A method for analyzing the effect of ethanol on the flavor properties of polypeptides

Abstract

The present invention provides a method for analyzing the effects of ethanol on the flavor properties of polypeptides, belonging to the field of food engineering technology. The method comprises three steps: sensory evaluation, molecular docking, and molecular dynamics simulation. The sensory evaluation step first uses quantitative descriptive analysis (QDA) to classify the flavor attributes of polypeptide samples. The flavor thresholds of the polypeptides in ethanol-water systems of varying volume ratios are then determined using flavor dilution analysis (TDA). This method combines sensory analysis with computational biology methods, preventing computer simulation results from being out of sync with actual results while providing a more in-depth explanation of the causes of flavor perception differences. It also reduces experimental errors caused by individual differences in sensory evaluation. The method is simple, uses readily available and inexpensive raw materials, and produces reliable results.

Description

Technical Field

[0001] The invention belongs to the technical field of food engineering, and particularly relates to an analysis method for the influence of ethanol on the flavor characteristics of polypeptides. Background Art

[0002] As the primary component of alcohol, ethanol has a crucial influence on its taste and flavor. Generally, higher ethanol concentrations increase consumers' perception of bitterness and sweetness, while reducing sourness. Furthermore, higher alcohol content increases the human perception of viscosity and density, leading to more pronounced sensations of heat, burning, tingling, and a "velvety" texture coating the mouth.

[0003] During the brewing process, microbial activity breaks down protein molecules into peptides. Due to their high molecular weight, most peptides are not distilled during distillation, remaining in the lees. Consequently, reports on peptides in alcohol have focused on non-distilled alcoholic beverages such as rice wine, beer, and wine. However, a small number of small-molecule peptides have been reported in baijiu (white liquor). This is because small-molecule peptides (molecular weight <1000 Da) are soluble in ethanol-water systems and are azeotropically distilled. Peptides encompass the five basic tastes: sour, sweet, bitter, salty, and umami. Peptides also play a crucial role in producing the characteristic flavors of fermented, pickled, and smoked products, imparting distinctive flavors to foods. However, research on peptides in alcohol has primarily focused on their activity and antioxidant properties, while research on their flavor is limited, warranting further investigation.

[0004] Currently, research on flavor compounds in liquor focuses primarily on the sense of smell, with less research on taste. The identification and study of key flavor components in liquor is a key focus for many researchers, but the limitations of quantitative evaluation methods for the flavor effects of flavor peptides have significantly hampered progress in flavor analysis.

[0005] At present, the research on flavor substances in wine is mostly focused on the screening and identification of bioactive peptides, such as Wu Jihong et al. [1] A peptide was isolated and identified from Gujing Luzhou-flavor liquor and Guojing Sesame-flavor liquor using a direct concentration method. Its amino acid sequence was identified using high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) and synthetic standards. A peptide with the amino acid sequence Pro-His-Pro (PHP) was obtained, which has antihypertensive activity. Jiaying Huo et al. [2]Six peptides were isolated and identified from Bandaojing Luzhou-flavor liquor, Caoyuanwang Qingxiang liquor, and Gujinggong Luzhou-flavor liquor. Their amino acid sequences were determined using HPLC-ESI-QTOF-MS. They are Lys-Tyr (KY), Ala-Cys-Phe (ACF), Asp-Cys-Asn (DCN), Lys-Val-Val-Ala (KVVA), Val-Cys-Trp-Asn (VCWN), and Trp-ILe-Lys-Lys (WIKK). All of these peptides exhibit antioxidant activity. However, little research has examined the effects of ethanol on the flavor properties of these peptides.

[0006] Food flavor analysis methods primarily include manual sensory analysis and instrumental analysis. Manual sensory evaluation places high demands on the taste aptitude of evaluators, and systematic training and assessment require reference to relevant standards. However, finding a sufficient number of qualified evaluators is often difficult, resulting in limited universality and accuracy in the evaluation results. Instrumental analysis lacks analysis of flavor synergy, and assessment of data accuracy is relatively limited.

