A salty taste enhancing peptide and its screening method, application and product

Abstract

The present invention discloses a salty taste-enhancing peptide, its screening method, application, and product, belonging to the field of food seasoning technology. This salty taste-enhancing peptide was screened from soy protein and binds to the TMC4 receptor protein. The salty taste-enhancing peptide is composed of one of the following peptides: DIF with an amino acid sequence and a molecular weight of 393.47 Da; QPR with an amino acid sequence and a molecular weight of 399.48 Da; MMR with an amino acid sequence and a molecular weight of 436.62 Da; and DPIY with an amino acid sequence and a molecular weight of 506.60 Da. Based on molecular docking results, four peptide segments, DIF, QPR, MMR, and DPIY, were screened and identified as potentially salty taste-enhancing peptides, each with a molecular weight less than 1000 Da. Sensory evaluation and electronic tongue analysis demonstrated that all four peptide segments exhibited a significant saltiness-enhancing effect and could be applied to food seasonings to address the issue of reducing salt without reducing saltiness. This invention provides a theoretical basis for discovering new sources of salty taste-enhancing peptides from soybeans and developing nutritious and healthy salty taste-enhancing peptides.

Description

Technical Field

[0001] The present invention relates to the technical field of food seasonings, and in particular to a salty taste enhancing peptide screened from soybean protein and capable of docking with a salty taste receptor TMC4, a screening method thereof, applications thereof, and a product containing the salty taste enhancing peptide. Background Art

[0002] Table salt (NaCl) is an important source of saltiness in our daily diet, giving food a pleasant flavor. However, excessive intake can lead to the development of related diseases such as hypertension and cardiovascular disease. Therefore, finding ways to "reduce salt without reducing saltiness" without changing the original sensory quality of the food has received widespread attention worldwide, and the development of salt substitutes has become a research hotspot. Salty peptides and salt-increasing peptides, as salt substitutes, have attracted much attention due to their advantages such as natural safety, wide availability, low taste threshold, and rich nutrition. Currently, traditional research on salty peptides mainly involves preparing them from natural foods through methods such as biosynthesis, fermentation, hydrolysis, enzymatic hydrolysis, and Maillard reaction. Although this method is relatively mature, it still has certain limitations in terms of efficiency, cost, and quality. Therefore, there is an urgent need to explore a scientific and effective screening method.

[0003] Compared to traditional peptide preparation and identification methods, virtual screening technology accurately identifies ideal bioactive compounds through computer simulation and prediction of interactions between biomolecules. This integration of computer technology and traditional methods significantly improves screening efficiency. The TMC4 receptor protein is a newly discovered voltage-dependent chloride channel. Studies in mice have shown that it is involved in the perception of high salt concentrations and exhibits a high-salt effect at low salt concentrations. Therefore, as a new salt receptor, it provides a new avenue for the exploration of salty-enhancing peptides. Soy protein is primarily composed of β-conglycinin (7S) and globulin (11S), accounting for 80-90% of the total soy protein content. Enzymatic hydrolysis of soy protein yields a large number of short peptides, many of which have been identified as associated with nutritional functions such as lipid-lowering, glucose-lowering, and antioxidant properties. However, limited research has been conducted on the use of virtual screening to identify salty-enhancing peptides from soy protein. Therefore, this study employed virtual screening combined with sensory evaluation to identify and screen salty-enhancing peptides.

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

[0005] In view of this, the present invention provides a salty taste enhancing peptide and a screening method, application and product thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a salty taste enhancing peptide, which is screened from soybean protein and binds to TMC4 receptor protein. The salty taste enhancing peptide is composed of one of the following peptides:

[0008] The amino acid sequence is DIF, and the molecular weight is 393.47 Da;

[0009] The amino acid sequence is QPR, and the molecular weight is 399.48Da;

[0010] The amino acid sequence is MMR, and the molecular weight is 436.62 Da;

[0011] The amino acid sequence is DPIY and the molecular weight is 506.60Da.

[0012] Furthermore, in the salty taste enhancing peptide, the peptide segment with the amino acid sequence of DIF undergoes molecular docking with the receptor TMC4, and the main binding sites are Ala158, Gln279, Thr280, His293, Ala283, Glu286, Ala584 and Ser585.

[0013] Furthermore, in the salty taste enhancing peptide, the peptide segment with the amino acid sequence QPR can be docked with the salty taste enhancing peptide TMC4 receptor based on molecular docking, and the main binding sites are Ala283, His293, Glu319, Glu323, Ala584 and Ser585.

