A mung bean protein salt-enhancing peptide, a screening and preparation method and application thereof

Abstract

The application provides a mung bean protein salty taste enhancing peptide, a screening and preparation method and application. The method comprises the following steps: step 1, carrying out step-by-step enzymolysis on mung bean protein by using alkaline protease and flavor protease, carrying out ultrafiltration grading, and screening an initial extract of the salty taste enhancing peptide by using an electronic tongue; step 2, further identifying polypeptide sequences by using LC-MS / MS; and step 3, screening polypeptides with strong interaction capability with salty taste receptors TMC4 and ENaC by combining biological activity prediction, target active fragment screening, safety and physicochemical property evaluation and molecular docking analysis. The salty taste enhancing peptide of the mung bean protein obtained by screening comprises polypeptides represented by RPFF, APDLRGY, WDDIGGL, FDGF and AEFF, and contains RP, RG, DD, DGF and FF characteristic peptide segments, respectively. The polypeptides are synthesized by using a solid phase, and the salty taste enhancing effect of the polypeptides is verified by using an electronic tongue. When 0.03% of the above synthesized polypeptides are added to a 0.4% NaCl solution, the salty taste enhancing rates are 90%, 62%, 34%, 52% and 42%, respectively. The polypeptides can significantly enhance salty taste perception of a solution under low-salt conditions, and achieve the effect of salt reduction without taste reduction.

Description

Technical Field

[0001] This invention belongs to the field of food processing and food additives technology, specifically relating to a mung bean protein salt-enhancing peptide, its screening and preparation method, and its application. Background Technology

[0002] Salt, primarily composed of sodium chloride (NaCl), is one of the most commonly used basic seasonings in food. Salt not only imparts a salty taste to food but also enhances its overall flavor profile and plays a vital role in regulating fluid balance and osmotic pressure, thus maintaining normal physiological functions. However, long-term excessive salt intake can easily induce hypertension and increase the burden on the kidneys and the risk of related diseases. While residents in Europe and America primarily consume salt from processed foods such as baked goods and meat products, a significant proportion of salt intake in my country comes from the addition of salt and salty seasonings during daily cooking. Currently, the average daily salt intake of adults in my country exceeds 10g, while the World Health Organization recommends that adults consume less than 5g of salt per day. Simply reducing the amount of added salt can lower sodium intake, but this often leads to insufficient saltiness and a decline in flavor, thus affecting consumer acceptance. Therefore, developing technologies that can maintain the saltiness and overall flavor of food while reducing salt usage is of great significance for the research and development of low-sodium foods and seasonings.

[0003] In recent years, utilizing flavor peptides, especially salty peptides or salt-enhancing peptides, to partially replace table salt has become an important research direction for achieving "reduced salt without sacrificing flavor." Compared with low-sodium salts replaced by metal salts, salt-enhancing peptides have advantages such as natural sources, better flavor harmony, and no introduction of aftertaste or potential health risks, thus attracting widespread attention. Enzymatic hydrolysis is a commonly used method for preparing food-derived peptides, characterized by mild conditions, high specificity, and relatively controllable processes, which can better preserve the nutritional and flavor characteristics of peptides.

[0004] Mung beans are a common edible legume resource in my country, boasting advantages such as high protein content, wide availability, and low cost. Mung bean protein is rich in various amino acids, with high levels of flavor-related amino acids such as glutamic acid and aspartic acid, showing potential as a source of flavor-enhancing peptides. However, current research on salt-enhancing peptides derived from mung bean protein is still limited. There is a lack of systematic screening of these peptides, clearly defined functional peptide sequences, and effective targeted screening methods, thus restricting their development and application in low-sodium foods and seasonings. Summary of the Invention

[0005] Technical Problem: In view of the problem that there is a lack of research on salt-enhancing peptides from mung bean protein in the existing technology, and a lack of clear functional peptide sequences and effective screening methods, this invention provides a salt-enhancing peptide from mung bean protein, its screening and preparation method and application, so as to obtain a mung bean protein-derived polypeptide with good salt-enhancing activity and salt-reducing potential.