[0007] As an automated and objective taste evaluation device, the electronic tongue has been widely used to investigate the flavor properties of peptides in foods. However, it has certain limitations when measuring the flavor of peptides in ethanol systems and the effects of ethanol on their flavor properties. Firstly, it requires a strict sample system. Ethanol concentrations of 20% or higher prevent the ability to measure aftertaste and rapidly reduce the sensor's lifespan. Furthermore, different sensors have different pH requirements: umami sensors (AAE) require a pH range of 4-7; sourness sensors (CA0) require a pH range of 2-5; and other sensors require a pH range of 2-8. However, it is well known that the ethanol content in liquor typically ranges from 38% to 65%. Therefore, the electronic tongue is not suitable for simulating measurements in ethanol-water systems with varying gradients in this experiment. Secondly, the potential of the taste sensor film in the electronic tongue is detected based on the phase change of a reference electrode. Therefore, taste sensors primarily rely on electrochemical detection principles. Consequently, the electronic tongue cannot be used to study the effects of ethanol on flavor properties.

[0008] This requires us to develop a new analytical method to study the effect of ethanol on the taste properties of peptides.

[0009] [1] Wu Jihong, Sun Baoguo, Zhao Mouming, et al. Discovery and research of angiotensin-converting enzyme inhibitory peptides in liquor[J]. Journal of Chinese Institute of Food Science and Technology, 2016, 16(9): 14-20.

[0010] [2]Jiaying Huo, Yuezhang Min, Huifeng Li, et al. The protective effects of peptides from Chinese baijiu on AAPH-induced oxidative stress inHepG2 cells via Nrf2 signaling pathway[J]. Food Science and Human Wellness, 2022, 11: 1527-1538. Summary of the Invention

[0011] In order to solve the above problems, the present invention provides a method for analyzing the effect of ethanol on the flavor characteristics of polypeptides.

[0012] In one aspect, the present invention provides a method for analyzing the effect of ethanol on the flavor properties of a polypeptide.

[0013] Specifically, the analysis method comprises the following steps:

[0014] (1) Sensory evaluation: Quantitative descriptive analysis (QDA) was used to classify the taste attributes of the peptide samples, and taste dilution analysis (TDA) was used to determine the taste threshold of the peptides in ethanol-water solution systems with different volume ratios.

[0015] (2) Molecular docking;

[0016] (3) Molecular dynamics simulation.

[0017] More specifically, the types of taste of the sample include sour, salty, umami, bitter and sweet.

[0018] More specifically, in step (1), the calculation method of the abscissa X and the ordinate Y of the CF linear fitting equation is:

[0019]

[0020]

[0021] More specifically, when the Y value of the CF linear fitting equation is 66.67%, the corresponding concentration ρ is the taste threshold of the polypeptide under this system.

[0022] More specifically, the sensory evaluation step is:

[0023] 1) Prepare standard taste compounds;

[0024] 2) Forming a sensory panel;

[0025] 3) Use QDA method to classify the taste of peptides: According to the triple standard deviation method (3σ), calculate the arithmetic mean of the evaluation results of multiple people ( ) and standard deviation (s). Values ​​≥+3s or ≤-3s in the evaluation results were considered abnormal and deleted.

[0026] 4) The threshold of the peptide in different solutions was determined using the TDA method combined with the CF linear fitting equation.

[0027] In some embodiments of the present invention, the arithmetic mean ( ) and standard deviation (s) are calculated as follows:

[0028]

[0029] More specifically, the molecular docking step includes:

[0030] a. Use software to obtain the three-dimensional structure of the peptide;

[0031] b. Screen receptors, process with software, and find active pockets;

[0032] c. Perform molecular docking and analysis.

