A red fish bone row fresh flavor peptide and its screening method and application

Abstract

The application discloses a red fish bone row umami peptide and a screening method and application thereof, relates to the technical field of umami substances, and can be combined with umami receptor protein T1R1 / T1R3, has the functions of umami and umami enhancement, and is one or several polypeptides shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. The umami peptide has a low umami threshold value, has a significant umami enhancement effect on low-concentration MSG, can replace or partially replace MSG, effectively improves food flavor, and realizes high-value utilization of red fish bone row by-products.

Description

Technical Field

[0001] This invention relates to the field of flavor substances technology, and more specifically to a red fish bone rib umami peptide, its screening method and application. Background Technology

[0002] Red snapper, also known as redfin snapper, is a flavorful marine fish. However, in current processing methods, the fish bones, a byproduct rich in high-quality protein and flavor-enhancing amino acids, are not fully utilized, leading to resource waste and environmental burden. In recent years, with the increasing attention given to environmental protection, the high-value utilization of aquatic product processing byproducts has become a research hotspot in the field of marine food science. Bio-enzymatic hydrolysis technology, as a mild, efficient, and green processing method, can directionally degrade the tightly structured collagen, myofibril protein, and other large molecules in fish bones into small-molecule peptides, free amino acids, and flavor-enhancing nucleotides through the hydrolysis of specific proteases.

[0003] Umami, as one of the five basic tastes, originates from the single stimulation of flavor nucleotides such as MSG. However, the complex umami perception in natural foods is actually the result of the synergistic effect of multiple flavor-active substances. Excessive intake of salt and MSG can have potential adverse effects on human health to some extent. Therefore, the nutritional and functional properties of umami peptides have received widespread attention in the condiment market. As a class of small-molecule flavor substances, umami peptides can not only directly activate T1R1 / T1R3 heterodimer receptors to produce umami, but also significantly lower the umami threshold and impart a lasting and rich flavor through synergistic effects with MSG, flavor nucleotides (IMP, GMP), etc.

[0004] Therefore, screening for highly active umami peptides from red carp bones is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This invention provides a red fish bone rib umami peptide, its screening method, and its application.

[0006] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0007] A red fish bone fillet umami peptide, wherein the red fish bone fillet umami peptide can bind to umami receptor proteins T1R1 / T1R3, and has the function of umami and enhancing umami, wherein the red fish bone fillet umami peptide is one or more polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0008] As a preferred technical solution, the main binding sites for molecular docking of the peptide segment with the amino acid sequence shown in SEQ ID NO: 1 to the umami receptor protein T1R1 / T1R3 are the amino acid residues Arg54, Tyr182, Gln181, Glu105, Glu148, Ala153, Thr154, Lys155, Ser104, Phe180, Asp215, Asp216 and Leu173 located on the T1R3 subunit; And / or, the main binding sites for molecular docking of the peptide with the amino acid sequence shown in SEQ ID NO: 2 with the umami receptor protein T1R1 / T1R3 are the amino acid residues Lys155, Val152, Met151, Thr154, Ala153, Ser104, Glu105, Glu148, Gln181, Asn150, Arg54, Asp216, Asp219, Asp218 and Arg220 located on the T1R3 subunit; And / or, the main binding sites for molecular docking of the peptide with the amino acid sequence shown in SEQ ID NO: 3 with the umami receptor protein T1R1 / T1R3 are the amino acid residues Lys155, Glu148, Thr154, Pro106, Ala153, Asp216, Asp219, Asp218, Asp215, Tyr182, Gln181 and Arg54 located on the T1R3 subunit; And / or, the peptide with the amino acid sequence shown in SEQ ID NO: 4 is molecularly docked with the umami receptor protein T1R1 / T1R3, with the main binding sites being the amino acid residues Lys155, Val152, Thr154, Ala157, Ala153, Val107, Pro106, Glu148, Glu217, Tyr182, Gln181, Asp219, Asp216, Arg220, and Arg54 located on the T1R3 subunit.

[0009] As a preferred technical solution, the threshold values ​​of the umami peptides with amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are 0.065 mg / mL, 0.065 mg / mL, 0.1 mg / mL and 0.25 mg / mL, respectively.

