A salty taste enhancing peptide screened from sea cucumber collagen and application thereof

Abstract

The application discloses a kind of salty taste enhancement peptide screened from sea cucumber collagen and application thereof, belong to food condiment technical field.The salty taste enhancement peptide provided in the application, its screening method includes the following steps: constructing TMC4 receptor protein model, and its reliability is evaluated;Computer simulation enzymatic sea cucumber collagen, obtain enzymatic peptide segment, select the sequence length 2-12 peptide segment to carry out biological activity prediction, obtain the peptide segment with potential biological activity, toxicity prediction is carried out to peptide segment, and non-toxicity peptide segment is screened;TMC4 receptor protein model is carried out molecular docking with non-toxicity peptide segment, sensory evaluation, electronic tongue analysis, and CSRH, KDINNRF with purity greater than 98% and molecular weight less than 1000Da are screened, again CSRH, KDINNRF are carried out molecular docking with TMC4 receptor protein model, and the docking binding site of both is obtained.The application provides a theoretical basis for discovering new salty taste enhancement peptide resources in marine organisms and developing nutrition and health salty taste enhancement peptide.

Description

Technical Field

[0001] This invention relates to the field of food seasoning technology, specifically to the screening of salty-enhancing peptides from sea cucumber collagen that can dock with the salty taste receptor TMC4 and their applications. Background Technology

[0002] Saltiness is one of the five basic tastes (sour, sweet, bitter, and umami). Salt, often called the "king of all flavors," is an essential seasoning in cooking. However, excessive intake can raise blood pressure and increase the risk of cardiovascular disease, stroke, and stomach cancer. In 2017, the number of deaths among hypertensive patients in China due to high-salt diets exceeded 1.5 million. To control daily salt intake among Chinese residents, a salt reduction plan for the food industry has been proposed, aiming to reduce the national average daily salt intake by 20% by 2030. Since the main component of salt is sodium, reducing salt intake means reducing sodium intake. Salt-enhancing peptides are oligopeptides that exhibit a salty taste, synthesized through enzymatic hydrolysis or from amino acids. Their molecular weight is typically 200–1500 Da. While salt-enhancing peptides themselves do not taste salty, when mixed with sodium chloride, they can enhance the perception of saltiness. They can also mask off-flavors in food and, to some extent, increase its nutritional value.

[0003] my country boasts abundant marine species resources, and marine animals contain a complex and diverse range of amino acid flavor compounds, making them excellent sources for the preparation of salty peptides and salty-enhancing peptides. In recent years, studies using proteases to hydrolyze short peptides from fish, dairy products, and various animal products have revealed that the hydrolyzed products produce a significant salty effect. Based on this research, we can enzymatically extract relevant salty-enhancing peptides from marine animals, ensuring the food retains its salty flavor while reducing sodium intake. Sea cucumbers, belonging to the class Holothuroidea of ​​the phylum Echinodermata, are important marine invertebrates. Due to their rich nutritional content and high practical value, they have attracted increasing attention. The body wall of sea cucumbers contains abundant protein (40.7%-63.3%), with collagen being the main component, possessing a series of important biological functions such as lowering blood pressure, anti-fatigue, and delaying aging. Current research on sea cucumbers mainly focuses on their related physicochemical properties, the functional characteristics of sea cucumber peptides, and quality changes during storage, while research on salty-enhancing peptides is relatively limited.

[0004] Therefore, how to screen out salt-enhancing peptides from sea cucumber collagen to replace table salt is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a salty-enhancing peptide screened from sea cucumber collagen and its application.

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

[0007] This invention provides a salty-tasting enhanced peptide, screened from sea cucumber collagen, capable of binding to the TMC4 receptor protein model, and composed of one of the following peptides:

[0008] 1) The amino acid sequence is CSRH;

[0009] 2) The amino acid sequence is KDINNRF.

[0010] Furthermore, the saltiness value of the peptide with the amino acid sequence CSRH is 3.01, and the saltiness value of the peptide with the amino acid sequence KDINNRF is 2.86.

[0011] Furthermore, the peptide with the amino acid sequence CSRH or KDINNRF is docked with the TMC4 receptor protein model based on molecular docking, and the main binding sites are Lys278, Leu282, Glu279, Gln279 and Glu92.

[0012] This invention provides a method for screening salty-tasting peptides, characterized by the following steps:

[0013] (1) Construct a TMC4 receptor protein model and evaluate its reliability;

[0014] (2) Computer simulation was used to enzymatically hydrolyze sea cucumber collagen to obtain enzymatically hydrolyzed peptides;

[0015] (3) Select enzymatically digested peptides with sequence lengths of 2-12 for bioactivity prediction, obtain peptides with potential bioactivity, search for peptide sequences with potential bioactivity through a protein library, retain unknown peptides, and perform toxicity prediction on unknown peptides to screen out non-toxic peptides.

