A screening method, application and product of a pea peptide with salty taste enhancing function

Abstract

The application discloses a salty taste enhancing peptide screened from a pea protein and a screening method thereof, and belongs to the technical field of food condiments. According to a molecular docking result, four peptide segments DRF, GRRY, VNRF and NRF with potential salty taste enhancing effects are screened, and the molecular weights of the four peptide segments are all less than 1000 Da. It is shown by sensory evaluation and electronic tongue analysis that the four peptide segments all have good salty taste enhancing effects, and can be applied to the field of food condiments to solve the purpose of'reducing salt without reducing salty taste'. The application provides a theoretical basis for discovering new salty taste enhancing peptide resources in peas and developing nutritious and healthy salty taste enhancing peptides.

Description

Technical Field

[0001] This invention relates to the field of food seasoning technology, specifically to a salty-enhancing peptide screened from pea protein that can dock with the salty taste receptor TMC4 and its application. Background Technology

[0002] Salt (NaCl) is a crucial component of the salty taste in our daily diet and plays a vital role in our lives. From a food perspective, saltiness typically comes from sodium ions, which are obtained through salt or other sodium-containing compounds. How to reduce salt content without compromising the sensory quality of food has become a widespread global concern.

[0003] Salt-enhancing peptides have attracted considerable attention as a natural, safe, readily available, low-taste-threshold, and nutrient-rich salt alternative. In recent years, computational methods have been widely applied to peptide screening and mechanism research. Compared to traditional methods, these methods, by simulating and predicting the interactions of biomacromolecules, can rapidly and accurately identify optimal bioactive compounds, potentially solving time and cost issues. Therefore, this study employs a combination of computer technology and sensory evaluation to rapidly screen salt-enhancing peptides. Transmembrane channel-like 4 (TMC4) is a voltage-dependent chloride ion channel mainly expressed in the posterior part of taste buds, responsible for salt taste perception. Mouse experiments have demonstrated its involvement in the perception of high concentrations of salt and its high-salt effect at low salt concentrations. As a novel salt taste receptor, TMC4 can reveal different signal transduction pathways, contributing to a deeper understanding of the diversity, complexity, and novelty of salt taste perception. Therefore, TMC4, as a novel specific salt receptor, provides a new avenue for exploring taste enhancement. Pea protein, as a high-quality plant protein, has attracted significant attention in the fields of plant-based foods and nutritional health due to its balanced amino acid composition, low allergenicity, and excellent functional properties. However, current research on the flavor properties of pea protein focuses on strategies for removing bitter peptides and identifying umami peptides, while exploration of pea protein as a direct source of salty-enhancing peptides is rarely reported. This study employs a systematic sequence screening method, focusing on two major seed storage globulins in pea protein: 7S globulin and 11S globulin, which together constitute the main body of pea storage proteins. Therefore, using pea protein as raw material, we employed high-throughput virtual screening technology to rapidly screen for potential salty-enhancing peptides.

[0004] Based on this, the present invention aims to use virtual screening technology to screen out novel salt-enhancing peptides from pea protein, providing a new strategy for achieving "reduced salt" without "reduced saltiness". Summary of the Invention

[0005] In view of this, the present invention provides a method, application and product for screening pea peptides with enhanced saltiness.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a salty-enhancing peptide, which is screened from pea protein and binds to the TMC4 receptor protein. The peptide is composed of one of the following: The amino acid sequence is DRF, and the molecular weight is 436.46 Da; The amino acid sequence is GRRY, and the molecular weight is 550.61 Da; The amino acid sequence is VNRF, and the molecular weight is 534.61 Da; The amino acid sequence is NRF, and the molecular weight is 435.48 Da.

