Eggshell membrane source antioxidant umami peptide and application thereof

Abstract

The invention discloses an eggshell membrane source antioxidant umami peptide and application thereof, the amino acid sequence of the eggshell membrane source umami peptide is at least one of CDDDF, CWDVYR and WHDCHR, and relates to the technical field of bioactive peptides. The eggshell membrane umami peptides with antioxidant activity are screened by using a method of combining virtual enzymolysis, machine learning, molecular docking and molecular dynamics simulation, and three eggshell membrane umami peptides are screened and can be combined with umami receptor T1R1 / T1R3 subunits, and the composite conformation is stable. Sensory evaluation and electronic tongue analysis prove that the eggshell membrane source antioxidant umami peptide has good umami characteristics, the umami threshold value is 0.04-0.10 mmol / L, the eggshell membrane source antioxidant umami peptide has DPPH free radical scavenging capacity, ABTS + free radical scavenging capacity and Fe < 3 + > reduction activity, a new raw material is provided for development of functional seasonings, and the eggshell membrane source antioxidant umami peptide has important application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioactive peptides, and particularly relates to an eggshell membrane source antioxidant umami peptide and application thereof. BACKGROUND

[0002] Eggshell membrane (ESM) is a porous network structure composed of highly cross-linked fibrous proteins, which contains 90% of proteins, 3% of lipids and 2% of carbohydrates, and also contains various minerals, nitrogen, uronic acid and other active ingredients, and has high nutritional value. The proteins in the eggshell membrane mainly exist in the form of glycoprotein, and other protein components include collagen, keratin, ovalbumin, osteonectin and the like, which have good bioactivity effects. The high protein composition makes it a high-quality resource for the preparation of functional bioactive peptides, and it has not been fully developed and utilized at present.

[0003] Eggshell membrane peptides refer to the general term of active peptide substances extracted from chicken eggshell membranes. Due to its unique biological activity and rich nutritional ingredients, it has shown wide application in many fields. In the field of biological medicine, eggshell membrane peptides are widely used in the maintenance and improvement of joint health, because they are rich in chondroitin sulfate, collagen peptides and other components, which can help protect joint cartilage, reduce joint pain and improve joint function. At the same time, it also contains various amino acids and trace elements, which can help improve human immunity and enhance disease resistance. In the field of beauty, collagen peptides in eggshell membrane can promote the regeneration and repair of skin cells, improve skin elasticity, reduce wrinkles and pigmentation, and improve skin nutrient metabolism, making the skin tender, smooth and elastic.

[0004] Umami peptides are a class of small molecular peptides with umami characteristics, with a molecular weight of usually 150-3000 Da. They can not only directly enhance the umami of food, making the taste more mellow and delicious, but also interact with other flavor substances to jointly build multi-level and colorful food flavors, meeting the development requirements of green food. Bioactive peptides with antioxidant effects are a class of natural antioxidants that can scavenge free radicals, reduce cell oxidative stress damage, promote cell metabolism and regeneration, and delay aging effects, and can be used as functional ingredients to control food oxidation and improve food health effects. Therefore, antioxidant umami peptides, as a combination of peptides with antioxidant properties and enhanced food umami, have a wide application prospect in the food industry and have important development significance.

[0005] With the development of bioinformatics, virtual enzymolysis, computer model screening and molecular docking technology are widely used in the screening and prediction of bioactive peptides. The umami peptides interact with umami receptor T1R1 / T1R3, activate the taste system cascade signal transmission and make the brain perceive umami. This provides an effective method for efficient screening of umami peptides and revealing the interaction between umami peptides and their receptors. Although there are many reports on the separation of antioxidant peptides from eggshell membranes, there are few studies on eggshell membrane peptides with antioxidant capacity and freshening effect. Therefore, it is of great significance to screen umami peptides with antioxidant activity from eggshell membranes based on modern bioinformatics technology, which not only provides a research direction for the high-value utilization of eggshell membranes, but also provides a new source for the preparation of umami peptides. SUMMARY

[0006] The technical problem to be solved: In view of the above technical problems, the purpose of the present application is to disclose an eggshell membrane-derived antioxidant umami peptide and its application. The amino acid sequence of the eggshell membrane-derived umami peptide is at least one of the following: CDDDF, CWDVYR and WHDCHR, which relates to the technical field of bioactive peptides. The present application screens eggshell membrane umami peptides with antioxidant activity by combining virtual enzymolysis, machine learning, molecular docking and molecular dynamics simulation. As a result, three kinds of eggshell membrane umami peptides are screened, which can all bind to the T1R1 / T1R3 subunit of umami receptor and have stable complex conformation. The eggshell membrane-derived antioxidant umami peptide disclosed in the present application has good umami characteristics after sensory evaluation and electronic tongue analysis verification. Its umami threshold is within 0.04-0.10 mmol / L, and it has DPPH free radical scavenging activity, ABTS+ free radical scavenging activity and Fe 3+ reducing activity, which provides a new raw material for the development of functional condiments and has important application value.

