A method for screening bifunctional peanut peptides with salty taste enhancement and antioxidant activity, applications and products

Abstract

The application discloses a salty taste enhancing peptide screened from peanut protein and a screening method thereof, and belongs to the technical field of food condiments. According to the molecular docking result, four peptide segments, SPDIY, DPSPR, QPGDY and SPPGER, with potential salty taste enhancing effect 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 effect, can be applied to the field of food condiments and solve the purpose of'reducing salt without reducing salty taste'. The peptide segment with the amino acid sequence of QPGDY is a bifunctional active peptide, has in-vitro antioxidant activity while reducing salt. The application provides a theoretical basis for discovering new salty taste enhancing peptide resources in peanuts 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 salt-enhancing peptide screened from peanut protein that can dock with the salt-taste receptor TMC4 and its application, while also possessing antioxidant activity. 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. The World Health Organization aims to reduce global daily salt intake by 30% by 2025. Therefore, how to "reduce salt without reducing saltiness" without compromising the sensory quality of food has received widespread attention worldwide.

[0003] Salt-enhancing peptides have attracted much 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 with 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. Peanut protein is an important plant protein, widely used in food and nutritional supplements due to its high nutritional value and strong functionality. However, there are currently no clear classification boundaries for peanut protein. This study primarily employed a systematic allergen nomenclature approach, focusing on the major seed storage proteins in peanuts: Arah1, Ara h2, Arah3, and Ara h6, which together account for 57%-65% of the total peanut protein content. Furthermore, we also included conaragenin A and conaragenin B in our study. Therefore, using peanut protein as raw material, we employed high-throughput virtual screening technology to rapidly screen for potential salt-enhancing peptides.

[0004] Based on this, the present invention aims to use virtual screening technology to screen out novel salt-enhancing peptides from peanut 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 peanut peptides that simultaneously enhance saltiness and have antioxidant properties.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a salty-tasting enhanced peptide that also has antioxidant effects. The peptide is screened from peanut protein and binds to the TMC4 receptor protein. The peptide is composed of one of the following:

[0008] The amino acid sequence is SPDIY, and the molecular weight is 593.63 Da;

[0009] The amino acid sequence is DPSPR, and the molecular weight is 570.6 Da;

[0010] The amino acid sequence is QPGDY, and the molecular weight is 578.57 Da;

[0011] The amino acid sequence is SPPGER, and the molecular weight is 641.67 Da.

[0012] Furthermore, the peptides with the amino acid sequences SPDIY, QPGDY, and DPSPR can dock with the TMC4 receptor via molecular docking, with the main binding sites being Lys136, Arg140, Gln143, Ala158, Glu161, Ser162, Ser165, Arg168, Leu268, Arg272, Ser275, and Gln279. Among these, Arg272, Glu161, and Arg168 occur most frequently and are key amino acid residues.

[0013] Furthermore, the peptide segment with the amino acid sequence SPPGER can dock with the TMC4 receptor via molecular docking, with the main binding sites being Gln143, Glu161, Ser162, Arg168, Ser165, Arg272, and Gln279. Among these, Ser165 and Gln279 occur most frequently and are key amino acid residues.

[0014] Furthermore, the amino acid sequence of the SPDIY peptide has a saltiness value of 4.04 and a saltiness enhancement threshold of 0.64 mmol / L, which can replace approximately 6.32% NaCl;

[0015] And / or, the amino acid sequence of the peptide with DPSPR has a saltiness value of 7.1 and a saltiness enhancement threshold of 0.16 mmol / L, and can replace about 87.1% of NaCl;

[0016] And / or, the peptide with the amino acid sequence QPGDY has a saltiness value of 4.42 and a saltiness enhancement threshold of 0.33 mmol / L, and can replace about 16.3% NaCl;

[0017] And / or, the amino acid sequence of the SPPGER peptide has a saltiness value of 4.49 and a saltiness enhancement threshold of 0.30 mmol / L, and can replace about 18.2% NaCl.

[0018] Furthermore, the peptide with the amino acid sequence QPGDY can scavenge at least one of DPPH free radicals and ABTS free radicals, and has antioxidant activity.

[0019] This invention provides a method for screening salty-tasting peptides, characterized by the following steps:

[0020] (1) A TMC4 salty taste receptor protein model was constructed using the AlphFold2 online server, and its reliability was evaluated.

