Fermented marine fish-derived umami peptides with salty taste enhancement effect and their application

Abstract

The present invention discloses a fermented marine fish-derived umami peptide with a salty taste enhancing effect and its application, and belongs to the field of food biotechnology. The amino acid sequence of the umami peptide is FDD or GIELE. The present invention screened out two umami polypeptides (FDD and GIELE) with a salty taste enhancing effect from the fermented marine fish water extract, analyzed their interaction and surface force with the T1R3 subunit, and found that both peptides can bind to the T1R3 subunit, and the composite conformation is stable. In addition, it was verified by sensory evaluation and electronic tongue analysis that both umami peptides have a salty taste enhancing effect, and the umami threshold is 0.0390625-0.09375 mg / mL, which provides a new peptide raw material for the development of salty and fresh condiments, and has important application value.

Description

Technical Field

[0001] The invention relates to the field of food biotechnology, in particular to fermented marine fish-derived umami peptides with salty taste enhancement effect and applications thereof. Background Art

[0002] As a new type of umami flavor enhancer, umami peptides can be extracted from food or synthesized from amino acids, and have the function of compensating or enhancing the original taste of food, increasing consumers' appetite, and showing high flavor activity. Umami peptides have a wide range of sources and have been identified and characterized in a variety of foods (such as beef, seafood, soybeans, peanuts, and soy sauce, etc.).

[0003] The unique umami taste of umami peptides is caused by the mutual binding of umami peptides and umami receptors. Eight umami receptors have been identified so far, and the most studied are several GPCRs that can bind umami substances. Among them, T1R1 / T1R3 is widely considered to be the main receptor for umami, with a ligand binding domain (VFTD) shaped like a Venus flytrap, which is the key domain for recognizing umami ligands. This domain has an upper and lower leaf structure connected by a hinge region. Therefore, in order to clarify the taste mechanism of umami peptides, researchers use molecular simulation and sensory evaluation to study the interaction between umami peptides and umami receptors.

[0004] Traditional fermented marine fish, as a solid-state naturally fermented fish product, has a unique flavor and is widely favored by consumers. During the fermentation process, marine fish undergo protein degradation, lipid degradation and other complex reactions under the action of proteases and microorganisms. Although umami peptides in fermented products have gradually attracted the attention of researchers in recent years, further research is needed on umami peptides in fermented marine fish products. Summary of the invention

[0005] The purpose of the present invention is to provide a fermented marine fish-derived umami peptide with a salty taste enhancement effect and its application, so as to solve the problems existing in the above-mentioned prior art. The umami peptides provided by the present invention can bind to the T1R3 subunit, and the composite conformation is stable, and they all have obvious salty and umami tastes, and the umami threshold is 0.0390625-0.09375 mg / mL, which provides a new peptide raw material for the development of salty and umami seasonings and has important application value.

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

[0007] The invention provides a fermented marine fish-derived umami peptide T1 with a salty taste enhancement effect, wherein the amino acid sequence is FDD.

[0008] The present invention also provides an application of the fermented marine fish-derived umami peptide T1 in improving the salty and umami taste of food.

[0009] The present invention also provides an application of the fermented marine fish-derived umami peptide T1 in preparing condiments.

[0010] The present invention also provides a condiment, the ingredients of which include the fermented marine fish-derived umami peptide T1.

[0011] The present invention also provides a fermented marine fish-derived umami peptide T3 with a salty taste enhancing effect, the amino acid sequence of which is shown in SEQ ID NO.2.

[0012] The present invention also provides an application of the fermented marine fish-derived umami peptide T3 in improving the salty and umami taste of food.

[0013] The present invention also provides an application of the fermented marine fish-derived umami peptide T3 in preparing condiments.

[0014] The present invention also provides a condiment, the ingredients of which include the fermented marine fish-derived umami peptide T3.

[0015] The invention also provides application of the seasoning in improving the salty and fresh taste of food.

[0016] The present invention discloses the following technical effects:

[0017] The present invention separates and identifies polypeptides in the fermented marine fish water extract by ultrafiltration, gel filtration chromatography and LC-MS / MS, and screens out two umami polypeptides (FDD and GIELE) with salty taste enhancement effect from the fermented marine fish water extract by sensory evaluation and electronic tongue analysis, and analyzes the interaction and surface force with the T1R3 subunit, and finds that the two umami peptides can bind to the T1R3 subunit, and the composite conformation is stable. In addition, the sensory evaluation and electronic tongue analysis verify that the two umami peptides have obvious salty taste enhancement effect, and the umami threshold is 0.0390625-0.09375 mg / mL.