[0016] (4) Molecular docking was performed between the TMC4 receptor protein model and the non-toxic peptide in step (3), and peptides with potential salt-enhancing effects were screened based on the molecular docking results.

[0017] (5) Perform sensory evaluation and electronic tongue analysis on the peptides in step (4) to obtain salty taste enhancement peptides with saltiness enhancement effect;

[0018] (6) Minimize the salty taste-enhancing peptide in step (5) and optimize the TMC4 receptor protein model in step (1);

[0019] (7) Molecular docking was performed on the minimized salty-enhancing peptide and the optimized TMC4 receptor protein model to obtain the docking binding site between the salty-enhancing peptide and the TMC4 receptor protein model.

[0020] Furthermore, the reliability evaluation in step (1) requires that the amino acid residues in the core region and the maximum allowable region account for more than 90% of the total protein.

[0021] Furthermore, the screening criterion for bioactivity prediction in step (3) is: prediction score > 0.5;

[0022] Furthermore, in step (4), the screening process involves selecting peptides with low energy values ​​in CDOCKER energy.

[0023] Furthermore, the minimization process in step (6) refers to minimizing the energy of the peptide segment;

[0024] Furthermore, in step (6), the optimization of the TMC4 receptor protein model requires removing the residual sequence and ligand groups from the TMC4 receptor protein model crystal and then performing Prepare Protein treatment.

[0025] Furthermore, in step (7), the molecular docking has docking pocket coordinates of x:19.253678, y:3.179322, z:-20.499577, and a radius of 38.

[0026] Furthermore, in step (7), the savory-enhancing peptide needs to be docked with the active pocket identified on the TMC4 receptor protein model for molecular docking.

[0027] The application of the saltiness-enhancing peptide provided by this invention in the preparation of seasonings and food.

[0028] Products containing the aforementioned salty-enhancing peptides selected from sea cucumber collagen.

[0029] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows: The present invention obtains two peptides, CSRH and KDINNRF, with a purity greater than 98% and a molecular weight less than 1000 Da. Both peptides have good saltiness-enhancing effects and are suitable as salt substitutes. The two peptides obtained by screening can be made into seasonings or added to food to achieve the purpose of "reducing salt without reducing saltiness". Attached Figure Description

[0030] 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.

[0031] Figure 1The attached figure shows the evaluation diagram of the TMC4 receptor protein model, where (a) is the Laplace conformation diagram, (b) is the Verify 3D diagram, and (c) is the ERRAT diagram;

[0032] Figure 2 The attached image shows the sensory evaluation results of the salty-enhancing peptides.

[0033] Figure 3 The attached figure is a schematic diagram of the docking of the peptide with the TMC4 receptor protein model molecule. A represents the interaction between CSRH and the TMC4 receptor, and B represents the interaction between KDINNRF and the TMC4 receptor. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] Example 1

[0037] (1) Constructing a TMC4 receptor protein model

[0038] The amino acid sequence of the TMC4 protein (NP_001138775) was obtained from the NCBI database and imported into the Alphafold2 server for modeling. During the modeling process, the Alphafold2 server established a minimum energy model based on molecular dynamics theory, thus obtaining the TMC4 receptor protein model.

[0039] (2) Reliability evaluation of the TMC4 receptor protein model

[0040] The TMC4 receptor protein model was evaluated using the Procheck procedure in SEVES v5.0 (https: / / servicesn.mbi.ucla.edu / SEVES / ). The evaluation results are as follows: Figure 1As shown in (a), 87.8% of the amino acid residues are located in the core region (optimal region), 8.3% are located in the additional allowed region, 3.3% are located in the maximum allowed region, and only 0.7% are located in the disallowed region. For the evaluation of the de novo protein model, amino acid residues located in the core and maximum allowed regions account for more than 90% of the total protein proportion. Therefore, the TMC4 receptor protein model constructed in this invention is reasonably designed. Verify 3D detection and ERRAT analysis were performed on the TMC4 receptor protein model. The Verify 3D detection results are as follows: Figure 1 As shown in (b), in the TMC4 receptor model, 80% of the amino acid residues had a mean 3D-1D fraction greater than 0.1, as indicated by ERRAT analysis. Figure 1 As shown in (c), the overall quality factor is 91.3043. Generally, a Verify3D score with an average 3D-1D score of amino acid residues > 0.1 and an overall quality factor score > 90 in ERRAT are considered reliable. Therefore, the TMC4 receptor protein model constructed in this invention passed the Verify 3D test and ERRAT analysis and can be used for subsequent experiments.

