Salty and fresh difunctional taste-active peptide as well as preparation method and application thereof

Abstract

The invention discloses a salty and fresh difunctional taste-active peptide as well as a preparation method and application thereof, and relates to the technical field of biology. The amino acid sequence of the salty and fresh difunctional taste-active peptide is as shown in any one of SEQ ID NO.1-4. According to the invention, a compound protease enzymolysis method is adopted to prepare enzymatic hydrolysate rich in salty and fresh dual-function taste-active peptides, a plurality of salty and fresh dual-function taste-active peptides are identified and screened out by utilizing polypeptiomics and a molecular docking technology, and meanwhile, a transmembrane structure domain of a taste receptor is evaluated and predicted; chemical bonds and docking sites for docking of the salty and fresh difunctional taste-active peptide and the taste receptor are analyzed, key chemical bonds and binding sites for interaction of the salty and fresh difunctional taste-active peptide and the salty receptor are summarized, and the salty taste and umami strength of the salty and fresh difunctional taste-active peptide and the salty receptor are evaluated. The salty and fresh dual-function flavor peptide screened by the invention has strong salty taste and fresh taste, can be used as a substitute of sodium salt, and is used for preparing low-salt and low-sodium food. The invention also provides a certain reference for rapidly and simply obtaining the salty and fresh dual-function taste-active peptide.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a salty and fresh 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 gradual improvement of healthy eating awareness, people have begun to realize the health problems derived from excessive intake of sodium salt. It is urgent to find a way to "reduce salt and sodium without reducing taste", and "reducing salt and sodium without reducing taste" has also attracted the attention of many experts and scholars. People's requirements for the taste of food have also improved, and gradually formed a trend of "taste first". Therefore, the development of sodium salt substitutes has become a hot topic of research. In view of current research, salty and fresh bifunctional flavor peptides can give food a strong salty and umami taste, achieving the effect of "reducing salt and sodium without reducing taste". In addition, salty and fresh bifunctional flavor peptides, as protein hydrolysates or amino acid synthesis, have strong salty and umami characteristics at the same time, which can significantly enhance the salty and umami taste of food. Salty and fresh bifunctional flavor peptides can reduce the amount of sodium salt added without changing the saltiness and umami intensity of food, and at the same time participate in and affect the formation of other flavors, produce synergistic effects with other flavor compounds, give food a strong taste, and reduce the use of sodium salt. Salty and fresh bifunctional flavor peptides also have certain nutrition, supplementing the amino acids needed by the human body. Therefore, the application of salty and fresh dual-functional flavor peptides in food has huge space and potential.

[0004] Meretrix lyrata, also known as "white clam", "qin clam", "Vietnamese clam", "white sea fat", etc., is a low-value marine shellfish abundant in my country's waters, but due to its small body and difficulty in processing, its utilization value is low. The meat of white clam is delicious and rich in nutrients. The mass fraction of protein is as high as 58.13% (dry basis), and the lipid content is only 12.86 (dry basis). The amino acid composition is complete. Its total amount of amino acids and essential amino acids is higher than that of oysters, and is equivalent to that of mussels. The total content of sweet and umami amino acids in the protein of white clam accounts for 59.63% of the total free amino acids, among which the content of alanine and glycine is higher than that of oysters and other shellfish. In addition, current research has discovered a variety of flavor peptides, but the flavor peptides currently prepared by protease hydrolysis using seafood as raw materials are mostly single-function flavor peptides, which only present umami taste or only salty taste. The present invention intends to obtain potential salty and fresh 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 purpose of the present invention is to provide a salty and fresh dual-functional flavor peptide and its preparation method and application to solve the problems existing in the above-mentioned prior art. The salty and fresh dual-functional flavor peptide screened by the present invention has strong salty and fresh taste, 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 fresh 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 fresh 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 fresh 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 fresh 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 fresh bifunctional flavor peptide P-1, the salty and fresh bifunctional flavor peptide P-2, the salty and fresh bifunctional flavor peptide P-3 and / or the salty and fresh 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 fresh bifunctional flavor peptide P-1, the salty and fresh bifunctional flavor peptide P-2, the salty and fresh bifunctional flavor peptide P-3 and / or the salty and fresh bifunctional flavor peptide P-4 in the preparation of condiments.

