Method for researching aroma synergistic effect based on S curve, trace thermophoresis and molecular dynamics and application

Abstract

The invention relates to a method for researching a synergistic effect of aroma based on an S curve, trace thermophoresis and molecular dynamics and application. The method comprises the following steps: 1) preparing ten groups of single citral and beta-ionone solutions with concentration gradient and a mixed solution of the two solutions, and performing sensory and S curve drawing; (2) preparing an OR52D1 olfaction receptor with a constant concentration, a single aroma substance and a mixture of the OR52D1 olfaction receptor and the single aroma substance, and measuring the sample mixture in a micro thermophoresis (MST) device; and 3) carrying out molecular dynamics research on the structural files of citral, beta-ionone and OR52D1. Compared with the prior art, the method provided by the invention combines subjective and objective researches, makes up for the defect of strong subjectivity of sensory experiments, and can more comprehensively research the synergistic effect of aroma from multiple angles.

Description

Technical Field

[0001] The present invention relates to the technical field of food flavor, and in particular to a method and application for studying the aroma synergy based on S-curve, microscale thermophoresis, and molecular dynamics. Background Art

[0002] With the development of science and technology, the interaction between olfactory receptors and flavor compounds has become a research hotspot. Understanding the interaction mechanism between olfactory receptors and flavor compounds helps to predict and control the flavor release or retention behavior of aroma components.

[0003] Human olfactory receptors are approximately encoded by 380 genes. The olfactory system can recognize and distinguish a large number of different odor molecules, starting from the interaction between olfactory receptors on the cilia of olfactory neurons and odor molecules. The recognition of aroma can be the result of the action of one or more olfactory receptors, and olfactory receptors have a certain number of active sites. From the perspective of food sensory science, olfactory perception plays a key role in food flavor, which is a key factor affecting food acceptance and consumption.

[0004] Currently, the main research methods for food aroma component synergy are the threshold method, the σ-τ method, the S-curve, and the OAV. These methods can initially determine the synergy between aroma substances and have a certain guiding role in the research of aroma systems, but they are all subjective and lack objective basis. Therefore, it is necessary to find a method that can objectively confirm sensory research. Summary of the Invention

[0005] The purpose of the present invention is to provide a method and application for studying the aroma synergy based on S-curve, microscale thermophoresis, and molecular dynamics. This method has high sensitivity, is easy to operate, and is fast and efficient in detection, making up for the lack of objective verification of the synergy of aroma substances.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The present invention provides a method for studying the aroma synergy based on S-curve, microscale thermophoresis, and molecular dynamics, and comprehensively uses the S-curve method, microscale thermophoresis method, and molecular dynamics method to analyze the aroma synergy.

[0008] Preferably, the S-curve method, microscale thermophoresis method, and molecular dynamics method are comprehensively used to analyze the aroma synergy of citral and β-ionone.

[0009] Preferably, it specifically includes the following steps: preparing solutions of citral, β-ionone and their mixed solutions with gradient concentrations and conducting sensory evaluation and S-curve plotting; preparing OR52D1 olfactory receptors with a constant concentration and single aroma substances or mixtures of two aroma substances, placing the sample mixture into a microscale thermophoresis device for measurement; conducting molecular dynamics studies on the structural files of citral, β-ionone and OR52D1.

[0010] Preferably, the analysis of aroma synergy using the S-curve includes the following steps:

[0011] S1. Solution preparation: Prepare solutions of citral and β-ionone with gradient concentrations, as well as their mixed solution, and set up each experimental group;

[0012] S2. Sniffing experiment: Have the snifters sniff the solutions of each experimental group one by one, record the experimental groups where no odor can be smelled, and determine the actual olfactory thresholds of citral, β-ionone and their mixture;

[0013] S3. Threshold calculation: Calculate the theoretical and experimental thresholds of citral and β-ionone;

[0014] S4: S-curve plotting: Use Origin software to plot an S-shaped curve with concentration as the abscissa and olfactory intensity as the ordinate;

[0015] S5. Synergy analysis: Analyze the synergy in olfactory perception between the mixed solution and the individual solutions by comparing the S-curves of the mixed solution and the individual solutions.