[0021] The reliability evaluation criteria are: pLDDT score > 70; the proportion of amino acid residues falling in the allowed region and the maximum allowed region of the whole protein is required to be higher than 90% for the model to be considered conformational and reliable; according to the Ramachandran diagram, the proportion of amino acid residues falling in the allowed region and the maximum allowed region of the whole protein is required to be higher than 90%;

[0022] (2) Computer simulation of enzymatic hydrolysis of peanut protein to obtain enzymatically hydrolyzed peptides with a sequence length of 4-8;

[0023] (3) The bioactivity of the enzymatically hydrolyzed peptides is predicted, and peptides with potential bioactivity are screened out. The physicochemical properties of the peptides are predicted, and non-toxic and water-soluble peptides are screened out.

[0024] The screening criterion for predicting bioactivity is: prediction score > 0.5;

[0025] The screening criteria for toxicity prediction are: Non-Toxin;

[0026] The screening criteria for water solubility prediction are: Good water solubility;

[0027] (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.

[0028] The TMC4 receptor protein needs to have water molecules removed and hydrogen atoms added in a CHARMM force field environment.

[0029] The coordinates of the active site for molecular docking are: X = -15.25, Y = 11, Z = 7.25, and the radius is 30.

[0030] The molecular docking site coordinates were selected by using the largest cavity in the TMC4 receptor protein as the docking pocket.

[0031] (5) Screen peptides with potential salt-enhancing effects based on molecular docking results, and synthesize peptides by solid-phase synthesis method;

[0032] The screening process involves selecting peptides with low energy values ​​from the CDOCKER energy database.

[0033] (6) Sensory evaluation and electronic tongue analysis were performed on the synthesized peptides to obtain the above-mentioned salty taste-enhancing peptides.

[0034] This invention provides the application of a salty-enhancing peptide in the preparation of seasonings and food.

[0035] The present invention provides a product containing the above-mentioned salty flavor-enhancing peptides screened from peanut protein.

[0036] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention screened four peptides with molecular weights less than 1000 Da—SPDIY, DPSPR, QPGDY, and SPPGER—from peanut protein. Sensory evaluation and electronic tongue analysis showed that all four peptides 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." Among them, the peptide with the amino acid sequence QPGDY is a bifunctional active peptide, which, while reducing salt, also has in vitro antioxidant activity. The new peptide sequences obtained by this invention provide new resources for the development of functional foods. Attached Figure Description

[0037] 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.

[0038] 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 pLDDT score of the modeling result, (c) is the Ramachandran diagram, and (d) is the Verify3D diagram;

[0039] Figure 2 This is a diagram showing the result of implementing the electronic tongue according to the present invention;

[0040] Figure 3 This is a graph showing the results of salinity value analysis in an embodiment of the present invention;

[0041] Figure 4 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;

[0042] Figure 5 The main interaction forces and their frequency of occurrence between the salty-enhancing peptide and TMC4 in the embodiments of the present invention are shown.

[0043] Figure 6 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.

[0044] Figure 7 The DPPH free radical scavenging rates of the four peptides in the embodiments of the present invention;

[0045] Figure 8 The ABTS free radical scavenging rate of the four peptides in the embodiments of the present invention is shown. Detailed Implementation

[0046] 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.

[0047] 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.

[0048] Example 1

[0049] Constructing a TMC4 receptor protein model

[0050] The amino acid sequence of TMC4 (NP_001138775) was obtained from the NCBI database. Using the NCBIBLAST tool online, amino acid sequences with less than 30% homology were identified. Therefore, the TMC4 amino acid sequence was modeled using the AI ​​de novo folding algorithm on the AlphFold2 online server, resulting in a TMC4 salty taste receptor protein model. The final 3D receptor protein model is shown below. Figure 1 (a).