[0007] Furthermore, the peptides with the amino acid sequences DRF, GRRY, VNRF, and NRF can dock with the TMC4 receptor via molecular docking, with the main binding sites being Arg140, Arg168, Arg272, Gln143, Leu139, Leu142, Lys136, Phe169, Glu161, Leu172, Ser165, Ser275, and His271. Among these, Arg168, Arg272, and Leu139 and Leu172 occur most frequently and are key amino acid residues. Furthermore, when the concentration of the DRF peptide is 0.2 g / L, its saltiness value in a 3.8 g / L NaCl solution is 4.40, which is equivalent to replacing about 4.6% of NaCl. And / or, when the concentration of the peptide with the amino acid sequence GRRY is 0.2 g / L, its saltiness value in a 3.8 g / L NaCl solution is 4.72, which is equivalent to replacing about 12.21% of NaCl; And / or, when the concentration of the peptide with the amino acid sequence VNRF is 0.2 g / L, its saltiness value in a 3.8 g / L NaCl solution is 4.21, which is equivalent to replacing about 0.24% of NaCl; And / or, when the concentration of the peptide with the amino acid sequence NRF is 0.2 g / L, its saltiness value in a 3.8 g / L NaCl solution is 4.27, which is equivalent to replacing about 1.67% of NaCl.

[0008] Furthermore, when the concentration of the DRF peptide is 0.5 g / L, its saltiness value in a 3.5 g / L NaCl solution is 6.69, which is equivalent to replacing approximately 59.16% of NaCl. And / or, when the concentration of the peptide with the amino acid sequence GRRY is 0.5 g / L, its saltiness value in a 3.5 g / L NaCl solution is 8.61, which is equivalent to replacing about 104.92% of NaCl; And / or, when the concentration of the peptide with the amino acid sequence VNRF is 0.5 g / L, its saltiness value in a 3.5 g / L NaCl solution is 5.32, which is equivalent to replacing about 26.49% of NaCl; And / or, when the concentration of the peptide with the amino acid sequence NRF is 0.5 g / L, its saltiness value in a 3.5 g / L NaCl solution is 6.27, which is equivalent to replacing about 49.09% of NaCl.

[0009] Furthermore, when the concentration of the DRF peptide is 0.8 g / L, its saltiness value in a 3.2 g / L NaCl solution is 7.35, which is equivalent to replacing approximately 74.86% of the NaCl. And / or, when the concentration of the peptide with the amino acid sequence GRRY is 0.8 g / L, its saltiness value in a 3.2 g / L NaCl solution is 7.95, which is equivalent to replacing about 89.22% of NaCl; And / or, when the concentration of the peptide with the amino acid sequence VNRF is 0.8 g / L, its saltiness value in a 3.2 g / L NaCl solution is 4.47, which is equivalent to replacing about 6.34% of NaCl; And / or, when the concentration of the peptide with the amino acid sequence NRF is 0.8 g / L, its saltiness value in a 3.2 g / L NaCl solution is 6.12, which is equivalent to replacing about 45.60% of NaCl.

[0010] This invention provides a method for screening salty-tasting peptides, characterized by the following steps: A TMC4 salty taste receptor protein model was constructed using the trRosetta online server, and its reliability was evaluated. The reliability evaluation was as follows: the overall quality score was 97.08%, significantly higher than the 95% threshold for a high-quality model, indicating that the model has excellent stereochemical rationality and a stable hydrophobic core structure; the model is considered conformationally reasonable and reliable if the proportion of amino acid residues falling within the allowed and maximum allowed regions of the entire protein is higher than 90%; according to the Ramachandran diagram, the proportion of amino acid residues falling within the allowed and maximum allowed regions of the entire protein is also higher than 90%. Computer simulations were used to enzymatically hydrolyze pea protein to obtain enzymatically hydrolyzed peptides with sequence lengths of 2-8. The bioactivity of the enzymatically hydrolyzed peptides is predicted, and peptides with potential bioactivity are screened out. Their physicochemical properties are predicted, and non-toxic, water-soluble peptides are screened out. The screening criterion for predicting bioactivity is: prediction score > 0.5; The screening criteria for toxicity prediction are: Non-Toxin; The screening criteria for water solubility prediction are: Good water solubility; (4) Molecular docking is performed between the TMC4 receptor protein model and the non-toxic, water-soluble peptide from step (3) to form a small molecule ligand-receptor protein complex system. The TMC4 receptor protein needs to have water molecules removed and hydrogen atoms added in a CHARMM force field environment. The coordinates of the active site for molecular docking are: X=-6.10, Y=17, Z=-31.20, and the radius is 30. The molecular docking site coordinates were selected by using the largest cavity in the TMC4 receptor protein as the docking pocket. (5) Screen peptides with potential salt-enhancing effects based on molecular docking results, and synthesize peptides by solid-phase synthesis method; The screening process involves selecting peptides with low energy values ​​from the CDOCKER energy database. (6) The synthesized peptides were subjected to electronic tongue and sensory evaluation analysis to obtain the above-mentioned salty taste-enhancing peptides.