[0016] Preferably, in step S1, the gradient concentration includes 10 -1 , 10 -2 , …, 10 -10 mol / L.

[0017] Preferably, in step S1, the solutions of citral and β-ionone with gradient concentrations are prepared from citral and β-ionone analytical standards.

[0018] Preferably, in step S2, the sniffing environment for the sniffing experiment is clean and odorless, and the conditions such as temperature and humidity are appropriate, and the sniffing method is unified. The sniffing method includes the sniffing time and the sniffing distance to ensure the consistency of the operation of each sniffer.

[0019] Preferably, in step S5, if the S-curve of the mixed solution moves to the left, that is, it can be smelled at a lower concentration, it indicates the existence of synergy; if it moves to the right, it may show a masking effect.

[0020] Preferably, the analysis of aroma synergy using the microscale thermophoresis method includes the following steps:

[0021] (1) Prepare the test solution of OR52D1 olfactory receptor protein and the test solutions of various aroma substances with gradient concentrations respectively;

[0022] (2) Mix the test solution of OR52D1 olfactory receptor protein and the test solutions of various aroma substances evenly to obtain the test solutions for the microscale thermophoresis instrument for each group;

[0023] (3) Use the microscale thermophoresis instrument to pre-test the test solution of OR52D1 olfactory receptor protein in step (1), and set the measurement parameters according to the pre-test results;

[0024] (4) According to the measurement parameters set in step (3), take a capillary to suck the test solutions for the microscale thermophoresis instrument for each group in step (2), and place them in the instrument to start the determination;

[0025] (5) Use the built-in software NT Analysis Software of the microscale thermophoresis instrument to calculate the dissociation constant Kd value, conduct data processing and analysis, and obtain the binding ability of the aroma substance and OR52D1 olfactory receptor protein and the thermodynamic parameters.

[0026] Preferably, in step (1), the test solution of OR52D1 olfactory receptor protein is obtained by diluting the test stock solution of OR52D1 olfactory receptor protein with a buffer solution, and the concentration of the test solution of OR52D1 olfactory receptor protein is 30 - 50 nM.

[0027] Preferably, in step (1), the test solutions of the aroma substances with gradient concentrations are obtained by diluting the test stock solution of the aroma substances with different degrees using a buffer solution, and the concentration of the test stock solution of the aroma substances is 0.5 - 1.5 mM.

[0028] Preferably, the buffer solution is 1X PBS, 0.05% BRIJ buffer solution; the pH of the 1X PBS is 7.4.

[0029] Preferably, the specific steps of step (1) are as follows:

[0030] Add the test stock solution of OR52D1 olfactory receptor protein to a certain amount of buffer solution to prepare a 40 nM test solution of OR52D1 olfactory receptor protein; Take 16 PCR tubes, number them 1 - 16, add 10 μL of buffer solution to tubes 2 - 16 respectively, then take 10 μL of the 1 mM test stock solution of the aroma substances and place it in PCR tubes 1 and 2. Take 10 μL from tube 2 and transfer it to tube 3, mix evenly and then take 10 μL and transfer it to tube 4; Dilute in this geometric progression until tube 16; Then take 10 μL of the prepared 40 nM concentration test solution of OR52D1 olfactory receptor protein and add it to tubes 1 - 16 respectively, and mix evenly to obtain the test solutions for the microscale thermophoresis instrument.

[0031] Preferably, the molecular dynamics method is used to analyze the aroma synergy, including the following steps:

[0032] A. Defining the research objective and model construction: First, the research objective of molecular dynamics simulation needs to be defined, including exploring the structural stability, reaction mechanism or kinetic properties of molecules; subsequently, an initial three-dimensional structure model of the target molecule is constructed or obtained according to the research objective;

[0033] B. Selecting the force field and simulation conditions: Selecting an appropriate force field model determines the calculation method of intermolecular interactions; at the same time, the environmental conditions of the simulation need to be set, including temperature, pressure, simulation time, and the positions and velocities of molecules are initialized to ensure that the simulation starts from a reasonable physical state;

[0034] C. Performing the simulation: Using molecular dynamics software, according to the set force field and conditions, the Newton's equations of motion are solved by numerical methods, and the positions and velocities of molecules are continuously updated to simulate the dynamic behavior of molecules;

[0035] D. Data collection: During the simulation process, the state data of the system are regularly recorded and saved, including the trajectories of molecules, energy changes, and interaction forces.

