Method for researching synergistic effect among aromas based on multiple dimensions of human sense-olfaction receptors

Abstract

The invention discloses a method for multi-dimensionally researching synergistic effect among aromas based on human sense-olfaction receptors, which comprises the following steps: firstly, measuring threshold values and interaction effects of aroma substances by using sensory experiments so as to characterize olfaction perception interaction among aroma compounds; constructing an olfactory receptor in-vitro heterologous expression cell model and an olfactory receptor expression element; carrying out olfaction receptor expression and determination; 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 receptor level. The method is a brand-new method for researching interaction among fragrances from multiple angles based on human sense organs and olfactory receptors, combines the advantages of high integration of human sense organs and high sensitivity of olfactory receptors, makes up for the defect that the interaction of flavor substances can be researched only through human sense organ experiments in the prior art, is intuitive and reliable in shown result, and is suitable for large-scale popularization and application. The applicability is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of food flavor, and particularly relates to an analysis method for the synergistic effect between two odor substances with different aroma types based on sensory experiments and olfactory receptor research. Background Art

[0002] The methods for studying the interaction between aromas are mainly based on sensory experiments, which can characterize the combined effect of two aromas at the macroscopic human sensory level. The previous research of the applicant's team identified the key flavor substances in complex food systems and the aroma interaction effects between their components from two levels of instrumental analysis and sensory analysis. More than 500 key aroma components were identified from natural products such as flowers, fruits, and teas. For example, we used headspace solid-phase microextraction combined with a mass spectrometry signal analysis system and aroma activity value method to comprehensively analyze the sweet, sour, and fruity substances in the key aroma compounds of sweet oranges, and found that diacetyl, limonene, myrcene, octanal, citral, linalool, decanal, etc. are its characteristic aroma components. In addition, the interaction laws between different characteristic aroma compounds were studied, and it was found that substances with the same or similar molecular structures and aroma types showed synergistic or additive effects, while substances with different structures and aroma types showed masking or antagonistic effects.

[0003] The systematic characterization of the olfactory receptor activation pattern has greatly promoted the in-depth understanding of food flavors by humans. It is worth noting that researchers have discovered the interaction between specific receptors and odorants, including the activation of OR5M3 and OR8D1 by the structural homologues furanone and sotolone respectively, and the selective response of OR8H1 to 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN), which is a characteristic flavor compound in Riesling wine. In addition, the development of olfactory receptor heterologous expression systems enables people to study the interaction between flavor compounds and receptors at the molecular level, providing important insights into the chemosensory mechanism. The construction of mammalian model cells and related research on olfactory receptor orphaning provide 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 verifying the synergistic effect between two different types of aroma substances based on sensory experiments and olfactory receptor signal detection, especially for studying the interaction between 2,3,5-trimethylpyrazine (TMP) and furfuryl alcohol (LA). 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 blank in the research on the interaction relationship of the binary aroma system of TMP and FA in the prior art. Summary of the Invention

[0005] The object of the present invention is to propose a method for verifying the synergistic effect between two different types of aroma substances based on sensory experiments and olfactory receptor signal detection research. This method combines the macroscopic human senses and the microscopic cell-receptor organically. First, people are asked to smell the aroma substances and the binary system after compounding, and then the aroma compounds are used to treat the model cells expressing the receptor. The S-curve method is used to fit the sensory data to obtain the change in the odor threshold, and a multifunctional microplate reader is used to detect the chemiluminescence change caused by the fluctuation of the cAMP production in the cells, so as to analyze the interaction between different aroma substances at the human olfactory sensory and receptor levels. This method complements and corroborates macroscopic experiments with microscopic experiments. The measurement method is scientific and reasonable, with high sensitivity, simple operation, fast and efficient detection, and small sample consumption, 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 existing technology.