[0010] Another object of the present invention is to provide a method for screening the above-mentioned umami peptides, comprising the following steps: (1) The red fish bones were hydrolyzed by a combination of complex protease and flavor neutral protease to obtain the hydrolysate; (2) After separation by ultrafiltration and purification by gel chromatography, the peptide structure was identified by LC-MS / MS to obtain peptides with potential biological activity. Their physicochemical properties were predicted, and non-toxic peptides with good solubility were screened out. (3) Construct a T1R1 / T1R3 receptor protein model and evaluate its reliability; (4) Molecular docking of the T1R1 / T1R3 receptor protein model with the peptide from step (2) to form a receptor protein-small molecule ligand complex; (5) Based on the molecular docking results, peptides with potential umami effects are screened to obtain umami peptides.

[0011] As a preferred technical solution In step (1), the mass ratio of complex protease to flavor neutral protease is 9:1, the enzymatic hydrolysis conditions are 45 ℃ for 147 min, and the enzyme is inactivated at 100 ℃ for 15 min after enzymatic hydrolysis. The molecular weight cutoff for ultrafiltration in step (2) is <3000 Da; In step (2), the gel chromatography column was dextran G-15, eluted with ultrapure water; the third peak component F3 was collected. The bioactivity prediction in step (2) uses the following screening criteria: an activity score greater than 0.5. The T1R1 / T1R3 receptor protein in step (4) was dehydrated and hydrogenated using Discovery Studio (2017).

[0012] In step (4), molecular docking involves semi-flexible docking of the peptide with the constructed umami receptor T1R1 / TIR3 model. The screening criteria for step (5) are: select peptides with docking interaction energy lower than -120.00 kcal / mol.

[0013] Another object of the present invention is to provide the application of the above-mentioned umami peptides in the preparation of food seasonings or food.

[0014] Another object of the present invention is to provide a food seasoning comprising the above-mentioned umami peptides.

[0015] As a preferred technical solution, the food seasoning also includes monosodium glutamate (MSG).

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention screened and identified four novel umami peptides from red fish bones and identified their binding sites and mechanisms of action with umami receptors T1R1 / T1R3, thus realizing the high-value utilization of red fish bone by-products.

[0017] (2) The umami peptide of the present invention has a low umami threshold and has a significant umami enhancement effect on low concentration MSG, which can effectively improve the flavor of food.

[0018] (3) The umami peptides of the present invention are suitable as a substitute for monosodium glutamate or for use in combination with monosodium glutamate, which can reduce the amount of monosodium glutamate used by about 30%, and meet the needs of condiment development for sodium reduction and umami enhancement and clean labeling. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 Gel chromatogram of the peptides obtained after ultrafiltration fractionation in Example 1; Figure 2 Sensory and electronic tongue results of the components separated by gel chromatography in Example 1; Figure 3 The identification results of F3 component separated by gel chromatography in Example 1, where A is the statistical result of polypeptide type and B is the statistical result of amino acid type; Figure 4 : 3D structural diagram of umami receptor T1R1 / TIR3; Figure 5 La-plot of umami receptors T1R1 / T1R3; Figure 6 : Conformation analysis results of peptide MRWNK; Figure 7 Results of CRLDPY conformational analysis of peptides; Figure 8 : Conformation analysis results of peptide RASYPRF; Figure 9 Conformation analysis results of peptide RRFNMVP; Figure 10 Electron cloud diagram of the docking interaction force between umami peptides and receptor proteins T1R1 / T1R3; Figure 11 Sensory profile of synthetic peptides; Figure 12 : Graph showing the flavor-enhancing effect of synthetic peptides on MSG. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] The reagents required for this invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0023] The term "red fish bone fillet" in this invention refers to the fish bone fillet (excluding the head) of a red fish after mechanical processing and removal of the muscle tissue from both sides. It mainly consists of vertebrae, ribs, and corresponding cartilage tissue. The red fish bone fillets used in the embodiments of this invention were provided by Dandong Taifeng Food Co., Ltd. (Dandong City, Liaoning Province), but this is not intended to limit the source.

[0024] Complex protease: Protamex® 1.6 Novozymes Investment Co., Ltd., enzyme activity 1.6 AU-N / g.