[0018] The invention provides a new peptide raw material for the development of salty and fresh seasonings and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 This is the GFC sensory evaluation chart of the gel filtration chromatography UF fraction;

[0021] Figure 2The secondary mass spectra and chemical structures of peptides T1, T2, T3 and T4;

[0022] Figure 3 The sensory evaluation diagram (A) and electronic tongue radar diagram (B) of the synthetic peptides;

[0023] Figure 4 It is a homology model diagram of T1R1 / T1R3, wherein A is a structural model diagram of receptor T1R1 / T1R3; B is a Ramachandran diagram of T1R1 / T1R3 receptor homology modeling;

[0024] Figure 5 It is the three-dimensional structure diagram of polypeptides T1 and T3;

[0025] Figure 6 This is a molecular docking analysis diagram of peptide T1 and umami taste receptor T1R1 / T1R3;

[0026] Figure 7 This is a molecular docking analysis diagram of peptide T3 and umami taste receptor T1R1 / T1R3;

[0027] Figure 8 Surface force analysis diagram of the interaction between peptides T1 and T3 and umami taste receptors T1R1 / T1R3;

[0028] Fig. 9 The changes of RMSD (A), Rg (B), RMSF (C) and H-Bond quantity (D) over time during the 100 ns molecular dynamics simulation;

[0029] Fig.10 This is a graph showing the change in free energy of the peptide T1 and T3 complex during a 100ns molecular dynamics simulation. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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 those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example

[0036] 1. Materials and Methods

[0037] 1. Preparation of fermented marine fish hydrolysate

[0038] The marine fish used in the present invention is a marine fish provided by a fish farm. The fish is euthanized by a professional using a manual blunt instrument method and then bled. The fish is dissected, visceral, cleaned, dried and set aside. The fish is evenly coated with coarse salt, stacked in layers in a fermentation box, and the gaps are filled with coarse salt, and pickled and fermented at a natural temperature for 16 days to obtain a fermented marine fish product. The fish meat of the fermented marine fish product is mixed with ultrapure water in a mass ratio of 1:4, homogenized, and then boiled for 20 minutes to extract peptides. The boiled homogenate is filtered using an 80-mesh filter cloth, and the filtrate is centrifuged (4°C, 15min, 10000rmp), the supernatant is collected, freeze-dried, and a fermented marine fish water extract is obtained, which is then stored at -80°C for standby use.

[0039] 2. Extraction of umami peptides

[0040] 2.1 Ultrafiltration (UF)

[0041] Existing studies have shown that the molecular weight of umami substances is mainly concentrated below 3KDa, so the present invention selects samples with a molecular weight of less than 3KDa in the water extract of fermented marine fish as the research object. The freeze-dried sample of the water extract is re-dissolved in ultrapure water, and centrifuged (10000rpm, 4°C, 20min) through an ultrafiltration centrifuge tube with a molecular weight cutoff of 3KDa, and the components with a molecular weight less than 3KDa are recovered, freeze-dried and stored at -80°C.

[0042] 2.2 Gel filtration chromatography (GFC) analysis

[0043] The ultrafiltration components were separated and purified using a Sephadex G-15 dextran gel column. The dextran gel powder was soaked in excess ultrapure water for 24 hours to swell, and the fully swollen filler was poured into a chromatography column (1.6 cm in diameter and 60 cm in length). The mobile phase was ultrapure water with an ultraviolet absorption wavelength of 214 nm. In order to make the dextran gel bind more firmly, the mobile phase was flushed through the gel filtration chromatography column at a flow rate of 2 mL / min for 5 hours. The ultrafiltration components with a molecular weight less than 3KDa were then re-dissolved in ultrapure water to a solution with a concentration of 20 mg / ml, filtered through a 0.45 μm filter, and loaded onto a Sephadex G-15 gel chromatography column (Solarbio, 16×60 cm), with a flow rate of 1 mL / min to ensure the stability and high efficiency of the elution process. The sample volume was set to 8 mL to meet the separation requirements, and each component was collected according to the spectral peak. Each fraction was collected, concentrated and freeze-dried, and sensory evaluation was performed to determine the fraction with the strongest umami taste.