[0006] The present invention provides a method for studying the synergistic effect between aromas based on multi-dimensional human senses-olfactory receptors, including the following steps:

[0007] S1: Measuring the aroma substance threshold and interaction effect using sensory experiments: Measure the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol respectively, and then measure the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol in the aroma system after mixing them respectively. Determine the aroma interaction effect by comparing the differences in the measured thresholds before and after mixing;

[0008] S2: Heterologous expression of olfactory receptors;

[0009] S3: Measuring the activation of cells by odorants:

[0010] S301: After heterologous expression of the receptor, the cells need to be incubated with 88% HBSS, 10% FBS, and 2% pGlosensor-22F (Promega Corporation, USA). After incubation, the pre-prepared odorants are added to the cells to obtain the final operating concentration to activate the olfactory receptors in the cells; the odorants include single odorants and binary system odorants;

[0011] S302: Perform chemiluminescence detection on the activated cells, and set a kinetic cycle to measure the chemiluminescence values of the cells at different time points within a certain period of time;

[0012] S4: Chemiluminescence data analysis: Statistically analyze the data obtained in S3 to obtain the response-concentration curve and the response-time curve, and characterize and study the interaction effect of the binary aroma system through the chemiluminescence changes when the cells are stimulated by single odorants and binary system odorants.

[0013] In some embodiments, the specific steps of measuring the aroma substance threshold and interaction effect using sensory experiments in S1 are:

[0014] S101: Determination of the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol: The aroma extract dilution analysis (AEDA) or / and the three-point forced-choice method was used to determine the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol, and the S-curve method was used for data analysis;

[0015] S102: Determination of the aroma interaction between 2,3,5-trimethylpyrazine and furfuryl alcohol: According to the method in S101, the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol in the aroma system after their mixture were respectively determined, and the aroma interaction effect was judged by comparing the differences in the measured thresholds before and after mixing.

[0016] In some embodiments, the sample size of the sensory experiment in S1 should be large enough (≥50), and the number of male and female volunteers should be the same.

[0017] In some embodiments, the specific steps of heterologous expression of olfactory receptors in S2 are as follows:

[0018] S201: Construction of olfactory receptor expression elements: Through polymerase chain reaction, the target gene encoding the olfactory receptor was amplified from human genomic DNA using specific primers and ligated into the expression vector using T4-DNA ligase;

[0019] S202: Transfection of olfactory receptors: The required olfactory receptor expression fragment was transfected into human non-olfactory model cells, and the cells were cultured under appropriate conditions;

[0020] S203: Detection of heterologous expression of olfactory receptors: Detection of the co-expression of the tag and the target gene, and the expression of the tag was observed and detected using an upright and inverted integrated microscope.

[0021] In some embodiments, the target genes in S2 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.

[0022] In some embodiments, the cells in S2 should be inoculated into a specific white 96-well plate (Thermo Scientific Nunc F96 MicroWell white, #13710).

[0023] In some embodiments, the cells need to be cultured for 18 - 24 h after transfection of the olfactory receptor expression fragment in S2.

[0024] In some embodiments, the olfactory receptors include, but are not limited to, OR5K1 receptors and OR2W1 receptors.

[0025] In some embodiments, the odorant in S3 is prepared from an odorant substance and a ligand solvent DMSO (ST038, Beyotime, Shanghai).

[0026] In some embodiments, the binary system odorant belongs to a premix. Before stimulating the cells, the mixture of the two odorant substances is first mixed.

[0027] In some embodiments, the preparation method of the binary system odorant is specifically as follows: two different odorants are mixed at a concentration ratio of 1:1 and different concentration gradients are set, with a range of 0.1 - 1000 μM.

[0028] In some embodiments, all the odorants in S3 are diluted from pure reagents with dimethyl sulfoxide (DMSO); specifically:

[0029] First, prepare a 2M stock solution of each odorant, and then dilute it successively to prepare a working solution with a suitable concentration;

[0030] The odorants required for the odor interaction experiment are prepared in advance. The stock solutions are premixed in the required proportions and then diluted into the working solution. 1 μL of the odorant is added to 99 μL of cell culture medium. In other words, the final ligand for stimulating the cells is the working solution with a concentration of 1 / 100.

[0031] In some embodiments, in S3, when adding the ligand to the cells cultured in a 96-well plate, the odorant of equal volume (1 μL) is added quickly at one time with a multi-channel pipette. Further preferably, a low-binding 10 μL pipette tip is used; accurate pipetting is required, and the cells cannot be touched during the sample addition process.