[0036] Molecular dynamics is a molecular simulation method based on classical mechanics. Different from molecular mechanics, molecular dynamics solves the states, behaviors and processes of molecules that change over time. This method simulates the process of molecular motion. It solves the Newton's equations of motion for each atom and the positions and velocities of each atom according to the instantaneous motion state of the molecules, and calculates various properties from a motion trajectory.

[0037] Preferably, in step A, the constructed or obtained molecular model should be verified to ensure its structural rationality and accuracy; for complex molecular systems, a combination of various experimental and theoretical data is required for comprehensive verification.

[0038] Preferably, Desmond in the Schrodinger software package is used for molecular dynamics analysis, specifically including the following steps:

[0039] Step 1) Click Task → Protein Preparation and Refinement → Protein Preparation Wizard to open the protein preparation window; enter 6LU7 in the PDB text box and click Import to import the protein structure into the working interface;

[0040] Step 2) Preprocess the complex structure, including correcting bond information, adding hydrogen atoms, capping with NME and ACE, deleting water molecules, and performing optimization, etc.;

[0041] Step 3) Click "Delete labels" to delete the labels and close the protein preprocessing window;

[0042] Step 4) Click "Tasks" → "Desmond" → "System Builder" to open the system building panel and display the "SystemBuilder" tab; ensure that the "Predefined" option under "Solvent model" is selected as "SPC";

[0043] Step 5) Select the box shape, "Orthorhomic", from the drop-down menu of the "Box shape" option; check the "Buffer" option after "Box size calculation method"; set the distance and angles in "Distance" and "Angles"; view the solvent box on the working page by checking "Snow boundary box"; click "Minimize Volume" to minimize the volume of the box and reposition the solvent;

[0044] Step 6) Switch to the "lons" tab, ensure that "Neutralize by adding" is selected; click "Recalculate"; check "Add salt" to simulate the in vivo environment; enter 0.15 in the "Salt concentration" text box; change the task name to "1bel-setup"; click "Run" to run the task;

[0045] Step 13) Click "Tasks" → "Desmond" → "Molecular Dynamics" to open the "Molecular Dynamics" window; select "Load from Workpace" from the drop-down menu of the "Model system" option and then click "Load";

[0046] Step 16) Set the simulation duration and recording interval in the "Simulation" option; check "Relax model system before simulation";

[0047] Step 19) Click "Advanced Options" to open the advanced options; switch to the "Output" tab and set "interval" to 1200 ps; click "OK" to close the advanced settings dialog box; click the task settings button and select "CPU" or "GPU" in "Processing unit".

[0048] Preferably, in step 4), a water model is mostly used for biomacromolecules, such as TIP3P, SPC, TI4PEW, etc. For non-accessible models of organic solvents, there are dimethyl sulfoxide, methanol, octanol, etc. If there is no required solvent model, it can be customized through Custom.

[0049] Preferably, in step 5), set the distance and angle in Distance and Angles and select the default values.

[0050] Preferably, in step 6), 0.15 indicates that the salt concentration is 0.15 mol / L, and sodium chloride is added by default.

[0051] Preferably, in step 8), under different simulation systems, different ensembles can be selected. Select the NPT ensemble for simulation, and set the temperature and pressure to the default values.

[0052] The present invention also provides an application of the method for studying aroma synergy based on S-curve, microscale thermophoresis, and molecular dynamics in the field of food flavor.

[0053] Preferably, by studying the synergy between different aroma substances, food flavor formulations that meet the tastes of consumers can be designed more accurately, improving the aroma quality and taste experience of foods.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] (1) The method for studying aroma synergy based on S-curve, microscale thermophoresis, and molecular dynamics of the present invention has high sensitivity, is easy to operate, and is fast and efficient in detection, making up for the deficiency of the lack of objective verification of the synergy of aroma substances.