[0044] 3. Identification of umami peptides

[0045] The best umami component purified by gel chromatography was prepared into a solution with mobile phase and filtered through a 0.45 μm filter membrane. The amino acid sequence of the component F2 purified by GFC was identified by liquid chromatography-tandem mass spectrometry (Thermo Fisher Scientific, MA, USA). After desalting the sample using a PepMap C18 desalting column (75 μm × 25 cm), 2 μL of the desalted sample was added to an Acclaim PepMap C18 column (75 μm × 25 cm). The mobile phases were solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile), the sample separation gradient was 60 min, the column flow rate was 350 nL / min, the column temperature was 40 °C, and the electrospray voltage was 2 kV. The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. Mass spectrometry parameter settings: (1) MS: scan range (m / z): 200-1500; resolution: 60,000; Normalized AGC target: 300%; maximum injection time: 25ms; (2) HCD-MS / MS: resolution: 15,000; Normalized AGC target: 50%; maximum injection time: 22ms; collision energy: 30%; dynamic exclusion time: 30s. The obtained fermented marine fish peptide sequence was subjected to De Novo detection and PEAKS library search, and the verifiable de novo sequence was selected as the final component of the purified fermented marine fish peptide.

[0046] 4. Prediction of potential sensory activity of peptides

[0047] By exhaustively querying the BIOPEP database, the number of fragments with specific taste activity types and the number of amino acid residues involved in the target peptide sequence are determined. The frequency of occurrence of biologically active fragments in the target peptide is obtained by analysis and calculation, thereby scientifically predicting its taste activity. This method not only improves the accuracy and reliability of the prediction of umami peptide bioactivity, but also provides strong data support for subsequent research work.

[0048] 5. Prediction of toxicity and physicochemical properties of peptides

[0049] ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to predict the toxicity of the identified umami peptides. The solubility of the sequenced peptides was predicted with the help of a server (http: / / www.innovagen.com / proteomicstools).

[0050] 6. Artificial synthesis of umami peptides

[0051] The sequence of the umami peptide has been determined to be synthesized by solid phase synthesis. Amino acids are added in sequence from the C-terminus to the N-terminus based on the shrinkage reaction between amino acids. After the peptide chain is completely connected, the resin is removed to obtain the desired peptide chain. The peptide is purified by reversed-phase high-performance liquid chromatography to make the peptide purity >97%. Purification conditions: mobile phase A is V(TFA) / V(water)=1 / 1000V, mobile phase B is V(TFA) / V(acetonitrile)=1 / 1000, the flow rate is set to 10mL / min, the retention time is 20-30min, and the purification preparation is performed twice. The sample purified by reversed-phase high-performance liquid chromatography is quickly frozen in liquid nitrogen and freeze-dried to obtain the final product. The synthesized pure peptide is then analyzed to study its taste characteristics.

[0052] 7. Sensory evaluation of synthetic peptides

[0053] The sensory evaluation panel consisted of six males and six females (25-30 years old) from the Fisheries Research Institute. Each member first received training to reach the standard of being able to easily identify basic tastes. The sensory evaluation was conducted in a sensory analysis laboratory (25±1°C). During the evaluation process, the mouth was rinsed with purified water, and the samples were then placed in the mouth for 2 minutes to evaluate their taste characteristics. All samples were dissolved in ultrapure water to prepare a 1 mg / mL aqueous solution, which was filtered and used for later use. The reference samples of the five basic tastes (sour, sweet, bitter, salty, and umami) were solutions of citric acid (0.08%), sucrose (1%), caffeine (0.08%), sodium chloride (0.35%), and monosodium glutamate (0.35%). The samples were evaluated using a 10-point intensity scale (1, no taste; 10, very strong taste), with the reference solution scoring 5.

[0054] The synthetic peptide was dissolved in ultrapure water to prepare a 1 mg / mL solution and diluted stepwise with ultrapure water at 1:1 (v:v). Panelists were asked to evaluate each diluted sample in a triangle test using deionized water as two blank controls. When there was a significant difference in taste between a certain level of dilution and the blank, the dilution level was recorded and defined as the taste dilution factor.