[0006] Technical solution: To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] This invention provides a mung bean protein saltiness-enhancing peptide, wherein the mung bean protein saltiness-enhancing peptide is selected from one or more of RPFF, APDLRGY, WDDIGGL, FDGF and AEFF.

[0008] This invention also provides a method for screening and preparing the mung bean protein salt-enhancing peptide, comprising the following steps:

[0009] (1) Prepare mung bean protein solution by adding water to mung bean protein, then hydrolyze it with endopeptidase and then with exopeptidase. Inactivate the enzyme, cool and centrifuge, and take the supernatant to obtain mung bean protein hydrolysate.

[0010] (2) After ultrafiltration and freeze-drying, mung bean protein hydrolysate was used to obtain mung bean protein peptide components with different molecular weights. The components with prominent salty taste were selected as the primary extract of salty mung bean protein peptides by electronic tongue determination.

[0011] (3) The primary extract of mung bean protein salt-enhancing peptides was analyzed by liquid chromatography-mass spectrometry to obtain multiple polypeptide sequences;

[0012] (4) The polypeptide sequences obtained in (3) were subjected to biological activity prediction, toxicity screening, solubility screening, salty taste active residue analysis and molecular docking screening with TMC4 receptor protein and ENaC receptor protein in sequence to obtain candidate salty taste peptide sequences.

[0013] (5) After classifying the candidate polypeptide sequences obtained in (4) according to their salty active fragments, select the polypeptide sequence with the lowest binding free energy to TMC4 and ENaC receptors from each category for solid-phase synthesis. Use an electronic tongue to analyze the saltiness value and saltiness enhancement effect to obtain mung bean protein saltiness enhancement peptide.

[0014] In step (1), the concentration of the mung bean protein solution is 6-8% (w / w), the amount of endopeptidase added is 4000-5000 U / g, the reaction temperature is 50-60℃, the reaction pH is 7.5-8.5, and the reaction time is 3-5 h. The amount of exopeptidase added is 300-500 U / g, the reaction temperature is 50-60℃, the reaction pH is 6.0-7.0, and the reaction time is 3-5 h. The enzyme inactivation temperature is 90-100℃, and the enzyme inactivation time is 10-15 min.

[0015] Specifically, step (4) involves using a peptide activity prediction tool to predict the peptide activity of the multiple peptide sequences obtained in step (3); (b) screening for salt-enhancing peptides based on a known salty peptide database; (c) screening for non-toxic peptide sequences using a toxicity prediction tool; (d) screening for water-soluble peptides using a water solubility prediction tool; and (e) finally screening for peptide sequences that can bind to TMC4, ENaC, and receptor proteins using molecular docking technology.

[0016] Furthermore, the preferred polypeptide sequences selected in (4) are classified according to peptides that have been verified to have salty characteristics, namely polypeptides containing RP characteristic peptides, polypeptides containing RG characteristic peptides, polypeptides containing DD characteristic peptides, polypeptides containing DGF characteristic peptides, and polypeptides containing FF characteristic peptides. Then, from each category, polypeptides with lower binding free energy to TMC4 and ENaC receptor proteins are selected for synthesis.

[0017] The above-mentioned mung bean protein salt-enhancing peptides are used in the preparation of low-salt foods and reduced-salt seasonings.

[0018] Beneficial effects

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention screened five mung bean protein source polypeptides with salt-enhancing activity, namely RPFF, APDLRGY, WDDIGGL, FDGF and AEFF;

[0021] (2) This invention establishes a method for screening and preparing mung bean protein salt-enhancing peptides based on double enzymatic hydrolysis, electronic tongue screening, liquid chromatography-mass spectrometry analysis, virtual screening and dual receptor molecular docking, which helps to improve the screening efficiency of mung bean protein salt-enhancing peptides.