[0033] More specifically, the molecular docking uses at least one of Discovery Studio, Autodock Vina, UCSF DOCK or DeepDock software.

[0034] In certain specific embodiments of the present invention, the software used in the molecular docking step is Discovery Studio.

[0035] Specifically, the data analysis of the molecular dynamics simulation includes at least one of root mean square deviation (RMSD), root mean square fluctuation (RMSF), the number of hydrogen bonds or the radius of gyration (Rg).

[0036] More specifically, the molecular dynamics simulation uses at least one of PyMOL, Gromacs, LAMMPS, AMBER or NAMD software.

[0037] In certain embodiments of the present invention, molecular dynamics simulations are performed using PyMOL and Gromacs software.

[0038] In another aspect, the present invention provides an application of the analytical method in exploring the effect of ethanol on the flavor properties of polypeptides.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] This method combines sensory analysis with computational biology methods, preventing discrepancies between computer simulations and actual results while providing a more in-depth explanation of the causes of differences in taste perception. Furthermore, it can reduce experimental errors caused by individual differences in sensory evaluation. Furthermore, the method is simple, the instrument is easy to operate, the evaluation results are highly accurate, and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a sensory evaluation taste classification and intensity radar chart of WIKK in Example 1 of the present invention;

[0042] Figure 2 These are the CF linear fitting graphs of WIKK in different ethanol-water systems according to Example 1 of the present invention; A is the CF linear fitting graph of WIKK in water; B is the CF linear fitting graph of WIKK in 38-degree ethanol-water; C is the CF linear fitting graph of WIKK in 46-degree ethanol-water; D is the CF linear fitting graph of WIKK in 54-degree ethanol-water; and E is the CF linear fitting graph of WIKK in 62-degree ethanol-water.

[0043] Figure 3 is a Ramachandran plot of the homology modeling model of the bitter taste receptor hTAS2R47 related to Example 1 of the present invention;

[0044] Figure 4 2D diagram of the interaction force and action site between WIKK and bitter taste receptor hTAS2R47;

[0045] Figure 5 3D diagram of the interaction force and action site between WIKK and bitter taste receptor hTAS2R47;

[0046] Figure 6 The root mean square deviation (RMSD) plot of the molecular dynamics simulation of WIKK and the bitter taste receptor hTAS2R47;

[0047] Figure 7 The root mean square fluctuation (RMSF) plot of the molecular dynamics simulation of WIKK and the bitter taste receptor hTAS2R47;

[0048] Figure 8 The figure shows the number of hydrogen bonds between WIKK and the bitter taste receptor hTAS2R47 from molecular dynamics simulation.

[0049] Figure 9 The radius of gyration (Rg) diagram of the molecular dynamics simulation of WIKK and the bitter taste receptor hTAS2R47.

[0050] Figure 10This is a radar chart showing the taste classification and intensity scores of WIKK using an electronic tongue. (The taste score of ultrapure water is normalized to 0.) DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0052] Example 1: A method for analyzing the effect of ethanol on the flavor properties of polypeptides

[0053] Experimental reagents:

[0054] Peptide standards (purity ≥98%), food grade, were synthesized by Shanghai Kepeptide Biotechnology Co., Ltd.; anhydrous citric acid: food grade, purchased from Lotus Health Industry Group Co., Ltd.; sucrose: food grade, purchased from Nanjing Ganzhiyuan Co., Ltd.; quinine: food grade, purchased from Shaanxi Haiyis Biotechnology Co., Ltd.; sodium chloride: food grade, purchased from China National Salt Shanghai Salt Co., Ltd.; monosodium glutamate: food grade, purchased from Lotus Health Industry Group Co., Ltd.; anhydrous ethanol: food grade, purchased from Henan Hanyong Alcohol Co., Ltd.

[0055] Experimental Materials:

[0056] The tasting cup was purchased from Xiamen Yudefeng Plastic Co., Ltd.; the 2 mL disposable plastic straw; the pipette gun; the vortex mixer; and the alcohol concentration meter were purchased from Guangzhou Suwei Electronic Technology Co., Ltd.