[0032] In some embodiments, the odorant substances include but are not limited to 2,3,5-trimethylpyrazine (TMP) that specifically responds to the olfactory receptor OR5K1 and furfuryl alcohol (LA) that responds to the olfactory receptor OR2W1.

[0033] In some embodiments, in S3, chemiluminescence is an index for detecting the change in olfactory receptor activity, which is achieved by measuring the amount of cAMP by the pGlosensor method.

[0034] In some embodiments, in S3, the incubation time required before measuring with an enzyme-linked immunosorbent assay (ELISA) reader is 2 h; when performing the test on the machine, the measurement interval for each well is 30 s, and the total measurement time is 10 min;

[0035] And / or, the optimal incubation temperature range is 20°C - 25°C, and the optimal temperature for measurement with an ELISA reader is 25°C.

[0036] The positive and progressive effects of the present invention are as follows:

[0037] Compared with the existing detection methods, the method of the present invention for studying the synergistic effect between aromas based on multi-dimensional research of human senses - olfactory receptors makes up for the deficiencies of the research technology on the interaction between aroma substances at the cell-receptor level, and innovatively combines the sensory description of aroma substances by the human body with the determination of the response of olfactory receptors at the cell level. The method of the present invention 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.

[0038] During the research process, the inventors also found that after 2,3,5-trimethylpyrazine (TMP) and furfuryl alcohol (FA) are mixed, they can synergistically enhance the activation level of OR5K1 and OR2W1, realizing the research on the synergistic effect of TMP and FA aroma substances at the cell-receptor level. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a graph showing the change in threshold value after equal-proportion mixing of TMP and FA in Example 1 of the present invention (AEDA-S curve method);

[0040] Figure 2 It is a schematic diagram of the response-concentration curve of OR5K1 to TMP and FA in Example 1 of the present invention;

[0041] Figure 3 It is a schematic diagram of the change in the response-concentration curve on the OR5K1 receptor after equal-proportion mixing of TMP and FA in Example 1 of the present invention;

[0042] Figure 4 It is a schematic diagram of the change in the response-time curve on the OR5K1 receptor after equal-proportion mixing of TMP and FA in Example 1 of the present invention;

[0043] Figure 5 It is a schematic diagram of the response-concentration curve of OR2W1 to TMP and FA in Example 2 of the present invention;

[0044] Figure 6 It is a schematic diagram of the change in the response-concentration curve on the OR2W1 receptor after equal-proportion mixing of TMP and FA in Example 2 of the present invention;

[0045] Figure 7 It is a schematic diagram of the change in the response-time curve on the OR2W1 receptor after equal-proportion mixing of TMP and FA in Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention will be specifically described below in conjunction with embodiments. The technical solutions of the present invention are 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 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 belong to 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.

[0047] The core instruments used in the following embodiments of the present invention include:

[0048] A microplate reader, an instrument of model Spark produced by TECAN of Switzerland.

[0049] Example 1 Study on the synergistic effect of 2,3,5-trimethylpyrazine and furfuryl alcohol based on sensory experiments and olfactory receptor OR5K1

[0050] 1. Sensory experiment

[0051] In this study, 25 adult males and 25 adult females were selected as evaluators.

[0052] In this study, a total of 4 groups of test samples were set up, and the preparation methods are as follows:

[0053] Group A (3-AFC): Fix the TMP concentration at 1000 μM, and the LA concentrations are set at 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1000 μM respectively. The reference sample is a 1000 μM TMP solution;

[0054] Group B (3-AFC): Fix the LA concentration at 100 μM, and the TMP concentrations are set at 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1000 μM respectively. The reference sample is a 100 μM LA solution;

[0055] Group C (AEDA): TMP and LA are mixed at equal concentrations, and the concentration gradients are 0.1 μM (0.1 μM TMP + 0.1 μM LA, the same below), 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1000 μM;

[0056] Group D (AEDA): Prepare TMP and LA solutions with concentrations of 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1000 μM respectively.