[0014] Furthermore, among the salty taste enhancing peptides, the peptide segment with the amino acid sequence MMR can be docked with the salty taste enhancing peptide TMC4 receptor based on molecular docking, and the main binding sites are Gln279, Glu286, His293, Glu319, Arg583, Ser585 and Asn588.

[0015] Furthermore, in the salty taste enhancing peptide, the peptide segment with the amino acid sequence DPIY can be docked with the salty taste enhancing peptide TMC4 receptor based on molecular docking, and the main binding sites are Ala158, Ala283, Glu286, His293, Glu319, Val322, Thr581, Phe582, Arg583, Ala584 and Ser585.

[0016] Furthermore, the saltiness value of the peptide segment with the amino acid sequence of DIF is 3.24;

[0017] and / or, the saltiness value of the peptide segment having the amino acid sequence QPR is 3.79;

[0018] and / or, the saltiness value of the peptide segment having the amino acid sequence of MMR is 4.00;

[0019] And / or, the saltiness value of the peptide segment having the amino acid sequence DPIY is 3.97.

[0020] The present invention provides a method for screening salty taste enhancing peptides, characterized by the following steps:

[0021] (1) Construct a TMC4 receptor protein model using the trRosetta online server and evaluate its reliability;

[0022] The reliability evaluation is as follows: according to the Ramachandran diagram, the proportion of amino acid residues falling in the allowed region and the maximum allowed region in the entire protein is required to be greater than 90%;

[0023] (2) Computer simulation of enzymatic hydrolysis of soy protein isolate to obtain enzymatic peptide segments with sequence lengths of 3-10;

[0024] (3) predicting the biological activity of the enzymatic peptides, screening for peptides with potential biological activity, and predicting their physical and chemical properties to screen for non-toxic and water-soluble peptides;

[0025] The screening criteria for biological activity prediction are: prediction score > 0.5;

[0026] The screening criteria for toxicity prediction are: Non Toxin;

[0027] The screening criteria for water solubility prediction are: Good water solubility;

[0028] (4) molecular docking the TMC4 receptor protein model with the non-toxic, water-soluble peptide segment of step (3) to form a receptor protein and small molecule ligand complex;

[0029] Among them, the TMC4 receptor protein needs to remove water molecules and add hydrogen atoms under the CHARMM force field environment;

[0030] The active site coordinates of the molecular docking are: X = -59.1, Y = 77, Z = 72.4, and the radius is 20;

[0031] The molecular docking selects the largest cavity in the TMC4 receptor protein as the docking pocket;

[0032] (5) Screening peptides with potential salt-enhancing effects based on the molecular docking results and synthesizing the peptides by solid-phase synthesis;

[0033] The screening comprises: selecting peptides with low energy values in CDOCKER energy;

[0034] (6) The synthesized peptides were subjected to sensory evaluation and electronic tongue analysis to obtain the salty taste enhancing peptides mentioned above.

[0035] The present invention provides a use of a salty taste enhancing peptide in the preparation of condiments and foods.

[0036] The present invention provides a product containing the salty taste enhancing peptide screened from soybean protein.

[0037] Compared with the existing technology, the beneficial effects achieved by the present invention are as follows: the present invention screened out four peptide segments, DIF, QPR, MMR and DPIY, with a molecular weight of less than 1000Da from the protein in soy protein. Sensory evaluation and electronic tongue analysis showed that these four peptide segments all have a good salt-enhancing effect. These four saltiness-enhancing peptides can be applied to food seasonings to achieve the purpose of "reducing salt" without "reducing saltiness". BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0039] Figure 1 The evaluation diagram of the TMC4 receptor protein model of the embodiment of the present invention, wherein (A) is the TMC4 receptor protein model, (B) is the Ramachandran diagram, (C) is the transmembrane channel region, and (D) is the Verify 3D diagram;

[0040] Figure 2 The statistical results of the amino acid sites of the peptide segments of the present invention docking with the TMC4 receptor protein;

[0041] Figure 3 Figures 2D and 3D interaction diagrams of the peptide DIF of an embodiment of the present invention docking with a TMC4 receptor protein model molecule, where A represents a 3D view of the peptide binding surface groove of the TMC4 receptor; B represents a 3D view of the peptide docking with the TMC4 receptor; and C represents a 2D view of the peptide docking with the TMC4 receptor.

[0042] Figure 4 Figures 2D and 3D interaction diagrams of the docking of the peptide QPR and the TMC4 receptor protein model molecule according to an embodiment of the present invention, wherein A represents the 3D view of the binding surface groove of the peptide and the TMC4 receptor; B represents the 3D view of the docking of the peptide and the TMC4 receptor; and C represents the 2D view of the docking of the peptide and the TMC4 receptor.