[0007] Technical scheme: An eggshell membrane-derived antioxidant umami peptide, which comprises peptide CDDDF, peptide CWDVYR and peptide WHDCHR, wherein the amino acid sequence of peptide CDDDF is Cys-Asp-Asp-Asp-Phe, the amino acid sequence of peptide CWDVYR is Cys-Trp-Asp-Val-Tyr-Arg, and the amino acid sequence of peptide WHDCHR is Trp-His-Asp-Cys-His-Arg.

[0008] Further, the umami threshold of the eggshell membrane-derived antioxidant umami peptide is 0.04-0.10 mmol / L.

[0009] Further, the peptide CDDDF, the peptide CWDVYR and the peptide WHDCHR all have DPPH free radical scavenging activity, ABTS+ free radical scavenging activity and Fe 3+ reducing activity.

[0010] The method for preparing the eggshell membrane-derived antioxidant umami peptide according to any one of the preceding items comprises the following steps: S1. performing virtual enzymolysis on a specific protein by using a BIOPEP-UWM tool to obtain a theoretical peptide sequence set; The proteases for the virtual enzymolysis include EC 3.4.22.2, EC 3.4.21.62, EC 3.4.22.32, EC.3.4.22.3, EC 3.4.21.1, EC.3.4.21.4, EC 3.4.21.1, EC 3.4.21.39, EC 3.4.23.1, EC 3.4.21.27, and EC 3.4.21.67. The specific proteins include lysyl oxidase-like 2 protein (E1C3U7), cysteine-rich ESM protein (CREMPs) (UPI0000E8213B), lysozyme C (P00698), collagen X (alpha 1 chain) (F1NRH2), and ovalbumin (P01012). S2. performing secondary screening by performing duplicate analysis and PeptideRanker scoring on the theoretical peptide sequence set, and evaluating water solubility, allergenicity, stability, and toxicity of the peptides to screen out potential umami peptides with good water solubility, no allergenicity, no toxicity, and good stability; S3. screening the potential umami peptides by iUmami-SCM, Umami_YYDS, and Tastepeptides_DM models; S4. evaluating the antioxidant activity of the potential umami peptides by a UniDL4BioPep deep learning model to screen out bifunctional umami peptides; S5. simulating the combination of the bifunctional umami peptides with umami receptors T1R1 / T1R3 by using molecular docking and molecular dynamics simulation technology to screen out candidate umami peptides; S6. synthesizing the candidate umami peptides by using a biological solid-phase synthesis method; verifying the umami characteristics and antioxidant activity of the candidate umami peptides to obtain the eggshell membrane-derived antioxidant umami peptide.

[0011] An eggshell membrane hydrolysate comprises one or more of the peptides CDDDF, CWDVYR, and WHDCHR according to claim 1.

[0012] The eggshell membrane-derived antioxidant umami peptide according to any one of the preceding items is applied to the preparation of food, food additives, and health products.

[0013] The eggshell membrane-derived antioxidant umami peptide according to any one of the preceding items is applied to improving umami and flavor of food.

[0014] Use of the eggshell membrane hydrolysate in the preparation of food, food additives and health products. Beneficial effects

[0015] 1. The present application uses machine learning and bioinformatics to screen out eggshell membrane umami peptides with antioxidant effect, which are peptide CDDDF (Cys-Asp-Asp-Asp-Phe), peptide CWDVYR (Cys-Trp-Asp-Val-Tyr-Arg), peptide WHDCHR (Trp-His-Asp-Cys-His-Arg), and peptide CDDDF, peptide CWDVYR and peptide WHDCHR can all bind to umami receptor T1R1 / T1R3 subunit to form stable complex conformation, and through sensory evaluation and electronic tongue analysis verification, peptide CDDDF, peptide CWDVYR and peptide WHDCHR all have good freshening effect, and the umami threshold is 0.04-0.10 mmol / L; in addition, peptide CDDDF, peptide CWDVYR and peptide WHDCHR all have high DPPH free radical and ABTS+ free radical scavenging capacity and Fe 3+ reducing activity, especially the DPPH free radical scavenging rate and ABTS+ free radical scavenging rate of 1 mg / mL eggshell membrane umami peptide are both higher than 90%.

[0016] 2. The present application can avoid the time-consuming and labor-consuming defects caused by traditional random proteinase cleavage combined with step-by-step separation and purification, and reduce the loss of polypeptides caused by complex experimental steps, thereby greatly shortening the screening time of umami peptides in eggshell membrane, and having the characteristics of high efficiency, high screening efficiency, high accuracy and repeatability.