[0057] In this study, a two-way test needs to be conducted on the first substance (TMP) and the second substance (LA) as follows:

[0058] Group A (3-AFC): At the start of the test, the assessor should be familiar with and firmly remember the olfactory sensation of the second substance, then smell the provided samples and record the sample numbers that can detect the second substance in the olfactory result record form. For the sample group that cannot detect the second substance at all, forced selection is required and the results of the forced selection should be recorded;

[0059] Group B (3-AFC): At the start of the test, the assessor should be familiar with and firmly remember the olfactory sensation of the first substance, then smell the provided samples and record the sample numbers that can detect the first substance in the olfactory result record form. For the sample group that cannot detect the first substance at all, forced selection is required and the results of the forced selection should be recorded;

[0060] Group C (AEDA): At the start of the test, the assessor should be familiar with and firmly remember the olfactory sensation of the first substance, then smell the provided samples in the order of decreasing concentration and record the sample numbers that can just not detect the first substance in the olfactory result record form; after the test on the first substance is completed, the assessor should be familiar with and firmly remember the olfactory sensation of the second substance, then smell the provided samples in the order of decreasing concentration and record the sample numbers that can just not detect the second substance in the olfactory result record form;

[0061] Group D (AEDA): The samples are arranged in descending order of concentration, and the assessor smells the samples in turn and records the sample numbers that can just not be smelled.

[0062] The experimental data is analyzed using the S-curve method to determine the olfactory threshold and odor interaction, and the fitting equation is P = 1 / (1 + e^(-(x - c) / D))

[0063] where P represents the detection probability after accidental correction, x represents the concentration of the odorant, c represents the olfactory threshold, and D represents the parameter defining the slope of the function.

[0064] For a binary system, two odorants A and B are mixed together, and the assessor smells the mixture to detect odorant A. The lg(c)-p curve is plotted and fitted, and the concentration values are added while maintaining the original ratio of A and B. The measured probability P(A) of detecting A is compared with the theoretical probability p(A) of detecting A alone. If P(A) is lower than p(A) when P = 0.5, then odorant B has a masking effect on A; if P(A) is higher than p(A), then B has a mutual enhancement or synergistic effect on A; if P(A) is equal to p(A), then there is no obvious interaction between A and B.

[0065] 2. Olfactory receptor expression

[0066] 1) Cell culture

[0067] ① First, culture the cells in a 10-cm dish until 100% confluent.

[0068] ② The 96-well plate can be pre-incubated overnight with 0.1 mg / mL poly-D-lysine (or poly-L-lysine).

[0069] 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 in 2 mL 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.

[0070] To prevent bacterial and mycoplasma contamination, 1 / 1000 levofloxacin can be added, and 1 / 500 double antibody can be supplemented during each passage.

[0071] 2) Construction of OR5K1 expression element

[0072] The target gene OR5K1 encoding the olfactory receptor is 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 is performed using Oxford Nanopore technology.

[0073] 3) Cell transfection

[0074] Cell transfection is performed using Lipofectamine TM 3000 transfection method.

[0075] 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 the transfection of a whole 96-well plate.

[0076] ① Preparation of transfection solutions A and B: Solution A is 500 μL Opti-MEM + 30 μL liposome, and solution B is 500 μL Opti-MEM + 18 μg DNA (6000 ng OR5K1 + 6000 ng 22F + 3000 ng mRTPs + 3000 ng Ga) + 30 μL P3000.

[0077] ② Mix solutions A and B, gently vortex and incubate for 15 - 20 min.

[0078] ③ Prepare a 15 ml centrifuge tube, add 10 ml of Opti-MEM to the tube, and add the mixed solution obtained in step 2 → vortex and mix well, then pour it into a pipette trough.

[0079] ④ Pour out the original culture 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.

[0080] ⑤ At 6 - 8 h after transfection, perform medium replacement / supplementation using a multi-channel pipette and a pipette trough.

[0081] ⑥ Observe 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.