[0051] Reliability evaluation of the TMC4 receptor protein model

[0052] The reliability of the TMC4 receptor protein model was evaluated based on the pLDDT score. A pLDDT score ≥ 90 indicated a high confidence level, while a pLDDT score between 70 and 90 indicated a good confidence level. The scoring results are as follows: Figure 1 As shown in (b), the pLDDT values ​​of the five top-ranked protein models (rank_1, rank_2, rank_3, rank_4, and rank_5) are 78.9, 77.1, 76.2, 74.3, and 72, respectively. Rank_1 has the highest pLDDT value (78.9), therefore, rank_1 was selected as the initial experimental model for further evaluation. The remaining percentage of the Ramachandran plot and ERRAT from SEVES v6.0 were used to evaluate the rank_1 model. The evaluation results are as follows: Figure 1 As shown in (c), approximately 92.7% of the amino acid residues are located in the largest allowed region, 6.7% are located in other allowed regions, only 0.2% are located in disallowed regions, and 0.5% are located in forbidden regions. Therefore, the TMC4 receptor structure constructed in this invention is reasonable, indicating that the model constructed by the AI ​​de novo folding algorithm has high reliability and can be used for subsequent research. Finally, the overall quality of the protein crystal structure was evaluated using the ERRAT method. Figure 1 d) The overall quality factor is 98.102%, indicating that the model is reliable. In conclusion, these results validate the reliability of the TMC4 protein model as a salty taste receptor.

[0053] Computer simulation of enzymatic hydrolysis of peanut protein

[0054] Protein subunits of Ara h1, Ara h2, Ara h3, Ara h6, conarachidin A, and conarachidin B were obtained from the NCBI GenBank database, with accession numbers AAL27476.1, QGA89299.1, ACH91862.1, AAM78596.1, AEL30377.1, and AAU21493.1, respectively. The obtained protein sequences were subjected to simulated enzymatic digestion using pepsin (EC3.4.23.1), trypsin (EC3.4.21.4), and chymotrypsin (EC3,4,21,1) on the ExPASyPeptideCutter online website, yielding a total of 307 peptides. Good biological activity is generally crucial for peptide research. Biological activity prediction was performed using the Peptide Ranker website. Each peptide's amino acid sequence was input, and a corresponding biological activity score was generated. A higher score indicates greater biological activity; typically, a biological activity score of 0.5 or higher is considered a bioactive peptide. Furthermore, non-toxicity is a fundamental requirement for any potential biomacromolecule, and good water solubility helps improve the bioavailability of peptides. The online website Innovagen was used to analyze the water solubility of peptides, categorizing them into "Good water solubility" and "Poor water solubility." The online website Toxin Pred was used to predict the toxicity of bioactive peptides. The final results are shown in Table 1, with a total of 19 highly bioactive, non-toxic, and well-water-soluble peptides identified. By cross-referencing reported amino acid sequences from databases and literature, we avoided the similarity and redundancy of short peptide sequences, while also considering the complexity, time, and cost of synthesizing long peptide chains. Therefore, we selected peptides with 4 to 8 amino acids as the focus of our research. A total of 13 potential salt-enhancing peptides were predicted, including the tripeptides QPDY, QPPR, and GQGR; the pentapeptides QPDGY, SPDIY, DPSPR, GPSPR, and ERPTF; the hexapeptides GPAGPR, SPPER, and GRPCVM; and the heptapeptide QSQRPPR.

[0055] Table 1. Bioactivity prediction and physicochemical property analysis of peptides

[0056]

[0057] Screening of potential salty-tasting peptides

[0058] To gain a deeper understanding of the interaction mechanism between peptides and the TMC4 salt-tasting receptor protein model, the TMC4 receptor was added to the DS software. Solvent water molecules were removed and hydrogen atoms were added under a 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 docking active positions were: X = -15.25, Y = 11, Z = 7.25, radius 30, and other parameters were set to default values. The CDOCKER molecular docking results are shown in Table 2. All 13 potential salt-enhancing peptides could enter the docking region of the TMC4 receptor and exhibited strong interactions. This may be because the TMC4 binding region has an open conformation and a large binding cavity. Notably, the docking energies for 4-5 amino acids with TMC4 ranged from -45.73 kcal / mol to -89.2205 kcal / mol, while the docking energies for 6-7 amino acids ranged from -53.2087 kcal / mol to -79.5969 kcal / mol. The docking energy typically reflects the stability of the ligand-receptor complex binding; a lower docking energy indicates a more stable binding and a higher potential for activity. Therefore, based on the docking energy analysis of 13 peptides, we selected the four peptides with the lowest docking energies—QPGDY, SPDIY, DPSPR, and SPPGER—for further investigation. Furthermore, these peptides were not documented in the literature, suggesting they may be undiscovered saltiness-enhancing peptides. Subsequently, these peptides were synthesized and characterized to investigate their flavor profiles.

[0059] Table 2. Molecular docking results of potential savory peptides with TMC4 receptor protein.