[0041] (3) Computer simulation of enzymatic hydrolysis of sea cucumber collagen

[0042] The amino acid sequence of sea cucumber collagen (serial number: APA22677.1) was found in the NCBI database. Then, using ExPASy Peptide Cutter software (http: / / web.expasy.org / peptide_cutter), simulated enzymatic digestion with pepsin (pH>2) and low-specificity chymotrypsin was performed, yielding 229 peptides. Peptides with sequence lengths of 2-12 were selected, and their bioactivity was predicted using the Peptide Ranker website. Peptides with a prediction score >0.5 were considered to have potential bioactivity. The BIO-PEP-UWM protein library was used to search for peptide sequences with potential bioactivity, retaining unknown peptides. A total of 11 unknown peptides with potential bioactivity were screened out. Toxin Pred was used to predict their toxicity, and the results are shown in Table 1. All 11 peptides showed no toxicity and can be used for subsequent experimental studies.

[0043] Table 1. Toxicity and hydrophilicity predictions for 11 peptide segments

[0044]

[0045]

[0046] (4) Molecular docking

[0047] To gain a deeper understanding of the interaction mechanism between 11 unknown peptides with potential biological activity screened from sea cucumber collagen and the TMC4 receptor protein model, molecular docking of the peptides and the TMC4 receptor protein model was performed using Discovery Studio software. The results are shown in Table 2. A lower CDOCKER energy value indicates a stronger binding affinity between the peptide and the TMC4 receptor protein model, suggesting a greater potential for saltiness enhancement. Therefore, based on the molecular docking results of the peptides and the TMC4 receptor protein model, CSRH, KDINNRF, DGATGPRG, and GADGVPGL were selected for subsequent research on saltiness-enhancing peptides.

[0048] Table 2. Molecular docking results of 11 peptides with the TMC4 receptor protein model.

[0049]

[0050] (5) Solid-phase synthesis of peptides

[0051] Nanjing Peptide Valley Biotechnology Co., Ltd. was commissioned to synthesize peptides using solid-phase synthesis.

[0052] (6) Sensory evaluation

[0053] The sensory evaluation team consisted of five healthy males and five females without taste disorders. They received sensory training in taste according to GB / T16291.1-2012. During training, food-grade solutions of 0.08% citric acid, 1% sucrose, 0.5% L-leucine, 0.35% sodium chloride, and 0.35% monosodium glutamate were used to test for sour, sweet, bitter, salty, and umami tastes, respectively. CSRH, KDINNRF, DGATGPRG, and GADGVPGL were each prepared into 0.05% solutions and evaluated using a scoring system from 0 to 10. 0 points indicated no taste, 10 points indicated a significant salt-enhancing effect from the synthetic peptides, and the standard was scored as 5 points. Using a 0.4% sodium chloride solution as a control, four peptides (CSRH, KDINNRF, DGATGPRG, and GADGVPGL) at concentrations of 0.05% were added to the 0.4% sodium chloride solution, respectively, and their salinizing effects were compared. The results showed that peptides CSRH and KDINNRF had a salinizing effect. Figure 2 As shown, CSRH was the most effective. However, since sensory evaluation results are greatly influenced by subjective factors, they cannot be taken as a final conclusion and further verification using an electronic tongue is needed.

[0054] (7) Electronic tongue analysis

[0055] The reference solution for the electronic tongue consisted of 30 mmol / L KCl and 0.3 mmol / L tartaric acid solution. Four synthetic peptides—CSRH, KDINNRF, DGATGPRG, and GADGVPGL—were dissolved in deionized water. After complete dissolution, the solutions were poured into a special beaker for the electronic tongue to identify the taste characteristics of the synthetic peptides. Using a 0.4% sodium chloride solution as a control, 0.05% of each of the four peptides was added to the 0.4% sodium chloride solution to compare their saltiness-enhancing effects. The results are shown in Table 3. Table 3 shows that the saltiness values ​​of peptides CSRH and KDINNRF were significantly higher than those of the 0.4% sodium chloride solution, while the saltiness values ​​of peptides DGATGPRG and GADGVPGL were both lower than those of the 0.4% sodium chloride solution, indicating that they did not have a saltiness-enhancing effect. Therefore, it can be demonstrated that CSRH and KDINNRF have a significant saltiness-enhancing effect and can be used as novel saltiness-enhancing peptides in sea cucumber collagen.

[0056] Table 3. Results of electronic tongue analysis of peptide segments.

[0057]

[0058] Note: Letters in the same column indicate significant differences between groups (P<0.05).

