Antioxidant umami dual functional peptide and application thereof

Abstract

This invention discloses an antioxidant umami bifunctional peptide and its applications. The amino acid sequence of the umami peptide is selected from AGDDAPRA, DDALRSQE, DDIKRVV, DDMEKIW, DKEGNGTVM, and SDMKHWP. The peptide provided by this invention has an extremely low sensory recognition threshold and highly efficient T1R1 / T1R3 taste receptor activation ability (ECG). 50 It has a concentration as low as 0.003 mM and also exhibits significant free radical scavenging activity (DPPH scavenging IC50). 50 (As low as 0.95 mM). This group of peptides solves the technical problems of existing flavoring agents having single function, weak sensory response, and easy oxidation and inactivation in complex systems. It can be widely used as a dual-function food ingredient in meat products, seasonings, and soups. While significantly enhancing the umami flavor of food, it effectively inhibits the oxidative degradation of lipids and proteins, and improves the sensory quality and shelf-life stability of food.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, and in particular to antioxidant umami bifunctional peptides and their applications. Background Technology

[0002] Umami is one of the basic human tastes, characterized by its deliciousness, persistence, and overall flavor enhancement. Currently, the industrial evaluation and enhancement of umami mainly relies on the exogenous addition of monosodium glutamate (MSG) and flavor nucleotides. However, single small-molecule flavor enhancers are insufficient to replicate the complex flavor profile produced by the biotransformation of natural ingredients.

[0003] Human perception of umami is primarily mediated by class C G protein-coupled receptors (GPCRs) on the surface of taste bud cells on the tongue, specifically the T1R1 / T1R3 heterodimer receptor. This receptor possesses a large extracellular domain known as the "Venus Flytrap" (VFT) domain. When an external ligand (such as L-glutamate or a flavor peptide) enters the VFT pocket of T1R1, it triggers a conformational shift from "open" to "closed" within the receptor. This conformational remodeling activates downstream G protein-coupled signaling pathways, including the activation of phospholipase Cβ2 (PLCβ2) and the resulting release of inositol triphosphate (IP3). IP3 acts on intracellular calcium stores, leading to the release of calcium ions (Ca). 2+ The instantaneous release and concentration increase of umami ultimately lead to the release of neurotransmitters through the TRPM5 ion channel, completing the transmission of umami signals to the central nervous system.

[0004] While some umami peptides have been reported in existing technologies, their types are limited, and the screening of umami peptides in existing technologies often overlooks their biological functional properties. During food storage, flavor substances are prone to deterioration due to free radical-induced oxidation reactions.

[0005] Therefore, developing a bifunctional molecule that can both activate receptors through molecular recognition and scavenge free radicals through redox reactions is currently the core challenge in the research and development of functional flavoring ingredients. Summary of the Invention

[0006] In view of this, the main objective of this invention is to provide a group of novel peptides with high receptor affinity, low umami recognition threshold, and significant antioxidant function. This addresses the problems in existing technologies such as weak sensory response of flavor components, limited functional properties, and performance degradation due to easy oxidation during food shelf life caused by a lack of biochemical stability.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an antioxidant umami bifunctional peptide or a pharmaceutically or food-grade acceptable salt thereof, wherein the amino acid sequence of the bifunctional peptide is any one or a combination of the following (1) to (6): (1) AGDDAPRA (AA-8); (2) DDALRSQE (DE-8); (3) DDIKRVV (DV-7); (4) DDMEKIW (DW-7); (5) DKEGNGTVM (DM-9); (6) SDMKHWP ((SP-7)).

[0008] In this invention, the bifunctional peptide can be obtained through artificial synthesis or by isolation and purification from natural protein hydrolysates.

[0009] The technical features of the bifunctional peptide in this invention are further described below: 1. Sequence Structure and Distribution Characteristics: The peptide chain length of the bifunctional peptide is 7 to 9 amino acid residues. The sequence conformation characteristics are as follows: at least one acidic amino acid center composed of Asp or Glu is distributed at the N-terminus or the beginning of the sequence, designed to form a hydrogen bond network with the T1R1 receptor compartment; a hydrophobic / aromatic center composed of Trp, Met, Val, or Pro, or other specific hydrophobic aliphatic amino acid residues are distributed at the C-terminus or the end of the sequence, designed to stabilize the receptor activation state through hydrophobic stacking and provide electron donors to scavenge free radicals.