[0011] This invention provides the application of a salty-enhancing peptide in the preparation of seasonings and food.

[0012] The present invention provides a product containing the above-mentioned salty flavor-enhancing peptides screened from pea protein.

[0013] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention screened four peptides (DRF, GRRY, VNRF, and NRF) with molecular weights less than 1000 Da from pea protein. Electronic tongue and sensory evaluation analyses showed that these four peptides all have good saltiness-enhancing effects. These four saltiness-enhancing peptides can be applied to food seasonings to achieve the goal of "reducing salt" without "reducing saltiness." The new peptide sequences obtained by this invention provide new resources for the development of functional foods. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 The following is an evaluation diagram of the TMC4 salty taste receptor protein model in an embodiment of the present invention, wherein (A) is the TMC4 receptor protein model, (B) is the transmembrane structure prediction, (C) is the Ramachandran diagram, (D) is the Verify 3D diagram, and (E) is the three-dimensional evaluation result of TMC4. Figure 2 This is a statistical result of the amino acid sites of the peptides docking with the TMC4 receptor protein in the embodiments of the present invention; Figure 3 This is a diagram showing the 2D and 3D interactions between four peptides and the TMC4 salty taste receptor protein molecule in an embodiment of the present invention. Figure 4 The frequency of amino acid sites at which the four peptides of this invention dock with the TMC4 receptor protein; Figure 5 This is a surface force analysis diagram showing the interaction between four peptides and the TMC4 receptor protein in an embodiment of the present invention; Figure 6 The figures show the results of four peptide electronic tongues and sensory evaluations in the embodiments of the present invention. (A) Salty taste value of electronic tongue for different concentrations of peptides, (B) Salty taste value of electronic tongue for different concentrations of peptides under the synergistic effect of NaCl, (C) Sensory evaluation under the synergistic effect of 0.5 g / L peptide + 3.5 g / L NaCl, (D) Response characteristics of electronic tongue for 0.8 g / L peptide, and (E) Response characteristics of electronic tongue under the synergistic effect of 0.8 g / L peptide + 3.2 g / L NaCl. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here. Example 1

[0018] Constructing a TMC4 receptor protein model The amino acid sequence of TMC4 (NP_001138775) was obtained from the NCBI database. Preliminary modeling using Swiss-MODEL showed low consistency (22.94%) among homologous protein models, lower than the 30% required for reliable models. Therefore, the deep learning-based trRosetta method was used for prediction. Through internal scoring and selection of the generated multiple models, we obtained the TMC4 receptor model with the highest confidence. The final 3D receptor protein model is shown below. Figure 1 (A)