[0060]

[0061] Solid-phase synthesis of peptides

[0062] Nanjing Peptide Valley Biotechnology Co., Ltd. was commissioned to synthesize peptides using solid-phase synthesis.

[0063] Taste characteristics of synthetic peptides and analysis of electronic tongue

[0064] To determine the saltiness intensity of synthetic peptides, the sensory evaluation panel used the taste dilution method to measure the saltiness threshold and then conducted taste descriptions. In ultrapure water, the four synthetic peptides only showed weak tastes and no detectable saltiness. From this, we can conclude that these four peptides do not have saltiness by themselves. However, it is worth noting that when mixed with NaCl solution, they all enhance the saltiness of the mixed solution. Specifically, QPGDY shows saltiness and weak sweetness, SPDIY shows saltiness, weak umami and weak sweetness, DPSPR shows saltiness and weak umami, and SPPGER shows saltiness, sweetness and weak umami. The ranking of the saltiness enhancement thresholds is as follows: DPSPR (0.16 mmol / L) < SPPGER (0.30 mmol / L) < QPGDY (0.33 mmol / L) < SPDIY (0.64 mmol / L). Using a 0.35% NaCl solution as a positive control, the saltiness enhancement effects of the four peptides were analyzed using an electronic tongue. As Figure 2 shown, the overall tastes of the four saltiness-enhancing peptides showed a similar trend. Saltiness was the main taste affecting the overall taste, followed by umami, and sourness was the weakest. The significant differences in acidity values may be related to the solubility and amino acid composition of the peptides. Research has shown that the solubility of peptides affects their dissociation behavior in water, leading to an increase in acidity. In addition, adding acidic residues and acetates during the polypeptide synthesis process is another key factor causing the increase in acidity values. In terms of the saltiness enhancement effect, the saltiness enhancement effect of DPSPR was the most significant, with the salt value increasing by 87.1%, followed by SPPGER (18.2%), QPGDY (16.3%) and SPDIY (6.32%).

[0065] Sensory evaluation

[0066] The sensory evaluation team consisted of 12 laboratory members (8 women and 4 men), aged 20-30, in good physical condition, with good taste and no bad habits. They underwent sensory training according to ISO 8586-1:2012, enabling them to determine the concentrations of five basic flavor solutions. The flavor reference solutions were prepared as follows: 0.08% quinine sulfate solution (bitter), 0.08% citric acid solution (acidic), 0.35% sodium chloride solution (salty), 0.35% monosodium glutamate solution (umami), and 1.00% sucrose solution (sweet). Team members conducted sensory evaluations on a scale of 0-6, where 0 represents no taste and 6 represents the strongest taste. Five reference solutions with different flavor intensities were prepared. The salty sensory evaluation reference solution was a 0.35% NaCl solution, with a salty score of 3 points. For every 0.1% increase or decrease in concentration, the salty score increased or decreased by 1 point. Team members were instructed to rinse their mouths with 30-40 ml of ultrapure water for 5 minutes between tests of two different samples. The synthetic peptides were dissolved in a 0.4% NaCl solution to prepare a 0.02% peptide solution. Different group members then sequentially scored and recorded the five flavor characteristics of the peptide solution. The results showed that all four peptides had a salt-enhancing effect. Sensory evaluation results are as follows: Figure 3 As shown in the figure, compared with the control group, the four peptide solutions at a concentration of 0.02% exhibited a stronger salty flavor profile. Among them, peptide DPSPR had the highest salty intensity (4.7), roughly equivalent to the salty intensity of the 0.45% NaCl solution. These results are largely consistent with those of the electronic tongue. In conclusion, a novel flavoring substance was developed by combining salty-enhancing peptides with NaCl, which can effectively reduce the intake of Na ions without reducing the salt strength.

