Salty and savory dual-functional flavor peptide and its preparation method and application

Abstract

The present invention discloses a salty and fresh bifunctional flavor peptide and its preparation method and application, and relates to the field of biotechnology. The amino acid sequence of the salty and fresh bifunctional flavor peptide is shown in any one of SEQ ID NO.1-4. The present invention adopts a composite protease enzymatic hydrolysis method to prepare an enzymatic hydrolyzate rich in salty and fresh bifunctional flavor peptides, and uses peptide omics and molecular docking technology to identify and screen out multiple salty and fresh bifunctional flavor peptides, while evaluating and predicting the transmembrane domains of taste receptors, and analyzing the chemical bonds and docking sites of the salty and fresh bifunctional flavor peptides docking with taste receptors, summarizing the key chemical bonds and binding sites of the interaction between the salty and fresh bifunctional flavor peptides and salty receptors, and evaluating their salty and umami intensity. The salty and fresh bifunctional flavor peptides screened by the present invention have strong saltiness and umami, can be used as a substitute for sodium salt, and are used to prepare low-salt and low-sodium foods. The present invention also provides a certain reference for quickly and easily obtaining salty and fresh bifunctional flavor peptides.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a salty and savory dual-functional flavor peptide and a preparation method and application thereof. Background Art

[0002] Saltiness and umami are extremely important flavors besides sourness, sweetness, and bitterness. They are the key to delicious food and play an indispensable role in food seasoning. Salt and sodium glutamate are important carriers of saltiness and umami respectively. Saltiness can enhance umami and umami can promote saltiness. They can complement each other to form a complex flavor, which can not only enhance the deliciousness of food, but also improve the quality of food. At the same time, salt has many functions such as extending the shelf life of food, and plays an important role in the food industry. In addition, Na in salt and sodium glutamate + It is an essential mineral element for the human body. It can regulate the body's water and salt metabolism and maintain the body's normal osmotic pressure. It is an indispensable substance in the body's metabolism. However, long-term excessive intake of sodium can lead to homeostasis disorders in the body's internal environment and increase the risk of heart disease, hypertension and other diseases. It is reported that a high-sodium diet may inhibit the expression of proteins related to cell autophagy, leading to damage to the intestinal barrier function and then triggering a series of diseases. "Reducing salt and sodium without reducing taste" has become a global fashion. However, if the amount of sodium salt is directly reduced without taking other measures, it will inevitably cause food taste disorders, lack of flavor, weakening of physical and chemical properties, shortened shelf life and other quality deterioration phenomena. Therefore, the current search for sodium salt substitutes is an important strategy to meet the challenge of "reducing salt and sodium without reducing taste".

[0003] With the increasing awareness of healthy eating, people are becoming more aware of the health risks associated with excessive sodium intake. Finding ways to reduce salt and sodium without sacrificing flavor is urgent, and this approach has garnered the attention of numerous experts and scholars. People's taste requirements for food have also risen, and a trend toward prioritizing flavor has gradually formed. Consequently, the development of sodium salt substitutes has become a hot topic of research. Current research suggests that salty and umami dual-functional flavor peptides can impart a rich salty and umami flavor to foods, achieving the goal of reducing salt and sodium without sacrificing flavor. Furthermore, salty and umami dual-functional flavor peptides, as protein hydrolysates or amino acid synthesis products, possess both rich salty and umami properties, significantly enhancing the saltiness and umami flavors of food. These dual-functional flavor peptides can reduce the amount of sodium added without altering the intensity of saltiness and umami. They also participate in and influence the formation of other flavors, synergizing with other flavor compounds to impart a rich flavor to foods while reducing the amount of sodium used. These dual-functional flavor peptides also have nutritional benefits, supplementing essential amino acids for the human body. Therefore, the application of salty and savory dual-functional flavor peptides in food has huge space and potential.

[0004] Meretrix lyrata, also known as "white clam," "qin clam," "Vietnamese clam," and "white sea fat," is a low-value marine shellfish abundant in my country's waters. However, due to its small size and difficulty in processing, its utilization value is low. The meat of the white clam is delicious and rich in nutrients, with a protein mass fraction of up to 58.13% (dry basis) and a lipid content of only 12.86 (dry basis). It has a complete amino acid profile, with both total amino acids and essential amino acids higher than those in oysters and comparable to those in mussels. The total content of sweet and umami amino acids in the protein of the white clam accounts for 59.63% of the total free amino acids, with alanine and glycine levels higher than those in oysters and other shellfish. Furthermore, current research has discovered a variety of flavor peptides, but most of the flavor peptides currently prepared from seafood through protease hydrolysis are single-function peptides, exhibiting only umami or salty flavors. The present invention intends to obtain potential salty and savory dual-functional flavor peptides by using white clams that are high in protein, low in lipids, and rich in flavor amino acids as raw materials through peptidomics and rapid virtual screening. Summary of the Invention

[0005] The present invention aims to provide a salty and savory dual-functional flavor peptide, its preparation method, and application, to address the above-mentioned problems in the prior art. The salty and savory dual-functional flavor peptide selected by the present invention has a strong salty and umami flavor and can be used as a substitute for sodium salt to prepare low-salt and low-sodium foods.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a salty and savory dual-functional flavor peptide P-1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a salty and savory dual-functional flavor peptide P-2, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides a salty and savory dual-functional flavor peptide P-3, the amino acid sequence of which is shown in SEQ ID NO.3.