[0019] Reliability evaluation of the TMC4 receptor protein model Ramachandran diagrams assess the chemical plausibility and stability of structural models by visualizing the distribution of dihedral angles (φ / ψ) along the protein backbone. Their core function is to analyze the distribution of allowed and disallowed regions. Based on Ramachandran... Figure 1 (C) shows that 99.8% of the amino acid residues in the TMC4 receptor model are located in the allowed regions. Among these, 94.3% are located in the most favorable regions, 5.2% in the additional allowed regions, and 0.3% in the loosely allowed regions. Only 0.2% are located in the forbidden regions, indicating that this protein structure model has high reliability and is suitable for further research. ERRAT evaluated the rationality of the three-dimensional structure of the TMC4 model by statistically analyzing the hydrophobic stacking of nonpolar side chains. Figure 1 (D) shows an overall quality score of 97.08%, significantly higher than the 95% threshold for high-quality models, indicating that the model possesses excellent stereochemical rationality and a stable hydrophobic core structure. The DeepTMHMM online tool was used to predict the transmembrane structure of the TMC4 protein, showing that the TMC4 receptor protein contains 10 transmembrane domains. Figure 1 B). Finally, the constructed 3D model of the TMC4 receptor was validated using SAVE 6.1. Figure 1 E). In summary, these results validate the reliability of the TMC4 protein model as a salty taste receptor.

[0020] Computer simulation of enzymatic hydrolysis of pea protein The pea 7S globulin sequence and the A and B chains of 11S globulin were obtained from the Uniprot database, accession numbers P02854, P02857, and P05693, respectively. Using the BIOPEP-UWM online website, the “ENZYME(S) ACTION” option was selected, and gamma-glutamyl endopeptidase (EC 3.4.21.19) and subtilisin (EC 3.4.21.62) were chosen to perform virtual enzymatic digestion on the obtained protein sequences, yielding a total of 230 peptides. Generally, good biological activity is key to peptide research. A higher PeptideRanker score indicates greater peptide biological activity; typically, a score of 0.5 or higher is considered an active peptide. The PeptideRanker online tool was used to predict peptide biological activity, selecting peptides with scores greater than 0.5. Strong hydrophilicity is beneficial for flavor release, while good water solubility, biocompatibility, and non-toxicity are crucial for the normal metabolism of peptides in the body. ToxinPred is specifically designed for predicting the toxicity of peptide compounds. This tool provides crucial support for the rational design of low-toxicity peptide drugs and the assessment of the safety of biologics. By evaluating the toxicological and physicochemical properties of peptides and cross-referencing amino acid sequences in databases and literature, we removed duplicate and published peptides. Considering the high cost and complexity of long-chain peptide synthesis, we selected 2-8 peptides for analysis. The final results are shown in Table 1, yielding 21 highly bioactive, non-toxic, and water-soluble peptides, including 2 dipeptides, 8 tripeptides, 4 tetrapeptides, 3 pentapeptides, 3 hexapeptides, and 1 heptapeptide.

[0021] Table 1. Prediction of bioactivity and analysis of physicochemical properties of peptides ; Screening of potential salty-tasting peptides To gain a deeper understanding of the interaction mechanism between the peptide and the TMC4 salty taste receptor protein model, The TMC4 receptor was added to the DS software, and solvent water molecules were removed and hydrogen atoms were added under the CHARMM force field. The constructed peptides were docked into the active cavity of the TMC4 structure using the CDOCKER tool in Discovery Studio software. The active docking positions were: X=-6.10, Y=17, Z=-31.20, radius 30, and other parameters were set to default values. The CDOCKER molecular docking results are shown in Table 2. All 21 potential salty-enhancing peptides could enter the docking region of the TMC4 receptor and exhibited strong interactions, with docking energies ranging from -99.40 kcal / mol to -30.79 kcal / mol. Compared to shorter peptide chains, longer peptide chains had lower docking energies. The average docking energies for 2-4 amino acid peptides and 5-7 amino acid peptides were -55.42 kcal / mol and -88.33 kcal / mol, respectively. Twenty-one peptides bound to the TMC4 receptor protein, with a total of 20 amino acid residues interacting with the peptides. The most frequently occurring amino acid residues included: Arg168, Arg272, His271, Leu139, Leu172, Phe169, and Ser165. The results are as follows... Figure 2 As shown. Typically, salt-enhancing peptides are low-molecular-weight, short peptide compounds that enhance the perception of saltiness and umami in food. The flavor characteristics of peptides are influenced by their amino acid composition, sequence, and spatial structure. Even with the same composition, sequence differences can alter their conformation, surface properties, and interaction with taste receptors, leading to significant differences in flavor characteristics. Among peptides that successfully docked with TMC4, arginine (R), phenylalanine (F), and glycine (G) are the three most core and crucial amino acids, and these peptides typically exhibit a strong salty taste. Considering the length of the peptide chain, the composition and distribution of amino acids, we selected four short peptides with relatively low docking energies: DRF, GRRY, VNRF, and NRF for further research. Furthermore, these peptides are not documented in the literature, suggesting they may be undiscovered salt-enhancing peptides.