[0025] Flavor neutral protease: Shandong Longkete Enzyme Preparation Co., Ltd., enzyme activity 100,000 u / g.

[0026] Example 1 1. Complex enzymatic hydrolysis 0.24% of a compound enzyme was added based on the weight of red fish bones. The compound enzyme consisted of a compound protease and a flavor neutral protease in a ratio of 9:1 (w / w), and was enzymatically hydrolyzed at 45 °C for 147 min.

[0027] After the enzymatic hydrolysis was completed, the enzyme was inactivated at 100 °C for 15 min. After the system cooled to room temperature, it was centrifuged at 4 °C and 8000 ×g for 10 min. The supernatant was then collected to obtain the enzymatic hydrolysate.

[0028] 2. Ultrafiltration separation The enzymatic hydrolysate was filtered through a 0.22 μm membrane to remove macromolecules, followed by ultrafiltration using a 3000 Da membrane at 4 °C. Fractions with molecular weights <3000 Da were collected and concentrated under reduced pressure at 50 °C and a vacuum of 0.09 MPa to 1 / 4 of their original volume. The concentrate was then placed in a [further context needed]. Pre-freeze at 80 ℃ for 12 h, then freeze-dry in a vacuum freeze dryer (Labconc, Free Zone 2.5 L, USA). Freeze-drying conditions: cold trap temperature: Red fish bone fillet umami crude peptides were obtained by freeze-drying at 60 ℃, vacuum degree: 1 Pa, and freeze-drying time of 48 h, and stored at -80 ℃.

[0029] 3. Gel chromatography purification The crude peptides of red fish bone ribs were redissolved in ultrapure water to prepare a 20 mg / mL solution, which was then separated and purified using dextran G-15.

[0030] Elution conditions: Injection volume 2 mL, ultrapure water as the eluent, UV detection wavelength 214 nm, flow rate 0.8 mL / min, 10 mL collected per fraction, column type: NmXK 16100-AF (Suzhou Nanomicro Technology Co., Ltd.). Each individual peak was collected and labeled F1-F3 (gel chromatogram as shown). Figure 1 As shown), each component was concentrated to 1 / 4 of its original volume under conditions of 50 °C and a vacuum of 0.09 MPa. The concentrate was then placed in... Pre-freeze at 80 ℃ for 12 h, then freeze-dry in a vacuum freeze dryer (Labconc, Free Zone 2.5 L, USA). Freeze-drying conditions: cold trap temperature: Freeze-dried red fish bone umami peptide powders were obtained by freezing at 60 ℃, vacuum degree: 1 Pa, and freeze-drying time of 48 h.

[0031] Example 2 1. The optimal components after gel chromatography separation were screened using sensory evaluation and electronic tongue. The sensory evaluation team consisted of 5 men and 5 women, aged 20-30. Prior to the evaluation, team members underwent basic taste sensory training to accurately distinguish between umami, sour, sweet, bitter, and salty flavors. Sensory evaluation tests were conducted in a sensory laboratory at 25 ± 2 ℃. Sensory evaluation was performed on four aspects: umami, fishy smell, odor, and acceptability (weighted at a ratio of 3:3:2:2). The final evaluation result was based on the total score of these four aspects. Specific scoring criteria are shown in Table 1.

[0032] The sensory evaluation method is as follows: 20 mL of 1 mg / mL solution was prepared from each of the lyophilized components F1-F3 using ultrapure water as sensory evaluation sample solutions. The sensory evaluation team then scored and evaluated each flavor. The score for each flavor was the average of the scores from all evaluators. The results are shown in [the table below]. Figure 2 As shown in the figure, component F3 had the strongest flavor-enhancing effect, followed by F2; at the same time, the scores of each component were higher than those of the control group.

[0033] The electronic tongue analysis method is as follows: Lyophilized components F1-F3 were reconstituted in ultrapure water to prepare 0.5 mg / mL solutions, which were then analyzed using an electronic tongue (Insent SA402B). The injection rate for the electronic tongue analysis was 10 s, the detection temperature was 25℃, and each peptide was tested four times. The data from the last three tests were used as the raw data. The results are shown below. Figure 2As shown in the figure, the F3 component had the highest umami response value, which was significantly different from the blank group (P<0.05).