[0056] (2) In the present invention, through the study of aroma synergy by the S-curve method, the S-type concentration-response curve of aroma substances is measured by the gradient dilution method, and the sensory synergy threshold can be objectively determined, breaking through the subjective deviation of the traditional threshold method that relies on artificial sensory evaluation and providing a repeatable quantitative benchmark for the determination of aroma synergy.

[0057] (3) In the present invention, through the study of aroma synergy by microscale thermophoresis, it makes up for the deficiency of the research technology of the interaction between olfactory receptor proteins and aroma substances. This method is simple and fast, the results are intuitive and reliable, and it has wide applicability, providing method support for objectively verifying the interaction between olfactory receptor proteins and aroma substances.

[0058] (4) In this invention, through molecular dynamics research on aroma synergy, the problem of the lack of research methods for the interaction of aroma substances is solved from a molecular perspective. The dynamic binding process of the aroma molecule-olfactory receptor complex is traced, and a reasonable explanation for the interaction between different aroma substances is given through molecular simulation research. This lays a foundation for further research on the interaction between aroma substances with different fragrance notes at the molecular level, and has good application prospects. Description of the Drawings

[0059] Figure 1-2 They are the S-curve results of citral and β-ionone respectively;

[0060] Figure 3 It is the microscale thermophoresis result diagram of OR52D1 and citral;

[0061] Figure 4 It is the microscale thermophoresis result diagram of OR52D1 and β-ionone;

[0062] Figure 5 It is the microscale thermophoresis result diagram of the mixture of OR52D1 and β-ionone and citral;

[0063] Figure 6 It is the microscale thermophoresis result diagram of the mixture of OR52D1 and citral and β-ionone;

[0064] Figure 7 It is the RMSD diagram of OR52D1 and citral;

[0065] Figure 8 It is the RMSD diagram of OR52D1 and β-ionone;

[0066] Figure 9 It is the RMSD diagram of the mixture of OR52D1 and β-ionone and citral;

[0067] Figure 10 It is the RMSD diagram of the mixture of OR52D1 and citral and β-ionone;

[0068] Figure 11 It is the RMSF diagram of OR52D1 and citral;

[0069] Figure 12 It is the RMSF diagram of OR52D1 and β-ionone;

[0070] Figure 13 It is the RMSF diagram of the mixture of OR52D1 and β-ionone and citral;

[0071] Figure 14 It is the RMSF diagram of the mixture of OR52D1 and citral and β-ionone;

[0072] Figure 15 Molecular dynamics ligand-protein contact map of OR52D1 and citral;

[0073] Figure 16 Molecular dynamics ligand-protein contact map of OR52D1 and β-ionone;

[0074] Figure 17 Molecular dynamics ligand-protein contact map of the mixture of OR52D1 and β-ionone and citral;

[0075] Figure 18 Molecular dynamics ligand-protein contact map of the mixture of OR52D1 and citral and β-ionone. Detailed implementation mode

[0076] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and gives detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0077] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present invention are conventional reagents, methods, instruments and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0078] Example 1: S-curve method

[0079] (1) Prepare solutions: Prepare a series of solutions with concentration gradients (from high to low, such as 10 -1 , 10 -2 , …, 10 -10 mol / L) of citral and β-ionone solutions, as well as their mixed solutions. Ensure that the volume of each group of solutions is the same to control variables.

[0080] (2) Olfactory experiment: Let trained olfactory discriminators smell each group of solutions one by one, and record the bottle numbers that cannot smell the odor. This step is to determine the actual olfactory thresholds of each compound and its mixture.

[0081] (3) Calculate the thresholds: According to the experimental data, calculate the theoretical thresholds (based on known data or assumptions) and experimental thresholds (the lowest concentration that cannot be smelled in the actual olfactory experiment) of citral and β-ionone.

[0082] (4) Draw the S-curve: Use Origin software to draw an S-shaped curve with the concentration as the abscissa and the olfactory intensity (which can be simplified to a binary variable of "can smell" and "cannot smell") as the ordinate. For the mixed solution, perform the same operation and observe the difference in the curve shape from that of the single compound curve.