[0010] 2. Sensory physical indicators: The umami recognition threshold of the peptide in deionized aqueous solution ranges from 0.044 mM to 0.142 mM. Its taste is predominantly umami, with some sequences exhibiting a significant synergistic sweetness perception.

[0011] 3. Receptor-ligand activation kinetics: In a cell evaluation model of heterologous expression of the T1R1 / T1R3 receptor, the dose-response curve induced by the peptide showed its half-maximal effective concentration (EC50). 50 The value ranges from 0.003 mM to 0.564 mM.

[0012] 4. Redox Biochemical Properties: The peptides exhibit the ability to scavenge free radicals within chemical systems. In the DPPH scavenging assay, its half-maximal inhibitory concentration (IC50) was [value missing]. 50 The lowest level can be as low as 0.95 mM; in ABTS + In the scavenging experiment, it demonstrated highly efficient hydrogen atom / single electron transfer activity.

[0013] In a second aspect, the present invention provides a composition containing the above-described bifunctional peptide or a pharmaceutically or food-grade salt thereof.

[0014] In a third aspect, the present invention provides the use of the above-described bifunctional peptide or a pharmaceutically or food-grade salt thereof in the preparation of seasonings or food additives.

[0015] Furthermore, the food additives include umami enhancers or food antioxidants.

[0016] In a fourth aspect, the present invention provides the use of the above-described bifunctional peptide or a pharmaceutically or food-grade salt thereof in enhancing the umami flavor of food.

[0017] In a fifth aspect, the present invention provides the use of the above-described bifunctional peptide or a pharmaceutically or food-grade salt thereof in the preparation of food ingredients that simultaneously possess both umami-enhancing and antioxidant functions.

[0018] In this invention, food ingredients are used to inhibit the oxidation of lipids and proteins in meat products, seasonings or soups, while enhancing their sensory umami intensity.

[0019] The present invention also provides a seasoning or food antioxidant or umami enhancer, comprising the above-described bifunctional peptide or a pharmaceutically or food-acceptable salt thereof.

[0020] The aforementioned bifunctional peptides or their pharmaceutically or food-acceptable salts can be added as flavor enhancers to meat products, seasonings, liquid soup bases, or artificial meat matrices.

[0021] The beneficial effects of this invention include at least the following: (1) Achieved high efficiency and precision in receptor activation: The peptides (such as SP-7) provided by this invention have achieved sub-micromolar activation efficacy for T1R1 / T1R3 receptors (EC). 50 = 0.003 mM), significantly better than conventionally reported food-derived umami peptides, solving the problem of insufficient molecular sensing intensity of traditional flavoring agents.

[0022] (2) Overcoming the bias in flavor intensity prediction: This invention introduces the UPS evaluation strategy, which correlates the relative abundance of peptides with the prediction threshold. The selected peptides have extremely high sensory weights in the actual food matrix, thus solving the technical bottleneck of the traditional model's failure to predict in complex peptide systems.

[0023] (3) It endows the flavoring components with significant bioactive functions: By introducing specific functional residues (such as the indole ring of tryptophan and the thioether group of methionine), the peptides can enhance the flavor and remove reactive oxygen species (ROS) through biochemical intervention, which significantly reduces the content of peroxidation products in food during storage.

[0024] (4) Improved stability of food sensory quality: Traditional specific antioxidants (such as L-glutathione) usually lack flavor properties or have unpleasant flavors, while traditional flavor enhancers lack antioxidant function. The peptides of this invention, through specific functional groups (such as the indole ring of tryptophan and the thioether group of methionine), exhibit excellent free radical scavenging ability at the millimolecular level (~1 mM level) while maintaining an extremely low umami threshold (0.044 mM). As a natural food-derived polypeptide, it can effectively scavenge reactive oxygen species (ROS) while significantly enhancing flavor, reducing the content of peroxidation products in food during storage, and achieving a dual breakthrough in flavor enhancement and antioxidant properties. Attached Figure Description

[0025] Figure 1 The dose-response fluorescence response curves for activating T1R1(A) and T1R3(B) receptors by six specific sequence peptides are shown.