[0059] (8) Analysis of interaction mechanisms based on molecular docking

[0060] First, ChemDraw 19.0 was used to convert the amino acid sequences of two potentially active peptides (CSRH, KDINNRF) into 2D structures. Then, Discovery Studio software was used to convert the 2D structures into 3D structures, followed by peptide minimization. Next, the TMC4 receptor protein model was optimized using Discovery Studio. The 3D structure of the TMC4 receptor protein model was imported into Discovery Studio, and residual sequences and ligand groups from the protein crystal were removed before "PrepareProtein" processing. Subsequently, CDOCKER software was used to perform molecular docking between the potentially active peptides (CSRH, KDINNRF) and the TMC4 receptor protein model. The docking pocket coordinates were x:19.253678, y:3.179322, z:-20.499577, with a radius of 38. The docking results are shown below. Figure 3 As shown, the docking sites of the peptides with the TMC4 receptor protein model are all located in the spatial region between the two methylene groups of the receptor protein. CSRH( Figure 3 a), KDINNRF ( Figure 3(b) The binding sites of the peptide to the TMC4 receptor protein model mainly include Lys278, Leu282, Glu279, Gln279, and Glu92, with Gln279, Glu92, and Lys278 appearing most frequently, indicating that Gln279, Glu92, and Lys278 are the major amino acid residues. Furthermore, the binding of the peptide to the TMC4 receptor protein model is mainly through hydrogen bonding and electrostatic interactions, with hydrogen bonding being the dominant force, thus revealing the flavor mechanism of the salt-enhancing peptide.

[0061] (9) Analysis of peptide purity and molecular weight

[0062] The purity and molecular weight of CSRH and KDINNRF were analyzed by high performance liquid chromatography and mass spectrometry. The molecular weight of CSRH was 501.56 Da and the purity was 98.64%; the molecular weight of KDINNRF was 906.02 Da and the purity was 99.38%.

[0063] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0064] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A salty taste enhancing peptide, characterized in that, The salty taste enhancing peptide is screened from sea cucumber collagen protein and binds to TMC4 receptor protein, and the salty taste enhancing peptide consists of one of the following peptides: 1) the amino acid sequence is CSRH; 2) the amino acid sequence is KDINNRF.

2. The salt taste enhancing peptide of claim 1, wherein The peptide segment with the amino acid sequence of CSRH or KDINNRF is docked with the TMC4 receptor protein model based on molecular docking, and the main binding site is Lys278, Leu282, Glu279, Gln279 and Glu92.

3. The salt taste enhancing peptide of claim 1, wherein The salty taste value of the peptide segment with the amino acid sequence of CSRH is 3.01, and the salty taste value of the peptide segment with the amino acid sequence of KDINNRF is 2.

86.

4. A method for screening a salty taste enhancing peptide, characterized by, The steps are as follows: (1) Construct a TMC4 receptor protein model and evaluate its reliability; The reliability evaluation requires that the proportion of amino acid residues located in the core region and the maximum allowed region to the entire protein is more than 90%; (2) Computer simulation of enzymatic hydrolysis of sea cucumber collagen protein to obtain enzymatic peptides; (3) Select the enzymatic peptide with a sequence length of 2-12 for biological activity prediction to obtain a peptide segment with potential biological activity, search the peptide segment sequence with potential biological activity through protein library, retain the unknown peptide segment, and predict the toxicity of the unknown peptide segment to screen out a non-toxic peptide segment; The screening standard for biological activity prediction is: prediction score > 0.5; (4) Molecular docking of the TMC4 receptor protein model and the non-toxic peptide segment in step (3), and screening of the peptide segment with potential salty taste enhancing effect according to the molecular docking result; The screening: select the peptide segment with low energy value in CDOCKERenergy; (5) Sensory evaluation and electronic tongue analysis of the peptide segment with potential salty taste enhancing effect to obtain the salty taste enhancing peptide of any one of claims 1-2; (6) Minimization of the salty taste enhancing peptide in step (5) and optimization of the TMC4 receptor protein model in step (1); The minimization refers to minimizing the energy of the peptide segment; The optimization requires removing the sequence and ligand group of the TMC4 receptor protein model crystal and performing PrepareProtein processing; (7) Molecular docking of the minimized salty taste enhancing peptide and the optimized TMC4 receptor protein model to obtain the docking binding site of the salty taste enhancing peptide and the TMC4 receptor protein model.

5. The method of claim 4, wherein the salt taste enhancing peptide is selected from the group consisting of SEQ ID NOs: 1 to 20. The molecular docking in step (7) has a pocket coordinate of x: 19.253678, y: 3.179322, z: -20.499577 and a radius of 38.

6. The method of claim 4, wherein the salt taste enhancing peptide is selected from the group consisting of SEQ ID NOs: 1 to 20. The molecular docking in step (7) requires the salty taste enhancing peptide to dock with the active docking pocket determined on the TMC4 receptor protein model.

7. Use of the salty taste enhancing peptide of claim 1 or 2 in the preparation of condiments and food.

8. A product containing the salty taste enhancing peptide screened from sea cucumber collagen protein according to claim 1 or 2.

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