[0010] The present invention also provides a salty and savory dual-functional flavor peptide P-4, the amino acid sequence of which is shown in SEQ ID NO.4.

[0011] The present invention also provides the use of the salty and savory bifunctional flavor peptide P-1, the salty and savory bifunctional flavor peptide P-2, the salty and savory bifunctional flavor peptide P-3 and / or the salty and savory bifunctional flavor peptide P-4 in the preparation of sodium salt replacement products.

[0012] The present invention also provides the use of the salty and savory bifunctional flavor peptide P-1, the salty and savory bifunctional flavor peptide P-2, the salty and savory bifunctional flavor peptide P-3 and / or the salty and savory bifunctional flavor peptide P-4 in the preparation of condiments.

[0013] The present invention also provides the use of the salty and savory bifunctional flavor peptide P-1, the salty and savory bifunctional flavor peptide P-2, the salty and savory bifunctional flavor peptide P-3 and / or the salty and savory bifunctional flavor peptide P-4 in improving the umami taste of food.

[0014] The present invention also provides the use of the salty and savory bifunctional flavor peptide P-1, the salty and savory bifunctional flavor peptide P-2, the salty and savory bifunctional flavor peptide P-3 and / or the salty and savory bifunctional flavor peptide P-4 in the preparation of low-salt and low-sodium foods.

[0015] The present invention also provides a sodium salt replacement product, comprising at least one of the above-mentioned salty and fresh bifunctional flavor peptide P-1, salty and fresh bifunctional flavor peptide P-2, salty and fresh bifunctional flavor peptide P-3 and salty and fresh bifunctional flavor peptide P-4.

[0016] The present invention also provides a seasoning, comprising at least one of the salty and savory bifunctional flavor peptide P-1, the salty and savory bifunctional flavor peptide P-2, the salty and savory bifunctional flavor peptide P-3, and the salty and savory bifunctional flavor peptide P-4.

[0017] The present invention also provides a method for preparing a salty and savory bifunctional flavor peptide, comprising the steps of synthesizing the salty and savory bifunctional flavor peptide by solid phase synthesis;

[0018] The amino acid sequence of the salty and savory bifunctional flavor peptide is shown in any one of SEQ ID NOs. 1-4.

[0019] The present invention discloses the following technical effects:

[0020] The present invention adopts a composite protease enzymatic hydrolysis method to prepare an enzymatic hydrolyzate rich in salty and savory bifunctional flavor peptides, and uses peptide omics and molecular docking technology to identify and screen a plurality of salty and savory bifunctional flavor peptides. At the same time, the transmembrane domains of taste receptors are evaluated and predicted, and the chemical bonds and docking sites of the salty and savory bifunctional flavor peptides docking with the taste receptors are analyzed. The key chemical bonds and binding sites of the interaction between the salty and savory bifunctional flavor peptides and the salty receptors are summarized, and their salty and savory flavor intensity is evaluated. The salty and savory bifunctional flavor peptides screened by the present invention have strong saltiness and savory taste, can be used as a substitute for sodium salt, and are used to prepare low-salt and low-sodium foods. The present invention also provides a certain reference for quickly and easily obtaining salty and savory bifunctional flavor peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Response surface diagram of enzyme addition amount and temperature;

[0023] Figure 2 is a contour plot of enzyme addition amount and temperature;

[0024] Figure 3 Response surface diagram of enzyme addition amount and time;

[0025] Figure 4 is a contour plot of enzyme addition amount and time;

[0026] Figure 5 is the response surface diagram of temperature and time;

[0027] Figure 6 is a contour plot of temperature and time;

[0028] Figure 7 The figure shows the bioinformatics screening of the savory and fresh bifunctional flavor peptides; A: a statistical diagram of the instability coefficient of the savory and fresh bifunctional flavor peptides; B: a statistical diagram of the isoelectric point of the savory and fresh bifunctional flavor peptides; C: a statistical diagram of the net charge of the savory and fresh bifunctional flavor peptides; D: a statistical diagram of the aliphatic coefficient of the savory and fresh bifunctional flavor peptides; E: a statistical diagram of the GRAVY of the savory and fresh bifunctional flavor peptides; F: a statistical diagram of the overall hydrophobicity of the savory and fresh bifunctional flavor peptides.