[0057] 1. Sensory evaluation of peptide WIKK

[0058] 1.1. Preparation of standard taste compounds

[0059] Aqueous solutions of standard taste compounds included: sour (citric acid solution, concentration 8.00 g / L (9 points), 4.50 g / L (5 points), 1.00 g / L (1 point)), sweet (sucrose solution, concentration 6.00 g / L (9 points), 3.75 g / L (5 points), 1.50 g / L (1 point)), bitter (quinine solution, concentration 1.40 × 10 -2 g / L(9 points), 7.70×10 -3 g / L (5 points), 1.40×10 -3g / L (1 point)), saltiness (sodium chloride solution, concentration 10.00g / L (9 points), 5.50g / L (5 points), 1.00g / L (1 point)), and umami (monosodium glutamate solution, concentration 20.00g / L (9 points), 10.00g / L (5 points), 0.20g / L (1 point)). All reagents are food grade and are scored on a 0-10 scale, with 0 representing no taste. Higher concentrations indicate a more pronounced flavor, leading to higher scores.

[0060] 1.2. Establish a sensory evaluation team

[0061] A sensory evaluation team was formed: the team members were from Beijing Technology and Business University, including 7 males and 8 females, a total of 15 people, all of whom were healthy and had no history of smoking or other behaviors that impaired taste in the past three months.

[0062] Training: Prepare aqueous solutions of the following standard taste compounds and conduct sensory training on the five basic tastes (sour, sweet, bitter, salty, and umami) for the panelists, referring to GB / T 6291.1-2012. Select 10 participants who have demonstrated proficiency in the training to serve as the final sensory evaluation panelists.

[0063] Sour: citric acid solution (1.00 g / L), sweet: sucrose solution (1.50 g / L), bitter: quinine solution (1.40 × 10 -3 g / L), salty: sodium chloride solution (1.00g / L), umami: monosodium glutamate solution (0.20g / L).

[0064] 1.3. Taste classification and intensity scoring of the small molecule peptide WIKK using QDA

[0065] Prepare a 1mg / mL WIKK aqueous solution as the sample solution to be evaluated. Place 2-3mL of the sample solution in your mouth for 10 seconds, spit it out, and rinse your mouth with purified water. Immediately after rinsing, taste the reference solution and rate the flavor and flavor intensity of the peptide solution.

[0066] Evaluation result abnormal value judgment: According to the triple standard deviation method (3σ), the arithmetic mean of the evaluation results of multiple people is calculated ( ) and standard deviation (s). Values ​​≥+3s or ≤-3s in the evaluation results are considered outliers and are deleted. The formula is as follows:

[0067]

[0068] 2. Determination of the taste threshold of WIKK in different ratios of ethanol-water systems

[0069] 2.1. Determination of the threshold value of WIKK aqueous solution using TDA

[0070] Samples were evaluated using taste dilution analysis (TDA). The synthesized peptides were dissolved in ultrapure water (1 mg / mL) and serially diluted 1:1 (v / v) with ultrapure water, vortexing for 30 seconds. Each sample was then placed into a tasting cup and randomly numbered with a three-digit number for sensory evaluation. Panelists were asked to select a cup with a different taste from each sample group (consisting of one sample containing the peptide and two blank controls without the peptide), record the cup number, and complete a questionnaire.

[0071] 2.2. Determination of the threshold value of WIKK in different ethanol aqueous solutions using TDA

[0072] Samples were evaluated using the taste dilution analysis (TDA) method. Aqueous ethanol solutions with volume fractions of 38%, 46%, 54%, and 62% were prepared and calibrated to 38°, 46°, 54°, and 62°, respectively, using an alcoholometer. The peptide (8 mg / mL) was dissolved in the aforementioned aqueous ethanol solutions and diluted 1:1 (v / v) with ultrapure water, vortexing for 30 seconds. The solutions were then placed in tasting cups, randomly numbered with three digits, and subjected to sensory evaluation. Panelists were asked to select a cup with a different taste from each sample group (consisting of one sample containing the peptide and two blank control samples without the peptide), record the tasting cup number, and complete a questionnaire.