[0022] (3) The mung bean protein salt-enhancing peptide obtained by the present invention has a good salt-enhancing effect and can be used in the development of low-salt foods and low-salt seasonings. Attached Figure Description

[0023] Figure 1 This is a secondary mass spectrum of peptide RPFF.

[0024] Figure 2 The secondary mass spectrum of peptide APDLRGY

[0025] Figure 3 This is a secondary mass spectrum of the peptide WDDIGGL.

[0026] Figure 4 This is the secondary mass spectrum of peptide AEFF.

[0027] Figure 5 This is the secondary mass spectrum of peptide AEFF.

[0028] Figure 6 Confidence interval plots for TMC4(a) and ENaC(b) receptor proteins;

[0029] Figure 7 The overall docking conformation and local binding sites of five mung bean protein saltiness-enhancing peptides RPFF, FDGF, AEFF, APDLRGY and WDDIGGL with saltiness receptors TMC4 and ENaC, respectively;

[0030] Figure 8 The electronic tongue saltiness value and saltiness enhancement rate of mung bean protein saltiness-enhancing peptides. Detailed Implementation Plan

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The described examples are merely 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.

[0032] The ultrafiltration method employed in this invention involves fractionating mung bean protein hydrolysate using ultrafiltration. The apparatus used is an MSM-Magic X4 experimental nanofiltration and ultrafiltration membrane separation device (Shanghai Mosu Scientific Instruments Co., Ltd.), and the ultrafiltration membranes are regenerated cellulose membranes with different molecular weight cutoffs. The mung bean protein hydrolysate is diluted with ultrapure water to a solid content of 3–5%, and fractionation is performed sequentially using ultrafiltration membranes with molecular weight cutoffs of 10 kDa, 3 kDa, 1 kDa, 500 Da, and 100 Da. During the operation, the pressure is maintained at 0.5 MPa, and the temperature is controlled at 25 °C. The obtained molecular weight fractions are freeze-dried and stored at −20 °C for subsequent analysis.

[0033] The specific parameters of the liquid chromatography-mass spectrometry (LC-MS) technique used in this invention are as follows: column pre-column specifications 150 μm·d × 50 mm, packing: Reprosil-Pur 120°C. 18 -AQ 3 μm, analytical column specifications 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18 AQ1.9 μm; liquid chromatography-mass spectrometry: Easy-nLC 1200 / QExactive (Thermo Fisher Scientific); chromatographic separation time: 66 min; mobile phase A: 0.1% FA; mobile phase B: 0.1% FA, 80% CAN; flow rate: 600 nL / min.

[0034] The electronic tongue used in this invention: This study employed an electronic tongue system (TS-5000Z, INSENT Inc., Japan) to analyze the taste characteristics of different samples. A lipid membrane-based taste sensor was used to detect saltiness (CT0). Before measurement, all sensors were activated in a reference solution for 24 hours according to the manufacturer's instructions. The sensor surface was cleaned with deionized water, and the electronic tongue system was then calibrated using a sodium chloride standard solution to verify the response performance of the saltiness sensor. Sample measurements were performed after confirming signal stability. The NaCl concentration for each sample group was 0.4%, and the peptide concentration was 0.3%. The measured saltiness values ​​were compared and analyzed.

[0035] The endonuclease (alkaline protease) and exonuclease (flavor protease) used below were purchased from Novozymes Enzyme Preparations Co., Ltd., and the mung bean protein powder was purchased from Yantai Oriental Protein Technology Co., Ltd.

[0036] Example 1: A method for preparing a primary extract of mung bean protein salt-enhancing peptides, the steps of which are as follows:

[0037] Step 1: Preparation of mung bean protein hydrolysate: Weigh 8g of mung bean protein powder, add 94g of water, and mix thoroughly to prepare a mung bean protein solution. Place the mung bean protein solution in a 95℃ water bath for 30 min to allow the protein to denature fully. After cooling the solution to room temperature, adjust the pH to 8.0, add 5000 U of alkaline protease, and hydrolyze in a 60℃ magnetically stirred water bath for 5 h. After the first step of hydrolysis, cool the hydrolysate to room temperature, adjust the pH to 7.0, add 500 U of flavor protease, and continue hydrolysis in a 50℃ magnetically stirred water bath for 3 h. After hydrolysis, inactivate the enzyme in a 98℃ water bath for 15 min, cool, centrifuge at 10,000 rpm for 15 min, and store the supernatant at -20℃ to obtain the mung bean protein hydrolysate.