[0083] (5) Analyze the synergistic effect: By comparing the S-curve of the mixed solution with that of the individual solutions, analyze the synergistic effect between the two in olfactory perception. If the S-curve of the mixed solution shifts to the left (i.e., it can be smelled at a lower concentration), it indicates the existence of a synergistic effect; if it shifts to the right, it may exhibit a masking effect.

[0084] Example 2: Analyze the aroma synergistic effect of citral and β-ionone by microscale thermophoresis

[0085] (1) Use a microscale thermophoretic instrument to pre-test the test solution of the OR52D1 olfactory receptor protein, and set the measurement parameters according to the pre-test results.

[0086] (2) Add the OR52D1 olfactory receptor solution to a certain amount of buffer solution to prepare a 40 nM receptor solution.

[0087] (3) Take 16 PCR tubes, numbered 1 - 16. Add 10 μL of buffer solution to tubes 2 - 16 respectively. Then add 10 μL of the 1 mM aroma substance test stock solution to tubes 1 and 2. Take 10 μL from tube 2 and transfer it to tube 3. After mixing evenly, take 10 μL and transfer it to tube 4; after mixing the solution in tube 4 evenly, take 10 μL and transfer it to tube 5; dilute in this geometric progression until tube 16; then take 10 μL of the prepared 40 nM concentration OR52D1 olfactory receptor test solution and add it to tubes 1 - 16 respectively, and mix evenly to obtain the microscale thermophoresis instrument test solution.

[0088] (4) Take 16 capillaries to aspirate the test samples of the aroma substances numbered 1 - 16 and the OR52D1 olfactory receptor protein, and place the samples in the instrument to measure the interaction between the aroma substances and the OR52D1 olfactory receptor protein.

[0089] (5) Use the built-in software NT Analysis Software of the microscale thermophoretic instrument to calculate the dissociation constant Kd value for data processing and analysis, and obtain the binding ability and thermodynamic parameters of the aroma substances and the OR52D1 olfactory receptor protein.

[0090] Table 1 shows the data analysis results of Example 2.

[0091] Table 1 shows the Kd values of the microscale thermophoresis experiments of OR52D1 with citral and β-ionone

[0092]

[0093] Example 3: Analyze the aroma synergistic effect of citral and β-ionone by molecular dynamics method

[0094] Molecular dynamics analysis was performed using Desmond in the Schrodinger software package, which specifically included the following steps:

[0095] Step 1) Click Task → Protein Preparation and Refinement → Protein Preparation Wizard to open the protein preparation window; enter 6LU7 in the PDB text box and click Import to import the protein structure into the working interface;

[0096] Step 2) Preprocess the complex structure, including correcting bond information, adding hydrogen atoms, capping with NME and ACE, deleting water molecules, and performing optimization;

[0097] Step 3) Click Delete labels to delete the labels and close the protein pretreatment window;

[0098] Step 4) Click Tasks → Desmond → System Builder to open the system construction panel and display the System Builder tab; ensure that the Predefined option under Solvent model is selected as SPC;

[0099] Step 5) Select the box shape, Orthorhomic, from the drop-down menu of the Box shape option; check the Buffer option after Box size calculation method; set the distance and angle in Distance and Angles; view the solvent box on the working page by checking Snow boundary box; click "Minimize Volume" to minimize the volume of the box and reposition the solvent;

[0100] Step 6) Switch to the lons tab, ensure that Neutralize by adding is selected; click Recalculate; check Add salt to add salt to simulate the in vivo environment; enter 0.15 in the Salt concentration text box; change the task name to 1bel-setup; click Run to run the task;

[0101] Step 7) Click Tasks → Desmond → Molecular Dynamics to open the Molecular Dynamics window; select Load from Workpace from the drop-down menu of the Model system option, and then click Load;

[0102] Step 8) Set the simulation duration and recording interval in the Simulation options; check Relax model system before simulation;

[0103] Step 9) Click Advanced Options to open the advanced options; switch to the Output tab, set the interval to be changed to 1200 ps; click OK to close the advanced settings dialog box; click the task settings button, and select CPU or GPU in the Processing unit.