[0082] 3. Ligand preparation

[0083] The two required aroma substances are 2,3,5-trimethylpyrazine (TMP) and furfuryl alcohol (FA). All odorants are diluted with dimethyl sulfoxide (DMSO) from pure reagents. First, prepare a 2 M stock solution of each odorant, and then dilute it successively to prepare an operating solution with an appropriate concentration. The odorants required for the odor interaction experiment are prepared in advance. The stock solutions are pre-mixed in the required ratio and then diluted into the operating solution. 1 μL of odorant is added to 99 μL of cell culture medium. In other words, the final ligand stimulating the cells is the operating solution with a concentration of 1 / 100. Finally, the obtained solutions are 0.1 μM FA + 0.1 μM TMP, 0.5 μM FA + 0.5 μM TMP,......, 1000 μM FA + 1000 μM TMP.

[0084] 4. Determine the activation of olfactory receptors using a TECAN Spark microplate reader

[0085] Cells transfected for 18 - 24 h can be used for the detection of specific response signals.

[0086] ① Prepare the incubation solution: Prepare 88% HBSS, 10% FBS, and 2% 22F reagent according to the kit requirements. That is, 8800 μl of HBSS, 1000 μl of FBS, and 200 μl of 22F reagent, vortex and mix well.

[0087] ② Pour out the culture medium in the 96-well plate, and use a multi-channel pipette to add the incubation solution obtained in step ①, and incubate in the dark for 2 h.

[0088] ③ Detect on the machine: The detection interval for each well is 30 s, and the total detection time is 10 min.

[0089] 5. Data processing

[0090] ① Baseline determination: Perform at least 3 rounds of detection without odor stimulation, and take the average of the last 3 detection results as the baseline (Base);

[0091] ② Response intensity measurement: The response intensity calculation formula is as follows:

[0092]

[0093] In the formula:

[0094] Lum: The response intensity of cells after stimulation by odor compounds, expressed as a multiple;

[0095] x: The reading of the experimental group well measured by the instrument at time t;

[0096] x0: The reading of the control group well measured by the instrument at time t;

[0097] ③ Use the Origin2019 data processing software to plot the response-time curve with a dot-line graph for the response values within 10 minutes 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. All data are normalized to the maximum value after subtracting the control group and are expressed as the mean ± SD (n = 4). RLU = relative light unit.

[0098] Example 2 Study on the synergistic effect of 2,3,5-trimethylpyrazine and furfuryl alcohol based on sensory experiments and olfactory receptor OR2W1

[0099] 1. Sensory experiments

[0100] In this study, 25 adult males and 25 adult females were selected as evaluators.

[0101] In this study, a total of 4 groups of test samples were set, and the preparation methods are as follows:

[0102] Group A (3-AFC): Fix the TMP concentration at 1000 μM, and set the LA concentrations at 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1000 μM. The reference sample is a 1000 μM TMP solution;

[0103] Group B (3-AFC): Fix the LA concentration at 100 μM, and set the TMP concentrations at 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1000 μM. The reference sample is a 100 μM LA solution;

[0104] Group C (AEDA): Mix TMP and LA at equal concentrations, and the concentration gradients are 0.1 μM (0.1 μM TMP + 0.1 μM LA, the same below), 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1000 μM;

[0105] Group D (AEDA): Prepare solutions of TMP and LA with concentrations of 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, and 1000 μM respectively.

[0106] In this study, two-way tests need to be conducted on the first substance (TMP) and the second substance (LA) as follows:

[0107] Group A (3-AFC): At the start of the test, the assessors should be familiar with and firmly remember the olfactory sensation of the second substance, then smell the provided samples and record the sample numbers that can smell the second substance in the smell result record form. For the sample groups that cannot smell the second substance at all, forced selection is required and the results of the forced selection should be recorded;

[0108] Group B (3-AFC): At the start of the test, the assessors should be familiar with and firmly remember the olfactory sensation of the first substance, then smell the provided samples and record the sample numbers that can smell the first substance in the smell result record form. For the sample groups that cannot smell the first substance at all, forced selection is required and the results of the forced selection should be recorded;

[0109] Group C (AEDA): At the start of the test, the assessors should be familiar with and firmly remember the olfactory sensation of the first substance, then smell the provided samples in the order of decreasing concentration and record the sample numbers that can just not smell the first substance in the smell result record form; after the test on the first substance is completed, the assessors should be familiar with and firmly remember the olfactory sensation of the second substance, then smell the provided samples in the order of decreasing concentration and record the sample numbers that can just not smell the second substance in the smell result record form;

[0110] Group D (AEDA): The samples are arranged in the order of decreasing concentration, and the assessors smell the samples in turn and record the sample numbers that can just not smell.