[0073] 2.3 Constructing the CF linear fitting equation

[0074] The X-axis value of the CF linear fitting equation is the logarithmic function value of the peptide concentration ρ with a base of 10. The calculation formula is:

[0075]

[0076] The sensory panel evaluation results were counted, with all results recorded as N (total), of which the correct results were N (correct). The ordinate Y value of the CF linear fitting equation represents the percentage P of sensory evaluators who can correctly identify the taste of the peptide. The calculation formula is:

[0077]

[0078] When Y=66.67%, the corresponding concentration value ρ is the taste threshold of the peptide under this system.

[0079] 3. Molecular Docking

[0080] The researchers screened bitter taste receptors using the UniProt (https: / / www.uniprot.org / ) database. Currently, 25 bitter taste receptors (TAS2Rs) have been discovered in human cells.

[0081] Discovery Studio (DS) 2019 software was used to perform molecular docking between WIKK and the taste receptor. First, the 2D structure of WIKK was drawn using ChemDraw 20.0 software. The 2D structure was further processed using the Prepare Ligands function in Discovery Studio to obtain the 3D structure of the peptide. Next, the taste receptor was imported into the PDB format. The receptor was processed for dehydration, ligand removal, and hydrogenation, and the active pocket was identified using FromReceptor Cavities in the software. Finally, molecular docking was performed using Dock Ligands (LibDock), and the docking results were analyzed.

[0082] 4. Molecular dynamics simulation

[0083] Molecular docking can produce false positive results, which greatly limits its application in flavor peptide screening. Analysis of peptide-receptor binding stability through molecular dynamics simulation can effectively reduce false positive results.

[0084] 4.1. Exploring and verifying the flavor properties of WIKK in water using molecular dynamics simulation

[0085] First, the taste receptor-WIKK ligand complex obtained by molecular docking was pre-processed, and the receptor and ligand were separated using PyMOL software and saved in pdb format and mol2 format respectively.

[0086] Afterwards, the mol2 format file of the small molecule peptide was processed using a virtual machine, and .itp format and .gro format files were obtained.

[0087] Finally, dynamic simulations were performed using Gromacs software under constant temperature, pressure, and periodic boundary conditions. The simulation system used the AMBER99SB-ILDN force field and the TIP3P water model. A topology file was created and a cubic box was constructed. In order to maintain charge balance in the simulation system, NaCl was added to the simulation box. + and Cl -Ions maintain charge balance in the simulation system. Before the dynamics simulation, the simulation system was minimized to eliminate close contacts between atoms. Energy minimization was achieved using the steepest descent method (a fast but non-convergent method) with 50,000 steps. After energy minimization, equilibrium simulations were performed for 100 ps using NVT temperature control (300 K, 2 fs) and NPT pressure control (0.1 MPa, 2 fs). Finally, a 50,000 ps molecular dynamics simulation of the taste receptor-WIKK ligand complex was performed (300 K, 2 fs; 0.1 MPa, 2 fs).

[0088] 4.2. Molecular dynamics simulations to investigate the effect of ethanol on the flavor properties of WIKK

[0089] First, the taste receptor-WIKK peptide ligand complex obtained by molecular docking was pre-processed, and the receptor and ligand were separated using PyMOL software and saved in pdb format and mol2 format respectively.

[0090] Afterwards, the mol2 format file of the small molecule peptide was processed using a virtual machine, and .itp format and .gro format files were obtained.