[0104] Figure 1 The experimental value is slightly higher than the theoretical value (deviation 14.5%), but their R 2 Both are close to 1, indicating that the S-curve model can accurately describe the dose-response relationship of citral, and the experimental data has strong reliability;

[0105] Figure 2 For the S-curve result of β-ionone, it can be seen from the figure that the theoretical value: 0.1300 mg / L (R 2 = 0.999, excellent fitting degree), experimental value: 0.0572 mg / L (R 2 = 0.995, high fitting degree);

[0106] From Figures 3-6 it can be seen that the microscale thermophoresis experimental data is stable and the results are reliable. The Kd ± standard deviation of the OR52D1-citral and OR52D1-β-ionone experiments are 1.06 ± 0.37 and 0.495 ± 0.18 μM respectively. From this, it can be obtained that the affinity between β-ionone and OR52D1 is the strongest, and the affinity between citral and OR52D1 is the second;

[0107] Figure 7 For the RMSD graph of OR52D1 and citral, it can be seen from the figure that OR52D1-citral reaches stability at 38.90 ns, and the RMSD value is OR52D1 stabilizes at

[0108] Figure 8 For the RMSD graph of OR52D1 and β-ionone, OR52D1-β-ionone reaches stability at 50.10 ns, and the RMSD value is OR52D1 stabilizes at

[0109] Figure 9 For the RMSD graph of the mixture of OR52D1 and β-ionone and citral, the RMSD value of (OR52D1-β-ionone)-citral It is shown that when citral and β-ionone coexist, the binding with OR52D1 may be more stable;

[0110] Figure 10 is the RMSD diagram of OR52D1 with a mixture of citral and β-ionone. The RMSD value of (OR52D1-citral)-β-ionone is

[0111] Figure 11 is the RMSF diagram of OR52D1 and citral. It can be seen that the protein of OR52D1-citral shows higher flexibility in residue regions such as 50-57AA, 180-195AA, 220-243AA, 260-275AA, etc.;

[0112] Figure 12 is the RMSF diagram of OR52D1 and β-ionone. The protein of OR52D1-β-ionone shows higher flexibility in residue regions such as 15-30AA, 80-95AA, 180-195AA, 220-243AA, 260-275AA, etc.;

[0113] Figure 13 is the RMSF diagram of OR52D1 with a mixture of β-ionone and citral. The protein of (OR52D1-β-ionone)-citral shows higher flexibility in residue regions such as 15-30AA, 80-95AA, 180-195AA, 260-275AA, etc.;

[0114] Figure 14 is the RMSF diagram of OR52D1 with a mixture of citral and β-ionone. The protein of (OR52D1-citral)-β-ionone shows higher flexibility in residue regions such as 130-145AA, 180-195AA, 260-275AA, etc.;

[0115] Figure 15 is the molecular dynamics ligand-protein contact diagram of OR52D1 and citral. In the OR52D1-citral binary system, molecular dynamics simulation shows that citral mainly forms hydrogen bonds with HIS184 (accounting for 57% of the simulation time), and additionally interacts with HIS184 (10%) and GLY202 (16%) through a water bridge, significantly enhancing the binding stability. In addition, citral also forms hydrophobic interactions with TYR111 and ALA206 to jointly maintain the ligand-protein binding;

[0116] Figure 16It is shown that in the OR52D1-β-ionone binary system, β-ionone forms stable hydrogen bonds with HIS184 (accounting for 98% of the simulation time), indicating that this residue is its key binding site. At the same time, β-ionone also has hydrophobic interactions with multiple residues such as VAL107, MET185, PHE262, LEU203, ALA206, MET210, and TYR111, jointly maintaining ligand-protein binding;

[0117] Figure 17 Figure 4 shows the molecular dynamics ligand-protein contact map of the OR52D1-β-ionone mixture and citral. In the (OR52D1-β-ionone)-citral system, citral forms hydrogen bonds with HIS108 (24%). Although the proportion is lower than that of HIS184 in the binary system, it is still an important site; at the same time, it has hydrophobic interactions with PHE162. This indicates that the presence of β-ionone changes the binding mode of citral, causing significant changes in its interacting residues and binding ratio;

[0118] Figure 18 It is shown that in the (OR52D1-citral)-β-ionone ternary system, the binding mode of β-ionone changes significantly: it forms a water-bridge-mediated hydrogen bond with SER261 (21%), and only PHE262 and TYR111 retain hydrophobic interactions. The newly emerged hydrophobic interaction with TYR181 (11%) and the positive charge interaction with GLU183 (15%) indicate that citral significantly changes the binding characteristics of β-ionone.