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

[0014] The present invention also provides the use of the salty and fresh bifunctional flavor peptide P-1, the salty and fresh bifunctional flavor peptide P-2, the salty and fresh bifunctional flavor peptide P-3 and / or the salty and fresh 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 salty and fresh bifunctional flavor peptide P-1, the salty and fresh bifunctional flavor peptide P-2, the salty and fresh bifunctional flavor peptide P-3 and the salty and fresh bifunctional flavor peptide P-4.

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

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

[0018] The amino acid sequence of the salty and fresh 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 fresh bifunctional flavor peptides, and uses peptide omics and molecular docking technology to identify and screen a plurality of 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 the taste receptors, summarizing the key chemical bonds and binding sites of the interaction between the salty and fresh bifunctional flavor peptides and the salty taste receptors, and evaluating their salty and umami intensity. The salty and fresh bifunctional flavor peptides screened by the present invention have strong salty and umami tastes, 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. 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0022] Figure 1 It is the response surface diagram of enzyme addition amount and temperature;

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

[0024] Figure 3 The response surface diagram for enzyme addition amount and time;

[0025] Figure 4 It 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 It is a bioinformatics screening diagram of salty and fresh bifunctional flavor peptides; wherein, A: a statistical diagram of the instability coefficient of salty and fresh bifunctional flavor peptides; B: a statistical diagram of the isoelectric point of salty and fresh bifunctional flavor peptides; C: a statistical diagram of the net charge of salty and fresh bifunctional flavor peptides; D: a statistical diagram of the aliphatic coefficient of salty and fresh bifunctional flavor peptides; E: a statistical diagram of the GRAVY of salty and fresh bifunctional flavor peptides; F: a statistical diagram of the overall hydrophobicity of salty and fresh bifunctional flavor peptides;

[0029] Figure 8 The distribution map of amino acids and fragments that give off a salty-umami taste;

[0030] Fig. 9 A statistical diagram of amino acids and fragments that give off a salty-umami taste;

[0031] Fig.10 This is the molecular weight distribution diagram of salty and fresh bifunctional flavor peptides;

[0032] Fig.11 It is a statistical diagram of the mass-charge ratio of salty and fresh bifunctional flavor peptides;

[0033] Fig.12 This is the amino acid composition diagram of the salty and fresh bifunctional flavor peptide;

[0034] Fig.13 This is the distribution diagram of the proportion of hydrophobic amino acids in the salty and fresh bifunctional flavor peptides;

[0035] Fig.14 This is the distribution diagram of the proportion of hydrophilic amino acids in the salty and fresh bifunctional flavor peptides;

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

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

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

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

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

[0041] Fig. 20 The results of the 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] Fig.21 The predicted map of the transmembrane domain of the taste receptor; wherein, A: amino acid composition map of TMC4; B: hydrophobicity distribution map of amino acids of TMC4; C: surface potential distribution map of TMC4; D: transmembrane domain distribution map of TMC4; E: amino acid composition map of T1R1 / T1R3; F: hydrophobicity distribution map of amino acids of T1R1 / T1R3; G: surface potential distribution map of T1R1 / T1R3; H: transmembrane domain distribution map of T1R1 / T1R3;

[0043] Fig. 22 This is the distribution diagram of the docking binding energy between the salty and fresh bifunctional flavor peptide and TMC4;

[0044] Fig.23 The distribution diagram of the docking binding energy between the salty and fresh bifunctional flavor peptides and T1R1 / T1R3;

[0045] Fig.24 A visualization diagram of the docking of LDWLAR and TMC4;

[0046] Fig.25 A visualization diagram of the docking of WGDLFK and TMC4;

[0047] Fig.26 A visualization diagram of the docking of LDTKFFK and TMC4;

[0048] Fig. 27 A visualization diagram of the docking of EDESLFL and TMC4;

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

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

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

[0052] Fig.31 A visualization diagram of the docking of EDESLFL and T1R1 / T1R3;

[0053] Fig.32 This is the result of electronic tongue measurement of salty and fresh bifunctional taste peptides;

[0054] Fig.33 This is the sensory scoring result of salty and fresh dual-functional flavor peptides;

[0055] Fig.34 This is the PCA analysis diagram of the sensory score of salty and fresh bifunctional 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0058] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are 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. The clams were washed and shelled, and the meat was taken out. After draining the water, it was packed into sample bags and then stored in a -20℃ refrigerator for later use.