[0029] Figure 8 The distribution of amino acids and fragments that contribute to salty-umami taste;

[0030] Figure 9 A statistical chart of amino acids and fragments that contribute to salty-umami taste;

[0031] Figure 10 This is the molecular weight distribution diagram of the salty and savory dual-functional flavor peptide;

[0032] Figure 11 This is a statistical diagram of the mass-charge ratio of the salty and savory bifunctional flavor peptides;

[0033] Figure 12 This is the amino acid composition diagram of the salty and savory dual-functional flavor peptide;

[0034] Figure 13 This is the distribution diagram of the proportion of hydrophobic amino acids in the salty and savory dual-functional flavor peptide;

[0035] Figure 14 This is the distribution diagram of the proportion of hydrophilic amino acids in the salty and savory dual-functional flavor peptide;

[0036] Figure 15 This is the distribution diagram of the acidic amino acids in the salty and savory dual-functional flavor peptides;

[0037] Figure 16This is the distribution diagram of the proportion of basic amino acids in the salty and savory dual-functional flavor peptides;

[0038] Figure 17 This is the motif-logo diagram of the salty and savory bifunctional flavor peptide with a peptide chain length of 5 to 7;

[0039] Figure 18 This is the motif-logo diagram of the salty and savory bifunctional flavor peptide with a peptide chain length of 8 to 10;

[0040] Figure 19 This is the motif-logo diagram of the salty and savory bifunctional flavor peptide with a peptide chain length of 11 to 13;

[0041] Figure 20 Results of structure prediction and evaluation of taste receptors; A: Schematic diagram of TMC4 salty taste receptor; B: Ramachandran diagram of TMC4; C: Schematic diagram of T1R1 / T1R3 umami taste receptor; D: Ramachandran diagram of T1R1 / T1R3;

[0042] Figure 21 Predicted diagram of the transmembrane domain of taste receptors; A: amino acid composition of TMC4; B: hydrophobicity distribution of amino acids of TMC4; C: surface potential distribution of TMC4; D: transmembrane domain distribution of TMC4; E: amino acid composition of T1R1 / T1R3; F: hydrophobicity distribution of amino acids of T1R1 / T1R3; G: surface potential distribution of T1R1 / T1R3; H: transmembrane domain distribution of T1R1 / T1R3;

[0043] Figure 22 The distribution diagram of the binding energy between the salty and savory bifunctional flavor peptide and TMC4;

[0044] Figure 23 The distribution diagram of the binding energy of the salty and savory bifunctional flavor peptides docking with T1R1 / T1R3;

[0045] Figure 24 A visual diagram of the docking of LDWLAR and TMC4;

[0046] Figure 25 A visual diagram of the docking of WGDLFK and TMC4;

[0047] Figure 26 A visual diagram of the docking of LDTKFFK and TMC4;

[0048] Figure 27 A visual diagram of the docking of EDESLFL and TMC4;

[0049] Figure 28A visualization diagram of the docking of LDWLAR with T1R1 / T1R3;

[0050] Figure 29 A visualization diagram of the docking of WGDLFK and T1R1 / T1R3;

[0051] Figure 30 A visualization diagram of the docking of LDTKFFK with T1R1 / T1R3;

[0052] Figure 31 A visualization diagram of the docking of EDESLFL with T1R1 / T1R3;

[0053] Figure 32 This is the result of electronic tongue measurement of salty and savory dual-functional taste peptides;

[0054] Figure 33 This is the sensory scoring result of the salty and savory dual-functional flavor peptide;

[0055] Figure 34 This is the PCA analysis diagram of the sensory score of salty and savory dual-functional flavor peptides. DETAILED DESCRIPTION

[0056] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0057] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0058] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0059] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0060] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0061] Example 1

[0062] 1. Materials and Methods

[0063] 1.1 Main materials and reagents

[0064] Fresh white clams were purchased from the local market in Mazhang, Zhanjiang City. After washing and shelling, the clams were taken out and the meat was drained and packed into sample bags. They were then stored in a -20℃ refrigerator for later use.

[0065] Acetonitrile (purity ≥99.9%) was mass spectrometry grade and purchased from Fisher Chemical; formic acid (purity ≥98.0%) and ammonium bicarbonate (purity ≥99.5%) were mass spectrometry grade and purchased from Sigma-Aldrich; dithiothreitol (purity ≥99%) and iodoacetamide (purity ≥99%) were analytical grade and purchased from Sigma-Aldrich. The remaining reagents were of domestic analytical grade and purchased from Guangzhou Chemical Reagent Factory.

[0066] 1.2 Instruments and Equipment

[0067] Thermo Lynx-6000 high-speed floor-standing centrifuge, Thermo Fisher Scientific, USA; Easy-nLC 1200 high-performance liquid chromatograph, Thermo Fisher Scientific, USA; QExactive TM Hybrid Quadrupole-Orbitrap TM Mass Spectrometer electrospray-ion trap-Orbitrap mass spectrometer, Thermo Fisher Scientific, USA; SPD121P-230 vacuum centrifugal concentrator, Thermo Fisher Scientific, USA; DH3600A electric heating constant temperature incubator, Tianjin Test Instrument Co., Ltd.