[0034] Table 1 Sensory Evaluation Weighting Table

[0035] 2. Identification of peptide structures by LC-MS / MS The peptide sequence of component F3, which exhibited good umami flavor after gel chromatography purification, was identified. 1.2 mg of the lyophilized sample was dissolved in 50 mmol / L NH4HCO3. The resulting solution was centrifuged at 16,000 × g for 10 min at 4 °C, and the supernatant was collected. 200 μL of the supernatant was mixed with 2 μL of DL-dithiothreitol (DTT, 10 mmol / L) and reacted in a 56 °C water bath for 1 h. Subsequently, 0.6 mol / L IAM solution was added to the sample until the concentration reached 55 mol / L, and the reaction was carried out at room temperature in the dark for 40 min. After the reaction, 1 mol / L DTT solution was added to neutralize any unreacted IAM, bringing the final concentration to 20 mmol / L. This yielded the final sample for instrumental testing.

[0036] The chromatographic separation system used a Reprosil-Pur 120 C18 reversed-phase column (3 μm, 100 μm × 180 mm) for elution at a flow rate of 600 nL / min in constant flow mode. The sample analysis time was 66 min. The chromatographic conditions were as follows: mobile phase A consisted of 0.1% (v / v) formic acid, and mobile phase B consisted of 0.1% (v / v) formic acid and 80% (v / v) acetonitrile. The full scan range of mass spectrometry was 100–1500 m / z. The resolution of the first-order mass spectrometry was 70,000, and the peptide fragments were broken up using a high-energy collisional dissociation method. The fragmented peptides were then analyzed by second-order mass spectrometry at a resolution of 17,500 and a fragmentation energy of 28 eV. Database analysis was used to obtain relevant information about the peptides from the second-order mass spectrometry data. The statistical results of peptide and amino acid types are as follows: Figure 3 As shown, a total of 5018 peptides were identified in the F3 fraction, with 4- to 6-peptides being the most numerous. The results are shown in [see attached table]. Figure 3 A. Amino acid types such as Figure 3 As shown in Figure B, alanine (A), aspartic acid (D), glutamic acid (E), isoleucine (I), proline (P), and threonine (T) are present in higher amounts.

[0037] 3. Activity prediction and screening of umami peptides A total of 5018 peptides were identified in fraction F3. The activity of each peptide was predicted using the Peptide Ranker tool, and the potential toxicity of the peptides was analyzed online using ToxinPred. Non-toxic sequences were selected for further screening. The water solubility of the peptides was assessed using the online tool Innovagen, and sequences with good water solubility were selected. 540 peptides with an activity score greater than 0.5, non-toxicity, and good solubility were obtained. The online website http: / / tastepeptides-meta.com / TPDM was used to verify whether they were umami peptides, and sequences showing as umami peptides were selected for further screening. The frequency of bioactive fragments of umami peptides was calculated using the online website https: / / biochemia.uwm.edu.pl / bioactivity-prediction. Finally, the novelty of these peptides was investigated online using https: / / www.uwm.edu.pl / biochemia / php / en / biopep. Based on this, 26 high-quality peptides with both clear bioactivity and excellent overall properties were finally obtained, and the results are shown in Table 2.

[0038] Table 2. Prediction results of peptides

[0039] Example 3 1. Homology modeling of umami receptors T1R1 / T1R3 The amino acid sequences of the umami receptors TIR1 / T1R3 were obtained from UniprotK (https: / / www.uniprot.org / ) (TIR1: Q7RTX1 and TIR3: Q7RTX0, with sequence similarities of 32% and 35%, respectively). Homology modeling was performed using a metabolic glutamate receptor as a template (PDB ID: 1EWK). Homology modeling of T1R1 / T1R3 was performed using SWISS-MODEL (https: / / swissmodel.expasy.org / ) based on sequence homology between the receptor and the template. The homology model of the umami receptors T1R1 / T1R3 is shown below. Figure 4 As shown, the left side represents the crystal structure of T1R1, and the right side represents the crystal structure of T1R3. The crystal structure of T1R1 on the left is in a closed state, while the crystal structure of T1R3 on the right is in an open state with a sufficiently large cavity to bind ligand molecules.