[0111] Use the S-curve method to analyze the experimental data, determine the olfactory threshold and odor interaction, and the fitting equation is P = 1 / (1 + e^(-(x - c) / D))

[0112] Among them, P represents the detection probability after accidental correction, x represents the concentration of the odorant, c represents the olfactory threshold, and D represents the parameter defining the slope of the function.

[0113] For a binary system, two odorants A and B are mixed together, and the assessor sniffs the mixture to detect odorant A. The lg(c)-p curve is plotted and fitted, and the concentration values are added while maintaining the original ratio of A and B. The measured probability P(A) of detecting A is compared with the theoretical probability p(A) of detecting A alone. If P(A) is lower than p(A) when P = 0.5, then odorant B has a masking effect on A; if P(A) is higher than p(A), then B has a mutual enhancement or synergistic effect on A; if P(A) is equal to p(A), then there is no obvious interaction between A and B.

[0114] 2. Olfactory receptor expression

[0115] 1) Cell culture

[0116] ① First, culture the cells in a 10 cm dish until 100% adherent.

[0117] ② The 96-well plates can be incubated with 0.1 mg / mL poly-D-lysine (or poly-L-lysine) overnight in advance.

[0118] ③ Passage the cells 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 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.

[0119] To prevent bacterial and mycoplasma contamination, 1 / 1000 levofloxacin agent can be added, and 1 / 500 double antibody can be supplemented during each passage.

[0120] 2) Construction of OR2W1 expression element

[0121] The target gene OR2W1 encoding the olfactory receptor is amplified from human genomic DNA using specific primers by polymerase chain reaction (PCR), and then it is 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 is performed using Oxford Nanopore technology.

[0122] 3) Cell transfection

[0123] Cell transfection is performed using the Lipofectamine 3000 transfection method.

[0124] The cells can be used for experiments when they can cover 70% or more of the bottom of the culture container. The following transfection protocol is for the transfection of an entire 96-well plate.

[0125] 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.

[0126] ② Mix solutions A and B, gently vortex and incubate for 15 - 20 min.

[0127] ③ Prepare a 15-ml centrifuge tube, add 10 ml of Opti-MEM to the tube, add the mixture obtained in step 2 → vortex and pour it into a pipette trough.

[0128] ④ Pour out the original culture 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.

[0129] ⑤ At 6 - 8 h after transfection, change or supplement the medium using a multi-channel pipette and a pipette trough.

[0130] ⑥ Observe the receptor expression under an upright and inverted microscope. Observe the cell morphology and quantity under bright field and the GFP expression under the green fluorescence channel.

[0131] 3. Ligand preparation

[0132] The two required aroma substances are 2,3,5-trimethylpyrazine (TMP) and furfuryl alcohol (FA). All odorants are diluted with dimethyl sulfoxide (DMSO) from pure reagents. First, prepare a 2 M stock solution of each odorant, and then dilute successively to prepare working solutions with appropriate concentrations. The odorants required for the odor interaction experiment are prepared in advance. Pre-mix the stock solutions in the required proportions and then dilute them into the working solutions. Add 1 μl of odorant to 99 μl of cell culture medium. In other words, the final ligand stimulating the cells is the 1 / 100 concentration of the working solution. Finally, the obtained solutions are 0.1 μM FA + 0.1 μM TMP, 0.5 μM FA + 0.5 μM TMP,..., 1000 μM FA + 1000 μM TMP.

[0133] 4. Determination of olfactory receptor activation using a TECAN Spark microplate reader

[0134] Cells transfected for 18 - 24 h can be used for the detection of specific response signals.