[0022] Table 2. Molecular docking results of potential salty flavor-enhancing peptides with TMC4 receptor protein. ; Molecular mechanism study of potential salty taste-enhancing peptides Four potential salt-enhancing peptides were subjected to multiple molecular docking experiments with TMC4 to determine the optimal binding posture for each ligand-receptor docking. The 2D and 3D models of the optimal docking postures of the four peptides with TMC4 are shown below. Figure 3As shown, the binding of four peptides (DRF, GRRY, VNRF, and NRF) to the TMC4 receptor is driven by a synergistic effect of hydrogen bonds, electrostatic interactions, C-H bonds, and hydrophobic interactions. All four peptides form electrostatic interactions with Arg272 and Arg168. GRRY and VNRF also form hydrogen bonds with Glu161, NRF with Ser275 and His271, and DRF with Leu142, Leu139, Gln143, and Arg140, forming a hydrogen bond network. The main active sites include residues such as Arg272, Arg168, Glu161, Ser165, Leu142, Leu139, Ser275, and His271, where hydrogen bonds and electrostatic interactions are dominant. To further elucidate the interactions between these four peptides and the TMC4 receptor protein, we analyzed the binding site of the salt-enhancing peptide to TMC4. The results are as follows: Figure 4 As shown, the four peptides dock with the TMC4 receptor protein, with the main binding sites being Arg140, Arg168, Arg272, Gln143, Leu139, Leu142, Lys136, Phe169, Glu161, Leu172, Pef169, Ser165, Ser275, and His271. Among these, Arg168, Arg272, and Leu139 and Leu172 appear most frequently and are key amino acid residues. Figure 5 For the six major surface interactions between the four peptides and TMC4, we found that the laterally stacked aromatic interactions were significantly stronger than the π electron density on the facet rings. All four peptides formed strong hydrogen bonds with TMC4, consistent with the results of energy interaction analysis. We also observed strong hydrophilicity and a high solvent-accessible surface area (SAS) of the peptides, possibly due to the presence of van der Waals forces.

[0023] In summary, we can demonstrate that DRF, GRRY, VNRF, and NRF are potential saltiness-enhancing peptides. Subsequently, these peptides were synthesized and characterized to investigate their flavor profiles.

[0024] Solid-phase synthesis of peptides Nanjing Peptide Valley Biotechnology Co., Ltd. was commissioned to synthesize peptides using solid-phase synthesis.