[0040] To further examine and confirm the reliability of the homology model, Laplace plots were generated using the online SAVESv6.0 (https: / / saves.mbi.ucla.edu / ) for evaluation. The Laplace plots are shown below. Figure 5As shown, the residue distribution in this model is extremely ideal: 99.4% of the residues are located in the allowed region, of which 87.8% are in the most favorable region, 10.1% are in the acceptable region, only 1.5% are in the generally allowed region, and only 0.5% are in the disallowed region. This residue distribution indicates that the umami receptor T1R1 / T1R3 homology model established in this study has extremely high accuracy and reliability. The model's conformation is spatially highly reasonable and can realistically reflect the structural characteristics of the umami receptor. Therefore, this model is fully capable of being used for subsequent molecular docking studies of umami peptides and umami receptors, providing a solid structural foundation for in-depth exploration of the molecular mechanism of interaction between umami peptides and umami receptors, and ensuring the scientific validity and effectiveness of molecular docking experiments.

[0041] 2. Molecular docking of umami peptides with umami receptors T1R1 / T1R3 First, Chem Draw (23.1.1) was used to visualize the umami peptide structure based on its sequence. Discovery Studio (2017) was used to pre-treat the receptor protein by dehydration and hydrogenation, and docking sites were defined. After optimization using the Minimization Ligand protocol, the peptide was semi-flexibly docked with the constructed umami receptor T1R1 / TIR3 model, and the interaction strength was assessed based on the docking energy. Then, the complex of the umami peptide and receptor protein was visualized in two and three dimensions, and interaction force cloud plots were analyzed. The docking results are shown in Table 3.

[0042] Table 3. Docking energy results of umami peptides with umami receptors T1R1 / T1R3

[0043] To elucidate the interaction mechanism between umami peptides and the umami receptors T1R1 / T1R3, molecular docking technology was used to screen 26 candidate peptides for those exhibiting strong interactions with the umami receptors. Of these 26 candidate peptides, 17 successfully docked with the receptor protein; these peptides are potential umami peptides. Lower docking energies indicate stronger affinity between the ligand (peptide) and the receptor, resulting in a more stable structure after binding. Among these peptides, MRWNK, CRLDPY, RASYPRF, and RRFNMVP showed docking energies below -120.00 kcal / mol, indicating strong binding affinity to the umami receptors T1R1 / T1R3 and stable structure after binding. Therefore, these four peptides were selected for further experiments to verify their activity and application potential as potential umami peptides.

[0044] 3. Interaction analysis between umami peptides and T1R3 To further clarify the interaction mechanism between MRWNK, CRLDPY, RASYPRF, RRFNMVP and the T1R1 / T1R3 receptor protein, conformational analysis was performed on MRWNK, CRLDPY, RASYPRF, and RRFNMVP from step 2. The results are attached. Figure 6 -Appendix Figure 9As shown, all four peptide ligands can be well embedded in the VFTD of T1R3, and the interaction forces between the receptor and the peptide are mainly hydrogen bonds, van der Waals forces and hydrophobic interactions. Visualization results of the docking between umami peptides and the receptor proteins T1R1 / T1R3 showed that the main binding sites for RRFNMVP with umami receptor proteins T1R1 / T1R3 were the amino acid residues Arg54, Tyr182, Gln181, Glu105, Glu148, Ala153, Thr154, Lys155, Ser104, Phe180, Asp215, Asp216, and Leu173 located on the T1R3 subunit; the main binding sites for RASYPRF with umami receptor proteins T1R1 / T1R3 were the amino acid residues Lys155, Val152, Met151, Thr154, Ala153, Ser104, Glu105, Glu148, Gln181, Asn150, Arg54, Asp216, and Leu173 located on the T1R3 subunit. p219, Asp218, and Arg220; the main binding sites for MRWNK to dock with the umami receptor protein T1R1 / T1R3 are the amino acid residues Lys155, Glu148, Thr154, Pro106, Ala153, Asp216, Asp219, Asp218, Asp215, Tyr182, Gln181, and Arg54 located on the T1R3 subunit; C RLDPY performs molecular docking with the umami receptor protein T1R1 / T1R3, with the main binding sites being the amino acid residues Lys155, Val152, Thr154, Ala157, Ala153, Val107, Pro106, Glu148, Glu217, Tyr182, Gln181, Asp219, Asp216, Arg220, and Arg54 located on the T1R3 subunit. The binding sites for both include a variety of amino acid residues such as Ala153, Ala157, Ala46, Arg151, Arg220, Arg54, Asn150, Asp108, Asp215, Asp216, Asp218, Asp219, Gln181, Gln221, Glu105, Glu148, Glu178, Glu217, Leu173, Leu245, Lys155, Met151, Phe180, Pro106, Pro244, Pro246, Pro57, Ser104, Ser109, Ser146, Ser173, Thr154, Thr179, Tyr182, Val107, and Val152, among which Glu148, Arg54, Lys155, and Asp215 are the main binding sites.Glutamic acid (Glu), arginine (Arg), lysine (Lys), and aspartic acid (Asp) are crucial for umami flavor presentation. The carboxyl group on the Glu side chain can form hydrogen bonds or ionic bonds with specific groups on the receptor, stabilizing peptide-receptor binding; the guanidinium group on Arg enhances binding specificity through interaction with the negatively charged groups on the receptor; the long side chain of Lys can penetrate deep into the receptor binding pocket, consolidating binding through non-covalent interactions such as van der Waals forces; the carboxyl group on Asp can also form hydrogen bonds, providing a key binding site for umami perception. The synergistic effect of these amino acid residues enables umami peptides to precisely bind to receptor proteins, triggering downstream signal transduction pathways and ultimately producing umami perception.