[0043] Figure 5Figures 2D and 3D interaction diagrams of the docking of the peptide MMR and the TMC4 receptor protein model molecule according to an embodiment of the present invention, wherein A represents a 3D view of the binding surface groove of the peptide and the TMC4 receptor; B represents a 3D view of the docking of the peptide and the TMC4 receptor; and C represents a 2D view of the docking of the peptide and the TMC4 receptor.

[0044] Figure 6 Figures 2D and 3D interaction diagrams of the docking of the peptide DPIY and the TMC4 receptor protein model molecule according to an embodiment of the present invention, wherein A represents a 3D view of the binding surface groove of the peptide and the TMC4 receptor; B represents a 3D view of the docking of the peptide and the TMC4 receptor; and C represents a 2D view of the docking of the peptide and the TMC4 receptor.

[0045] Figure 7 This is a diagram showing the sensory evaluation results of an embodiment of the present invention;

[0046] Figure 8 This is a diagram showing the results of electronic tongue analysis according to an embodiment of the present invention;

[0047] Figure 9 This is a diagram of the salinity value analysis results of an embodiment of the present invention. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0050] Example 1

[0051] Construction of TMC4 receptor protein model

[0052] The amino acid sequence of TMC4 (NP_001138775) was obtained from the NCBI database and searched using the NCBI BLAST tool online. A TMC4 amino acid sequence with a homology of less than 30% was found. Therefore, a TMC4 salty taste receptor protein model was constructed using the trRosetta online server using an AI de novo folding algorithm. The final 3D model is shown in Figure 2. Figure 1 (A).

[0053] Reliability evaluation of TMC4 receptor protein model

[0054] The constructed TMC4 salty taste receptor model was evaluated using the residual percentage of the Ramachandran plot and ERRAT in SEVES v6.0. Figure 1 As shown in (B), about 92.7% of the amino acid residues are located in the largest allowed region, 6.7% of the amino acid residues are located in other allowed regions, only 0.2% of the amino acid residues are located in the disallowed region, and 0.5% of the residues are located in the prohibited region. This shows that the model constructed by the AI de novo folding algorithm has high reliability and can be used for subsequent research. The transmembrane domain of TMC4 was predicted using the DeepTMHMM online tool. Figure 1 As shown in (C), there are 10 transmembrane regions in the protein structure of the invention. The ERRAT analysis results are as follows Figure 1 As shown in (D), the overall quality factor is 98.102. The above results indicate that the TMC4 receptor protein model constructed in the present invention can be used for subsequent experiments.

[0055] Computer simulation of enzymatic hydrolysis of soybean protein

[0056] The soybean 7S globulin protein sequences (α-conglycinin α (alpha-subunit), α' (alpha prime subunit), and β (beta-subunit) subunits, accession numbers BAB64304.1, BAB64303.1, and BAB64306.1, respectively) were obtained from the NCBI GenBank database. The obtained protein sequences were virtually hydrolyzed using alkaline protease (EC 3.4.21.62) and trypsin (EC 3.4.21.4) using the "ENZYME(S)ACTION" program in BIOPEP-UWM. A total of 208 peptides were generated. Peptides with sequence lengths of 2-10 were selected from these peptides for bioactivity prediction using the Peptide Ranker website. Each peptide's amino acid sequence was input and assigned a corresponding bioactivity score. A higher score indicates greater bioactivity. Generally, a peptide with a bioactivity score of 0.5 or higher was considered active. A total of 33 peptides with potential bioactivity were screened, as shown in Table 1. The solubility of peptides in water was analyzed using the online website Innovagen, which categorizes peptide solubility into "Good water solubility" and "Poor water solubility." The toxicity of active peptides was predicted using the online website ToxinPred. The results, shown in Table 2, indicate that all 13 peptides were nontoxic and well-soluble in water.

