Preparation method of umami peptide from procambarus clarkii and composite freshness enhancement application

Abstract

The invention relates to preparation and application of umami peptide from procambarus clarkii, and belongs to the technical field of food biology. A preparation method of umami peptides from procambarus clarkia is characterized by comprising the following steps: firstly, separating and purifying polypeptides in a shrimp meat taste extract, predicting peptide activity through a database, carrying out molecular docking and molecular dynamics simulation method characterization, and finally identifying two potential umami peptides, namely LAEPLTFN and YGGEFPARPDN; the umami peptide is artificially synthesized, the fresh property of the umami peptide is verified through sensory evaluation and an electronic tongue method, and the synergistic effect of the umami peptide and other fresh substances is further evaluated. The umami peptide obtained by the method has a composite freshness increasing effect, enriches a fresh water source umami peptide library, and provides a new umami peptide raw material for development of composite freshness increasing seasonings.

Description

Technical Field

[0001] The present invention relates to the preparation and application of umami peptides derived from Procambarus clarkii, and belongs to the field of food biotechnology. Technical Background

[0002] Umami peptides are a class of oligopeptides obtained by enzymatic hydrolysis of animal or plant proteins. They are easily digested and absorbed in the human body and can quickly provide essential amino acids and other nutrients for the human body. Umami peptides are rich in sources and can be isolated from beef, peanuts, mushrooms, clams, soy sauce, etc. Among them, animal-derived umami peptides have good palatability, flavor and biological activities. The preparation method and polypeptide spatial structure of umami peptides will both affect their umami effect.

[0003] Currently, the preparation processes of food-derived umami peptides mainly include separation and purification, identification and synthesis evaluation. Traditional screening and identification methods are mainly based on structure-activity relationships or the frequency of umami fragments, which are prone to difficulties in separation and purification, long time consumption, and the identified peptide segments having no umami taste, etc. Therefore, the combined technology of machine learning, molecular docking, and sensory evaluation technology is widely used because it can screen umami peptides quickly and in a high-throughput manner. It studies the interaction mechanism between umami substances and umami receptors, calculates the interaction strength, and thus conducts molecular dynamics simulation to screen umami peptides. At the same time, proteomics, because it can qualitatively and quantitatively analyze peptides in samples comprehensively and identify and evaluate a large number of potential umami peptides in a short time, is usually applied in combination with technologies such as machine learning.

[0004] Procambarus clarkii, also known as crayfish, has excellent taste, mainly showing umami and strong flavor, and has a reasonable amino acid composition, rich in umami amino acids and unsaturated fatty acids, and has good flavor and nutritional value. It is a major economic shrimp species in China. Currently, there are no reports on umami peptides derived from Procambarus clarkii. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and compound umami enhancement application of umami peptides derived from Procambarus clarkii. The umami peptides obtained by this method have a compound umami enhancement effect, enrich the umami peptide library of fresh water sources, and provide new umami peptide raw materials for the development of compound umami seasonings.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is a preparation method of umami peptides derived from Procambarus clarkii, which is characterized by including the following steps: First, separate and purify the polypeptides in the shrimp meat flavor extract, predict the peptide activity through a database, and characterize them by methods such as molecular docking and molecular dynamics simulation. Finally, two potential umami peptides (amino acid sequences are LAEPLTFN and YGGEFPARPDN respectively) are identified; synthesize the above umami peptides, verify their umami characteristics by methods such as sensory evaluation and electronic tongue, and further evaluate the synergistic effect with other umami substances.

[0007] The specific steps are as follows:

[0008] 1. Sample preparation: After the Procambarus clarkii is euthanized, it is washed and steamed in boiling water until the central temperature of the shrimp reaches 90 °C, and then cooled to room temperature; the shrimp tails are mixed with ultrapure water at a ratio of 1 g:4 mL, homogenized at 15000 r / min for 2 min, then hot water extracted for 30 min, and after cooling and centrifuging (10000 r / min, 4 °C, 2 min), the supernatant is the shrimp meat flavor extract (Shrimp meatextract, SE), which is stored in a -80 °C refrigerator;

[0009] 2. Extraction of umami peptides:

[0010] 2.1 Ultrafiltration: Separate the proteins below 3 kDa in the shrimp meat flavor extract (SE). The shrimp meat flavor extract (SE) is ultrafiltered through a nanofiltration membrane separation device equipped with a 3 kDa ultrafiltration membrane. This process needs to be carried out at 4 °C and 20 Bar pressure, and all the filtrates (UF) are collected and freeze-dried to obtain a freeze-dried sample;

[0011] 2.2 Gel filtration chromatography analysis: The freeze-dried sample is prepared into a polypeptide solution of 10 mg / mL and separated through a Sephadex G-15 gel column. During this period, the ultraviolet absorption intensity of the eluent needs to be monitored at 220 nm; the eluent is pure water, the flow rate is 0.75 mL / min, and the sample loading volume is 3 mL; the 4 fractions obtained are named P1, P2, P3, and P4 in the order of the peak elution time, and the test solution is collected and freeze-dried for subsequent analysis and determination to obtain the freeze-dried shrimp meat flavor extract to be tested after gel filtration;

[0012] 2.3 Amino acid analysis in umami peptides: Take 1 mL of the shrimp meat flavor extract to be tested (SE), add 100 μL of the derivatization solution (the derivatization solution is: ethanol:phenylisothiocyanate:water:triethylamine volume ratio = 7:1:1:1), and derivatize for 30 min; then add 0.9 mL of mobile phase B and mix well; Instrument conditions: detection wavelength 254 nm; oven temperature 40 °C; injection volume 10 μL; mobile phase A is: an aqueous acetonitrile solution with a volume concentration of 80%, and mobile phase B is: 0.1 mol / L anhydrous sodium acetate-acetonitrile (97:3, V / V); the elution program is shown in Table 1; the flow rate is 1 mL / min; external standard quantification is carried out using a known concentration of amino acid standard mixed solution;

[0013] Table 1, Mobile phase elution program

[0014] Time / min 1 14 29 30 37 38 45 Mobile phase A / % 0 20 40 100 100 0 0 Mobile phase B / % 100 80 60 0 0 100 100