[0038] Step 2: Preparation of primary salty peptide extract from mung bean protein: The mung bean protein hydrolysate obtained in Step 1 was diluted to a solids concentration of less than 3%, and then subjected to ultrafiltration using an ultrafiltration membrane separation device. Based on molecular weight, the hydrolysate was separated into five components: Component I (>10 kDa), Component II (3 kDa–10 kDa), Component III (1 kDa–3 kDa), Component IV (1 kDa–500 Da), and Component V (<500 Da). After freeze-drying for 72 h, the five components were measured using an electronic tongue, and the component with the highest saltiness value was selected as the primary salty peptide extract from mung bean protein.

[0039] Example 2: A screening method for saltiness enhancement of mung bean protein, the steps of which are as follows:

[0040] Step 1: The primary extract of mung bean protein saltiness-enhancing peptides obtained in Example 1 was identified using liquid chromatography-mass spectrometry (LC-MS / MS) to obtain multiple polypeptide sequences. The secondary mass spectra of representative peptides RPFF, APDLRGY, WDDIGGL, FDGF, and AEFF are shown below. Figures 1 to 5 As shown. The specific parameters of the liquid chromatography-mass spectrometry (LC-MS) technique are as follows: column pre-column specifications: 150 μm id × 50 mm, packing: Reprosil-Pur 120C18-AQ 3 μm; analytical column specifications: 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18AQ 1.9 μm; LC-MS instrument: Easy-nLC 1200 / QExactive (Thermo Fisher Scientific); chromatographic separation time: 66 min; mobile phase A: 0.1% FA; mobile phase B: 0.1% FA, 80% CAN; flow rate: 600 nL / min.

[0041] Step 2: Preliminary screening of the multiple peptide sequences obtained in Step 1. First, the Peptide Ranker online tool was used to predict the potential biological activity of the peptides, and peptides with a score greater than 0.5 were selected for further analysis. Second, based on known umami-active fragments included in the BIOPEP-UWM database, components with a target fragment accounting for no less than 25% of the peptide were selected for further analysis. Then, the ToxinPred tool was used to predict the toxicity of the candidate peptides, and non-toxic peptides were selected. Further screening for water solubility and sensitization was conducted using online tools, selecting peptide sequences with good solubility and low sensitization. Finally, 34 candidate peptide sequences were obtained for subsequent research.

[0042] Step 3: Obtain the amino acid sequence of the salty taste receptor TMC4 from the Uniprot database (Uniprot ID: Q7Z404) and the amino acid sequence of ENaC from the PDB database (PDB ID: 6WTH). Then, construct three-dimensional structural models of the TMC4 and ENaC receptor proteins using the Swiss-model online tool. Perform Ramachandran plot analysis on the constructed three-dimensional models using the SAVES online tool to evaluate the reliability of the models. The confidence intervals for the TMC4 and ENaC receptor proteins are shown below. Figure 6 As shown.

[0043] Step 4: Three-dimensional structural models of the peptides screened in Step 2 were established using Chem Draw 23.1.1 and Chem 3D 23.1.1, and energy minimization was performed using the MM2 force field. Molecular docking of the peptides with the TMC4 and ENaC receptor models obtained in Step 3 was performed using AutoDock Vina 1.5.6. Molecular visualization and interaction analysis were then performed using the Plip online tool and PyMol software.