[0055] 8. Synthetic peptide electronic tongue analysis

[0056] The taste characteristics of the synthetic peptides were verified by the electronic tongue taste analysis system TS-5000Z (INSENT, Japan). The five sensors are AAE, CT0, CA0, C00 and AE1, representing taste, saltiness, sourness, bitterness and astringency, respectively. The synthetic peptide samples were dissolved in ultrapure water, prepared into 1 mg / mL aqueous solutions, and measured using an electronic tongue. Each sample was measured four times in parallel, and the average value was calculated three times.

[0057] 9. Molecular docking

[0058] Since the three-dimensional structure of the umami taste receptor T1R1 / T1R3 is not clear, it is necessary to construct its three-dimensional structure through homology modeling. Homology modeling includes three steps, namely model construction, optimization and evaluation. The homology model of the umami taste receptor T1R1 / T1R3 was constructed using the SWISS-MODEL online server (https: / / swissmodel.expasy.org / ). The amino acid sequences of T1R1 and T1R3 (T1R1: Q7RTX1, T1R3: Q7RYX0) were retrieved from UniProKB (https: / / www.uniprot.org / uniprotkb), and the metabotropic glutamate receptor (PDB ID: 1EWK) was selected as the modeling template. SAVESv6.0 (https: / / saves.mbi.ucla.edu / ) was used to build a Ramachandran plot to evaluate the model, and the reliability of the model was evaluated by calculating the Ramachandranran plot of the optimized receptor model.

[0059] The structure of the umami peptide was constructed and optimized using online tools. Autodock Vina was used to perform molecular docking of the umami peptide and the umami receptor T1R1 / T1R3. The docking process was considered to be semi-flexible docking (the peptide conformation was flexible and the protein structure was rigid). The docking pocket grid size was 70*70*70, the active center coordinates were (x, y, z): (32.654, -5.612, 36.141), and the default grid spacing was The best docking results were evaluated based on the docking energy, and the conformation with the lowest binding free energy was selected as the most likely binding mode for further analysis. In addition, PyMOL and Discovery Studio2020Client were used for visualization analysis and interaction force analysis between umami peptides and T1R1 / T1R3.

[0060] 10. Molecular dynamics simulation of umami peptide and umami receptor complex

[0061] The GROMACS software (2020.3) was used to perform a 100 ns molecular dynamics simulation of the complexes of the six umami peptides obtained by molecular docking and the corresponding umami receptors. First, the 3D model of the umami receptor T1R1 / T1R3 and the umami peptide was placed in the Amber99SB-ILDN force field combined with the TIP3P water model, a water box with a boundary of 1.2 nm was established, and Na +and Cl- to balance the charge in the system. Before the formal dynamics simulation, the complex was energy minimized using the conjugate gradient algorithm, and then further balanced using an isothermal (300K) system (NVT) and an isobaric (1 standard atmosphere) system (NPT). Finally, a 100ns molecular dynamics simulation was performed at ambient temperature 300K and pressure 1atm. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), changes in hydrogen bonds between T1R3 and the peptide in the complex, and the free energy of the MD simulation trajectory were then analyzed.

[0062] 2. Results

[0063] 1. Ultrafiltration and gel chromatography separation results

[0064] The present invention performs preliminary separation on the fermented marine fish water extract through an ultrafiltration tube with a molecular cutoff of 3000Da, and obtains two components with different molecular weight ranges, which are recorded as UF-1 (MW>3000Da) and UF-2 (MW<3000Da). According to the condition that the molecular weight of the umami peptide is less than 3000Da, UF-2 is selected for gel filtration chromatography (GFC) for the next step of separation.

[0065] After GFC separation of the UF components, a total of 4 components were obtained, which were marked as F1, F2, F3, and F4, with retention times ranging from 20 to 200 minutes, among which F4 had the longest retention time. The sensory evaluation results of the GFC components showed that ( Figure 1 ), the four components separated by GFC have obvious differences. The four components are dominated by umami, followed by salty, sweet and sour, and bitterness is weak. The order of umami intensity is F2>F1>F3>F4, and F2 has the highest umami value (4.0 points). Analysis found that the change trend of salty and sweet taste in the taste profile is consistent with umami, that is, the components with higher umami taste also have higher sweetness, which may be because when salty, sweet and umami substances coexist, the tastes promote each other and produce interactions. Therefore, the F2 component was selected for the next step of separation and purification to obtain a component with higher polypeptide purity.