[0015] 2.4 Taste dilution analysis and electronic tongue characterization: 5 mL of the fractions (P1, P2, P3, P4) separated in step 2.2 were diluted with pure water in equal volumes to obtain test samples, which were then presented to sensory evaluation personnel, and the triangle test method was used for evaluation; if the evaluators considered that there was a difference in the taste between the test sample and pure water, equal volume dilution was carried out again, and this cycle was repeated until the taste of the test sample disappeared, and the dilution factor was the TDA; the electronic tongue was based on a 0.025% MSG solution, the polypeptide fraction concentration was 1 mg / mL, and five independent replicates were performed for each sample;

[0016] 3. Identification of umami peptides: 2 mL of the fractions (P1, P2, P3, and P4) with significantly stronger umami in step 2.2 were fully hydrated, dithiothreitol (DTT) was added to a final concentration of 10 mM, and incubated at 56 °C for 1 h; subsequently, iodoacetamide (IAM) was added to a final concentration of 50 mM, and incubated again in the dark for 40 min; the product was immediately treated through a desalting column, the eluate was collected and freeze-dried for further testing, and the test substance was dissolved in ultrapure water;

[0017] The polypeptide was separated from the above-mentioned test substance by an Easy-nLC 1200 high performance liquid chromatography system; solvent A (0.1% formic acid by volume) and solvent B (80% acetonitrile and 0.1% formic acid by volume) were used as eluents, and a 120-min gradient elution program was used to separate the test substance (sample), and the elution process was as follows: 0.01 - 2 min (3% - 8% solvent B), 2 - 10 min (8% - 12% solvent B), 10 - 80 min (12% - 25% solvent B), 80 - 94 min (25% - 35% solvent B), 94 - 112 min (35% - 60% solvent B), 112 - 117 min (60% - 95% solvent B), and 117 - 120 min (95% solvent B); the fragmentation mode of the first-stage mass spectrometry was high energy collision dissociation (HCD); the resolution, dynamic gain control value (AGC), ion injection time, and error value of the second-stage mass spectrometry were 1.5×10 4 、1×10 5 、50 ms and 100 m / z, the shortest pre-scan time was 8×10 3 ,and the intensity threshold was 1.6×10 5; The obtained polypeptide fingerprint map was identified by Peaks Studio 10.6 (Thermo, Waltham, MA, USA), and the obtained polypeptide fingerprint map was identified by Peaks Studio 10.6 (Thermo, Waltham, MA, USA) to obtain potential umami peptides from Procambarus clarkii (an umami peptide derived from Procambarus clarkii);

[0018] 4. Prediction of Umami Peptide Activity

[0019] 4.1 Molecular Docking

[0020] The amino acid sequences of T1R1 (Uniprot: Q7RTX1) and T1R3 (Uniprot: Q7RTX0) were derived from the NCBI database; sequence alignment was performed in the PDB database using the protein BLAST tool to obtain the human calcium-sensing receptor (PDB ID: 5K5S) for homology modeling of the receptor; Modeler v9.19 was used for the modeling and optimization of the receptor protein, and the rationality of the receptor model was further evaluated by the PROCHECK Verify 3D program and Ramachandran plot provided by SAVES v6.0 serves; for the convenience of running molecular docking, the T1R1 / T1R3 dimer was assembled and optimized by PyMol; AutoDock vina characterized the interaction mode between the umami peptide and T1R1 / T1R3 by the semi-flexible docking method; among them, the three-dimensional conformations of the polypeptide and the receptor were both subjected to energy minimization and converted to the pdbqt format; the following parameters were used to construct the grid box to completely cover the entire protein surface: Other parameters were set to default values; after 100 docking attempts, the complex conformation with the lowest energy was selected for further optimization; that is, it was first assigned the Amber14 force field and processed for 1000 steps using the Steepest Descent Method, and then processed for 5000 steps using the Conjugate Gradient Method. The final result was visualized and analyzed using Discovery Studio 2019;

[0021] 4.2 Molecular dynamics simulation: The umami peptides obtained in step 4.1 with low binding energy to the T1R1 / T1R3 receptor were further characterized by MD; using the Gromacs software package, the three-dimensional conformation of the complex and the free monomer were encapsulated in a TIP (three-site transferable intermolecular potential) water molecule model under the Amber14 force field; and the energy of the simulation system was minimized using the steepest descent method, and then MD simulations were performed for 100 ns under the conditions of constant number of particles, volume and temperature and constant number of particles, pressure and temperature (NPT); in addition, the number of hydrogen bonds formed between the ligand and the receptor was obtained by the VMD (visual molecular dynamics) software package by restricting the distance intercept and the angle intercept values to and 30°, respectively; the identified polypeptide sequences were obtained;

[0022] 4.3 Identification and verification of synthetic peptides: The identified polypeptide sequences (amino acid sequences are LAEPLTFN and YGGEFPARPDN, respectively) obtained in step 4.2 were synthesized into peptides by solid-phase synthesis, and the purity and molecular weight of the synthetic peptides were determined by HPLC and LC-MS, respectively;

[0023] 4.4 Sensory evaluation of synthetic peptides: The experimental group consisted of 20 adults (10 males and 10 females, aged 22-28 years) who had received sensory training according to GB / T16291.1-2012 and had more sensory evaluation experience; the umami threshold of the synthetic peptides was determined by the triangle test method. The experiment was carried out at an environment with a temperature of 25±2 °C and a humidity of 55±5%. The umami peptides synthesized in 4.3 (there were 3 coded samples, 2 of which were the same, and the inspectors needed to screen out the different samples) were all randomly coded with three digits. The initial concentration of the synthetic peptide solution was prepared at 1 mg / ml and gradually diluted step by step at a ratio of 1:1 (v / v) until the group members could not distinguish it from the pure water area;

[0024] 4.5 Electronic tongue characterization of synthetic peptides: The taste threshold was measured using deionized water as the solvent, and the umami polypeptides synthesized in 4.3 were measured; the concentration of the synthetic peptides was set at 0.2 mg / mL, and the concentrations of the MSG standard solutions were 0.025%, 0.05%, 0.1%, 0.2%, 0.3% and 0.4% (w / v), respectively. Each sample was measured five times independently;

[0025] 5. Data analysis: Statistical analysis was performed using the SPSS17.0 statistical software package (mathematical statistical calculations were required for all indicators in this study); statistical significance was determined by one-way analysis of variance and then Tukeys or Dunnets tests were performed; SCIEX OS1.5 and ProteinPilot 5.0.2 were used for data collection and processing.