[0119] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A method for studying aroma synergy based on S-curve, microthermophoresis, and molecular dynamics, characterized in that: The S-curve method, microthermophoresis method and molecular dynamics method were used to analyze the aroma synergistic effect, which specifically included the following steps: preparing groups of citral, β-ionone solutions with concentration gradients and their mixed solutions, and drawing sensory and S-curves; preparing constant concentrations of OR52D1 olfactory receptors and a single aroma substance, and a mixture of two aroma substances, and placing the sample mixture in a microthermophoresis device for measurement; and conducting molecular dynamics research on the structural files of citral, β-ionone and OR52D1.

2. The method for studying aroma synergy based on S-curve, microthermophoresis and molecular dynamics according to claim 1, characterized in that: The analysis of aroma synergy using the S-curve includes the following steps: S1. Solution preparation: prepare citral and β-ionone solutions with gradient concentrations, as well as mixed solutions of the two, and set up various experimental groups; S2. Sniffing test: The olfactory examiner sniffs the solutions of each experimental group one by one, records the experimental groups that cannot smell the odor, and determines the actual olfactory threshold of citral, β-ionone and their mixture; S3. Calculate thresholds: calculate theoretical and experimental thresholds of citral and β-ionone; S4: Draw an S-curve: Use Origin software to draw an S-shaped curve with concentration as the horizontal axis and olfactory intensity as the vertical axis; S5. Analyze synergistic effect: By comparing the S curve of the mixed solution with the S curve of the single solution, analyze the synergistic effect of the two in olfactory perception.

3. The method for studying aroma synergy based on S curve, microthermophoresis and molecular dynamics according to claim 2, characterized in that: In step S2, the sniffing environment of the sniffing experiment is clean and odorless, and the temperature and humidity conditions are suitable, and the sniffing method is unified, and the sniffing method includes sniffing time and sniffing distance to ensure that each olfactory identifier operates consistently; In step S5, if the S curve of the mixed solution moves to the left, that is, it can be smelled at a lower concentration, it indicates that there is a synergistic effect; If it moves to the right, it may have a masking effect.

4. The method for studying aroma synergy based on S-curve, microthermophoresis and molecular dynamics according to claim 1, characterized in that: The analysis of aroma synergy using microthermophoresis includes the following steps: (1) preparing the OR52D1 olfactory receptor protein test solution and the aroma substance test solutions of various groups with gradient concentrations respectively; (2) mixing the OR52D1 olfactory receptor protein test solution and each group of aroma substance test solutions evenly to obtain each group of micro-thermophoresis test solutions; (3) using a micro-thermophoresis instrument to pre-test the OR52D1 olfactory receptor protein test solution in step (1), and setting measurement parameters according to the pre-test results; (4) According to the measurement parameters set in step (3), take the capillary tube to absorb each group of micro-thermophoresis test liquid in step (2), place it in the instrument and start the measurement; (5) The dissociation constant Kd value was calculated using the built-in software NT Analysis Software of the micro-thermophoresis instrument, and the data was processed and analyzed to obtain the binding ability and thermodynamic parameters of the aroma substance and the OR52D1 olfactory receptor protein.

5. The method for studying aroma synergy based on S curve, microthermophoresis and molecular dynamics according to claim 4, characterized in that: In step (1), the OR52D1 olfactory receptor protein test solution is obtained by diluting the OR52D1 olfactory receptor protein test stock solution with a buffer solution, and the concentration of the OR52D1 olfactory receptor protein test solution is 30-50 nM; In step (1), the aroma substance test solution with gradient concentration is obtained by diluting the aroma substance test stock solution to different degrees with a buffer solution, and the concentration of the aroma substance test stock solution is 0.5-1.5 mM; The buffer solution is 1X PBS, 0.05% BRIJ buffer solution; The pH of the 1X PBS is 7.

4.