[0066] 2. Identification of umami peptides by mass spectrometry chromatography

[0067] The present invention screens umami peptides based on the conditions that the proportion of glutamic acid and aspartic acid in the peptide segment is greater than 25% and the molecular weight is less than 1500Da. As shown in Table 1, a total of 4 peptides were identified, and their amino acid sequences were Phe-Asp-Asp (FDD), Glu-Asp-Glu-Ile (EDEI, SEQ ID NO.1), Gly-Ile-Glu-Leu-Glu (GIELE, SEQ ID NO.2), Glu-Leu-Pro-Asp-Gly-Gln (ELPDGQ, SEQ ID NO.3), and their molecular weights were 395.1328Da, 504.2067Da, 559.2853Da, and 657.3009Da, respectively. The 4 identified peptides all contain glutamic acid and aspartic acid, and the highest proportion is about 67%, indicating that glutamic acid and aspartic acid are the key amino acids for the umami taste of peptides, which contribute to the formation of the umami taste of fermented marine fish. The secondary mass spectra and chemical structures of the 4 peptides are shown in Figure 1. Figure 2 As shown in Figure 4, all four peptides are short peptides with a molecular weight of <1 kDa.

[0068] Table 1 Identification of umami peptides in fraction F2

[0069]

[0070] 3. Peptide physicochemical properties and toxicity prediction results

[0071] The solubility of peptides is positively correlated with their flavoring effect. The lower the solubility, the lower the umami, salty and sweet tastes. The higher the solubility, the higher the various flavors and the easier it is to penetrate into food, thus giving the food a better taste. In addition, good water solubility, safety and non-toxicity are the key to the normal metabolism of peptides in the body. The water solubility and toxicity prediction results of the peptides identified in this study are shown in Table 2. All four peptides have good water solubility and are non-toxic and safe.

[0072] Table 2 Peptide physicochemical properties and toxicity prediction results

[0073]

[0074] 4. Prediction of umami peptide taste activity

[0075] The present invention introduces four umami peptides into the BIOPEP database tool, and all four umami peptides have umami taste, and the results are shown in Table 3. The peptide segment with the highest frequency of umami active fragments is EDEL, with an occurrence frequency of up to 2.0000, and the umami fragments it contains are D, E, ED, DE, EDE, EL and DEL, followed by FDD and ELPDGQ, with occurrence frequencies of 1.0000 and 0.6667 respectively, while the occurrence frequency of GIELE is second only to ELPDGQ, which is 0.6, and the umami fragments it contains are E and EL. The peptide segment with the highest frequency of bitter active fragments is GIELE, followed by ELPDGQ.

[0076] The peptides of the present invention contain hydrophobic amino acids with obvious bitter characteristics, such as Leu, Gly, Ile, Pro and Phe. However, when glutamic acid or aspartic acid is present in the polypeptide sequence, the bitterness is significantly reduced. The BIOPEP database provides certain references and theoretical basis for the study of flavor characteristics, but the actual flavor characteristics of the polypeptide still need to be further verified through sensory evaluation of synthetic peptides and flavor descriptive analysis.

[0077] Table 3 Prediction of taste activity of identified umami peptides

[0078]

[0079] Note: / represents the amino acid fragment contained in the peptide that is not found in the corresponding flavor in the database.

[0080] 5. Sensory evaluation and electronic device analysis of synthetic peptides

[0081] The sensory evaluation results of the four synthetic peptides are as follows Figure 3 As shown in A. Among the five basic tastes of synthetic peptides, umami is the most prominent, followed by bitterness, saltiness and sweetness, and sourness is the weakest. Among the four synthetic peptides, ELPDGQ has the highest umami intensity, with a score of 6.75 points, followed by EDEI (6.25 points), then FDD (4.75 points) and GIELE (4.75 points). As can be seen from the figure, all synthetic peptides show a certain bitterness and sourness, which may be due to the introduction of impurities in the synthesis process of the peptides, but has no effect on the umami. Previous studies have shown that the umami taste of peptides is related to glutamic acid and aspartic acid residues in the sequence, and the umami peptides obtained in the present invention also have glutamic acid and aspartic acid.