[0026] The umami peptides derived from Procambarus clarkii obtained by the above method are characterized in that the amino acid sequences are LAEPLTFN and YGGEFPARPDN.

[0027] Furthermore, the umami peptides derived from Procambarus clarkii obtained by the above method are characterized in that:

[0028] 1) They have good affinity with the umami receptor T1R1 / T1R3. The docking energies of Pep11 with the umami receptor T1R1 / T1R3 are -7.346 kcal / mol and -7.912 kcal / mol respectively; the docking energies of Pep18 with the umami receptor T1R1 / T1R3 are -7.992 kcal / mol and -9.442 kcal / mol respectively;

[0029] 2) Complex stability: The Pep11-T1R1 / T1R3 complex reaches stability after 20 ns, and its RMSD value is 0.379 nm; the Pep18-T1R1 / T1R3 complex reaches stability only within 10 ns, and its RMSD value is 0.346 nm;

[0030] 3) They have obvious umami and sweetness, and the taste thresholds of the polypeptides are 0.258 mg / mL and 0.379 mg / mL.

[0031] The application of the umami peptides derived from Procambarus clarkii obtained by the above method is characterized in that it is applied in one of the following three aspects: 1) Application in the field of food processing;

[0032] 2) Application in improving the umami of food;

[0033] 3) Application as or in the preparation of flavor bases and food additives, and the specific application performance is to be used in combination with sodium glutamate.

[0034] The present invention uses machine learning and virtual screening technologies to discover umami peptides from the protein sequence of Procambarus clarkii for the first time, enriching the umami peptide library from freshwater sources, enhancing the economic value of Procambarus clarkii, and providing a new reference for studying the umami mechanism of umami peptides.

[0035] All kinds of terms and phrases used in the present invention have the general meanings well-known to those skilled in the art.

[0036] The beneficial effects of the present invention are as follows: Based on liquid chromatography-mass spectrometry (LC-MS) peptideomics combined with molecular docking and molecular dynamics, screening is carried out according to aspects such as the stability of molecular conformation and the ease of binding to the umami receptor T1R1 / T1R3. The umami peptides obtained by this method have a compound umami enhancement effect. Through sensory evaluation and electronic tongue verification of their umami results, it is found that they have a synergistic effect with common umami seasonings and can effectively block bitterness and sourness. Enriching the umami peptide library of fresh water sources, it can be used as a umami base material or compounded with existing seasonings to form a new type of seasoning, which has important application value and provides new umami peptide raw materials for the development of compound umami seasonings. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a distribution diagram of each fraction in UT of the present invention.

[0038] Figure 2 It is an electronic tongue characterization diagram of each fraction in UT of the present invention.

[0039] Figure 3 It is a homologous modeling and result verification diagram of the T1R1 / T1R3 receptor of the present invention, where: the homology degree of T1R1 / T1R3 with the template sequence (A), the three-dimensional structures of T1R1 and T1R3 (B, C), and the corresponding Ramachandran plots (D, E).

[0040] Figure 4 It is a molecular docking result diagram of six identified polypeptides in the active site of the T1R1 / T1R3 receptor of the present invention.

[0041] Figure 5 It is a diagram of hot spot amino acids during the docking process of the umami peptide of the present invention with T1R1 (A) and T1R3 (B).

[0042] Figure 6 It is a surface force analysis diagram of the interaction between T1R1 of the present invention and six umami peptides.

[0043] Figure 7 It is a surface force analysis diagram of the interaction between T1R3 of the present invention and six umami peptides.

[0044] Figure 8 It is a diagram of the changes in RMSD (A-F) and RMSF (G-L) during the MD process of the complex of the present invention.

[0045] Figure 9 It is a diagram of the electronic tongue taste response intensity (A) and taste behavior (B) of Pep11 and Pep 18 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0047] For the instruments, reagents, and materials involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods and detection methods existing in the prior art.

[0048] Example 1

[0049] Experiment: Preparation, Screening, and Verification of Umami Peptides Derived from Procambarus clarkii

[0050] I. Materials and Methods

[0051] 1. Sample Preparation

[0052] Procambarus clarkii used in the present invention was purchased from Wushang Supermarket in Hubei. Professional personnel euthanized the shrimp and then washed them. They were steamed in boiling water until the central temperature of the shrimp reached 90°C and then cooled to room temperature. A certain mass of shrimp tails was mixed with ultrapure water at a ratio of 1:4 (g / mL), homogenized at 15000 r / min for 2 min, and then hot water extracted for 30 min. After cooling and centrifuging (10000 r / min, 4°C, 2 min), the supernatant was the shrimp meat extract (SE), which was stored in a -80°C refrigerator.

[0053] 2. Extraction of Umami Peptides

[0054] 2.1 Ultrafiltration

[0055] To collect as many flavor components as possible, proteins with a molecular weight below 3 kDa in the shrimp meat extract (SE) were separated. The shrimp meat extract (SE) was ultrafiltered through a nanofiltration membrane separation device equipped with a 3 kDa ultrafiltration membrane. This process was carried out at 4°C and a pressure of 20 Bar, and all the filtrates (UF) were collected and freeze-dried to obtain a freeze-dried sample.

[0056] 2.2 Gel Filtration Chromatography Analysis

[0057] The freeze-dried sample was formulated into a 10 mg / mL polypeptide solution, which was separated by a Sephadex G-15 gel column. During this process, the ultraviolet absorption intensity of the eluent was monitored at 220 nm. The eluent was pure water, the flow rate was 0.75 mL / min, and the sample loading volume was 3 mL. The four fractions obtained were named P1, P2, P3, and P4 in the order of the peak elution time. The test solution was collected and freeze-dried for subsequent analysis and determination to obtain the freeze-dried shrimp flavor extract to be tested after gel filtration. Through taste dilution analysis;

[0058] 2.3 Amino acid analysis of taste peptides

[0059] Take 1 mL of the shrimp flavor extract SE to be tested, add 100 μL of the derivatizing solution (volume ratio of ethanol: phenylisothiocyanate: water: triethylamine = 7:1:1:1), and derivatize for 30 min. Then add 0.9 mL of mobile phase B and mix well. Instrument conditions: detection wavelength 254 nm; oven temperature 40 °C; injection volume 10 μL; mobile phase A: 80% acetonitrile aqueous solution by volume concentration, mobile phase B: 0.1 mol / L anhydrous sodium acetate - acetonitrile (97:3, V / V); elution program is shown in Table 1; flow rate 1 mL / min. External standard method was used for quantification with a known concentration of amino acid standard mixed solution (including Met, Asp, Ser, Gly, Glu, Thr, Ala, Lys, Tyr, Pro, Val, Leu, Phe, Ile, His, Arg, and Cys); the samples (the taste intensities of the four fractions after gel filtration in 2.2 were determined based on the amino acid content data) were obtained.