[0091] Finally, dynamic simulations were performed using Gromacs software under constant temperature, pressure, and periodic boundary conditions. The simulation system used the AMBER99SB-ILDN force field and the TIP3P water model. A topology file was created, a cubic box was constructed, and a certain number of ethanol molecules were added to the cubic box. In order to maintain the charge balance of the simulation system, NaCl was added to the simulation box through Na + and Cl - Ions maintain charge balance in the simulation system. Before the dynamics simulation, the simulation system was energy minimized to eliminate close contacts between atoms. Energy minimization was achieved using the steepest descent method (a fast but non-convergent method) with 50,000 steps. After energy minimization, equilibrium simulations were performed for 100 ps using NVT temperature control (300 K, 2 fs) and NPT pressure control (0.1 MPa, 2 fs). Finally, a 50,000 ps molecular dynamics simulation of the taste receptor-small molecule peptide ligand complex was performed (300 K, 2 fs; 0.1 MPa, 2 fs).

[0092] 5. Experimental Results

[0093] 5.1. WIKK's Taste Classification and Intensity Scoring Results

[0094] Evaluation result abnormal value judgment: According to the triple standard deviation method (3σ), the arithmetic mean of the evaluation results of multiple people is calculated ( ) and standard deviation (s). Values ​​≥+3s or ≤-3s in the evaluation results are considered outliers and are deleted. The formula is as follows:

[0095]

[0096] The results are shown in Table 1:

[0097] Table 1 Taste scoring results of small molecule peptide WIKK

[0098]

[0099] WIKK's taste radar chart, such as Figure 1 As shown in the WIKK flavor radar chart, WIKK has a distinct bitter taste, while the scores for the other flavor attributes are relatively low. Therefore, it can be preliminarily determined that WIKK is a bitter peptide.

[0100] 5.2. WIKK threshold results in different systems

[0101] In the CF linear fitting curve, when Y is 66.67%, the corresponding small molecule peptide concentration ρ is the taste threshold under this condition. Figure 2 A shows that the threshold of WIKK in water is 0.04 mg / mL; Figure 2 B shows that the threshold of WIKK in 38-degree ethanol water is 2.45 mg / mL; Figure 2 C shows that the threshold of WIKK in 46-degree ethanol water is 2.95 mg / mL; Figure 2 D shows that the threshold of WIKK in 54-degree ethanol water is 0.53 mg / mL; Figure 2 E shows that the threshold value of WIKK in 62-degree ethanol water is 0.79 mg / mL.

[0102] Molecular docking results

[0103] 5.3.1. Screening of Bitter Taste Receptors

[0104] As shown in Table 2, the table contains information summary of 25 bitter receptors (hTAS2Rs), the model names corresponding to the bitter receptors (information comes from the Uniprot database and the AlphaFold 2 homology modeling platform), and the scores of WIKK molecular docking with these 25 bitter receptors.

[0105] Table 2 Summary and scoring of 25 bitter taste receptors (hTAS2Rs)

[0106]

[0107] According to the scores, it can be seen that hTAS2R47 has the highest score, and therefore, this receptor is the taste receptor most likely to bind to WIKK.

[0108] 5.3.2. Quality Assessment of Bitterness Model

[0109] The Ramachandran plot shows the theoretically possible conformations of amino acid residues. Its primary purpose is to assess the quality of homology modeling. Therefore, the Ramachandran plot can be used to determine the plausibility of the model P59541 for the hTAS2R47 receptor.

[0110] From the Laplace diagram, we can see that the model P59541 is reasonable. Figure 3 It can be seen that the proportion of amino acid residues in the model that fall within the allowed region and the maximum allowed region accounts for more than 90% of the entire protein. Therefore, the conformation of the model conforms to the rules of stereochemistry.

