating concentration and activate the olfactory receptors in the cells. S4: Measuring chemiluminescence with a microplate reader: Perform chemiluminescence detection on the cells activated in S3, and measure the luminescence values of the cells at different time points within a certain time by setting a kinetic cycle. S5: Analysis of chemiluminescence data: Statistically analyze the data obtained in S4 to obtain a response-concentration curve and a response-time curve, and characterize and study the interaction effect of the binary odor system through the chemiluminescence changes when the cells are stimulated by a single odorant and a binary system odorant.

2. The method for studying the mutual antagonism between aromas based on olfactory receptors according to claim 1, wherein The target genes described in S1 are OR5K1 and OR2W1, the expression vector is pGL4.29, and the detection tag is the green fluorescent protein GFP-tag.

3. The method for studying the antagonistic interaction between aromas based on olfactory receptors according to claim 1, wherein In S1, the expression of the olfactory receptor protein is observed using an upright and inverted integrated microscope.

4. A method for studying the mutual antagonism between aromas based on olfactory receptors according to claim 1, characterized in that, The olfactory receptors include, but are not limited to, OR5K1 olfactory receptor and OR2W1 olfactory receptor.

5. A method for studying the mutual antagonism between aromas based on olfactory receptors according to claim 2, characterized in that In S1, the target gene encoding the olfactory receptor is amplified from human genomic DNA using polymerase chain reaction with specific primers and ligated into the vector pGL4.29 using T4-DNA ligase.

6. A method for studying the mutual antagonism between aromas based on olfactory receptors according to claim 1, characterized in that The odorants in S2 are prepared from odor substances and the ligand solvent DMSO.

7. A method for studying the mutual antagonism between aromas based on olfactory receptors according to claim 1, characterized in that, The binary system odorant in S2 belongs to a pre-mixture, and the mixture of the two odor substances is first mixed before stimulating the cells.

8. A method for studying the mutual antagonism between aromas based on olfactory receptors according to claim 1, characterized in that, The odor substances include, but are not limited to, 2,3,5-trimethylpyrazine that specifically responds to the OR5K1 olfactory receptor and L-menthol that responds to the OR2W1 olfactory receptor.

9. A method for studying the mutual antagonism between aromas based on olfactory receptors according to claim 7, characterized in that, The specific preparation method of the binary system odorant is: Mix two different odorants at a concentration ratio of 1:1 and set different concentration gradients in the range of 0.1 - 1000 μM.

10. A method for studying the mutual antagonism between aromas based on olfactory receptors according to claim 1, characterized in that, The chemiluminescence in S4 is an index for detecting the change in the activity of olfactory receptors and is achieved by measuring the amount of cAMP using the pGlosensor method.

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