[0060] Table 1, Mobile phase elution program

[0061] Time / min 1 14 29 30 37 38 45 Mobile phase A / % 0 20 40 100 100 0 0 Mobile phase B / % 100 80 60 0 0 100 100

[0062] 2.4 Taste dilution analysis and electronic tongue characterization

[0063] The fractions (P1, P2, P3, P4) separated in step 2.2 were each diluted with pure water in equal volume to 5 mL to obtain test samples, which were then presented to sensory evaluation personnel and evaluated using the triangle test method. If the evaluators considered that there was a difference between the taste of the test sample and pure water, equal volume dilution was performed again, and this cycle was repeated until the taste of the test sample disappeared. The dilution factor was the TDA. The electronic tongue was based on a 0.025% MSG solution, the concentration of the polypeptide fraction was 1 mg / mL, and each sample was measured five times independently. This step was to further corroborate the results of 2.3, that is, P2 and P3 had the strongest umami taste, so umami peptides were identified in P2 and P3 subsequently.

[0064] 3. Identification of umami peptides

[0065] Select 2 mL of the fractions (P1, P2, P3, and P4) with significantly stronger umami taste in Step 2.2 for full hydration (only P2 and P3 with the strongest umami taste were selected for full hydration), add dithiothreitol (DTT) to a final concentration of 10 mM, and incubate at 56 °C for 1 h. Subsequently, add iodoacetamide (IAM) to a final concentration of 50 mM and incubate again in the dark for 40 min. The product was immediately treated with a desalting column, the eluate was collected and lyophilized for further testing, and it was dissolved in ultrapure water to obtain the test substance.

[0066] The polypeptide was separated from the above-mentioned test substance by an Easy-nLC 1200 high-performance liquid chromatography system. Using solvent A (0.1% formic acid by volume) and solvent B (80% acetonitrile and 0.1% formic acid by volume) as eluents, a 120-min gradient elution program was used to separate the test substance, and the elution process was as follows: 0.01 - 2 min (3% - 8% solvent B), 2 - 10 min (8% - 12% solvent B), 10 - 80 min (12% - 25% solvent B), 80 - 94 min (25% - 35% solvent B), 94 - 112 min (35% - 60% solvent B), 112 - 117 min (60% - 95% solvent B), and 117 - 120 min (95% solvent B). The fragmentation mode of the first-stage mass spectrometry was high-energy collision-induced dissociation (HCD). The resolution, dynamic gain control value (Automatic gain control, AGC), ion injection time, and error value of the second-stage mass spectrometry were 1.5×10 4 、1×10 5 、50 ms, and 100 m / z, the shortest pre-scan time was 8×10 3 ,the intensity threshold was 1.6×10 5 . The obtained polypeptide fingerprint map was identified by Peaks Studio 10.6 (Thermo, Waltham, MA, USA), and the obtained polypeptide fingerprint map was identified by Peaks Studio 10.6 (Thermo, Waltham, MA, USA) to obtain potential umami peptides from Procambarus clarkii (the activity needs to be further analyzed and determined to obtain an umami peptide derived from Procambarus clarkii).

[0067] 4. Prediction of Umami Peptide Activity

[0068] 4.1 Molecular Docking

[0069] The amino acid sequences of T1R1 (Uniprot: Q7RTX1) and T1R3 (Uniprot: Q7RTX0) were derived from the NCBI database. Sequence alignment was performed in the PDB database using the Protein BLAST tool (this is a computer prediction of the reaction process at the molecular level) to obtain the human calcium-sensing receptor (PDB ID: 5K5S) for homology modeling of the receptor. Modeler v9.19 was used for the modeling and optimization of the receptor protein, and the rationality of the receptor model was further evaluated by the PROCHECK Verify 3D program and Ramachandran plot provided by SAVES v6.0 serves. To facilitate the operation of molecular docking, the T1R1 / T1R3 dimer was assembled and optimized by PyMol. AutoDock vina characterized the interaction mode of umami peptides with T1R1 / T1R3 by semi-flexible docking method. Among them, the three-dimensional conformations of the polypeptide and the receptor were both subjected to energy minimization and converted to the pdbqt format. The following parameters were used to construct the grid box to completely cover the entire protein surface: Other parameters were set to default values. After 100 docking attempts, the conformation of the complex with the lowest energy was selected for further optimization. That is, it was first assigned the Amber14 force field and processed for 1000 steps using the Steepest Descent Method, and then processed for 5000 steps using the Conjugate Gradient Method. The final result was visualized using Discovery Studio 2019. Umami peptides with higher binding energy during the computer-simulated molecular docking process were obtained.

[0070] 4.2 Molecular dynamics simulation

[0071] The potential umami peptides obtained in step 4.1 with low binding energy to the T1R1 / T1R3 receptor (here are the potential umami peptides with low binding energy during the computer-simulated molecular docking process in 4.1) were further characterized by MD. Using the Gromacs software package, the three-dimensional conformation of the complex and the free monomer were encapsulated in the TIP (three-site transferable intermolecular potential) water molecule model under the Amber14 force field. And the energy of the simulation system was minimized using the Steepest Descent Method, and then 100 ns of MD simulation was carried out under the conditions of constant number of particles, volume and temperature and constant number of particles, pressure and temperature (NPT). In addition, the number of hydrogen bonds formed between the ligand and the receptor was determined by the VMD (visual molecular dynamics) software package by restricting the distance intercept and the angular intercept values to Obtained at 0° and 30°. The identified polypeptide sequences were obtained (here, the polypeptide sequences with strong umami effects were obtained).

[0072] 4.3 Identification and Verification of Synthetic Peptides

[0073] The identified polypeptide sequences obtained in step 4.2 (amino acid sequences were LAEPLTFN and YGGEFPARPDN respectively) were synthesized into polypeptides by solid-phase synthesis, and the purity and molecular weight of the synthetic peptides were determined by HPLC and LC-MS respectively.