[0068] 1.3 Experimental methods

[0069] 1.3.1 Preparation of white scallop meat hydrolysate

[0070] The shellfish flesh was broken up using a beater and accurately weighed to 300.0 g. Deionized water was added for homogenization, followed by homogenization at 12,000 rpm for 180 s (60 s homogenization, 60 s rest, three cycles). The pH of the enzymatic hydrolysis system was adjusted to neutral conditions, and the compound protease was added according to the ratio of enzyme activity to substrate protein, followed by enzymatic hydrolysis. After the enzymatic hydrolysis was complete, the hydrolyzate was placed in a boiling water bath to inactivate the enzyme for 15 min, cooled to room temperature, centrifuged to remove the precipitate, and then filtered through gauze to collect the supernatant for later use.

[0071] 1.3.2 Response surface optimization experiment

[0072] Taking the degree of hydrolysis as the response index, the addition amount of composite protease, time and temperature were selected as experimental factors (Table 1). According to the Box-Behnken principle, a suitable experimental design was selected for process optimization.

[0073] Table 1. Level coding table of response surface factors for optimization of white clams enzymatic hydrolysis process

[0074]

[0075] 1.3.3 Determination of hydrolysis degree

[0076] The total ammonia nitrogen content of shellfish meat (T1) and enzymatic hydrolysate (T2) was determined by formaldehyde titration, and the total protein nitrogen content of shellfish meat (P1) was determined by Kjeldahl method. The degree of hydrolysis was calculated according to the following formula:

[0077] Degree of hydrolysis = (T2-T1) / P1×100.

[0078] 1.3.4 Liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection of peptide sequences

[0079] Based on the results of response surface optimization, enzymatic hydrolysis of white clams was conducted under conditions that exhibited strong salty and umami flavors. The resulting white clams hydrolyzate, which exhibited a strong salty and umami flavor, was then processed through an ultrafiltration system (3000 Da) to obtain a hydrolyzate containing fractions <3000 Da. Furthermore, the <3000 Da fraction was reductively alkylated using dithiothreitol and iodoacetamide, desalted using a self-packed desalting column, and loaded onto an LC column (0.15 mm × 150 mm, RP-C18, Column Technology Inc.). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in 80% acetonitrile in water. The flow rate was 600 nL / min, and the total analysis time was 66 min. The LC gradient program was as follows: 0–2 min, linear gradient from 4% to 8% mobile phase B; 2–35 min, linear gradient from 8% to 28% mobile phase B; 35–55 min, linear gradient from 28% to 40% mobile phase B; 55–56 min, linear gradient from 40% to 95% mobile phase B; 56–66 min, mobile phase B maintained at 95%. LC-MS / MS data were acquired and generated into mass spectrum raw files. These files were opened with Xcalibur and searched using PEAKS Studio 10.6 software to obtain total ion current chromatograms and peptide sequences.

[0080] 1.3.5 Screening and bioinformatics analysis of savory and savory dual-functional flavor peptides

[0081] Based on the results of the taste peptide database (BIOPEP-UWM) and molecular docking (AutoDockTools), a total of 152 salty and umami bifunctional flavor peptides were screened, and the potential salty and umami amino acids or peptides of the salty and umami bifunctional flavor peptides were calculated and evaluated. At the same time, their isoelectric point (Expasy) and charge (Expasy), instability coefficient (Protparam), fat coefficient (Protparam), GRAVY (Protparam) and overall hydrophobicity (PepDraw) were calculated. Subsequently, Alphafold2 was used to predict the structure of the salty and umami bifunctional flavor peptides, and the model with the highest credibility was selected for molecular docking.

[0082] 1.3.6 Prediction of taste receptor structure and transmembrane domain

[0083] TMC4 (TransMembrane Channel-like 4) and T1R1 / T1R3 (Taste receptor type 1 member 1 / Taste receptor type 1 member 3) were selected as salty and umami receptors, respectively. The receptor protein sequences were obtained from the uniprot database. Swiss-Model, Discovery Studio, and Alphafold2 software were then used to predict their 3D structures, respectively. Ramachandran plots (PROCHECK) and SAVES (https: / / saves.mbi.ucla.edu / ) were used to evaluate and optimize their structures. The most appropriate model was selected for molecular docking. TMHMM-2.0 was used to calculate the transmembrane domains of the receptor proteins and analyze the positions of their N-termini.

[0084] 1.3.7 Molecular Docking of Salty and Fresh Bifunctional Taste Peptides with Salty Taste Receptors

[0085] Based on the structures of the salty and savory bifunctional taste peptide and taste receptor predicted by 1.3.5 and 1.3.6, the binding mode and energy after the ligand (salty and savory bifunctional taste peptide) binds to the receptor protein (TMC4 or T1R1 / T1R3) were predicted using AutoDockVina (version 1.5.6) software, which also showed the flexible conformation of the salty and savory bifunctional taste peptide and the rigid structure of the protein. The center coordinates of the docking box were set to x = 188.9, y = 149.8, z = 137.7, and the size of the box was x = 86.1, y = 68.3, z = 62.8. During the AutoDock Vina docking process, the docking pocket grid size was 90 × 90 × 90, and the default grid spacing was Furthermore, the stability of the interaction between the savory and umami bifunctional flavor peptide and taste receptor is reflected in their docking energy, with lower docking binding energies indicating more stable binding modes. Therefore, the configuration with the lowest docking binding energy was selected as the most suitable binding mode for analysis. PyMOL and Discovery Studio 2022 software were used to visualize the savory and umami bifunctional flavor peptide-taste receptor binding complex.