[0025] Electronic tongue analysis of synthetic peptides To further explore the flavor characteristics of potential salt-enhancing peptides, the peptides were prepared into solutions of different concentrations, and their basic flavor characteristics were measured. Then, the peptide solutions of different concentrations were mixed with NaCl solution to prepare a series of mixed solutions with a concentration of 4 g / L, and their flavor was analyzed using an electronic tongue. Figure 6(A) shows the electronic tongue saltiness values ​​of peptides at different concentrations. When the peptide concentration is 0.2 g / L, the saltiness values ​​of DRF and GRRY are 0.48 and 2.65, respectively, while the saltiness values ​​of VNRF and NRF are negative, at -1.27 and -2.25. With increasing peptide concentration, the saltiness values ​​of all four peptides increase significantly, showing a concentration-dependent relationship. When the peptide concentration is 0.8 g / L, the saltiness values ​​of DRF, GRRY, VNRF, and NRF are 3.47, 3.77, 2.15, and 2.75, respectively. GRRY has the highest saltiness value and exhibits the highest saltiness value at all concentrations. Besides saltiness, peptides themselves also possess other tastes, such as... Figure 6 As shown in (D), all four polypeptides exhibit high levels of saltiness, umami, and rich flavor, but are accompanied by a strong bitterness, possibly due to the presence of hydrophobic amino acids. VNRF has the highest bitterness and lowest saltiness, possibly because it contains two hydrophobic amino acids, valine and phenylalanine, which result in a prominent bitterness that masks or inhibits the saltiness, thus reducing the saltiness of the solution. The synergistic saltiness-enhancing effect of different concentrations of polypeptides in the NaCl complex system is shown in Figure [Figure number missing]. Figure 6As shown in (B), compared with 0.4% NaCl, all peptides showed varying degrees of saltiness enhancement with increasing concentration. Except for VNRF, which showed no significant saltiness enhancement at a concentration of 0.2 g / L, the saltiness values ​​of the other peptides combined with NaCl were significantly higher than those of the control group 0.4% NaCl. Specifically, when the concentrations of peptides DRF, GRRY, VNRF, and NRF were 0.2 g / L, their saltiness values ​​in a 3.8 g / L NaCl solution were 4.40, 4.72, 4.21, and 4.27, respectively, equivalent to replacing approximately 4.6%, 12.21%, 0.24%, and 1.67% of NaCl, respectively. When the concentrations of the four peptides were 0.5 g / L, their saltiness values ​​in a 3.5 g / L NaCl solution were 6.69, 8.61, 5.32, and 6.27, respectively, equivalent to replacing approximately 59.16%, 104.92%, 26.49%, and 49.09% of NaCl, respectively. When the concentrations of the four peptides were 0.8 g / L, their saltiness values ​​in a 3.2 g / L NaCl solution were... The saltiness values ​​of the NaCl solutions at concentrations of 4.0 g / L were 7.35, 7.95, 4.47, and 6.12, respectively, equivalent to replacing approximately 74.86%, 89.22%, 6.34%, and 45.60% of the NaCl, respectively. Compared with the 4.0 g / L NaCl control group, GRRY showed the most significant saltiness enhancement effect, followed by DRF, while VNRF had the weakest enhancement effect. When the concentrations of peptides GRRY, VNRF, and NRF were 0.8 g / L combined with NaCl, the saltiness value was lower than that at a peptide concentration of 0.5 g / L. This may be because the increased peptide concentration enhances the interaction between hydrophobic residues in the peptide chain, increasing the bitterness value of the solution and masking the saltiness, thus weakening the enhancement effect on saltiness perception. The electronic tongue characteristics of the peptides combined with NaCl are as follows: Figure 6 As shown in (E), the saltiness, umami, and richness of the peptides after mixing with NaCl were significantly higher than those in the control group with 0.4% NaCl. The significant enhancement of umami, synergistically with the saltiness, provided a richer and fuller flavor profile. Regarding the saltiness-enhancing effect, the synergistic saltiness-enhancing ability of the peptides on NaCl, from largest to smallest, was GRRY, DRF, NRF, and VNRF.