[0074] 4.4 Sensory Evaluation of Synthetic Peptides

[0075] The experimental group consisted of 20 adults (10 males and 10 females, aged 22 - 28 years) who had received sensory training according to GB / T16291.1 - 2012 and had rich sensory evaluation experience. The umami threshold of the synthetic peptides was determined by the triangle test method. The experiment was carried out under the conditions of a temperature of 25 ± 2°C and a humidity of 55 ± 5%. All the umami peptides synthesized in 4.3 (there were 3 coded samples, 2 of which were the same, and the inspector needed to screen out the different sample) were randomly coded with three digits. The initial concentration of the synthetic peptide solution was prepared at 1 mg / ml and gradually diluted step by step at a ratio of 1:1 (v / v) until the group members could no longer distinguish it from the pure water area. This step was to characterize the taste characteristics of the umami peptides from Procambarus clarkii screened by us. The same principle applied to the electronic tongue. There was no next step that required this result.

[0076] 4.5 Characterization of Synthetic Peptides by Electronic Tongue

[0077] Using deionized water as the solvent, the taste threshold was measured for the umami polypeptides synthesized in 4.3. The concentration of the synthetic peptides was set at 0.2 mg / mL, and the concentrations of the MSG standard solutions were 0.025%, 0.05%, 0.1%, 0.2%, 0.3%, and 0.4% (w / v) respectively. Each sample was measured five times independently.

[0078] 5. Data Analysis

[0079] Statistical analysis was performed using the SPSS17.0 statistical software package (mathematical statistical calculations were required for all indicators in this study). Statistical significance was determined by one-way ANOVA and then Tukeys or Dunnets tests were carried out. SCIEX OS 1.5 and ProteinPilot5.0.2 were used for data collection and processing (ProteinPilot is a component in SCIEX OS used for qualitative analysis of peptide segments and was used in the identification of umami peptides).

[0080] II. Results and Discussion

[0081] 1. Free Amino Acid Content in Taste Peptides

[0082] FromFigure 1 As shown, four main fractions (P1, P2, P3, and P4) were enriched and distilled. As shown in Table 2, UF (the filtrate in step 2.1) contains more than 90% of free amino acid FAAs, which can be considered to be the most flavor-active part. Compared with other fractions, P2 and P3 have relatively higher fresh and sweet amino acid contents, which are 0.29±0.03 and 0.43±0.01 mg / g, respectively, and may express a stronger fresh and sweet taste. Arginine Arg has a higher content in the above fractions, which are 9.82±0.03 and 6.08±0.78 mg / g, respectively. As a bitter amino acid, it has been shown in previous studies to have auxiliary freshness activity, so it can be preliminarily considered that P2 and P3 have richer taste than P1 and P4.

[0083] Table 2. Composition of free amino acids in flavor peptides of Procambarus clarkii

[0084]

[0085] 2. TDA and electronic tongue characterization

[0086] As shown in Table 3, UF (the filtrate in step 2.1) has good umami, sweetness and partial saltiness, and the P2 and P3 fractions have good umami properties. Figure 2 As shown in the figure, compared with 0.025% monosodium glutamate MSG, P2 and P3 have stronger umami and richness, while P1 and P4 mainly present bitterness and strong aftertaste. Therefore, it can be considered that the P2 and P3 fractions contain umami peptides from Procambarus clarkii.

[0087] Table 3, TDA (dilution factor) and taste characteristics of each fraction

[0088] Fraction Taste description TDA Taste threshold (mmol / L) UF Umami, sweetness and saltiness 128 0.287 P1 Sweetness 32 0.558 P2 Umami, sweetness and saltiness 64 0.312 P3 Umami and sweetness 64 0.323 P4 Bitterness and saltiness 16 0.464

[0089] 3. Identification and activity prediction of umami peptides

[0090] The protonated amide bonds in the polypeptide are broken by collision-induced dissociation (CID) or high-energy collision dissociation (HCD) technology, and the b-type and y-type ions generated in the process can be used to determine the amino acid sequence of the polypeptide. The present invention uses PEAKS and NOVOR algorithms to analyze protein fragments with a confidence level (CL) greater than 90% in the test results, of which the former identified 1409 polypeptides and the latter identified 227. In order to eliminate possible deviations in the algorithm as much as possible, the results are cross-compared to obtain peptides with the highest CL possible.

[0091] As shown in Table 4, 21 peptide segments with molecular weights in the range of 800 - 1700 Da were screened out through the BIOPEP-UWM database, and their umami activities were predicted. These polypeptides usually contain 8 - 12 amino acids and all contain umami amino acids such as Glu and Asp. However, the umami activity values of different polypeptides are not consistent. Among them, the predicted umami activity value of Pep18 (YGGEFPARPDN) is 0.6316, which is the highest among the 21 polypeptides. Followed by Pep11 (LAEPLTFN) with an activity value of 0.5714. It is thus speculated that the content of umami amino acids in the peptide sequence is proportional to the umami characterization, while when the types of umami amino acids are more than four, this characteristic is not manifested.

[0092] Table 4, Prediction of Taste Characteristics and Thresholds of Peptide Sequences

[0093]

[0094]

[0095] 4. Molecular Docking between T1R1 / T1R3 Receptors and Umami Peptides

[0096] The T1R1 / T1R3 receptor needs to be constructed through homology modeling technology. It can be seen from Figure 3 that by comparing the target sequences, the crystal structure of the active form (5K5S) of the extracellular domain of the human calcium-sensing receptor obtained from the NCBI database has the highest similarity, which are 35.68% and 35.19% respectively. And the calculation results of the Ramachandran plot show that only 0.34% of the amino acid residues are in the disallowed region. Therefore, the T1R1 / T1R3 model constructed in this study can be used for molecular docking simulation.

[0097] As can be seen from Table 5, the binding energies of the identified twenty-one umami peptides when docked with the T1R1 / T1R3 dimer are all lower than -5 kcal / mol, and the binding energies with T1R3 are all lower than those with T1R1, indicating that umami peptides are more likely to bind to the VFTD domain in T1R3 to present umami. Among them, the binding energies of Pep4, Pep8, Pep9, Pep11, Pep14, and Pep18 with T1R1 / T1R3 are relatively low, and they are more likely to form complexes with stable conformations.