[0065] Acetonitrile (purity ≥99.9%) was of mass spectrometry grade and purchased from Fisher Chemical; formic acid (purity ≥98.0%) and ammonium bicarbonate (purity ≥99.5%) were of mass spectrometry grade and purchased from Sigma-Aldrich; dithiothreitol (purity ≥99%) and iodoacetamide (purity ≥99%) were of 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 Centrifuge 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 meat was broken up using a beater and 300.0 g was accurately weighed, deionized water was added for homogenization, and then homogenized at 12000 rpm for 180 s (homogenization for 60 s, rest for 60 s, and cycled 3 times), and the pH value of the enzymatic hydrolysis system was adjusted to a neutral condition, and a composite protease was added according to the ratio of enzyme activity to substrate protein, followed by enzymatic hydrolysis. After the enzymatic hydrolysis was complete, the enzymatic hydrolyzate was placed in a boiling water bath to boil and inactivate the enzyme for 15 min, cooled to room temperature, centrifuged to remove the precipitate, and then filtered with gauze to take the supernatant for standby 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 degree of hydrolysis

[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 nitrogen determination. 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, the enzymatic hydrolysis of white mussels was tested under conditions with strong salty and umami taste. The obtained white mussel hydrolysate with strong salty and umami taste was then treated by an ultrafiltration system (3000Da) to obtain the hydrolysate of <3000Da components. In addition, the <3000Da components were reductively alkylated using dithiothreitol and iodoacetamide, and desalted using a self-filled desalting column. The obtained sample was loaded on an LC column (0.15mm×150mm, RP-C18, Column Technology Inc.). Among them, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid-80% acetonitrile aqueous solution, flow rate: 600nL / min, total analysis time: 66min. The relevant LC gradient program is as follows: 0-2min, linear gradient of mobile phase B from 4% to 8%; 2-35min, linear gradient of mobile phase B from 8% to 28%; 35-55min, linear gradient of mobile phase B from 28% to 40%; 55-56min, linear gradient of mobile phase B from 40% to 95%; 56-66min, mobile phase B is maintained at 95%. The samples were collected by LC-MS / MS to generate mass spectrum Raw files, which were opened with Xcalibur and searched by PEAKS Studio10.6 software to obtain the total ion current chromatogram and peptide sequence of the samples.

[0080] 1.3.5 Screening and bioinformatics analysis of salty and savory bifunctional flavor peptides

[0081] Based on the results of the taste peptide database (BIOPEP-UWM) and molecular docking (AutoDockTools), a total of 152 salty-savory bifunctional flavor peptides were screened, and the potential salty and umami amino acids or peptides of the salty-savory 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-savory 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 receptortype 1member 1 / Taste receptor type 1member 3) were selected as the receptors for salty and umami taste, respectively. The receptor protein sequences were obtained from the uniprot database, and their 3D structures were predicted using Swiss-Model, Discovery Studio and Alphafold2 software, respectively. Ramachandran diagrams (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, and TMHMM-2.0 was used to calculate the transmembrane domains of the receptor proteins, and the positions of their N-termini were analyzed.

[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 fresh bifunctional taste peptides and taste receptors predicted by 1.3.5 and 1.3.6, the binding mode and energy of the ligand (salty and fresh bifunctional taste peptide) and the receptor protein (TMC4 or T1R1 / T1R3) were predicted using AutoDockVina (version 1.5.6) software, and the flexible conformation of the salty and fresh bifunctional taste peptide and the rigid structure of the protein were also shown. 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 In addition, the stability of the interaction between the salty and fresh bifunctional flavor peptides and taste receptors is reflected in their docking energy, and the lower the docking binding energy, the more stable their binding mode. Therefore, the configuration with the lowest docking binding energy was selected as the most suitable binding mode for analysis, and the salty and fresh bifunctional flavor peptide-taste receptor binding complex was visualized and analyzed using PyMOL and Discovery Studio 2022 software.

[0086] 1.3.8 Determination of salty and umami taste of salty and umami bifunctional 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 electronic tongue taste sensors CTO (salty) and AAE (umami) were used to measure the saltiness and umami intensity of the salty and umami bifunctional flavor peptides, respectively.

[0088] 1.3.9 Sensory evaluation of salty and savory dual-functional flavor peptides

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

[0090] 20.0mL of salty and umami dual-functional flavor peptide (1.0mg / mL) was dispensed into clean and transparent cups and randomly coded with 3 digits. After equilibration at room temperature for 30 minutes, 20 trained food major students (aged between 20 and 30 years old) who were familiar with taste peptides were organized to conduct sensory evaluation and score of the salty and umami intensity of the salty and 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 multiple test was used for significant difference test (P < 0.05 indicated significant difference). The graphs were drawn using Origin 2023 software. expressed in the form of .