[0086] 1.3.8 Salty and umami taste determination of salty and umami dual-functional flavor peptides

[0087] The salty and umami bifunctional flavor peptides screened in the early stage were synthesized by solid-phase synthesis, and then prepared into 1.0 mg / mL aqueous solutions and filtered using a 0.45 μm filter membrane. The salty and umami intensities of the salty and umami bifunctional flavor peptides were measured using the electronic tongue taste sensors CTO (salty) and AAE (umami), respectively.

[0088] 1.3.9 Sensory Evaluation of Salty and Fresh Dual-Functional Flavor Peptides

[0089] A sensory evaluation of the salty and umami bifunctional flavor peptides was conducted using a quantitative descriptive analysis method. The intensity of saltiness and umami was assessed using a 10-point scale: very weak (0-2 points); weak (2-4 points); moderate (4-6 points); strong (6-8 points); and very strong (8-10 points). The saltiness / umami intensity of a 0.01 mg / mL NaCl / MSG standard solution was recorded as "0 points," and the saltiness / umami intensity of a 1.0 mg / mL NaCl / MSG standard solution was recorded as "10 points."

[0090] 20.0 mL of a salty-umami dual-functional flavor peptide (1.0 mg / mL) was dispensed into clean, transparent cups and randomly assigned a three-digit code. After equilibration at room temperature for 30 minutes, 20 trained food science students (aged 20-30 years) familiar with taste peptides were asked to perform a sensory evaluation and score the saltiness and umami intensity of the salty-umami dual-functional flavor peptide. All samples were evaluated under the same conditions.

[0091] 1.4 Data Statistics and Analysis

[0092] All samples were tested three times, and the data were analyzed using SPSS 17.0 software. Duncan's multiple test was used to test for significant differences (P < 0.05 indicated a significant difference). The graphs were drawn using Origin 2023 software. .

[0093] 2. Results and Analysis

[0094] 2.1 Establishment of response surface model and variance analysis

[0095] Based on the above single-factor test results, the enzyme addition amount, temperature and time can significantly affect the efficiency of the composite protease hydrolysis of white clams protein, and the saltiness and umami intensity, short peptide content and hydrolysis degree of the hydrolyzate are positively correlated. Therefore, the enzyme addition amount, time and temperature were selected as single response surface factors, and the hydrolysis degree was used as the response value. The response surface experimental design was carried out using Design-Expert.13 software to explore the influence of single factors on the enzymatic hydrolysis efficiency of white clams and optimize the efficiency of protease hydrolysis of white clams. The experimental data were fitted with a quadratic multivariate regression, and the quadratic regression equation of the hydrolysis degree (DH) with respect to the enzyme addition amount (A), enzymatic hydrolysis temperature (B) and enzymatic hydrolysis time (C) was obtained as follows: DH = 24.61 + 1.22A-1.27B + 0.71C-0.73AB + 0.17AC-0.15BC-0.80A 2 -4.57B 2 -1.51C 2 .

[0096] The shape of the contour lines can effectively reflect the significance of the interaction between single factors on the enzymatic hydrolysis efficiency, and the steepness of the 3D graph reflects the effects of enzyme addition, temperature and time on the hydrolysis degree of white clams. Figures 1 to 6 The interaction between the three single factors of enzyme addition, temperature, and time of enzymatic hydrolysis affects the degree of hydrolysis of white mussel hydrolysate. Among the single factors, the slopes of the response surface curves of enzyme addition and temperature, and temperature and time show a relatively steep trend, while the slope of the response surface curve of enzyme addition and time shows a relatively gentle trend, indicating that the interaction between enzyme addition and temperature, and temperature and time, has a significant effect on the degree of hydrolysis, while the interaction between enzyme addition and time has no significant effect on the degree of hydrolysis. In addition, with the increase of enzymatic hydrolysis temperature, the degree of hydrolysis shows a significant upward trend, while the surface curve of enzyme addition and time shows a relatively gentle trend, indicating that increasing the enzyme addition, extending the time, or excessively high temperature all have an adverse effect on the degree of hydrolysis. Therefore, it is speculated that the degree of hydrolysis is easily affected by the enzymatic hydrolysis temperature, and that excessively high temperature will reduce the activity of the composite protease, or even inactivate it, thereby inhibiting the enzymatic hydrolysis reaction. Based on the above analysis, the optimal enzymatic hydrolysis conditions for white mussel protein hydrolysis using the composite protease were determined to be: 5000 U / g enzyme, 47.77°C temperature, and 4.30 h. The predicted degree of hydrolysis reached 25.39%. To verify the reliability of the predicted results, the experiment was repeated three times under the optimal conditions, resulting in a degree of hydrolysis of 25.86% for white mussel protein hydrolyzed by the composite protease, with minimal deviation from the predicted value. This demonstrates that the model can effectively predict the efficiency of protease hydrolysis of white mussel protein.