[0098] Table 5, Binding Energies of Twenty-One Umami Peptides with T1R1 / T1R3

[0099]

[0100] 5. Interaction Forces between T1R1 / T1R3 Receptors and Umami Peptides

[0101] Umami peptides mainly form complexes with T1R1 / T1R3 through hydrogen bonds or electrostatic interactions between hydrophilic amino acids. Among the hydrophilic amino acid residues that interact with the umami receptor, approximately 88.93% (mass percentage, the number of hydrophilic amino acid residues) and 94.29% of the amino acids bind to T1R1 and T1R3 through hydrogen bonds, respectively. Figure 4 As shown, compared with the N-terminal residues, the C-terminal residues of umami peptides are more likely to interact with the docking pocket located on the surface of T1R1 / T1R3. As can be seen from Table 6 and Table 7, carbon-hydrogen bonds and alkyl bonds also make important contributions to stabilizing the conformation of the complex, followed by salt-bridges and charge-charge interactions.

[0102] As Figure 5 shown, a total of 38 amino acid residues are significant during the binding process with T1R1, mainly Arg, Pro, and Cys. Cys66, Arg81, Glu285, Arg307, His308, Pro313, Arg371, and Pro376 are the hot-spot amino acids during the docking process, and the above residues are the main binding sites for forming the Pep-T1R1 complex. Similarly, there are 45 amino acid residues during the Pep-T1R3 binding, mainly Arg and Pro, and its main binding sites are Arg64, Phe65, Ser67, Asp307, Leu308, Arg357, Glu358, and Leu385. In addition, the Pep-T1R1 / T1R3 complex often involves some specific amino acid residues in the formation of non-polar interactions. For example, in T1R1, Ser, Glu, Leu, and His mainly form carbon-hydrogen bonds, while Val and Arg tend to form alkyl bonds; in T1R3, Ala, Asp, Glu, and Ser mainly form carbon-hydrogen bonds, while Leu and Arg tend to form alkyl bonds.

[0103] Meanwhile, some special structures involving multiple hydrogen bonds or salt bridge bindings also have strong stability. Pep11-T1R1 has an "EE" structure (Thr374, Met375) and an "EEE", which is composed of a salt bridge (Glu285), hydrogen bonds (Gly314 and His308), and a carbon-hydrogen bond (Pro31); there are two "EEE" structures in Pep11-T1R3; the Pep18-T1R1 complex has a typical "EEE" structure, which is composed of a salt bridge (Arg317), a hydrogen bond (Gly314), and a carbon-hydrogen bond (Arg307); Pep18-T1R3 has a "DD" structure (Arg64, Phe65, Ser66, and Ser67) and an "EVDD" structure, which is composed of a hydrogen bond (Ile309 and His308) and Pi-Alkyl (Val288). These strong interactions will enable umami peptides to have better taste performance in receptors.

[0104] Table 6, Amino Acid Residues of Polypeptides with Interaction Forces with T1R1

[0105]

[0106]

[0107]

[0108] Table 7, Amino Acid Residues of Polypeptides with Interaction Forces with T1R3

[0109]

[0110]

[0111]

[0112] 6. Surface Interaction Forces between T1R1 / T1R3 Receptors and Umami Peptides

[0113] The surface interaction forces between umami peptides and the T1R1 / T1R3 complex are as Figure 6 、 Figure 7 shown. DS plotted the contour maps of five surface interaction forces, including aromatic interaction (Aromatic), hydrogen bond (H-bond), hydrophobic interaction (Hydrophobicity), charge interaction (Charge), and solvent-accessible surface area (Solvent-accessibility surface, SAS).

[0114] Aromatic is judged according to the ratio of the local electrostatic interaction strength between protons on the benzene ring at the edge of the structure to the π - electron density on the benzene ring on the surface of the structure. The Aromatic cloud map shows that the electrostatic interaction between protons on the edge ring of the Pep - T1R1 / T1R3 complex is stronger than the π - electron density on the surface ring, and the interaction strength of the T1R3 complex is significantly higher than that of the T1R1 complex; the H - bond cloud map shows that the H - bond cloud maps of the Pep18 - T1R1 / T1R3 and Pep11 - T1R1 / T1R3 complexes are darker in color, indicating stronger interactions and higher product stability; the Hydrophobicity cloud map shows that the Pep - T1R3 complex is more hydrophilic than the Pep - T1R1 complex, which may be due to the rich hydrophilic amino acid residues in T1R3; SAS simulates the binding between solvent molecules and the van der Waals surface of the receptor. The SAS cloud map shows that the SAS of the binding domain between the umami peptide and T1R1 is higher, indicating that the polypeptide and the receptor can also bind effectively through van der Waals forces; compared with other surface forces, the degree of ionization in the region between T1R3 and the umami peptide is much lower, indicating that the six umami peptides do not rely on this interaction force during the binding process with the receptor.

[0115] In summary, during the formation of the complex between the umami peptide of Procambarus clarkii and T1R1 / T1R3, the surface forces involved mainly include Aromatic, H - bond, Hydrophobicity, and SAS, all of which contribute to the formation of umami. Due to the good affinity of Pep11 and Pep18 with the umami receptor, they may have stronger umami - presenting activity in practical applications. According to research, the hydrophobicity of polypeptides usually shows a strong positive correlation with their bitter activity. Pep11 - T1R1 / T1R3 and Pep18 - T1R3 not only have lower binding energies, but also their hydrophobicity is significantly lower than that of other polypeptides, which can be used as one of the bases to explain their stronger umami effect.

[0116] 7. Molecular Dynamics (MD) Simulation

[0117] Due to the limitations of the Autodock algorithm, during the binding of T1R1 / T1R3 with the umami peptide, only the conformation of the ligand will change partially to fit the spatial conformation of the active site in the receptor (semi - flexible docking), so the accuracy is limited. And the intermolecular force analysis is based on the best conformation determined by molecular docking, and examines the changes of the above - mentioned conformation in TIP (a professional term, a force field model) within a certain time. Therefore, to further explore and determine the binding stability of the Pep - T1R1 / T1R3 complex, MD (Molecular Dynamics) analysis was used, with a simulation time of 50 ns, and the results are as Figure 8 shown.

[0118] The root mean square deviation value RMSD is used to evaluate the time required for the complex to reach a stable state in the system. The smoother the RMSD curve, the more stable the complex formation. As Figure 8 shown in A-F below, the Pep4, Pep9, and Pep11-T1R1 / T1R3 complexes all reached stability after 20 ns, with RMSD values of 0.414, 0.370, and 0.379 nm, respectively; the Pep8, Pep14, and Pep18-T1R1 / T1R3 complexes reached stability within only 10 ns, with RMSD values of 0.407, 0.376, and 0.346 nm, respectively.