[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 composite protease hydrolysis of white mussel protein, and the saltiness and umami intensity, short peptide content and hydrolysis degree of the enzymatic hydrolysate 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 mussels and optimize the efficiency of protease hydrolysis of white mussels. The experimental data were fitted by quadratic multivariate regression, and the quadratic regression equation of the degree of hydrolysis (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. Figure 1 to Figure 6 The interaction between the three single factors of enzyme addition, temperature and time of enzymatic hydrolysis affects the hydrolysis degree of white shellfish hydrolysate. Among the single factors, the slope of the response surface of enzyme addition and temperature, temperature and time shows a relatively steep trend, while the slope of the response surface of enzyme addition and time shows a relatively gentle trend, indicating that the interaction of enzyme addition and temperature, temperature and time has a significant effect on the hydrolysis degree, while the interaction of enzyme addition and time has no significant effect on the hydrolysis degree. In addition, with the increase of enzymatic hydrolysis temperature, the hydrolysis degree shows a significant upward trend, while the surface of enzyme addition and time shows a relatively gentle trend, indicating that increasing the enzyme addition, extending the time or too high temperature all have an adverse effect on the hydrolysis degree. Therefore, it is speculated that the hydrolysis degree is easily affected by the enzymatic hydrolysis temperature, and too 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 the composite protease hydrolysis of white mussel protein were obtained: enzyme addition 5000U / g, temperature 47.77℃, time 4.30h, and the predicted value of hydrolysis degree was as high as 25.39%. In order to verify the reliability of the experimental prediction results, the experiment was repeated 3 times under the optimal enzymatic hydrolysis conditions, and the hydrolysis degree of the composite protease hydrolyzed white mussel protein was 25.86%, which was less than the predicted value. It can be seen 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 the conditions with the strongest salty and umami taste, and detected and identified by LC-MS / MS. A total of 650 peptides (Denovo Score ≥ 80) were detected in the hydrolyzate of the <3000Da component, of which 152 were salty and umami bifunctional flavor peptides, and their peptide chain length was 5 to 13 amino acids, which is the ideal length for salty and umami bifunctional flavor peptides. According to the peptide chain length of the polypeptide, the 152 salty and umami bifunctional flavor peptides were divided into three groups, namely Length (5 to 7) with 40 peptides, Length (8 to 10) with 68 peptides, and Length (11 to 13) with 44 peptides. The three groups of salty and umami bifunctional flavor peptides were subjected to bioinformatics analysis, and the results are as follows: Figure 7 to Figure 12 As shown. Proteins with an instability index greater than 40 are unstable and easily mutated, while the instability coefficients of most salty and fresh bifunctional flavor peptides are less than 40, indicating that these salty and fresh bifunctional flavor peptides are highly stable and not easily degraded ( Figure 7As the length of the peptide chain increases, the isoelectric point of the salty and fresh bifunctional flavor peptide shifts toward the alkaline direction, while the net charge content decreases. The salty and fresh bifunctional flavor peptide with length (5-7) has the lowest isoelectric point and the highest net charge content ( Figure 7 In addition, the aliphatic index is the relative volume of the aliphatic side chain (alanine, valine, isoleucine and leucine) occupied by the molecule, and GRAVY (Grand average of hydropathicity) is also an important indicator for evaluating the hydrophobicity of the molecular side chain structure, and its value is distributed between -2.0 and 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 fresh bifunctional flavor peptide ( Figure 7 D and E). However, with the increase of peptide chain length, the overall hydrophobicity of the salty and fresh bifunctional flavor peptides increased significantly, but the overall hydrophobicity values ​​of these salty and fresh bifunctional flavor peptides were mainly distributed below 20 kcal / mol, indicating that they are highly hydrophilic and easily soluble in aqueous solution ( Figure 7 F). Current studies have shown that the structure of salty 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 give salty and umami bifunctional flavor peptides strong hydrophilicity, resulting in them having a good moisturizing effect in the mouth. Therefore, in order to analyze the effects of the 152 salty and umami bifunctional flavor peptides on salty and umami taste, they need to be further analyzed.