[0111] 5.3.3. Interaction force and binding site of WIKK with the bitter taste receptor hTAS2R47

[0112] Figure 4 and Figure 5 The two-dimensional and three-dimensional images of WIKK binding to the bitter taste receptor hTAS2R47 show that the interactions between WIKK and the bitter taste receptor hTAS2R47 mainly include hydrogen bonding, π-π conjugation, and π-alkyl conjugation. The main interaction sites are: ASN251, ARG254, TRP88, HIS65, TYR85, GLU151, ILE147, ILE82, and ARG81.

[0113] Molecular dynamics simulation experiments

[0114] RMSD determination results

[0115] The root mean square deviation (RMSD) indicates the distance between the same atoms in different structures. The RMSD of a protein can reveal the positional changes between the conformation of the protein during the simulation and the initial conformation. The larger the RMSD value, the greater the degree to which the target molecule deviates from the reference molecule. The changing trend of the RMSD of the protein and the ligand is also an important indicator for judging whether the simulation has reached stability. For this study, the RMSD of hTAS2R47 can not only characterize the stability of its protein structure, but also further characterize whether the ligand has a certain depolymerization effect on the protein. Figure 6 The results show that in different ethanol-water systems, the binding stability of the bitter taste receptor hTAS2R47 to WIKK follows the order of 62°≈54° > 46° > 38°. Therefore, within a certain range, the higher the ethanol concentration, the more persistent and stable the human body's perception of WIKK bitterness.

[0116] 5.4.2. RMSF measurement results

[0117] Individual amino acid residues in a protein-ligand complex play a crucial role in the stability of the complex. The fluctuation of amino acid residues can be judged by the root mean square fluctuation (RMSF) parameter, which explains the average deviation of each amino acid from the reference position over time. Amino acids or amino acid groups with high RSMF values ​​indicate that the complex has greater flexibility, while low RMSF values ​​indicate less flexibility. In addition, frequent fluctuations indicate poor stability. Figure 7 It can be seen that the order of flexibility of the complexes judged by amino acids or amino acid groups in the ethanol-water system is: 62°≈54°≈46°≈38°.

[0118] During the simulation, the ligand will form a certain number of hydrogen bonds with the protein. The intermolecular hydrogen bond analysis tool provided by GROMACS can be used to extract the distribution of the number of hydrogen bonds between the protein and the ligand over the simulation time. The hbond program uses geometric criteria to determine hydrogen bonds. When the distance between the hydrogen bond donor and acceptor is less than 3.5 angstroms and the angle between the hydrogen-donor and acceptor is less than 30 degrees, it is considered a hydrogen bond. The addition of the -ac parameter can automatically calculate the average lifetime of hydrogen bonds (forward lifetime), which can be used as an indicator to measure the stability of hydrogen bonds. Figure 8 It can be seen that as the ethanol content increases, the number of hydrogen bonds between the bitter taste receptor hTAS2Rs and WIKK also increases.

[0119] 5.4.4. Rg determination results

[0120] The radius of gyration (Rg) can characterize the compactness of protein structure. It can also be used to characterize the change in the looseness of the protein peptide chain during the simulation. During the entire molecular dynamics simulation, the lower and more consistent the fluctuation, the higher the rigidity and compactness of the system, and the more stable the protein structure. Unique conformational changes or folding of proteins may lead to greater changes in Rg. Figure 9 As can be seen, the Rg values ​​of proteins in water, 54°, and 62° ethanol-water systems did not change significantly from the beginning to the end of the stabilization process, with Rg values ​​ranging from 2.3-2.4 (water system) and 2.5-2.7 (54° and 62° ethanol-water systems), respectively. However, the protein in 38° ethanol-water and 46° ethanol-water systems did not bind stably to the ligand. This conclusion is consistent with the RMSD results.