[0045] The binding forces between ligands and receptors in the spatial structure significantly affect umami intensity. Hydrogen bonds and van der Waals forces, as the strongest intermolecular interactions, can effectively reduce peptide-receptor binding energy; hydrophobic interactions act as auxiliary forces to improve complex stability. The synergistic effect of these three factors may enhance the umami perception of umami peptides. Increased solvent-accessible surface area is a characterization of conformational dynamics or dissociation at the binding interface, driven by entropy effects and solvation energy. Van der Waals forces maintain structural compactness and limit excessive expansion of the SASA, ensuring the rationality of the docking conformation. Six interaction forces exist between MRWNK, CRLDPY, RASYPRF, RRFNMVP, and the receptor in step 2; the results are shown in […]. Figure 10 Docking energy analysis revealed that the interaction between the umami peptide and T1R3 is primarily driven by hydrogen bonding and hydrophobic contact. Although the umami peptide exhibits strong hydrophilicity due to its abundance of hydrophilic groups such as -NH2, -COOH, and -OH, its nonpolar side chains can still form stable hydrophobic interactions with the receptor. The high SASA value is consistent with its hydrophilic characteristics, indicating that the peptide chain maintains moderate solvent exposure within the binding pocket and does not undergo nonspecific hydrophobic collapse. In summary, the synergistic effect of hydrogen bonding and hydrophobic interactions constitutes the core molecular force for the recognition of T1R1 / T1R3 by the umami peptide.

[0046] 4. Solid-phase synthesis of peptides We commissioned Nanjing Peptide Valley Biotechnology Co., Ltd. to synthesize four peptides, MRWNK, CRLDPY, RASYPRF, and RRFNMVP, using solid-phase synthesis, with a purity >98%.

[0047] 5. Sensory evaluation to verify the taste characteristics of the synthetic peptides. The panel consisted of 5 men and 5 women, aged 20-30. Prior to the evaluation, panel members underwent basic sensory training to accurately distinguish between umami, sour, sweet, bitter, and salty tastes. Sensory evaluation tests were conducted in a sensory laboratory at 25 ± 2 ℃. Evaluators were required to provide sensory evaluations based on sweet, salty, sour, bitter, and umami tastes, with a 1-minute break between each sample. Samples were evaluated using a scoring system from 0 to 10. Five basic taste compounds were selected to establish a taste evaluation benchmark: umami, sweet, bitter, sour, and salty tastes corresponded to MSG solution (0.35 g / 100 mL), sucrose solution (1 g / 100 mL), L-isoleucine solution (0.25 g / 100 mL), citric acid solution (0.08 g / 100 mL), and sodium chloride solution (0.35 g / 100 mL), respectively. The intensity of taste perception at the above concentrations was uniformly calibrated to a benchmark value of 5 points. The scoring details are shown in Table 4.