[0026] Sensory evaluation The sensory evaluation team consisted of 16 laboratory personnel to ensure a sufficient sample size and statistically significant results. To ensure the breadth and representativeness of the results, we selected 8 women and 8 men, aged 20 to 30 years. All participants gave informed consent to participate in the sensory evaluation study and received sensory training, including training in taste and olfactory sensitivity. The saltiness, umami, and richness values ​​of the 0.4% NaCl solution were set to 4. Synthetic peptides were dissolved in a 0.32% NaCl solution to prepare a 4 g / L mixed solution. The entire sensory evaluation process was conducted in the sensory evaluation room, with the temperature controlled at 25 ± 2℃ and the humidity at 60%. To avoid fatigue and residual effects that might interfere with the experimental results, team members were required to rinse their mouths with purified water and rest for 2 minutes before evaluating different samples. Figure 6 (B) It can be seen that when the polypeptide concentration is 0.5 g / L and forms a complex system with 3.5 g / L NaCl, the saltiness enhancement rate reaches its maximum value. Therefore, this concentration was chosen for sensory evaluation, and the results are as follows: Figure 6 (C) shows that the sensory scores for saltiness of the four peptides combined with NaCl were significantly higher than those of the control group with 0.4% NaCl, namely: DRF (7.20), GRRY (7.61), VNRF (5.50), and NRF (6.26). Peptides GRRY and DRF showed the best saltiness enhancement effect. The sensory scores for umami and richness of the four peptides were consistent with the trend of saltiness score. The umami scores of peptides GRRY and DRF were 5.8 and 5.21, and the richness scores were 5.53 and 5.15, respectively. In summary, the sensory evaluation of the four peptides combined with NaCl was consistent with the results of the electronic tongue, and peptides GRRY and DRF had the most significant saltiness enhancement effect. In conclusion, the development of novel flavor substances by combining saltiness-enhancing peptides with NaCl can effectively reduce NaCl concentration without reducing the saltiness intensity. + Intake.

[0027] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pea-derived salty-enhancing peptide, characterized in that: The salty-enhancing peptide is screened from pea protein and binds to the TMC4 receptor protein. The salty-enhancing peptide is composed of one of the following peptides: 1) amino acid sequence DRF, molecular weight 436.46 Da; 2) amino acid sequence GRRY, molecular weight 550.61 Da; 3) amino acid sequence VNRF, molecular weight 534.61 Da; 4) amino acid sequence NRF, molecular weight 435.48 Da. Alternatively, it may be a derived peptide with more than 90% homology to the above sequences and retaining the salty-enhancing activity.

2. The four salty-tasting enhanced peptides according to claim 1, characterized in that, The peptides with the amino acid sequences DRF, GRRY, VNRF, and NRF can dock with the TMC4 receptor via molecular docking. The main binding sites are Arg140, Arg168, Arg272, Gln143, Leu139, Leu142, Lys136, Phe169, Glu161, Leu172, Ser165, Ser275, and His271. Among these, Arg168, Arg272, and Leu139 and Leu172 have the highest frequency and are key amino acid residues.

3. The saltiness-enhancing peptide according to claim 1, characterized in that, The saltiness-enhancing peptide exhibits saltiness-enhancing activity in NaCl solutions at concentrations of 0.2 g / L to 0.8 g / L, ranging from 3.2 g / L to 3.8 g / L.

4. A method for screening salty-tasting peptides, characterized in that, Includes the following steps: (1) Construct a 3D structural model of the TMC4 salty taste receptor protein and evaluate its rationality; (2) Perform simulated enzymatic digestion on the amino acid sequence derived from plant protein to obtain a short peptide library; (3) Perform activity prediction, toxicity and water solubility screening on the short peptide library to obtain candidate active peptides; (4) Perform molecular docking between the TMC4 salty taste receptor protein model and the candidate active peptides, and screen for target peptides with potential salt-enhancing effects based on docking energy; (5) Synthesize the target peptides and verify their salt-enhancing activity through sensory evaluation and / or electronic tongue analysis.

5. The application of the saltiness-enhancing peptide according to any one of claims 1-3 in the preparation of seasonings and food.

6. A product containing the salty-enhancing peptide screened from pea protein as described in any one of claims 1-3.

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