[0057] Table 1 PeptideRanker prediction scores of potential bioactive peptides

[0058]

[0059] Table 2 Predicted toxicity and physicochemical properties of 13 peptides

[0060]

[0061] Molecular docking

[0062] To further understand the interaction mechanism between 13 potential salt-enhancing peptides screened from soy protein and the TMC4 receptor protein model, the TMC4 receptor was added to the DS software. Under the CHARMM force field environment, solvent water molecules were removed and hydrogen atoms were added. The constructed peptides were docked into the active cavity of the TMC4 structure using CDOCKER within Discovery Studio software. The active position for docking was set at: X = -59.1, Y = 77, Z = 72.4, with a radius of 30, and all other parameters were left to default. Molecular docking results using CDOCKER showed that all 13 potential salt-enhancing peptides were able to bind to the TMC4 cavity pocket and form stable complexes. Figure 2 Thirteen potential salt-enhancing peptides interact with binding sites around the TMC4 subunit, totaling 19 binding sites. Dipeptides (MR, PR, GR, and EF) primarily bind to the TMC4 receptor through Arg294, Glu319, Glu323, Arg583, and Ser585, with Glu319 appearing most frequently. Tripeptides (MMR, PGR, DIF, DDF, QPR, and PGS) primarily bind to the receptor through Glu286, His293, Glu319, Ala584, and Ser585, with Ser585 forming multiple hydrogen bonds with the ligand. Pentapeptide (NPQPR) and octapeptide (PHHADADF) primarily bind through hydrogen bonds via Ala283, Arg294, Ala286, Arg294, and Arg583. Glu286, His293, Glu319, and Ser585 appeared frequently during docking, suggesting they are potential key receptor binding sites. The docking results of 14 potential salt-enhancing peptides with TMC4 are shown in Table 3. Generally, lower CDOCKER energy values indicate stronger binding ability and higher affinity of the peptide to the TMC4 receptor, and potentially higher flavor intensity. Therefore, based on the molecular docking results of peptides with TMC4 and the amino acid structures, DIF, QPR, MMR, and DPIY were selected for subsequent research on salty taste-enhancing peptides.

[0063] Table 3 Molecular docking results of potential salt-enhancing peptides and TMC4 receptor protein

[0064]

[0065] Interaction mechanism of molecular docking

[0066] Interaction of four peptides with TMC4 salty taste receptor protein: Figure 3 As shown, the tripeptide DIF mainly interacts through hydrogen bonds and hydrophobic interactions, among which Thr280, Ala283, and Ser585 bind to carbon-hydrogen bonds, form hydrogen bonds with Gln279, and interact with Ala158, His293, and Ala584 in the receptor through hydrophobic interactions. Figure 4 As shown in Figure 3, QPR mainly interacts with Ala283, His293, Ala584, and Ser585 amino acid residues through hydrogen bonds. Figure 5 As shown in the figure, the interaction between MMR and TMC4 receptor protein is mainly through forming hydrogen bonds with the receptor through Glu319, Glu286, Ser584, Asn588 and Gln279. In addition to forming hydrogen bonds with the ligand, Glu286 and Glu319 also have electrostatic interactions. Figure 6 As shown, the interaction between the ligand DPIY and the TMC4 receptor protein is primarily based on hydrogen bonds and hydrophobic interactions. It forms hydrogen bonds with His293, Thr581, Ala584, and Ser585, while Ala158, Ala283, Val322, Glu319, Arg583, and Phe582 interact with the receptor through hydrophobic interactions. During the docking process described above, we found that conventional hydrogen bonds, carbon-hydrogen bonds, electrostatic interactions, and hydrophobic interactions are the primary forces stabilizing ligand-receptor binding, with hydrogen bonds and hydrophobic interactions being the primary factors.

[0067] Solid-phase peptide synthesis

[0068] Nanjing Peptide Valley Biotechnology Co., Ltd. was commissioned to synthesize peptides through solid-phase synthesis.

[0069] Sensory evaluation

[0070] The sensory evaluation panel consisted of 12 laboratory members to ensure sufficient sample size and statistically significant results. To ensure broad and representative results, eight of the panelists were female and four were male, aged between 20 and 30 years. The panelists were in good physical condition, had good tastes, and no unhealthy habits. They underwent sensory training according to ISO 8586-1:2012 to enable them to judge the concentrations of five basic flavor solutions. The flavor reference solutions were prepared as follows: 0.08% (w / v) quinine sulfate solution (bitter), 0.08% (w / v) citric acid solution (sour), 0.40% (w / v) sodium chloride solution (salty), 0.35% (w / v) monosodium glutamate solution (umami), and 1.00% (w / v) sucrose solution (sweet). These samples were sensory evaluated on a scale of 0–10, with 0 representing no flavor and 10 representing the strongest flavor. Five flavor intensity sample solutions were prepared, with the salt content of 0.4% (w / v) NaCl solution being assigned a score of 5. For every 0.1% (w / v) increase or decrease in concentration, the saltiness score increased or decreased by 2 points. Panelists were asked to rinse their mouths with 30-40 ml of ultrapure water for 5 minutes between testing two different samples. The synthetic peptides were dissolved in 0.4% (w / v) sodium chloride solution to prepare a 0.02% (w / v) solution, and then different panelists scored and recorded the five flavors in turn. The results showed that all four peptides had a salt-enhancing effect, such as Figure 7 As shown in the results, MMR has the most significant salt-enhancing effect. However, because the results of sensory evaluation are greatly influenced by subjective factors, they cannot be used as a final conclusion and require further verification using an electronic tongue.