[0048] Table 4 Sensory Evaluation Criteria

[0049] Sensory evaluation was performed on the four peptides synthesized in step 4, as follows: 20 mL of each of the four peptides was prepared using ultrapure water to create a 1 mg / mL solution as sensory evaluation sample. The sensory evaluation panel scored each peptide, and the score for each taste was the average of the scores from all evaluators. The results are shown in [Figure number missing]. Figure 8 .

[0050] Depend on Figure 11 The sensory evaluation results of the four synthetic peptides showed significant differences in their flavor characteristics. CR6 had the highest umami intensity (4.40 points), the fewest off-flavors, and a pure taste, demonstrating its potential as a highly efficient umami enhancer. RR7 had the second highest umami intensity (3.90 points), with a balanced and harmonious distribution of various flavor attributes, making it suitable for complex food systems where flavor balance is crucial. MR5 had a moderate umami intensity (3.75 points), but the best effect in suppressing bitterness and sourness, resulting in a mild and pleasant taste, suitable for applications seeking a refreshing flavor. RA7 had the weakest umami intensity (3.40 points), with relatively prominent sourness and bitterness, poor flavor harmony, and its overall quality needs further optimization. Although the umami scores of all four synthetic peptides were lower than the standard (5.00 points), their umami intensity had reached or exceeded the average level of common commercial umami peptides, showing promising application prospects.

[0051] 6. Umami Threshold of Synthetic Peptides The umami threshold of the synthetic peptide was determined using a taste dilution analysis method. First, a 1 mg / mL sample solution of the synthetic peptide was prepared and then serially diluted 1:1 (v / v) with ultrapure water. Evaluators tested the synthetic peptide solutions at different dilution levels in ascending order of concentration, using a three-point test method for differentiation. When the evaluators could clearly distinguish a particular dilution level of the sample solution from the blank control (ultrapure water), the corresponding concentration of the sample solution was identified as the umami threshold of that synthetic peptide. The results are shown in Table 5.

[0052] Table 5 Umami Thresholds of Synthetic Peptides

[0053] The results showed that the umami thresholds of MRWNK, CRLDPY, RASYPRF, and RRFNMVP were 0.1 mg / mL, 0.25 mg / mL, 0.065 mg / mL, and 0.065 mg / mL, respectively, all significantly lower than that of monosodium glutamate (0.3 mg / mL). This confirms that these peptides can be perceived at lower concentrations, exhibiting superior umami efficiency compared to traditional umami enhancers. Descriptive flavor analysis revealed that these peptides are predominantly umami-based, with a subtle saltiness and a mild sweetness. The three flavors are harmoniously integrated, resulting in a rounded and full-bodied taste without any unpleasant aftertaste, presenting a richer and more diverse flavor profile than monosodium glutamate. This complex flavor characteristic allows them to exhibit a more natural and three-dimensional umami enhancement effect in food systems. Combined with their low threshold advantage, they demonstrate significant application potential in the field of salt reduction and umami enhancement.

[0054] 7. Synergistic flavor-enhancing effect of synthetic peptides To analyze the flavor-enhancing effect of synthetic peptides on monosodium glutamate (MSG), 0.15%, 0.25%, and 0.35% MSG solutions were used as references. Then, 0.2 mg / mL synthetic peptide solutions were prepared using these MSG solutions, and the flavor-enhancing effect was determined using an electronic tongue assay. The results are as follows: Figure 12As shown in the figure, the umami intensity produced by the combination of each peptide with monosodium glutamate (MSG) exhibits a typical nonlinear synergistic effect. Under low concentration MSG (0.15%), the four synthetic peptides showed the most significant umami enhancement efficiency, with MRWNK (55.32%), CRLDPY (44.52%), RASYPRF (49.83%), and RRFNMVP (50.33%), indicating that the synthetic peptides have a more prominent taste-enhancing effect in the low MSG concentration system. As the MSG concentration increases, the umami enhancement effect shows a significant decreasing trend. Under 0.35% MSG conditions, the umami enhancement efficiency of each peptide decreases to the 15-35% range, indicating that the synergistic effect has a concentration-dependent characteristic. Notably, the umami intensity value of the MRWNK + 0.35% MSG combination reached 10.72 (relative unit), significantly surpassing the single 0.35% MSG solution (8.97), achieving a technological breakthrough of reducing MSG usage by nearly 30%.