[0044] Step 5: Based on the currently verified salty peptides RP, RG, DD, DGF, and FF, candidate polypeptide sequences were divided into five categories: polypeptides containing the RP characteristic peptide, polypeptides containing the RG characteristic peptide, polypeptides containing the DD characteristic peptide, polypeptides containing the DGF characteristic peptide, and polypeptides containing the FF characteristic peptide. Based on the molecular docking results of the candidate polypeptides with TMC4 and ENaC receptor proteins, polypeptides with lower binding energies were screened from each category for chemical synthesis. The amino acid sequence information of the screened mung bean protein saltiness-enhancing peptides is shown in Table 1, and the molecular docking results with the two receptor proteins are as follows: Figure 7 As shown.

[0045] Table 1. Amino acid sequence information of mung bean protein salt-enhancing peptides

[0046]

[0047] Example 3: Electronic tongue evaluation and salt-enhancing effect evaluation of mung bean protein salt-enhancing peptides. The steps are as follows:

[0048] The five peptides obtained in Example 2 were synthesized using a solid-phase synthesis method. The purity of the synthesized peptides was greater than 95%. The saltiness-enhancing effect of the mung bean protein saltiness-enhancing peptides was evaluated as follows:

[0049] (1) Evaluation of electronic tongue: Peptide RF4, peptide AY7, peptide WL7, peptide FF4, peptide AF4 and NaCl were mixed with ultrapure water to prepare a mixed solution. The concentration of peptide was 0.03% (w / w) and the concentration of NaCl was 0.4% (w / w). Electronic tongue was then used for measurement.

[0050] (2) Evaluation of saltiness enhancement effect: by Figure 8It was found that all five peptides had varying degrees of saltiness-enhancing effect. The saltiness value of RPFF peptide was 8.12±0.04, significantly higher than that of 0.4% NaCl solution (4.71±0.00, p<0.05), followed by peptide AY7 (7.24±0.02). The saltiness values ​​of peptides FF4, AF4, and WL7 were 6.89±0.02, 6.50±0.01, and 6.16±0.00, respectively, lower than RF4 but still higher than that of 0.4% NaCl solution. Further analysis showed that peptide RF4 had the most significant saltiness-enhancing effect at 90%, while peptides AY7, FF4, and AF4 had saltiness-enhancing rates of 62%, 52%, and 42%, respectively, all significantly enhancing the perceived saltiness intensity of NaCl solution. Peptide WL7 had a saltiness-enhancing rate of 34%, lower than the other four peptides, but still possessed a certain saltiness-enhancing effect. The above five mung bean protein salt-enhancing peptides all showed good salt-enhancing potential and can be used in the development of low-sodium foods.

[0051] Comparative Example 1: Evaluation of the effect of exonuclease hydrolysis time on the flavor value of mung bean protein peptides. The procedure is as follows:

[0052] This comparative example uses the same method as Example 1 for preparing mung bean protein salty peptide hydrolysate, the only difference being the hydrolysis time of the exonuclease.

[0053] Specifically, the denaturation treatment of mung bean protein and the first step of alkaline protease hydrolysis were carried out according to the method of step 1 in Example 1. After the first step of hydrolysis, the solution was cooled to room temperature, the pH was adjusted to 7.0, 500 U of flavor protease was added, and the solution was hydrolyzed for 2 h, 3 h and 5 h under magnetic stirring water bath at 50 ℃. Then, the enzyme was inactivated by heating in a water bath at 98 ℃ for 15 min. After cooling, the solution was centrifuged at 10,000 rpm for 15 min and the supernatant was collected.

[0054] The remaining steps, including the electronic tongue measurement method, were the same as in Example 1. The test results are shown in Table 2.

[0055] The results showed that when the enzymatic hydrolysis time of the exonuclease was extended from 2 h to 3 h, the saltiness and umami values ​​of the obtained mung bean protein peptides were significantly improved; however, when the hydrolysis time was further extended to 5 h, no further significant improvement in saltiness and umami values ​​was observed. These results indicate that under the conditions of this experiment, enzymatic hydrolysis with the exonuclease for 3 h yields a better hydrolysis effect.