[0026] Figure 2 The contact residues and binding forces of umami peptides with umami receptors T1R1(A) and T1R3(B) complexes.

[0027] Figure 3 To scavenge DPPH free radicals (A) and ABTS from peptides + Free radical (B) dose-response curve. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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.

[0029] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0030] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Acquisition and Precise Screening of Bifunctional Peptides (1) Acquisition of the peptide library: In this embodiment, protein-rich chicken meat was used as raw material. After appropriate enzymatic hydrolysis and thermal modification, a matrix containing abundant peptides was obtained. After the sample was treated with a 5 kDa ultrafiltration membrane, peptide fingerprint analysis was performed using a Nano-LC / Q-Orbitrap-MS system. The focus of this invention is on the structure and function of the obtained peptides. The specific enzymatic hydrolysis and thermal reaction processes can adopt conventional techniques in the field and do not constitute a limitation of this invention.

[0031] (2) Bioinformatics prediction and virtual screening Database filtering: The tens of thousands of sequences identified by mass spectrometry were compared with the TastePeptidesDB database to exclude known umami peptides that have already been reported.

[0032] Umami Attribute Prediction: Candidate sequences were scored from multiple dimensions using integrated online tools. First, a Q-value model was used for initial screening; then, online prediction tools such as Tastepeptides_DM, Umami_YYDS, UMPed-FRL, and iUmami-SCM were employed to comprehensively evaluate the umami probability of peptides, retaining high-probability candidate sequences.

[0033] Molecular docking screening: High-throughput molecular docking of candidate peptides was performed using Smina software. Sequences with binding energies below -7 kcal / mol were screened, targeting the VFT domain of the human T1R1 / T1R3 receptor, to ensure they possess the structural basis for binding to umami receptors.

[0034] Threshold prediction: The sensory threshold (Ti) of each candidate peptide is predicted online using Umami IP (http: / / tastepeptides-meta.com / Umami_IP), providing kinetic parameters for subsequent UPS calculations.

[0035] UPS strategy evaluation method: Calculate the UPS value of each peptide segment ( ).in, : Relative abundance of peptide i; : Umami threshold of peptide i; The minimum ratio between the relative abundance of peptides in potential umami peptides and the umami prediction threshold. ; The total number of all potential umami peptides in the sample; : Peptide interaction matrix coefficients; : Global scale factor of interaction effects; : Characteristic vector representing the physicochemical properties of the sample matrix; Correction function; For umami peptide screening indicators, A higher value indicates a greater contribution of the peptide to the umami flavor of the sample.

[0036] Based on descending UPS values, six core peptides with high sensory contribution potential were identified.

[0037] Example 2: Sensory Threshold Verification Experiment Based on 3-AFC Method Evaluation team composition: Recruit 10 professionally trained evaluators (5 men and 5 women) to conduct the evaluation in a standard sensory evaluation laboratory (ISO 8589).

[0038] Three-point forced selection (3-AFC) procedure: Prepare a series of serially diluted synthetic peptide solutions (purity >98%). Provide three sets of samples each time, including two sets of ultrapure water controls and one set of target peptide solution.

[0039] The evaluators must identify samples with differences in taste. The concentration is increased from low to high until the evaluator correctly identifies the sample twice in a row.

[0040] Data processing: The identification threshold is calculated as the geometric mean of the concentration of the evaluator's last incorrect response and the concentration of the first correct response.

[0041] Validation results: Sensory evaluation confirmed that all the sequences had low umami recognition thresholds (as shown in Table 1). Among them, DKEGNGTVM (DM-9) had a threshold of 0.044 mM, exhibiting extremely high umami intensity. AGDDAPRA (AA-8) had a threshold of 0.066 mM and showed significant flavor enhancement weight in the tested matrix.

[0042] Table 1. Flavor characteristics, recognition thresholds, and ranking among reported umami peptides of the six peptides.