[0055] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0056] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A red fish bone rib umami peptide, characterized in that, The red fish bone umami peptide can bind to the umami receptor protein T1R1 / T1R3, and has the function of umami and enhancing umami. The red fish bone umami peptide is one or more polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

4.

2. The umami peptide according to claim 1, characterized in that, The main binding sites for molecular docking of the peptide with the amino acid sequence shown in SEQ ID NO: 1 with the umami receptor protein T1R1 / T1R3 are the amino acid residues Arg54, Tyr182, Gln181, Glu105, Glu148, Ala153, Thr154, Lys155, Ser104, Phe180, Asp215, Asp216 and Leu173 located on the T1R3 subunit; And / or, the main binding sites for molecular docking of the peptide with the amino acid sequence shown in SEQ ID NO: 2 with the umami receptor protein T1R1 / T1R3 are the amino acid residues Lys155, Val152, Met151, Thr154, Ala153, Ser104, Glu105, Glu148, Gln181, Asn150, Arg54, Asp216, Asp219, Asp218 and Arg220 located on the T1R3 subunit; And / or, the main binding sites for molecular docking of the peptide with the amino acid sequence shown in SEQ ID NO: 3 with the umami receptor protein T1R1 / T1R3 are the amino acid residues Lys155, Glu148, Thr154, Pro106, Ala153, Asp216, Asp219, Asp218, Asp215, Tyr182, Gln181 and Arg54 located on the T1R3 subunit; And / or, the peptide with the amino acid sequence shown in SEQ ID NO: 4 is molecularly docked with the umami receptor protein T1R1 / T1R3, with the main binding sites being the amino acid residues Lys155, Val152, Thr154, Ala157, Ala153, Val107, Pro106, Glu148, Glu217, Tyr182, Gln181, Asp219, Asp216, Arg220, and Arg54 located on the T1R3 subunit.

3. The umami peptide according to claim 1, characterized in that, The threshold values ​​of the umami peptides with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are 0.065 mg / mL, 0.065 mg / mL, 0.1 mg / mL and 0.25 mg / mL, respectively.

4. The method for screening umami peptides according to any one of claims 1-3, characterized in that, Includes the following steps: (1) The red fish bones were hydrolyzed by a combination of complex protease and flavor neutral protease to obtain the hydrolysate; (2) After separation by ultrafiltration and purification by gel chromatography, the peptide structure was identified by LC-MS / MS to obtain peptides with potential biological activity. Their physicochemical properties were predicted, and non-toxic peptides with good solubility were screened out. (3) Construct a T1R1 / T1R3 receptor protein model and evaluate its reliability; (4) Molecular docking of the T1R1 / T1R3 receptor protein model with the peptide from step (2) to form a receptor protein-small molecule ligand complex; (5) Based on the molecular docking results, peptides with potential umami effects are screened to obtain umami peptides.

5. The method for screening umami peptides according to claim 4, characterized in that, In step (1), the mass ratio of complex protease to flavor neutral protease is 9:1, the enzymatic hydrolysis conditions are 45 ℃ for 147 min, and the enzyme is inactivated at 100 ℃ for 15 min after enzymatic hydrolysis. The molecular weight cutoff for ultrafiltration in step (2) is <3000 Da; In step (2), the gel chromatography column was dextran G-15, eluted with ultrapure water; the third peak component F3 was collected. The bioactivity prediction in step (2) uses the following screening criteria: an activity score greater than 0.

5. In step (4), the T1R1 / T1R3 receptor protein was dehydrated and hydrogenated using Discovery Studio. In step (4), molecular docking involves semi-flexible docking of the peptide with the constructed umami receptor T1R1 / TIR3 model. The screening criteria for step (5) are: select peptides with docking interaction energy lower than -120.00 kcal / mol.

6. The use of any one of the umami peptides described in claims 1-3 in the preparation of food seasonings or food products.

7. A food seasoning, characterized in that, The food seasoning contains the umami peptides described in any one of claims 1-5.

8. The food seasoning according to claim 7, characterized in that, The food seasoning also includes monosodium glutamate (MSG).

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