[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 flavor peptides decreased with the increase of peptide chain length, and in the structures of these salty and umami bifunctional flavor peptides, the salty and umami amino acids and fragments were mainly acidic amino acids and their related fragments ( Figure 8-Figure 9 The average molecular weight of the salty and fresh bifunctional flavor peptides with length (5-7) is 800Da, the average molecular weight of the salty and fresh bifunctional flavor peptides with length (8-10) is 1100Da, and the average molecular weight of the salty and fresh bifunctional flavor peptides with length (11-13) is 1300Da, but their m / z values ​​are all distributed in the range of 500( Figure 10-11 ). In addition, the hydrophilic and hydrophobic amino acids of the salty and fresh bifunctional flavor peptides 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%, and the salty and fresh bifunctional flavor peptides are rich in Leu, Asp, Glu, Lys, Val and Phe, which can provide abundant docking active sites for the salty and fresh bifunctional flavor peptides to bind to taste receptors ( Figure 12 to Figure 16 ).

[0101] The characteristic amino acids at the N-terminus and C-terminus of salty and fresh bifunctional flavor peptides are as follows Figure 17 to Figure 19 As shown in the figure, in the structure of the salty and fresh bifunctional taste peptide, the N1 terminus is mainly Leu and Thr, the N2 and N3 termini are mainly Asp, Leu, Ala, and Val; the C2 terminus is mainly Leu, Val, Ala, and Phe, and the C1 and C3 termini are mainly Asp, Lys, Arg, Leu, Val, and Phe. Therefore, the N-terminus and C-terminus of the salty and fresh bifunctional taste peptide are mainly polar amino acids and hydrophobic amino acids. It is reported that polar amino acids can provide abundant cations and anions (or highly electronegative atoms) for the salty and fresh bifunctional taste peptide, so that the salty and fresh bifunctional taste peptide can form stable ionic bonds and hydrogen bonds with the taste receptors, while hydrophobic amino acids can bind to the taste receptors through hydrophobic interactions and can provide part of the hydrogen atoms for the salty and fresh bifunctional taste peptide to form stable hydrogen bonds. Therefore, polar amino acids and hydrophobic amino acids are the key amino acids of the salty and fresh bifunctional taste peptide, and it is further speculated that these amino acids can synergistically bind to the taste receptors. In addition, in order to maintain the stability of its own structure, polar amino acids and hydrophobic amino acids in the salty and fresh bifunctional flavor peptide structure are arranged regularly in an intermittent manner. If N1 is Leu in the salty and fresh bifunctional flavor peptide structure, 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 the N-terminus, which may be a symmetrical pattern of arrangement of polar and hydrophobic amino acids, stabilizing the structure of the salty and fresh bifunctional flavor peptide. Therefore, polar and hydrophobic amino acids are easily recognized by taste receptors and can provide abundant binding active sites for salty and fresh bifunctional flavor peptides. It is further speculated that salty and fresh bifunctional flavor peptides 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 Fig. 20 As shown in the figure, TMC4 and T1R1 / T1R3 receptor models were constructed by computer de novo folding method, and they were used as salty and umami receptors for molecular docking. 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 special attention paid to the identification of transmembrane domains. The results are shown in the figure. Fig.21As shown. In the taste receptor structure, their hydrophilic and hydrophobic amino acids are evenly distributed and account for about 50% of the total amino acids, so their surface potential is evenly distributed, and they are all stable protein models. In addition, both taste receptors are rich in Leu, Ala, Val, Ser, Arg, and Glu, and the salty and fresh bifunctional taste peptides are also rich in hydrophobic amino acids and polar amino acids. Therefore, it is speculated that in the process of docking with the salty and fresh bifunctional taste peptides, these hydrophobic amino acids can provide hydrophobic interactions and hydrogen atoms to form hydrogen bonds for the taste receptors, while polar amino acids can provide ionic bonds and hydrogen bonds for the taste receptors, promoting the combination of taste receptors and salty and fresh bifunctional taste peptides to form a stable complex. In addition, TMC4 and T1R1 / T1R3 are typical transmembrane structure proteins, and they have 8 and 13 transmembrane domains, respectively. Among them, the N-terminus of TMC4 is on the inner side of the cell membrane, while the N-terminus of T1R1 / T1R3 is on the outer side of the cell membrane, and the starting and ending positions of their transmembrane domains are shown in Table 2. Current studies have shown that the transmembrane domain is widely considered to be a conserved domain in the products of the transmembrane 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 bifunctional flavor peptides and taste receptors