[0097] 2.2 Bioinformatics analysis of salty and savory dual-functional flavor peptides

[0098] Based on the results of response surface optimization, enzymatic hydrolysis of white clams was carried out under conditions that maximized saltiness and umami. LC-MS / MS was then used for detection and identification. A total of 650 peptides (Denovo Score ≥ 80) were detected in the hydrolyzed fractions <3000Da, of which 152 were salty and umami bifunctional flavor peptides. Their peptide chains were 5 to 13 amino acids long, which is the ideal length for salty and umami bifunctional flavor peptides. The 152 salty and umami bifunctional flavor peptides were divided into three groups based on their peptide chain length: 40 peptides with lengths of 5 to 7, 68 peptides with lengths of 8 to 10, and 44 peptides with lengths of 11 to 13. Bioinformatics analysis was performed on the salty and umami bifunctional flavor peptides in these three groups. The results are as follows: Figures 7 to 12 As shown. Proteins with an instability index greater than 40 are unstable and easily mutated, while the instability coefficients of most salty and savory bifunctional flavor peptides are lower than 40, indicating that these salty and savory bifunctional flavor peptides are highly stable and not easily degraded ( Figure 7With the increase of peptide chain length, the isoelectric point of the salty and savory bifunctional flavor peptide shifts toward the alkaline direction, while the net charge content decreases. The salty and savory bifunctional flavor peptide with length (5-7) has the lowest isoelectric point and the highest net charge content ( Figure 7 B and C). In addition, the aliphatic index is the relative volume of the molecule occupied by the aliphatic side chains (alanine, valine, isoleucine and leucine), and GRAVY (Grand average of hydropathicity) is also an important indicator for evaluating the hydrophobicity of the molecular side chain structure, and its value ranges from -2.0 to 2.0. The larger the value of the aliphatic index and GRAVY, the stronger the hydrophobicity of the molecular side chain structure, but the length of the peptide chain does not affect the hydrophobicity of the side chain structure of the salty and savory bifunctional flavor peptide ( Figure 7 D and E). However, as the peptide chain length increases, the overall hydrophobicity of the salty and savory bifunctional flavor peptides increases significantly, but the overall hydrophobicity values of these salty and savory bifunctional flavor peptides are mainly distributed below 20 kcal / mol, indicating that they are highly hydrophilic and easily soluble in aqueous solution ( Figure 7 Current research indicates that the structure of savory and umami bifunctional flavor peptides is rich in acidic amino acids (such as aspartic acid and glutamic acid). These acidic amino acids are polar amino acids, which endow them with strong hydrophilicity, resulting in their excellent moisturizing effect in the mouth. Therefore, in order to analyze the salty and umami effects of the 152 savory and umami bifunctional flavor peptides, further analysis is needed.

[0099] 2.3 Structural characteristics analysis of salty and savory dual-functional flavor peptides

[0100] The salty and umami scores of the salty and umami bifunctional peptides decreased with the increase of peptide chain length. In the structures of these salty and umami bifunctional peptides, the amino acids and fragments that produce salty and umami tastes are mainly acidic amino acids and their related fragments ( Figures 8 and 9 The average molecular weight of the salty and fresh bifunctional flavor peptides of length (5-7) is 800Da, the average molecular weight of the salty and fresh bifunctional flavor peptides of length (8-10) is 1100Da, and the average molecular weight of the salty and fresh bifunctional flavor peptides of length (11-13) is 1300Da, but their m / z values are all distributed in the range of 500( Figures 10 and 11 In addition, the hydrophilic and hydrophobic amino acids of the salty and savory bifunctional flavor peptide are evenly distributed, each accounting for about 50% of the total amino acids, while the content of acidic and basic amino acids is as high as 10%. The salty and savory bifunctional flavor peptide is rich in Leu, Asp, Glu, Lys, Val and Phe. These amino acids can provide abundant docking active sites for the salty and savory bifunctional flavor peptide to bind to taste receptors ( Figures 12 to 16 ).

[0101] The characteristic amino acids at the N-terminus and C-terminus of the salty and savory dual-functional flavor peptides are as follows Figures 17 to 19 As shown in the structure of the savory bifunctional flavor peptide, the N1 terminus is primarily composed of Leu and Thr, the N2 and N3 termini are primarily composed of Asp, Leu, Ala, and Val; the C2 terminus is primarily composed of Leu, Val, Ala, and Phe, and the C1 and C3 termini are primarily composed of Asp, Lys, Arg, Leu, Val, and Phe. Therefore, the N- and C-termini of the savory bifunctional flavor peptide are primarily composed of polar and hydrophobic amino acids. It has been reported that polar amino acids provide abundant cations and anions (or highly electronegative atoms) to the savory bifunctional flavor peptide, allowing it to form stable ionic and hydrogen bonds with taste receptors. Hydrophobic amino acids, on the other hand, bind to taste receptors through hydrophobic interactions and contribute some hydrogen atoms to form stable hydrogen bonds. Therefore, polar and hydrophobic amino acids are key amino acids in savory bifunctional flavor peptides, and it is further speculated that these amino acids may act synergistically to bind to taste receptors. In addition, in order to maintain the stability of its own structure, polar amino acids and hydrophobic amino acids in the structure of the salty and savory bifunctional flavor peptide are arranged in a regular, spaced manner. If N1 in the salty and savory bifunctional flavor peptide structure is Leu, there will be at least one polar amino acid (such as aspartic acid) at the N2 and N3 positions. On the contrary, if N1 is Thr, the N2 and N3 positions are likely to be hydrophobic amino acids. In addition, the distribution of amino acids at the C-terminus has a similar structure to that of the N-terminus, which may be a symmetrical pattern of arrangement of polar and hydrophobic amino acids, stabilizing the structure of the salty and savory bifunctional flavor peptide. Therefore, polar and hydrophobic amino acids are easily recognized by taste receptors and can provide rich binding active sites for the salty and savory bifunctional flavor peptide. It is further speculated that the salty and savory bifunctional flavor peptide can form ionic bonds, hydrogen bonds and hydrophobic interactions with taste receptors.