[0121] Comparative Example 1: Using an electronic tongue to detect the effect of ethanol on the taste characteristics of polypeptides

[0122] Six sensors for sourness (CA0), bitterness (C00), astringency (AE1), umami (AAE), saltiness (CT0), and sweetness (GL1) were pretreated in a reference solution (an aqueous solution containing 0.045g of L-tartaric acid and 2.2365g of KCl) for 24 hours before analysis. Sweetness was measured separately from the other tastes. Before testing, the electronic tongue sensor underwent a self-test and calibration. The program was set to a 30-second taste collection time, a 30-second aftertaste collection time, and a 300-second cleaning time. The test was conducted at 25°C. The five basic tastes were measured four times (sweetness was repeated five times as recommended in the workbook), and the final three responses were selected and marked as valid data.

[0123] The experimental results were analyzed using Excel and Origin software. Figure 10 As shown, the sample numbered CCS is ultrapure water, and the taste score of ultrapure water is normalized to 0. Figure 10 As can be seen, when ultrapure water is used as the reference solution, the electronic tongue cannot accurately measure the bitterness of small molecule peptides, but it scores higher for sourness and saltiness. Furthermore, the intensity of other flavors also deviates significantly from the sensory evaluation results. Therefore, traditional electronic tongue flavor measurement methods are not applicable to the embodiments of this patent.

[0124] In addition, due to the limitations mentioned in the background technology, it is impossible to use the electronic tongue to measure the WIKK threshold in 38°, 46°, 54°, and 62° ethanol aqueous solution systems and to subsequently study and analyze the effect of ethanol on the taste characteristics of WIKK.

[0125] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for analyzing the effect of ethanol on the flavor properties of polypeptides, characterized in that: The method comprises the following steps: (1) Sensory evaluation: The sensory evaluation step includes: 1) Prepare standard taste compound solutions; 2) Establish a sensory evaluation team; 3) Use quantitative descriptive analysis to classify and score the taste intensity of peptide samples: prepare peptide aqueous solution as the sample solution to be evaluated, and calculate the arithmetic mean of the taste evaluation results of multiple people according to the triple standard deviation method 3σ Compared with the standard deviation s, values ​​≥+3s or ≤-3s in the evaluation results were considered as outliers and deleted; (2) The taste threshold of the peptide in ethanol-water systems with different volume concentrations was determined by using the flavor dilution analysis method combined with the CF linear fitting equation; the abscissa X value of the CF linear fitting equation is the logarithmic function value of the peptide concentration ρ with a base of 10, and the calculation formula is: Among them, the sensory panel evaluation results are counted, and the total number of results is recorded as N (total), of which the correct result is N (correct). The vertical coordinate Y value of the CF linear fitting equation represents the percentage P of sensory evaluators who can correctly identify the taste of the peptide. The calculation formula is: When Y=66.67%, the corresponding concentration value ρ is the taste threshold of the peptide under this system; (3) Molecular docking: Use software to perform molecular docking between peptides and taste receptors; (4) Molecular dynamics simulation: Molecular dynamics simulation is used to analyze the binding stability between peptides and receptors, and molecular dynamics simulation is used to explore the effect of ethanol on the taste properties of peptides; The volume concentration of the ethanol aqueous solution is 0-62%; The types of taste of the sample include sour, salty, umami, bitter and sweet.

2. The analysis method according to claim 1, characterized in that In step (3), the molecular docking step includes: a. Use software to obtain the three-dimensional structure of the peptide; b. Screen receptors, process with software, and find active pockets; c. Use software to perform molecular docking and analysis.

3. The analysis method according to claim 2, characterized in that The molecular docking uses at least one of DiscoveryStudio, Autodock Vina, UCSF DOCK or DeepDock software.

4. The analysis method according to claim 1, characterized in that In step (4), the molecular dynamics simulation is performed using at least one of PyMOL, Gromacs, LAMMPS, AMBER or NAMD software for data processing and analysis; The data processing and analysis includes at least one of root mean square deviation, root mean square fluctuation, number of hydrogen bonds or radius of gyration.

5. Application of the analytical method according to claim 1 in investigating the effect of ethanol on the flavor properties of polypeptides.

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