[0135] ① Prepare the incubation solution: Prepare 88% HBSS, 10% FBS, and 2% 22F reagent according to the kit requirements. That is, 8800 μl of HBSS, 1000 μl of FBS, and 200 μl of 22F reagent. Vortex and mix well.

[0136] 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.

[0137] ③ Detection on the instrument: The measurement interval for each well is 30 s, and the total measurement time is 10 min.

[0138] 5. Data processing

[0139] ① Baseline determination: Conduct no less than 3 rounds of detections when no odor stimulation is added, and take the average value of the last 3 detection results as the baseline (Base);

[0140] ② Response intensity determination: The response intensity calculation formula is as follows:

[0141]

[0142] In the formula:

[0143] Lum: The response intensity of cells after odor compound stimulation, expressed as a multiple;

[0144] x: The reading of the experimental group well measured by the instrument at time t;

[0145] x0: The reading of the control group well measured by the instrument at time t;

[0146] ③ Use Origin2019 data processing software to plot the response-time curve using 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. All data are normalized to the maximum value after subtracting the control group, and are expressed as the mean ± SD (n = 4). RLU = relative light unit.

[0147] In the examples of the present invention, the thresholds and odor interaction modes of TMP and LA were determined through sensory experiments:

[0148] Table 1 (AEDA-S curve method)

[0149] Substance Threshold Interaction mode TMP 6191.99 μM / FA 17405.24 μM / FA + TMP 15118.89 μM Synergistic

[0150] Table 2 (3-AFC-S curve method)

[0151] Substance Threshold Interaction mode TMP 6191.99 μM / FA 17405.24 μM / FA + TMP 6884.78 μM Synergistic

[0152] Combining Table 1-2 and Figure 1 (the threshold change diagram of the equal-proportion mixture of TMP and FA in Example 1) shows that when TMP and FA are mixed in equal proportion, the two show a synergistic effect. At Figure 1In [the case where] TMP and FA are mixed in equal proportions, the moving trend of the S curve of FA is not obvious, and the measured recognition threshold is less than the theoretical recognition threshold, D (experimental threshold / theoretical threshold) = 0.87, showing a synergistic effect.

[0153] In the embodiments of the present invention, cell experiments were conducted to determine the EC 50 values of the specific receptor OR5K1 of TMP and the receptor OR2W1 of FA for TMP and FA:

[0154] Table 3

[0155] Receptor Ligand <![CDATA[EC 50 value]]> OR5K1 TMP 27.67 ± 2.02 μM OR5K1 FA NA OR2W1 TMP NA OR2W1 FA 294.26 ± 81.05 μM

[0156] Combined with Table 3 and Figures 2 - 7 (Schematic diagrams of the response-concentration curves of OR5K1 / OR2W1 to TMP and FA and the schematic diagrams of the changes in the response-concentration curves and response-time curves on the OR5K1 / OR2W1 receptor after equal-proportion mixing of TMP and FA in Examples 2-4 / Examples 5-6 are the schematic diagrams of Example 1 / 2 respectively), it can be seen that in Table 3 and Figures 2 - 4 , OR5K1 specifically responds to TMP and does not respond to FA. Compared with the group treated with TMP alone, the chemiluminescence level of cells slightly increases when the same concentration of FA is mixed with TMP, which confirms that FA enhances the activation of OR5K1 by TMP at the same concentration; while Table 3 and Figures 5 - 7 The results show that OR2W1 specifically responds to FA and does not respond to TMP. Compared with the group treated with TMP alone, the activation level of OR2W1 increases in the presence of the same concentration of TMP.

[0157] Based on Tables 1-3 and Figures 1 - 7 it can be known that after 2,3,5-trimethylpyrazine TMP and furfuryl alcohol FA are mixed, they can synergistically enhance the activation level of OR5K1 and OR2W1, and the interaction effect and synergistic mechanism between TMP and FA are studied at the cell-receptor level.

[0158] 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 expressly listed, or also includes elements inherent to such process, method, article or device.