6. The method for studying aroma synergy based on S-curve, microthermophoresis and molecular dynamics according to claim 4, characterized in that: The specific steps of step (1) are as follows: The OR52D1 olfactory receptor protein test stock solution was added to a certain amount of buffer solution to prepare a 40nM OR52D1 olfactory receptor protein test solution; 16 PCR tubes were taken, numbered 1-16, and 10μL of buffer solution was added to tubes 2-16 respectively, then 10μL of 1mM aroma substance test stock solution was taken and placed in PCR tubes 1 and 2, 10μL was taken from tube 2 to tube 3, and 10μL was pipetted into tube 4 after mixing evenly; the solution was diluted to tube 16 in the same proportion; then 10μL of the prepared 40nM OR52D1 olfactory receptor protein test solution was taken to tubes 1-16, and mixed evenly to obtain the micro-thermophoresis test solution.

7. The method for studying aroma synergy based on S-curve, microthermophoresis and molecular dynamics according to claim 1, characterized in that: The molecular dynamics method is used to analyze the aroma synergy, including the following steps: A. Clarify the research objectives and model construction: First, it is necessary to clarify the research objectives of molecular dynamics simulation, including exploring the structural stability, reaction mechanism or kinetic properties of the molecule; then, construct or obtain the initial three-dimensional structural model of the target molecule according to the research objectives; B. Select force field and simulation conditions: Selecting a suitable force field model determines the calculation method of molecular interactions. At the same time, it is necessary to set the simulation environment conditions, including temperature, pressure, simulation time, and initialize the position and velocity of the molecules to ensure that the simulation starts from a reasonable physical state. C. Execute simulation: Use molecular dynamics software to solve Newton's equations of motion numerically according to the set force field and conditions, continuously update the position and velocity of molecules, and simulate the dynamic behavior of molecules; D. Data collection: During the simulation process, the system status data is regularly recorded and saved, including the trajectories of molecules, energy changes, and interaction forces.

8. The method for studying aroma synergy based on S-curve, microthermophoresis and molecular dynamics according to claim 7, characterized in that: In step A, the constructed or obtained molecular model should be verified to ensure its structural rationality and accuracy; for complex molecular systems, comprehensive verification combining multiple experimental and theoretical data is required.

9. The method for studying aroma synergy based on S-curve, microthermophoresis and molecular dynamics according to claim 7, characterized in that: Molecular dynamics analysis was performed using Desmond in the Schrodinger software package, which included the following steps: Step 1) Click Task → Protein Preparation and Refinement → Protein Preparation Wizard to open the protein preparation window; enter 6LU7 in the PDB text box and click Import to import the protein structure into the working interface; Step 2) preprocessing the complex structure, including correcting bond information, adding hydrogen atoms, capping with NME and ACE, and deleting water molecules for optimization; Step 3) Click Delete labels to delete the labels and close the protein pretreatment window; Step 4) Click Tasks → Desmond → System Builder to open the System Builder panel and display the System Builder tab. Make sure SPC is selected in the Predefined option under Solvent model. Step 5) Select the box shape, Orthorhomic, from the drop-down menu of the Box shape option; check the Buffer option after the Box size calculation method; set the distance and angle in Distance and Angles; view the solvent box on the work page by checking the Snow boundary box; click "Minimize Volume" to minimize the volume of the box, and the solvent is repositioned; Step 6) Switch to the lons tab and make sure Neutralize by adding is checked; Click Recalculate; check Add salt to add salt to simulate the in vivo environment; enter 0.15 in the Salt concentration text box; change the task name to 1bel-setup; Click Run to run the task; Step 7) Click Tasks→Desmond→Molecular Dynamics to open the Molecular Dynamics window. Select Load from Workspace from the Model system drop-down menu and click Load. Step 8) Set the simulation duration and recording interval in the Simulation option; check Relax model system before simulation; Step 9) Click Advanced Options to open the advanced options; switch to the Output tab and change the interval to 1200ps; click OK to close the Advanced Settings dialog box; click the Task Settings button and select CPU or GPU in Processing unit.

10. Application of the method for studying aroma synergy based on S-curve, microthermophoresis and molecular dynamics as claimed in any one of claims 1 to 9 in the field of food flavor.

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