[0102] 2.4 Analysis of taste receptor structure prediction and evaluation results

[0103] like Figure 20 As shown in the figure, TMC4 and T1R1 / T1R3 receptor models were constructed using a de novo folding method, and molecular docking was performed using them as salty and umami receptors, respectively. In order to obtain more information about the transmembrane structure of taste receptors, the amino acid sequence of taste receptors needs to be analyzed, with particular attention paid to the identification of the transmembrane domain. The results are shown in the figure. Figure 21As shown in Figure 2 , the hydrophilic and hydrophobic amino acids in the taste receptor structure are evenly distributed, accounting for approximately 50% of the total amino acids. This results in a uniform surface potential distribution, and both represent stable protein models. Furthermore, both taste receptors are rich in Leu, Ala, Val, Ser, Arg, and Glu, and the savory bifunctional taste peptide is also rich in hydrophobic and polar amino acids. Therefore, it is speculated that during docking with the savory bifunctional taste peptide, these hydrophobic amino acids provide hydrophobic interactions and hydrogen atoms for hydrogen bonding, while the polar amino acids provide ionic and hydrogen bonds, promoting the binding of the taste receptor and the savory bifunctional taste peptide to form a stable complex. Furthermore, TMC4 and T1R1 / T1R3, as typical transmembrane proteins, possess eight and 13 transmembrane domains, respectively. The N-terminus of TMC4 is on the inner side of the cell membrane, while that of T1R1 / T1R3 is on the outer side. The start and end positions of their transmembrane domains are shown in Table 2. Current research shows that the transmembrane domain is widely considered to be a conserved domain in the products of the transmembrane structure family of proteins, and plays a vital role in the structure and function of proteins.

[0104] Table 2 The start and end positions of the transmembrane domains of TMC4 and T1R1 / T1R3

[0105]

[0106]

[0107] 2.5 Analysis of the binding energy and docking results of salty and savory dual-functional taste peptides docked with taste receptors

[0108] Molecular docking is a computer-generated method that simulates the recognition and binding processes between two or more molecules. Semi-flexible docking was performed using Auto Dock Vina. By simulating the spatial structure and energy complementarity of molecules, the interaction mechanism between savory and savory bifunctional flavor peptides and taste receptors can be investigated. A total of 152 savory and savory bifunctional flavor peptides were screened from enzymatic hydrolysates of white clams and subsequently docked with taste receptors. The docking results showed that all 152 savory and savory bifunctional flavor peptides stably bound to taste receptors, with an average docking energy of -7.3 kca / mol. Furthermore, although docking binding energy is thought to be related to peptide chain length, ranking the docking binding energies of savory and savory bifunctional flavor peptides of different amino acid sequence lengths does not directly assess their taste intensity. Compared to short-chain salty and savory bifunctional peptides, long-chain peptides may have more action sites, with multiple amino acid residues capable of being recognized by taste receptors. However, the average docking binding energy of long-chain peptides is slightly higher than that of short-chain peptides. This may be because short-chain peptides have smaller molecular size and less steric exclusion, making them easier to be recognized by the active regions of taste receptors and bind to their active sites. Therefore, the shorter the salty peptide chain, the lower its docking binding energy and the more stable the complex formed with the taste receptor.

[0109] Table 34 Typical salty and savory dual-functional flavor peptides

[0110]

[0111]