[0082] Figure 3The B of shows the electronic tongue radar maps of 4 synthetic peptides. The results indicate that the umami intensity ranking of the 4 umami peptides from high to low is FDD, EDEI, GIELE, and ELPDGQ. FDD has the highest umami intensity, followed by EDEI. This shows that the results of the electronic tongue are different from those of the sensory evaluation. This may be because there are taste interactions in the sensory evaluation, such as the synergistic effect between umami and saltiness, and the inhibitory effect between umami and bitterness, which the electronic tongue cannot detect. Further research on its bitterness inhibition will be carried out later. Generally speaking, sensory evaluation is easily affected by various factors, such as individual differences and environmental factors, resulting in differences in sensory evaluation and taste description. Therefore, it is necessary to combine sensory evaluation with the electronic tongue to analyze the taste characteristics of peptides.

[0083] 6. Descriptive evaluation of synthetic peptides

[0084] To further study the taste characteristics of synthetic peptides, members of the sensory evaluation panel were required to evaluate the taste thresholds of 4 peptides using taste dilution analysis (TDA) and describe their tastes. The taste thresholds and taste descriptions of the 4 synthetic peptides are shown in Table 4. The results show that all peptides have umami, and the thresholds of the 4 peptides are lower than that of monosodium glutamate. This indicates that the umami of the 4 synthetic peptides is stronger than that of monosodium glutamate. The ranking of the sensory evaluation thresholds of the polypeptides is FDD (0.0390625 mg / mL) < ELPDGQ (0.0625 mg / mL) < GIELE (0.09375 mg / mL) = EDEI (0.09375 mg / mL). In addition, the taste descriptions of the synthetic peptides are more complex, mostly showing umami, saltiness, and sweetness. Among them, polypeptide T3 (GIELE) has obvious umami and saltiness, while polypeptide T1 (FDD) has stronger saltiness than umami. The sequence of the polypeptide, the length of the peptide chain, and the conformation of the polypeptide may all affect the taste characteristics of the polypeptide. Generally speaking, peptides with a molecular weight lower than 1500 Da can cause umami or make it stronger. The number of carbon atoms in the amino acid structure of umami peptides is between 4 and 6, and the umami is more obvious. This is one of the reasons for the lower thresholds of the four polypeptides.

[0085] Table 4 Taste dilution analysis of synthetic peptides

[0086]

[0087] 7. Molecular docking of umami peptides with umami receptor T1R1 / T1R3

[0088] The amino acid sequences of T1R1 and T1R3 were input into the SWISS-MODEL online server. The sequence identities of the metabotropic glutamate receptor (PDBID: 1EWK) with T1R1 and T1R3 were 23.20% and 23.00% respectively, and it was used as the template protein for constructing the T1R1 / T1R3 structure. The results of the homology modeling of T1R1 / T1R3 are as follows Figure 4 As shown in A, the left side is T1R1 and the right side is T1R3. Figure 4 It can be seen from A that in the T1R1 / T1R3 model, T1R1 is in a closed conformation, while T1R3 is in an open conformation and has a ligand binding domain (VFTD) shaped like a Venus flytrap. Studies have shown that the VFTD domain in the T1R3 subunit is the main domain for distinguishing and recognizing umami taste. Umami peptides can enter this structure and bind to it, thereby triggering an umami response. In addition, SAVESv6.0 was used to evaluate the optimized model. Ramachandran Figure Figure 4 As shown in B, this shows that 99.4% of the amino acid residues in the T1R1 / T1R3 receptor model are located in reasonable regions (87.7% are located in the "most favorable" region, 9.9% are located in the "extra allowed" region, and 1.8% are located in the "generous allowed" region). In contrast, 0.6% of the amino acid residues are located in the unallowed region. These results show that the T1R1 / T1R3 receptor model is reasonable and successfully constructed, and can be used for the next step of molecular docking analysis.