[0108] Molecular docking is a method that uses computers to simulate the recognition and mutual binding process between two or more molecules. Auto Dock Vina was used for semi-flexible docking, and by simulating the spatial structure and energy complementarity of molecules, the interaction mechanism between salty and fresh bifunctional flavor peptides and taste receptors can be studied. A total of 152 salty and fresh bifunctional flavor peptides were screened from the enzymatic hydrolysates of white clams, and then molecular docking with taste receptors was performed. According to the results of molecular docking, all 152 salty and fresh bifunctional flavor peptides can stably bind to taste receptors, and their average binding energy of docking is mainly distributed in -7.3kca / mol. In addition, although the docking binding energy is considered to be related to the length of the peptide chain, the docking binding energy ranking of salty and fresh bifunctional flavor peptides with different amino acid sequence lengths cannot directly evaluate their taste intensity. Compared with short-chain salty and fresh bifunctional taste peptides, long-chain peptides may have more action sites, and multiple amino acid residues can be recognized by taste receptors, but 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 small molecular volume and small spatial exclusion, and are easily recognized by the active area 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 fresh bifunctional flavor peptides

[0110]

[0111]

[0112] In addition, the results of the ligand-receptor interactions of four typical salty and fresh bifunctional flavor peptides (Table 3) were visualized and analyzed. Figure 22 to Figure 31As shown. By analyzing the binding interaction mode and binding site of salty and fresh bifunctional flavor peptides with taste receptors, it was found that the chemical bonds for their binding include ionic bonds, hydrophobic interactions and hydrogen bonds, and hydrophobic interactions and hydrogen bonds are the key chemical bonds for their binding. In the docking binding process, the hydrogen bonds for the docking binding of salty and fresh bifunctional flavor peptides and taste receptors are evenly distributed, and the length of hydrogen bonds can directly affect the stability of their binding complexes. However, the peptide chain length, structure and physicochemical properties of salty and fresh bifunctional flavor peptides can significantly affect the docking results, and the differences are mainly reflected in the chemical bonds and binding sites of salty and fresh bifunctional flavor peptides-taste receptors. Therefore, based on the results of the interaction between salty and fresh bifunctional flavor peptides and taste receptors, the key sites for the interaction between salty and fresh bifunctional flavor peptides and TMC4 through hydrophobic interactions are Ile426 and Leu520, while the key sites for the interaction with TMC4 through hydrogen bonds are Arg506, Gln527, Leu520, Gln524, and Glu525. In addition, the key sites for the salty-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-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 taste peptides (LDWLAR, WGDLFK, LDTKFFK, EDESLFL) Figure 32 to Figure 34As shown. The four typical salty and savory bifunctional flavor peptides all have strong salty and savory tastes. Among them, LDWLAR, WGDLFK and LDTKFFK have the strongest salty and savory taste intensity, the highest sensory scores of salty and savory taste, and their salty taste intensity is stronger than the same concentration of NaCl, while the savory taste intensity is about 40% of the same concentration of monosodium glutamate (MSG). Compared with other salty and savory bifunctional flavor peptides, although EDESLFL has weaker salty and savory taste intensity and lower salty and savory taste sensory scores, its salty taste intensity is still stronger than the same concentration of NaCl, and the savory taste intensity is about 30% of the same concentration of MSG. In addition, based on the sensory scores of salty and savory taste of the four typical salty and savory bifunctional flavor peptides, their salty and savory taste scores are mainly different from the main component PC1, while the difference in the main component PC2 is small, which further proves that LDWLAR, WGDLFK and LDTKFFK have similar intensity of salty and savory taste, and all have strong salty and savory taste. Therefore, the four typical salty and umami bifunctional 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

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

1.

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

2.

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

3.

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

4.

5. Use 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 / or the salty and fresh bifunctional flavor peptide P-4 according to claim 4 in the preparation of sodium salt substitute products.

6. Use 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 / or the salty and fresh bifunctional flavor peptide P-4 according to claim 4 in preparing condiments.

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

8. Use of the salty and fresh bifunctional flavor peptide P-1 as claimed in claim 1, the salty and fresh bifunctional flavor peptide P-2 as claimed in claim 2, the salty and fresh bifunctional flavor peptide P-3 as claimed in claim 3 and / or the salty and fresh bifunctional flavor peptide P-4 as claimed in claim 4 in the preparation of low-salt and low-sodium foods.

9. A sodium salt replacement product, characterized in that: It 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 fresh dual-functional flavor peptide, characterized in that: The method comprises the steps of synthesizing the salty and fresh bifunctional flavor peptide by solid phase synthesis; The amino acid sequence of the salty and fresh bifunctional flavor peptide is shown in any one of SEQ ID NOs. 1-4.

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