[0056] Table 2. Results of different reaction times for flavor proteases: salty / umami comparison

[0057]

[0058] Comparative Example 2: A comparative experiment on the docking results of peptides and protein receptor molecules under the condition of increasing the Peptide Ranker screening threshold. The steps are as follows:

[0059] This comparative example uses the same screening method for mung bean protein salt-enhancing peptides as in Example 2, the only difference being the screening threshold for PeptideRanker activity prediction.

[0060] Specifically, in step 2 of Example 2, the screening criteria for predicting the potential biological activity of peptides were adjusted from a PeptideRanker score greater than 0.5 to a Peptide Ranker score greater than 0.7. The remaining screening steps, including the screening of the proportion of target active fragments based on the BIOPEP-UWM database, toxicity prediction, water solubility and sensitization prediction, as well as the subsequent receptor protein structure construction and molecular docking, are the same as in Example 2.

[0061] The peptides obtained from this comparative screening were molecularly docked with TMC4 and ENaC receptor proteins, respectively. The results showed that a higher bioactivity score does not necessarily correspond to a lower receptor binding free energy, indicating that simply increasing the activity prediction threshold does not effectively improve the binding ability of candidate peptides to salty taste receptors. Therefore, setting the PeptideRanker activity prediction screening threshold to greater than 0.5 is more conducive to enriching candidate peptides with salt-enhancing potential, thereby improving the accuracy and effectiveness of the screening method. The screening results are shown in Table 3.

[0062] Explanation of the results of the examples and comparative examples

[0063] In summary, this invention focuses on the preparation and screening of salt-enhancing peptides from mung bean protein, constructing a complete technical solution consisting of enzymatic hydrolysis regulation, molecular weight fractionation, virtual multidimensional screening, and receptor verification. Specifically, Example 1, through synergistic enzymatic hydrolysis with endonucleases and exonucleases, combined with ultrafiltration fractionation and electronic tongue evaluation, successfully obtained a low-molecular-weight peptide extract with a significant salt-enhancing effect, providing a reliable material basis for subsequent precise screening. Example 2, building upon this, further introduced LC-MS / MS identification, activity prediction, target active fragment screening, safety and physicochemical property screening, and multi-receptor molecular docking analysis, achieving systematic and efficient screening of salt-enhancing peptides and obtaining multiple candidate peptides with strong interaction capabilities with salt-taste receptors. Example 3, through the synthesis of the screened peptides and electronic tongue verification, further confirmed that the peptides have a good salt-enhancing effect under low-salt conditions.

[0064] Comparative experiments verified the present invention from different technical aspects. Comparative Example 1, by changing the enzymatic hydrolysis time of the flavor protease, showed that the hydrolysis time of the exonuclease affects the flavor characteristics of the salty-enhancing peptides from mung bean protein. Too short a reaction time is not conducive to obtaining the ideal salty and umami effects, thus demonstrating the rationality of the enzymatic hydrolysis conditions adopted in the present invention. Comparative Example 2, by increasing the Peptide Ranker activity prediction threshold, showed that simply increasing the prediction score cannot guarantee that the peptide has a better binding ability with the salty taste receptor, further verifying the necessity and effectiveness of the screening threshold and multi-index joint screening strategy adopted in the present invention in enriching salty-enhancing peptides.

[0065] Therefore, it can be seen that the present invention, through the systematic design and verification of key process parameters and screening strategies, can stably obtain mung bean protein-derived peptides with good saltiness-enhancing effects.

[0066] The above embodiments are merely preferred embodiments of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Any modifications, improvements, or equivalent substitutions made to the present invention by those skilled in the art without departing from the technical concept and solutions of the present invention should fall within the scope of protection of the present invention.

[0067] Table 3. Screening information for 34 mung bean protein saltiness-enhancing peptides

[0068]

Claims

1. A mung bean protein salt-enhancing peptide, characterized in that, The mung bean protein saltiness-enhancing peptide is selected from one or more of RPFF, APDLRGY, WDDIGGL, FDGF, and AEFF.