[0159] 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 multi-dimensional research on the synergistic effect between aromas based on human senses - olfactory receptors, characterized in that, It includes the following steps: S1: Using sensory experiments to determine the aroma substance thresholds and interaction effects: Respectively determine the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol, and then respectively determine the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol in the aroma system after their mixing. Determine the aroma interaction effect by comparing the differences in the measured thresholds before and after mixing; S2: Heterologous expression of olfactory receptors; S3: Odorant-activated cell assay: S301: After heterologous expression of the receptor, the cells need to be incubated with 88% HBSS, 10% FBS, and 2% pGlosensor-22F. After incubation, the pre-prepared odorant is added to the cells to obtain the final operating concentration, and the olfactory receptors in the cells are activated; The odorants include single odorants and binary system odorants; S302: Conduct chemiluminescence detection on the activated cells, and by setting a kinetic cycle, measure the chemiluminescence values of the cells at different time points within a certain period; S4: Chemiluminescence data analysis: Statistically analyze the data obtained in S3 to obtain a response-concentration curve and a response-time curve, and characterize and study the interaction effect of the binary aroma system through the chemiluminescence changes when the cells are stimulated by single odorants and binary system odorants.

2. The method for studying the synergistic effect between aromas based on multi-dimensional human sensory-olfactory receptors according to claim 1, characterized in that, The specific steps for using sensory experiments to determine the aroma substance thresholds and interaction effects in S1 are: S101: Determination of the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol: Use the aroma extract dilution analysis method or / and the three-point choice forced method to determine the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol, and use the S-curve method for data analysis; S102: Determination of the aroma interaction between 2,3,5-trimethylpyrazine and furfuryl alcohol: According to the method in S101, respectively determine the olfactory thresholds of 2,3,5-trimethylpyrazine and furfuryl alcohol in the aroma system after their mixing, and determine the aroma interaction effect by comparing the differences in the measured thresholds before and after mixing.

3. A method for studying the synergistic effect between aromas based on multi-dimensional research of human sensory-olfactory receptors according to claim 1, characterized in that, The specific steps for heterologous expression of olfactory receptors in S2 are: S201: Construction of olfactory receptor expression elements: Through polymerase chain reaction, use specific primers to amplify the target gene encoding the olfactory receptor from human genomic DNA, and use T4-DNA ligase to ligate it into the expression vector; S202: Transfect the olfactory receptor: Transfect the required olfactory receptor expression fragment into human non-olfactory model cells, and culture the cells under appropriate conditions; S203: Detect the heterologous expression of the olfactory receptor: Detect the co-expression of the tag and the target gene, and use an upright and inverted integrated microscope to observe the expression of the tag.

4. A method for studying the synergistic effect between aromas based on multi-dimensional human sensory-olfactory receptors according to claim 3, characterized in that, In S2, the target genes are OR5K1 and OR2W1, the expression vector is pGL4.29, and the detection tag is the green fluorescent protein GFP-tag.

5. A method for studying the synergistic effect between aromas based on multi-dimensional research of human sensory-olfactory receptors according to claim 1, characterized in that, In S3, the odorant is prepared from an odor substance and a ligand solvent DMSO.

6. A method for studying the synergistic effect between aromas based on multi-dimensional human sensory-olfactory receptors according to claim 1 or 5, characterized in that, The binary system odorant belongs to a pre-mixture, and before stimulating the cells, the mixture of the two odor substances is first mixed.

7. A method for studying the synergistic effect between aromas based on the multi-dimensional research of human sensory-olfactory receptors according to claim 6, 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, with a range of 0.1 - 1000 μM.

8. A method for studying the synergistic effect between aromas based on multi-dimensional research of human sensory-olfactory receptors according to claim 6, characterized in that, The odor substances include, but are not limited to, 2,3,5-trimethylpyrazine to which the OR5K1 olfactory receptor specifically responds and furfuryl alcohol to which the OR2W1 olfactory receptor responds.

9. A method for studying the synergistic effect between aromas based on multi-dimensional research of human sensory-olfactory receptors according to claim 1, characterized in that, The chemiluminescence in S3 is an index for detecting changes in olfactory receptor activity and is achieved by measuring the amount of cAMP by the pGlosensor method.

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