[0043] Note: *The reported umami peptide data were obtained from the TastePeptidesDB database (accessed on January 30, 2025).

[0044] Example 3: Verification experiment of the activation effect of taste receptors at the molecular level (calcium flow experiment) The intensity of the sensing signal between peptides and human T1R1 / T1R3 receptors was quantified using a high-throughput cell evaluation system. 1) Cell Culture and Transfection: HEK293T cells capable of heterologous Gα16 / gust44 protein were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2. Cells were seeded in 96-well black-walled clear plates, and human T1R1 / T1R3 receptor plasmids were transfected into the cells using Lipofectamine 2000 after 24 hours.

[0045] 2) Calcium ion monitoring: Discard the culture medium, add sample loading buffer containing Fluo-4 AM fluorescent dye (4 μM), and incubate in the dark for 1 hour. Use a multi-mode microplate reader (excitation 485 nm, emission 535 nm) to capture fluorescence intensity in real time. Inject at 10 seconds a concentration ranging from 10% of the sensory recognition threshold of each peptide. -3 A series of peptide dilutions, ranging from 100 mM to 100 mM, were continuously monitored for 120 seconds.

[0046] 3) Measurement of kinetic parameters: Calculate fluorescence change rate Using Origin software, the dose-response curve was fitted with a three-parameter logistic equation to calculate the half-maximal effective concentration (EC50). 50 ).

[0047] 4) Verification results ( Figure 1 EC of DDMEKIW (DW-7) 50 The EC50 of SDMKHWP (SP-7) is 0.007 mM. 50 With a value of 0.003 mM, it exhibits the highest activation sensitivity.

[0048] Example 4: Molecular docking simulation and binding mechanism analysis experiment 1) Receptor model construction and parameter setting: Human T1R1 and T1R3 receptor sequences were obtained from the UniProt database (Q7RTX1 and Q7RTX0). The grid parameters were set in the VenusFlytrap (VFT) active pocket region of the T1R1 receptor using the molecular docking program Smina.

[0049] 2) Evaluation of docking results: All screened candidate peptides showed binding energies below -7 kcal / mol to the T1R1 / T1R3 receptor, indicating high binding affinity. Peptides such as DDMEKIW (DW-7) were also included. Figure 2 The diagram shows a typical skeleton connection pattern.

[0050] 3) Interaction force analysis: Hydrogen bond network: Docking results show that Asn150, Gln181, Tyr181 and Tyr182 in the T1R1 receptor are key binding residues that form a durable and stable hydrogen bond network with the peptide backbone.

[0051] Hydrophobic interactions and salt bridges: Hydrophobic side chains in the peptide sequence (such as tryptophan Trp in DW-7) are deeply embedded in the hydrophobic pocket formed by Ala157 and Val152; at the same time, the salt bridge formed between the acidic amino acid (Asp / Glu) and the receptor residue further stabilizes the binding conformation.

[0052] 4) Experimental conclusion: The above-mentioned molecular-level interactions lock the receptor in a closed active state, verifying the molecular mechanism of peptide activation of umami receptors at the atomic level.

[0053] Example 5: Biochemical-level verification experiment of antioxidant activity The biological functional properties of peptides were evaluated using various free radical scavenging models.

[0054] Considering the extremely low throughput of traditional cuvettes (10 mm optical path), the colorimetric reactions in this embodiment were all performed using a high-throughput 96-well microplate system (approximately 3 mm optical path). Due to the shortened optical path, according to the Lambert-Beer law, the absolute sample concentration required to achieve a 50% inhibition rate will increase accordingly; therefore, the overall IC... 50 The absolute numerical values ​​appear higher than those of the cuvette system. To ensure the rigor of the evaluation, this embodiment introduced L-glutathione (GSH, one of the strongest known endogenous small molecule antioxidants) as a positive control under strictly identical microenvironmental conditions, and assessed its actual antioxidant potential through relative potency.

[0055] (1) DPPH free radical scavenging experiment: Prepare a 0.1 mM DPPH ethanol solution. Mix 100 μL of peptide solutions of different concentrations (1-25 mM) with 100 μL of DPPH solution (A1), and mix 100 μL of peptide solutions of the same concentration with 100 μL of anhydrous ethanol (A2). Mix 100 μL of deionized water with 100 μL of DPPH solution (A3). After thoroughly mixing the above mixtures, incubate at room temperature in the dark for 30 minutes, and then measure the absorbance of each group at a wavelength of 517 nm.