[0119] RMSF is usually used to measure the fluctuations of individual amino acid residues of Pep-T1R1 / TIR3 in MD simulations. The RMSF value is proportional to the degree of fluctuation of amino acid residues in the protein. As Figure 8 shown in G-J below, the fluctuations of the six umami peptide complexes are similar, and the fluctuation range is between 0 - 0.6 nm, with relatively stable structures.

[0120] 8. Sensory Verification of Synthetic Peptides

[0121] Since acidic amino acids may remain in the process of synthesizing the target peptide, resulting in the detection of sour or astringent tastes in the synthetic peptide, which affects the characterization of its taste properties, sensory evaluation combined with analysis is required.

[0122] As shown in Table 8, both Pep11 and Pep18 mainly exhibit umami taste, followed by fishy and sour tastes, and also slightly salty taste. Among them, the umami taste perceptible to humans of Pep18 is higher than that of Pep11. The sensory evaluation thresholds of Pep11 and Pep18 in aqueous solution are between 0.254 and 0.319 mmol / L, which are lower than the threshold of the umami hexapeptide WDDMEK from Trachinotus ovatus (0.034 mmol / L).

[0123] As Figure 9 shown, Pep11 and Pep18 have different degrees of synergistic effects with 0.025% - 0.4% MSG solution, and the coexistence of Pep11 and Pep18 with MSG will reduce the bitterness of the overall solution and enhance the umami taste.

[0124] Table 8, Taste Thresholds and Toxicity Predictions of Pep11 and 18

[0125]

[0126] Generally speaking, combining molecular docking and molecular dynamics simulation, it was determined that Pep-11 (LAEPLTFN) and Pep-18 (YGGEFPARPDN) could bind to the umami receptor T1R1 / T1R3, and their complex conformations were tight and stable. In addition, both umami peptides derived from Procambarus clarkii had obvious umami flavors, could be synergistically applied with MSG to enhance umami and reduce bitterness, and had the potential to be applied in the food industry.

[0127] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a umami peptide derived from Procambarus clarkii, characterized in that The method includes the following steps: First, separate and purify the polypeptides in the shrimp flavor extract, predict the peptide activity through a database, and characterize them by molecular docking and molecular dynamics simulation methods. Finally, two potential umami peptides are identified, and their amino acid sequences are LAEPLTFN and YGGEFPARPDN respectively. Synthesize the above umami peptides artificially, verify their umami characteristics by sensory evaluation and electronic tongue methods, and further evaluate their synergistic effects with other umami substances.