[0089] In order to further study the interaction mechanism between umami peptides and receptors T1R1 / T1R3, the peptides T1 (FDD) and T3 (GIELE) with umami taste were firstly modeled and optimized. Figure 5 As shown. The docking software AutodockVina was used to realize the molecular docking of umami peptides with T1R1 / T1R3. After the molecular docking results were processed by Pymol software, the surface model diagram of the interaction was output. The software Discoveystudio2020Client was used to analyze the interaction between different umami peptides and umami receptors T1R1 / T1R3, and ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to predict the toxicity of the identified umami peptides. The docking energies of the four umami peptides when docked with T1R1 / T1R3 molecules are shown in Table 5. The results showed that the docking energies of the four umami peptides were between -6.901 and -6.064 kcal / mol, and the average docking energy was -6.622 kcal / mol. Previous studies have shown that lower docking energy indicates a stronger affinity between the ligand and the receptor, and a more stable structure after binding, which may be more likely to produce umami. The binding energies of the four umami peptides to T1R3 in the present invention are all low. Therefore, the molecular docking interactions between the four umami peptides and T1R1 / T1R3 are considered to be stable.

[0090] Table 5 Molecular docking energy and toxicity prediction of 4 umami peptides and receptors T1R1 / T1R3

[0091]

[0092] Figure 6-Figure 7 The binding mode between these umami peptides and T1R1 / T1R3 receptors was further demonstrated intuitively. The docking results showed that both peptides T1 and T3 can enter the binding pocket located in the T1R3 cavity VFTD, and their binding sites show basically the same characteristics. This may be because the binding domain of T1R1 is in a closed state, while the binding domain of T1R3 is in an open conformation, and its binding site is large enough to be more easily bound to umami peptides. The binding of umami peptides to T1R3 subunits is mainly through hydrogen bonds and hydrophobic interactions.

[0093] In the present invention, there are six kinds of interaction forces between umami peptides and T1R3. Conventional hydrogen bonds and carbon-hydrogen bonds are the main interaction forces between umami peptides and T1R3, followed by alkyl, π-π stacking and π-alkyl, while π-donor hydrogen bonds only appear in DEEYPDLS, such as Figure 6 As shown. Among them, the benzene ring in the peptide structure forms hydrophobic interactions with the receptor amino acid residues through π-π stacking or π-alkyl interaction, which helps to improve the umami effect of the peptide. In addition, the benzene ring in the peptide structure can also form a hydrophobic interaction with nearby hydrophobic amino acids, further enhancing the affinity of the umami peptide with the umami receptor T1R1 / T1R3. In addition, all hydrogen bond distances are short This indicates that umami peptides have a strong binding force to the taste receptor pocket and the conformation of the complex is stable. According to Table 6, there are 16 amino acid residues in T1R3 that play a key role in the interaction with umami peptides, and these amino acid residues are mainly connected to umami peptides through hydrogen bond interactions. Among them, the active residues mainly include SER170, SER169, SER147, SER146, GLY168, HIS145, GLN389, GLU301, ALA302, LEU304 and TYR218, among which HIS145, SER147, SER170, ALA169, SER146, GLY168 and GLU301 appear more frequently in all interactions with umami peptides than other amino acid residues, which indicates that SER, HIS, ALA, GLY and GLU contribute the most to the molecular taste mechanism of umami peptides.

[0094] Table 6 Key binding sites of umami peptides and umami receptors T1R1 / T1R3

[0095]

[0096] 8. Analysis of the docking interaction between umami peptides and umami receptors

[0097] like Figure 8As shown, there are six main interaction surface forces between umami peptides and receptors T1R1 / T1R3, namely aromatic interactions, hydrogen bonds, intercalated charges (IC), hydrophobicity, ionization, and solvent accessible surface (SAS).

[0098] An important reason for the bitter taste is the hydrophobicity of the peptide. The proportion of hydrophobic amino acids in the polypeptides T1 and T3 of the present invention is relatively low, so the bitterness is small and the umami taste is stronger. In this study, it can be seen that the SAS in the region where the umami peptide binds to T1R3 is higher, which may be due to the presence of van der Waals forces in the binding between them. However, the IC and ionization of the region where the umami peptide binds to T1R3 are very small, which may have little effect on the binding of the umami peptide to T1R3. This shows that the interaction surface forces between the umami peptide and the receptor T1R1 / T1R3 are mainly aromatic interactions, hydrogen bonds, hydrophilicity and SAS.