[0067] Study on the molecular mechanism of salty taste-enhancing peptides

[0068] Four salt-enhancing peptides were subjected to multiple molecular docking experiments with TMC4 to determine the optimal binding posture for each ligand-receptor docking. The docking energies indicated that all four salt-enhancing peptides could enter the active cavity of TMC4 with relatively low energies. The average docking energies of the pentapeptide and hexapeptide were -84.4025 kcal / mol and -79.5969 kcal / mol, respectively, indicating that the hexapeptide binds to TMC4 more readily than the pentapeptide. However, although SPDIY had the lowest docking energy with TMC4, sensory evaluation and electronic tongue analysis showed that DPSPR had the most significant salt-enhancing effect. This suggests that docking energy cannot be used as the sole criterion for evaluating saltiness intensity. To further elucidate the interaction between these four peptides and the TMC4 receptor protein, we analyzed the binding sites of the salt-enhancing peptides with TMC4. Figure 4As shown, the binding sites of the pentapeptides (SPDIY, QPGDY, and DPSPR) to TMC4 include Lys136, Arg140, Gln143, Ala158, Glu161, Ser162, Ser165, Arg168, Leu268, Arg272, Ser275, and Gln279. Among these, Arg272, Glu161, and Arg168 occur most frequently and are key amino acid residues. The main binding sites of the hexapeptide SPPGER to TMC4 are Gln143, Glu161, Ser162, Arg168, Ser165, Arg272, and Gln279, with Ser165 and Gln279 being key amino acid residues. Analysis... Figure 5 We found that the binding process mainly occurs through hydrogen bonding and electrostatic interactions. Specifically, the hydrogen bond ratio at the major active sites (Arg272, Glu161, Gln279, Arg168, and Ser165) is 60%–80%. Therefore, we can hypothesize that hydrogen bonding is the most important non-covalent interaction between the peptide and the TMC4 receptor. The optimal docking postures of the four peptides with TMC4 are shown in two-dimensional and three-dimensional diagrams. Figure 6 As shown, all four peptides form hydrogen bonds with Ser165, Arg272, and Gln279, and electrostatic interactions (salt bridges) with Glu161 and Arg168. The P residue benzene ring of the ligand SPDIY interacts hydrophobically with Arg272 through its alkyl group. In summary, we can demonstrate that the four peptides SPDIY, DPSPR, QPGDY, and SPPGER have significant salt-enhancing effects, laying the foundation for screening novel salt-enhancing peptides from peanut protein.

[0069] Determination of antioxidant activity

[0070] We evaluated the antioxidant activity of four salt-enhancing peptides by studying their free radical scavenging abilities against DPPH and ABTS. The DPPH and ABTS free radical scavenging rates were determined according to GB / T3900-2020, "Determination of Antioxidant Activity of Peptides: DPPH and ABTS Methods". Figure 7QPGDY and DPSPR exhibited excellent DPPH radical scavenging capabilities, with QPGDY's DPPH scavenging effect being dose-dependent (p<0.05). At a concentration of 500 μg / mL, QY5 achieved a radical scavenging rate of 60.33%. ABTS radical scavenging is also commonly used to assess antioxidant capacity. SPDIY and QPGDY both showed ABTS radical scavenging activity in the concentration range of 100–500 μg / mL, exhibiting significant dose-dependency (p<0.05). At a concentration of 500 μg / mL, SPDIY (50.00%) and QPGDY (51.67%) showed significantly higher ABTS radical scavenging rates than the other two peptides. Figure 8 Therefore, it can be shown that the peptide QPGDY can scavenge at least one of DPPH free radicals and ABTS free radicals, and has good antioxidant activity.

[0071] 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. The application of a peanut-derived salty flavor-enhancing peptide in the preparation of seasonings, characterized in that: The saltiness-enhancing peptide is screened from peanut protein and binds to the TMC4 receptor protein. The saltiness-enhancing peptide is composed of one of the following peptides. composition: 1) The amino acid sequence is SPDIY, and the molecular weight is 593.63 Da; 2) The amino acid sequence is DPSPR, and the molecular weight is 570.6 Da; 3) The amino acid sequence is QPGDY, and the molecular weight is 578.57 Da; 4) The amino acid sequence is SPPGER, and the molecular weight is 641.67 Da.

2. The application of the peanut-derived salty flavor-enhancing peptide according to claim 1 in the preparation of seasonings, characterized in that, The peptides with the amino acid sequences SPDIY, QPGDY, and DPSPR are molecularly docked with the receptor TMC4, with the main binding sites being Lys136, Arg140, Gln143, Ala158, Glu161, Ser162, Ser165, Arg168, Leu268, Arg272, Ser275, and Gln279.

3. The application of the peanut-derived salty flavor-enhancing peptide according to claim 1 in the preparation of seasonings, characterized in that, The amino acid sequence of the SPPGER peptide is used for molecular docking with the receptor TMC4, with the main binding sites being Gln143, Glu161, Ser162, Arg168, Ser165, Arg272, and Gln279.

Images