2. The preparation method of an umami peptide derived from Procambarus clarkii according to claim 1, characterized in that the specific steps are as follows: 1). Sample preparation: Wash the Procambarus clarkii after death, steam it in boiling water until the central temperature of the shrimp reaches 90 °C, and cool it to room temperature. Mix the shrimp tails and ultrapure water at a ratio of 1 g:4 mL, homogenize at 15000 r / min for 2 min with a homogenizer, then extract with hot water for 30 min. After cooling and centrifugation, the supernatant is the shrimp flavor extract, which is stored in a -80 °C refrigerator. 2). Extraction of umami peptides: 2.1). Ultrafiltration: Separate the proteins with a molecular weight below 3 kDa in the shrimp flavor extract. Ultrafilter the shrimp flavor extract through a nanofiltration membrane separation device equipped with a 3 kDa ultrafiltration membrane. This process needs to be carried out at 4 °C and a pressure of 20 Bar, and collect all the filtrates and freeze-dry them to obtain a freeze-dried sample. 2.2). Gel filtration chromatography analysis: Prepare the freeze-dried sample into a polypeptide solution with a concentration of 10 mg / mL, and separate it through a Sephadex G-15 gel column. During this period, monitor the ultraviolet absorption intensity of the eluent at 220 nm. The eluent is pure water, the flow rate is 0.75 mL / min, and the sample loading volume is 3 mL. Name the 4 fractions obtained in the order of the peak elution time as P1, P2, P3, and P4 respectively. Collect the test solution and freeze-dry it for subsequent analysis and determination to obtain the freeze-dried test shrimp flavor extract for gel filtration. 2.3). Amino acid analysis of umami peptides: Take 1 mL of the test shrimp flavor extract, add 100 μL of the derivatization solution. The derivatization solution is: ethanol:phenylisothiocyanate:water:triethylamine volume ratio = 7:1:1:1, and derivatize for 30 min. Then add 0.9 mL of mobile phase B and mix well. Instrument conditions: detection wavelength 254 nm; oven temperature 40 °C; injection volume 10 μL; mobile phase A is: 80% acetonitrile aqueous solution by volume concentration, mobile phase B is: 0.1 mol / L anhydrous sodium acetate - acetonitrile; the elution program is shown in Table 1; flow rate 1 mL / min; use an external standard method for quantification with a known concentration of amino acid standard mixed solution. Table 1, Mobile phase elution program 2.4) Taste dilution analysis and electronic tongue characterization: 5 mL of the fractions (P1, P2, P3, P4) separated in step 2.2) were each diluted with pure water to the same volume to obtain test samples, which were then presented to sensory evaluation panelists, and the triangle test method was used for evaluation; if the panelists considered that there was a difference in taste between the test sample and pure water, equal-volume dilution was performed again, and this cycle was repeated until the taste of the sample disappeared, and the dilution factor was the TDA; the electronic tongue was calibrated with a 0.025% MSG solution, and the polypeptide fraction concentration was 1 mg / mL. Five independent replicates were performed for each sample. 3) Identification of umami peptides: 20 mL of the fraction with significantly stronger umami taste in step 2.2) was fully hydrated, dithiothreitol (DTT) was added to a final concentration of 10 mM, and the mixture was incubated at 56 °C for 1 h; subsequently, iodoacetamide was added to a final concentration of 50 mM, and incubation was continued in the dark for 40 min; the product was immediately treated with a desalting column, the eluate was collected and freeze-dried for further analysis, and the resulting substance was dissolved in ultrapure water for testing. The polypeptide was separated from the above-mentioned analyte by an Easy-nLC 1200 high-performance liquid chromatography system; solvent A and solvent B were used as eluents, where solvent A was formic acid with a volume concentration of 0.1%, and solvent B was acetonitrile with a volume concentration of 80% and formic acid with a volume concentration of 0.1%; a 120-min gradient elution program was used to separate the analyte, and the elution process was as follows: 0.01 - 2 min (3% - 8% solvent B), 2 - 10 min (8% - 12% solvent B), 10 - 80 min (12% - 25% solvent B), 80 - 94 min (25% - 35% solvent B), 94 - 112 min (35% - 60% solvent B), 112 - 117 min (60% - 95% solvent B), and 117 - 120 min (95% solvent B); the fragmentation mode of the first-stage mass spectrometry was high-energy collision-induced dissociation; the resolution, dynamic gain control value, ion injection time, and error value of the second-stage mass spectrometry were 1.5×10 4 、1×10 5 、50 ms, and 100 m / z, respectively, and the shortest pre-scan time was 8×10 3 , and the intensity threshold was 1.6×10 5 ; the obtained polypeptide fingerprint was identified by Peaks Studio 10.6, and the potential umami peptides from Procambarus clarkii were obtained; 4) Prediction of umami peptide activity 4.1) Molecular docking The amino acid sequences of T1R and T1R3 were derived from the NCBI database; sequence alignment was performed in the PDB database using the Protein BLAST tool to obtain the human calcium-sensing receptor for homology modeling of the receptor; Modeler v9.19 was used for the modeling and optimization of the receptor protein, and the PROCHECK Verify 3D program and Ramachandran plot provided by SAVES v6.0 serves were used to further evaluate the rationality of the receptor model; for the convenience of running molecular docking, the T1R1 / T1R3 dimer was assembled and optimized by PyMol; AutoDock vina characterized the interaction mode of umami peptides with T1R1 / T1R3 by semi-flexible docking method; among them, The three-dimensional conformations of both the polypeptide and the receptor were subjected to energy minimization and converted to the pdbqt format; a grid box was constructed using the following parameters to fully cover the entire protein surface: Other parameters were set to their default values; after 100 docking attempts, the complex conformation with the lowest energy was selected for further optimization; that is, it was first assigned the Amber14 force field and processed with the steepest descent method for 1000 steps, and then processed with the conjugate gradient method for 5000 steps. The final results were visualized and analyzed using Discovery Studio 2019; 4.2) Molecular dynamics simulation: The umami peptides obtained in step 4.1) with low binding energy to the T1R1 / T1R3 receptor were further characterized by MD; using the Gromacs software package, the three-dimensional conformation of the complex and the free monomer were encapsulated in the TIP water molecule model under the Amber14 force field; and the energy of the simulation system was minimized using the steepest descent method, and then 100 ns of MD simulation was carried out under the conditions of constant number of particles, volume and temperature and constant number of particles, pressure and temperature; in addition, the number of hydrogen bonds formed between the ligand and the receptor was obtained by the VMD software package by restricting the distance intercept and the angle intercept values to and 30°, respectively; the identified polypeptide sequences were obtained; 4.3) Identification and verification of synthetic peptides: The identified polypeptide sequences obtained in step 4.2) were synthesized into peptides by solid-phase synthesis. The amino acid sequences were LAEPLTFN and YGGEFPARPDN, respectively. The purity and molecular weight of the synthetic peptides were determined by HPLC and LC-MS, respectively. 4.4) Sensory evaluation of synthetic peptides: The experimental group consisted of 20 adults who had received sensory training in accordance with GB / T 16291.1-2012 and had extensive sensory evaluation experience; the umami threshold of the synthetic peptides was determined by the triangle test method. The experiment was carried out at a temperature of 25 ± 2 °C and a humidity of 55 ± 5%. The umami peptides synthesized in 4.3) were all randomly coded with three digits. The initial concentration of the synthetic peptide solution was prepared at 1 mg / ml and gradually diluted 1:1 (v / v) until the group members could no longer distinguish it from the pure water area. 4.5) Electronic tongue characterization of synthetic peptides: Deionized water was used as the solvent to determine the taste threshold, and the synthetic umami peptides in 4.3) were measured. The concentration of the synthetic peptide was set at 0.2 mg / mL, and the concentrations of the MSG standard solutions were 0.025%, 0.05%, 0.1%, 0.2%, 0.3%, and 0.4% (w / v), respectively. Five independent replicates were performed for each sample. 5) Data analysis: Statistical analysis was performed using the SPSS 17.0 statistical software package; statistical significance was determined by one-way analysis of variance followed by Tukey's or Dunnett's test; SCIEX OS 1.5 and ProteinPilot 5.0.2 were used for data collection and processing.

3. The umami peptide derived from Procambarus clarkii obtained by the method according to claim 1, characterized in that: The amino acid sequences are LAEPLTFN and YGGEFPARPDN.

4. The umami peptide derived from Procambarus clarkii according to claim 3, characterized in that: With good affinity for the umami receptor T1R1 / T1R3, the docking energies of Pep11 with the umami receptor T1R1 / T1R3 are -7.346 kcal / mol and -7.912 kcal / mol respectively; the docking energies of Pep18 with the umami receptor T1R1 / T1R3 are -7.992 kcal / mol and -9.442 kcal / mol respectively.

5. The umami peptide derived from Procambarus clarkii according to claim 3, characterized in that: Complex stability: The Pep11-T1R1 / T1R3 complex reaches stability after 20 ns, and its RMSD value is 0.379 nm; the Pep18-T1R1 / T1R3 complex reaches stability only within 10 ns, and its RMSD value is 0.346 nm.

6. The umami peptide derived from Procambarus clarkii according to claim 3, characterized in that: Having obvious umami and sweet tastes, the taste thresholds of the polypeptide are 0.258 mg / mL and 0.379 mg / mL.

7. Use of the umami peptide derived from Procambarus clarkii obtained by the method according to claim 1, characterized in that: Applications in the field of food processing.

8. Use of the umami peptide derived from Procambarus clarkii obtained by the method according to claim 1, characterized in that: Applications in enhancing the umami of foods.

9. Use of the umami peptide derived from Procambarus clarkii obtained by the method according to claim 1, characterized in that: Applications as or in the preparation of flavor bases and food additives.

10. Use of the umami peptide derived from Procambarus clarkii according to claim 9, characterized in that: The specific application performance is manifested as being used in combination with monosodium glutamate.

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