2. The method for screening and preparing mung bean protein salt-enhancing peptides according to claim 1, characterized in that, Includes the following steps: S1. Mix mung bean protein with water to prepare a mung bean protein solution, then hydrolyze it with an endopeptidase, followed by an exopeptidase to inactivate the enzymes, cool, centrifuge, and collect the supernatant to obtain mung bean protein hydrolysate; S2. The mung bean protein hydrolysate was ultrafiltered and freeze-dried to obtain mung bean protein peptide components of different molecular weights. The primary extract of mung bean protein saltiness peptide with prominent salty characteristics was obtained by electronic tongue screening. S3. The primary extract of mung bean protein salt-enhancing peptides was analyzed by liquid chromatography-mass spectrometry to obtain the peptide sequence; S4. The polypeptide sequences obtained in S3 were subjected to biological activity prediction, toxicity screening, solubility screening, salty active fragment analysis, and molecular docking screening with TMC4 and ENaC salty receptors to obtain candidate salty peptide sequences. S5. After classifying the candidate polypeptide sequences obtained from S4 according to their salty active fragments, representative polypeptides were selected for solid-phase synthesis, and the saltiness value and saltiness enhancement effect were analyzed using an electronic tongue to screen and obtain the mung bean protein saltiness enhancement peptide.

3. The method for screening and preparing mung bean protein salt-enhancing peptides according to claim 2, characterized in that: The mass ratio of mung bean protein to water in S1 is 1:11 to 1:13; the endopeptide is selected from one or more alkaline proteases, wherein the amount of endopeptide added is 4000 to 5000 U / g, the reaction temperature is 50 to 60℃, the reaction pH is 7.5 to 8.5, and the reaction time is 3 to 5 h.

4. The method for screening and preparing mung bean protein salt-enhancing peptides according to claim 2, characterized in that: The exonuclease is selected from one or more flavor proteases. The amount of exonuclease added is 300-500 U / g, the reaction temperature is 50-60℃, the reaction pH is 6.0-7.0, and the reaction time is 3-5 h. The enzyme inactivation temperature is 90-100℃, and the enzyme inactivation time is 10-25 min.

5. The method for screening and preparing mung bean protein salt-enhancing peptides according to claim 2, characterized in that: In step S3, liquid chromatography-mass spectrometry was used to identify the polypeptide sequence of the primary extract of mung bean protein saltiness peptide.

6. The method for screening and preparing the mung bean protein salt-enhancing peptide as described in claim 2, characterized in that: The specific operation of S4 is as follows: the peptide sequence obtained in S3 is used to predict peptide activity using a peptide activity prediction tool to screen out peptide sequences with a biological activity greater than 0.5; potential salt-enhancing peptides are screened based on a known salty peptide database to screen out peptide sequences in which the target active fragment accounts for no less than 25% of the peptide; then, non-toxic peptide sequences are screened using a toxicity prediction tool, and water-soluble peptide sequences are screened using a water solubility prediction tool; finally, peptide sequences that can bind to TMC4 and ENaC salty taste receptor proteins are screened using molecular docking technology.

7. The method for screening and preparing the mung bean protein salt-enhancing peptide as described in claim 2, characterized in that: Screening was performed on proteins with binding free energies of -6.0 to -9.5 kcal·mol⁻¹. -1 Furthermore, the free energy for the peptide to bind to the ENaC receptor protein is -7.5 to -10.5 kcal·mol⁻¹. -1 The polypeptide sequence.

8. The method for screening and preparing the mung bean protein salt-enhancing peptide as described in claim 2, characterized in that: In step S5, candidate salt-enhancing peptide sequences are classified into peptides containing RP characteristic peptides, peptides containing RG characteristic peptides, peptides containing DD characteristic peptides, peptides containing DGF characteristic peptides, and peptides containing FF characteristic peptides according to their salty taste active fragments. Then, based on the molecular docking results, peptides with lower binding free energy to TMC4 and ENaC salty taste receptors are selected from each category for solid-phase synthesis.

9. The application of the mung bean protein salt-enhancing peptide according to claim 1 in the preparation of low-sodium foods or low-sodium seasonings.

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