[0071] Electronic tongue analysis

[0072] Dissolve the four synthetic peptides DIF, QPR, MMR, and DPIY in deionized water. After the solution is completely dissolved, pour it into a beaker dedicated to the electronic tongue to identify the taste characteristics of the synthetic peptides. Using 0.4% (w / w) sodium chloride solution as a control, the four peptides were added to 0.4% (w / v) sodium chloride solution to prepare 0.02% (w / v) solutions to compare the saltiness of the peptides. The results are as follows: Figure 8 As shown. It can be seen that saltiness is the main factor affecting the overall flavor of the peptide, followed by freshness, while sourness has the weakest response (p<0.05). The significant difference in acidity compared to sodium chloride solution may be related to the composition of solubility. In terms of increasing the saltiness level, MMR and DPIY have the most significant effects, while DIF has the worst effect. All solutions show obvious umami and have a certain synergistic effect with saltiness and umami. The saltiness enhancement effect of the four peptides is shown as follows Figure 9As shown. Typically, when the electronic tongue exhibits a high negative response to a substance, it indicates a relatively low sensitivity to that flavor or chemical, or a weak flavor attribute. Compared to a 0.4% sodium chloride solution, the saltiness values of each peptide solution at a 0.02% (w / w) concentration were negative, indicating that the four peptides themselves lack saltiness. However, when mixed with a 0.4% sodium chloride solution, the 0.02% (w / w) concentration of the four peptides all exhibited a significant salt enhancement effect. MMR had the strongest salt enhancement ability, increasing salt by 40.35%, followed by DPIY (39.30%) and QPR (32.98%), while DIF (13.68%) had the weakest salt enhancement effect (p < 0.05). The saltiness values are generally consistent with the molecular docking results mentioned above. The lower the docking energy of the peptide binding to the TMC4 receptor, the more stable the complex conformation and the stronger the salt enhancement effect. This is generally consistent with the results of the sensory evaluation analysis. Therefore, it can be proved that DIF, QPR, MMR and DPIY have significant saltiness-enhancing effects and can be used as new saltiness-enhancing peptides in soy protein.

[0073] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only 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 in the scope of protection of the present invention.

Claims

1. Use of a salty taste enhancing peptide in the preparation of condiments and foods, characterized in that: The salty taste enhancing peptide is screened from soybean protein and binds to TMC4 receptor protein. The salty taste enhancing peptide is composed of one of the following peptides: The amino acid sequence is DIF, and the molecular weight is 393.47 Da; The amino acid sequence is QPR, and the molecular weight is 399.48Da; The amino acid sequence is MMR, and the molecular weight is 436.62 Da; The amino acid sequence is DPIY and the molecular weight is 506.60Da.

2. The use of a salty taste enhancing peptide according to claim 1 in preparing condiments and foods, characterized in that: In the salty taste enhancing peptide, the peptide segment with the amino acid sequence of DIF undergoes molecular docking with the receptor TMC4, and the main binding sites are Ala158, Gln279, Thr280, His293, Ala283, Glu286, Ala584 and Ser585.

3. The use of a salty taste enhancing peptide according to claim 1 in preparing condiments and foods, characterized in that: In the salty taste enhancing peptide, the peptide segment with the amino acid sequence of QPR undergoes molecular docking with the receptor TMC4, and the main binding sites are Ala283, His293, Glu319, Glu323, Ala584 and Ser585.

4. The use of a salty taste enhancing peptide according to claim 1 in preparing condiments and foods, characterized in that: In the salty taste enhancing peptide, the peptide segment with the amino acid sequence of MMR undergoes molecular docking with the receptor TMC4, and the main binding sites are Gln279, Glu286, His293, Glu319, Arg583, Ser585 and Asn588.

5. The use of a salty taste enhancing peptide according to claim 1 in preparing condiments and foods, characterized in that: In the salty taste enhancing peptide, the peptide segment with the amino acid sequence DPIY undergoes molecular docking with the receptor TMC4, and the main binding sites are Ala158, Ala283, Glu286, His293, Glu319, Thr581, Phe582, Val322, Arg583, Ala584 and Ser585.

Images