[0099] 9. Analysis of molecular dynamics (MD) simulation results

[0100] To further prove the degree and stability of the binding of umami peptides to umami receptors T1R1 / T1R3, molecular dynamics simulation was used to analyze them with a simulation time of 100 ns. Fig. 9 and 10 As shown. RMSD is an indicator for evaluating the stability of protein-ligand complexes. The smoother the RMSD curve, the more stable the complex formation. Fig. 9 As shown in A, the four umami peptide complexes fluctuated greatly before 20ns, with a fluctuation range of less than 0.5nm. After 20ns, they began to gradually stabilize and basically reached equilibrium at 50ns. The fluctuation range was within the standard fluctuation range (0.08-014nm), which indicated that the four umami peptides could form a stable complex conformation with the umami receptor T1R1 / T1R3.

[0101] Rg is used to characterize the compactness and stability of the structure. The smaller the Rg, the more compact and stable the protein structure. The larger the Rg, the looser the protein structure. Fig. 9 As shown in B, similar to RMSD, the four umami peptide complexes fluctuated greatly in the first 20 ns. Then they gradually stabilized and basically reached equilibrium at 50 ns. This shows that the addition of the four umami peptides will not cause significant changes in the overall conformation of the protein, and the corresponding umami peptide complexes have good overall compactness and stability.

[0102] Fig. 9Figure C shows the RMSF results of the umami peptide complex. The RMSF curve indicates the degree of fluctuation of the amino acid residues in the protein during the kinetic simulation. The RMSF value is proportional to the degree of fluctuation of the amino acid residues in the protein. It can be seen that there is no significant difference in the RMSF curves of the four umami peptide conjugates overall. Among them, the 100-150, 350-380 and 400-420 amino acid residues showed obvious fluctuations, especially the 350-380 residues with the largest fluctuation, and the fluctuation range was close to 1.0nm. However, these fluctuations did not significantly affect the main structure of the protein. This may be because these amino acid residues are located on the periphery of the T1R1 / T1R3 protein and interact with other molecules in the complex, resulting in some fluctuations during the kinetic simulation.

[0103] To investigate the hydrogen bonding characteristics of the binding sites in the umami peptide-T1R1 / T1R3 complex, Fig. 9 It can be seen from D that the number of hydrogen bonds in the ELPDGQ-T1R1 / T1R3, GIELE-T1R1 / T1R3, FDD-T1R1 / T1R3, and EDEI-T1R1 / T1R3 complexes remained stable at 5 or more during the entire simulation process, which is sufficient to show that the four umami peptides can form highly stable complexes with the T1R1 / T1R3 receptor.

[0104] like Fig.10 The free energy changes of each umami peptide complex during the 100ns molecular dynamics simulation. The purple / blue spots reflect the minimum energy value, indicating that the structure is the most stable. In contrast, the red / yellow spots represent unstable structures. It can be seen that in the free energy landscape of the peptide T1 and T3 complexes, an almost single smooth minimum energy group can be observed. This indicates that the complex formed by the T1R1 / T1R3 receptor and the peptides T1 and T3 exhibits good stability.

[0105] In summary, molecular dynamics simulation verified that peptides T1 and T3 can bind to the umami taste receptor T1R1 / T1R3, and the corresponding complex conformations are tight and stable, which is consistent with the molecular docking results.

[0106] 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. An application of fermented marine fish-derived umami peptide T1 in improving the salty and umami taste of food, characterized in that: The amino acid sequence of the fermented marine fish-derived umami peptide T1 is FDD.

2. An application of fermented marine fish-derived umami peptide T1 in preparing condiments, characterized in that: The amino acid sequence of the fermented marine fish-derived umami peptide T1 is FDD.

3. A seasoning, characterized in that: The ingredients of the seasoning include fermented marine fish-derived umami peptide T1; the amino acid sequence of the fermented marine fish-derived umami peptide T1 is FDD.

4. Use of the condiment as claimed in claim 3 in improving the salty and fresh taste of food.

5. A fermented marine fish-derived umami peptide T3 having a salty taste enhancing effect, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

6. Use of the fermented marine fish-derived umami peptide T3 as claimed in claim 5 in improving the salty and umami taste of food.

7. Use of the fermented marine fish-derived umami peptide T3 as claimed in claim 5 in preparing condiments.

8. A seasoning, characterized in that: The ingredients of the seasoning include the fermented marine fish-derived umami peptide T3 described in claim 5.

9. Use of the condiment as claimed in claim 8 in improving the salty and fresh taste of food.

Images