[0056] The absorbance was measured at a wavelength of 517 nm (A1), with deionized water as a blank control (A3). .

[0057] (2) ABTS + Free radical scavenging experiment: Mix 7 mM ABTS solution with 2.45 mM potassium persulfate and let stand in the dark for 12-16 hours to generate ABTS. + Stock solution. Dilute the stock solution with ethanol before use to maintain its absorbance at 734 nm at 0.70 ± 0.02.

[0058] Mix equal volumes of peptide series solutions (1-25 mM) with ABTS + Dilute the solution and react for 10 minutes, then measure the absorbance at 734 nm.

[0059] (3) Data fitting The peptide concentration (IC50) at which clearance reaches 50% was calculated using nonlinear regression. 50 ).

[0060] Verification results: such as Figure 3 As shown in Tables 2 and 3, DKEGNGTVM (DM-9) exhibits excellent reducing activity in the aqueous reaction system. The sulfur atom in the methionine (Met) side chain of the sequence can undergo oxidation transformation, thereby scavenging pro-oxidative free radicals in the system. DDMEKIW (DW-7) showed a high IC50 value in the free radical scavenging experiment of DPPH. 50 = 0.95 mM, indicating that the indole ring of the tryptophan side chain acts as a highly efficient proton donor, achieving effective quenching of DPPH free radicals. It should be noted that although the absolute clearance value of the food-derived peptide of this invention is lower than that of the positive control (L-glutathione, IC50) used as a pure drug, it still achieves significant quenching. 50 (0.01~0.05 mM), but its IC 50 It achieved an extremely low concentration range of 0.95–1.16 mM. In the field of natural food-derived peptides, the IC50 at the 1 mM level... 50 It exhibits extremely high intrinsic antioxidant activity. This proves that the peptide of the present invention, while acting as an extremely sensitive umami enhancer, also possesses excellent biochemical preservative potential, perfectly achieving a combination of dual functional properties.

[0061] Table 2. Antioxidant activity (DPPH) analysis of six umami peptides

[0062] Table 3. Antioxidant activity of six umami peptides (ABTS) + Free radical scavenging analysis

[0063] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0064] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antioxidant umami bifunctional peptide or a pharmaceutically or food-grade acceptable salt thereof, characterized in that, The amino acid sequence of the bifunctional peptide is any one or a combination of the following (1) to (6): (1) AGDDAPRA; (2) DDALRSQE; (3) DDIKRVV; (4) DDMEKIW; (5) DKEGNGTVM; (6) SDMKHWP.

2. A composition containing the bifunctional peptide of claim 1 or a pharmaceutically or food-grade salt thereof.

3. The use of the bifunctional peptide as described in claim 1 or a pharmaceutically or food-grade acceptable salt thereof in the preparation of flavorings or food additives.

4. The use of the bifunctional peptide as described in claim 1 or its pharmaceutically or food-grade acceptable salt in enhancing the umami flavor of food.

5. The use of the bifunctional peptide as described in claim 1 or a pharmaceutically or food-grade acceptable salt thereof in the preparation of food antioxidants.

6. The use of the bifunctional peptide as described in claim 1 or a pharmaceutically or food-grade acceptable salt thereof in the preparation of umami enhancers.

7. The use of the bifunctional peptide as described in claim 1 or a pharmaceutically or food-grade acceptable salt thereof in the preparation of food ingredients that simultaneously possess both umami-enhancing and antioxidant functions.

8. A condiment, characterized in that, Includes the bifunctional peptide of claim 1 or a pharmaceutically or food-grade acceptable salt thereof.

9. A food antioxidant, characterized in that, Includes the bifunctional peptide of claim 1 or a pharmaceutically or food-grade acceptable salt thereof.

10. A flavor enhancer, characterized in that, Includes the bifunctional peptide of claim 1 or a pharmaceutically or food-grade acceptable salt thereof.

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