[0112] In addition, the results of the ligand-receptor interaction of four typical salty and fresh bifunctional taste peptides (Table 3) were visualized and analyzed. Figures 22 to 31As shown. Analysis of the binding interaction modes and binding sites of the savory bifunctional flavor peptide with taste receptors revealed that the chemical bonds involved in their binding include ionic bonds, hydrophobic interactions, and hydrogen bonds, with hydrophobic interactions and hydrogen bonds being the key chemical bonds. During the docking process, the hydrogen bonds between the savory bifunctional flavor peptide and the taste receptor were evenly distributed, and the length of the hydrogen bonds directly affected the stability of the binding complex. However, the peptide chain length, structure, and physicochemical properties of the savory bifunctional flavor peptide significantly influenced the docking results, with the differences primarily reflected in the chemical bonds and binding sites of the savory bifunctional flavor peptide-taste receptor binding. Therefore, based on the results of the interactions of the savory bifunctional flavor peptide with taste receptors, the key sites for the savory bifunctional flavor peptide to interact with TMC4 via hydrophobic interactions are Ile426 and Leu520, while the key sites for the savory bifunctional flavor peptide to interact with TMC4 via hydrogen bonds are Arg506, Gln527, Leu520, Gln524, and Glu525. In addition, the key sites for the salty and savory bifunctional flavor peptide to interact with T1R1 / T1R3 through hydrophobic interactions are Leu51 and Ile244 of the T1R1 chain and Arg220 and Leu242 of the T1R3 chain, while the key sites for the salty and savory bifunctional flavor peptide to interact with T1R1 / T1R3 through hydrogen bonds are Ser109, Asn150, Ser217, Ser107, Asp243 of the T1R1 chain and Lys155, Ser224, and Glu240 of the T1R3 chain.

[0113] 2.6 Evaluation of salty and umami taste intensity of salty and umami dual-functional flavor peptides

[0114] The salty and umami intensity and their scores of four typical salty and umami bifunctional flavor peptides (LDWLAR, WGDLFK, LDTKFFK, EDESLFL) Figures 32 to 34As shown in the figure, the four typical salty-savory bifunctional flavor peptides all possess strong salty and umami flavors. Among them, LDWLAR, WGDLFK, and LDTKFFK possess the strongest salty and umami intensity and the highest sensory scores for saltiness and umami. Their saltiness intensity is stronger than that of NaCl at the same concentration, while their umami intensity is approximately 40% that of monosodium glutamate (MSG) at the same concentration. Compared with the other salty-savory bifunctional flavor peptides, although EDESLFL has weaker salty and umami intensity and lower salty and umami sensory scores, its saltiness intensity is still stronger than that of NaCl at the same concentration, and its umami intensity is approximately 30% that of MSG at the same concentration. In addition, based on the sensory scores of saltiness and umami of the four typical salty-savory bifunctional flavor peptides, their saltiness and umami scores mainly differ in principal component PC1, while there is little difference in principal component PC2, further demonstrating that LDWLAR, WGDLFK, and LDTKFFK have similar saltiness and umami intensities and all possess strong saltiness and umami flavors. Therefore, the four typical salty and umami dual-functional flavor peptides (LDWLAR, WGDLFK, LDTKFFK, and EDESLFL) all have strong salty and umami tastes, and can be used as substitutes for sodium salt (table salt and monosodium glutamate) to prepare low-salt and low-sodium foods.

[0115] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A salty and savory dual-functional flavor peptide P-1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A salty and savory dual-functional flavor peptide P-2, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. A salty and savory dual-functional flavor peptide P-3, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

4. A salty and savory dual-functional flavor peptide P-4, characterized in that: The amino acid sequence is shown in SEQ ID NO.

4.

5. Use of the savory and fresh bifunctional flavor peptide P-1 according to claim 1, the savory and fresh bifunctional flavor peptide P-2 according to claim 2, the savory and fresh bifunctional flavor peptide P-3 according to claim 3, and / or the savory and fresh bifunctional flavor peptide P-4 according to claim 4 in the preparation of a sodium salt substitute product.

6. Use of the savory and fresh bifunctional flavor peptide P-1 according to claim 1, the savory and fresh bifunctional flavor peptide P-2 according to claim 2, the savory and fresh bifunctional flavor peptide P-3 according to claim 3, and / or the savory and fresh bifunctional flavor peptide P-4 according to claim 4 in preparing a condiment.

7. Use of the savory and fresh bifunctional flavor peptide P-1 according to claim 1, the savory and fresh bifunctional flavor peptide P-2 according to claim 2, the savory and fresh bifunctional flavor peptide P-3 according to claim 3, and / or the savory and fresh bifunctional flavor peptide P-4 according to claim 4 in improving the umami taste of food.

8. Use of the savory and fresh bifunctional flavor peptide P-1 according to claim 1, the savory and fresh bifunctional flavor peptide P-2 according to claim 2, the savory and fresh bifunctional flavor peptide P-3 according to claim 3, and / or the savory and fresh bifunctional flavor peptide P-4 according to claim 4 in preparing low-salt and low-sodium foods.

9. A sodium salt replacement product, characterized in that: The invention comprises at least one of the salty and fresh bifunctional flavor peptide P-1 according to claim 1, the salty and fresh bifunctional flavor peptide P-2 according to claim 2, the salty and fresh bifunctional flavor peptide P-3 according to claim 3, and the salty and fresh bifunctional flavor peptide P-4 according to claim 4.

10. A method for preparing a salty and savory dual-functional flavor peptide, characterized in that: The method comprises the steps of synthesizing the salty and savory bifunctional flavor peptide by solid phase synthesis; The amino acid sequence of the salty and savory bifunctional flavor peptide is shown in any one of SEQ ID NOs. 1-4.

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