[0017] 3. The eggshell membrane umami peptide of the present application has antioxidant activity of scavenging free radicals, and has the effects of antioxidant property and enhancing food umami, which provides a new raw material basis for the development of umami agents and condiments, and also provides good technical support for the screening and development of umami peptides, and has a wide application prospect in functional foods and condiments. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 3 is a 3D structure diagram of peptide CDDDF, peptide CWDVYR and peptide WHDCHR, wherein A represents peptide CDDDF, B represents peptide CWDVYR, and C represents peptide WHDCHR; Figure 2Molecular docking of peptide CDDDF, peptide CWDVYR, peptide WHDCHR with umami receptor, wherein A1 represents the three-dimensional structure of T1R1 / T1R3-CDDDF complex, A2 represents the three-dimensional structure of T1R1 / T1R3-CWDVYR complex, A3 represents the three-dimensional structure of T1R1 / T1R3-WHDCHR complex, B1 represents the two-dimensional structure of T1R1 / T1R3-CDDDF complex, B2 represents the two-dimensional structure of T1R1 / T1R3-CWDVYR complex, B3 represents the two-dimensional structure of T1R1 / T1R3-WHDCHR complex, C1 represents the two-dimensional LigPlot molecular interaction of T1R1 / T1R3-CDDDF complex, C2 represents the two-dimensional LigPlot molecular interaction of T1R1 / T1R3-CWDVYR complex, C3 represents the two-dimensional LigPlot molecular interaction of T1R1 / T1R3-WHDCHR complex; Figure 3 Molecular dynamics simulation of peptide CDDDF, peptide CWDVYR, peptide WHDCHR with umami receptor, wherein A represents the RMSD plot, B represents the Rg plot; Figure 4 Solid phase synthesis reverse high performance liquid chromatogram of peptide CDDDF, peptide CWDVYR, peptide WHDCHR, wherein A represents peptide CDDDF, B represents peptide CWDVYR, C represents peptide WHDCHR; Figure 5 Solid phase synthesis mass spectrum of peptide CDDDF, peptide CWDVYR, peptide WHDCHR, wherein A represents peptide CDDDF, B represents peptide CWDVYR, C represents peptide WHDCHR; Figure 6 Sensory evaluation radar chart of peptide CDDDF, peptide CWDVYR, peptide WHDCHR; Figure 7 Peptide CDDDF, peptide CWDVYR, peptide WHDCHR umami-enhancing effect; Figure 8 Electronic tongue radar chart of peptide CDDDF, peptide CWDVYR, peptide WHDCHR; Figure 9 Antioxidant activity evaluation of peptide CDDDF, peptide CWDVYR, peptide WHDCHR. DETAILED DESCRIPTION

[0019] The application will be further described in conjunction with the following examples, which are intended to explain the application but not limit the application to the following examples: Example 1

[0020] The method for screening potential umami peptides from eggshell membrane comprises the following steps: Step 1. In vitro enzymatic hydrolysis of the main proteins of chicken eggshell membrane (Lysyl oxidase-like 2, CREMPs, Lysozyme C, Collagen X, Ovalbumin) was performed using the BIOPEP-UWM online enzymatic hydrolysis program, and 11 hydrolytic enzymes in the UniProt database were used for processing. The virtual enzymatic proteases include EC. 3.4.22.2, EC, 3.4.21.62, EC, 3.4.22.32, EC. 3.4.22.3, EC.3.4.21.1, EC. 3.4.21.4, EC.3.4.21.1, EC.3.4.21.39, EC. 3.4.23.1, EC. 3.4.21.27, EC.3.4.21.67; The results of enzymatic hydrolysis showed that there were significant differences in the peptide spectrum produced by different enzymes, indicating that the specificity of the enzyme had an important influence on the hydrolysis product. A total of 6365 peptides were obtained. Based on the characteristics of umami peptides consisting of 2-9 amino acids, 3693 potential bioactive peptides were screened, and a theoretical database of eggshell membrane-derived peptides was constructed. Step 2. The PeptideRanker tool was used to predict the biological activity of the eggshell membrane protein peptides with amino acid length of 2-9 screened out, and 1287 peptide sequences were obtained. Step 3. The water solubility, allergenicity, stability and toxicity of the 1287 peptides were evaluated, and 69 peptides with good water solubility, no allergenicity, no toxicity and good stability were screened out. Step 4. The umami characteristics of the 69 peptides with good water solubility and no allergenicity were predicted using the composite machine learning model (iUmami-SCM, Umami_YYDS and Tastepeptides_DM), and the umami potential of the peptide sequences was comprehensively evaluated. The prediction results showed that 17 peptides had umami potential. Step 5. Toxicity detection and human intestinal absorption prediction were performed on the 17 peptides, and the results showed that 12 peptides were predicted to be non-toxic (III level toxicity) and had good human intestinal absorption capacity (HIA+), as shown in Table 1. Step 6. The antioxidant activity of the 12 peptides was predicted using the deep learning model UniDL4BioPep, and 6 peptides showed antioxidant potential, as shown in Table 1.

[0021] Table 1. Antioxidant activity evaluation and umami score of peptides

[0022] As shown in Table 1, the three peptides CDDDF, CWDVYR, and WHDCHR had the highest predicted antioxidant activity scores, at 0.8751, 0.9914, and 0.9199, respectively, significantly higher than other candidate peptides. Based on the overall umami score, CDDDF, WHDCCHR, and CWDVYR were ultimately selected as the subjects for subsequent experiments.

[0023] Example 2 Molecular docking targeted screening of umami peptides The binding interaction between umami peptides and umami receptors T1R1 / T1R3 was simulated using molecular docking technology with AutoDock software, and candidate umami peptides were obtained through targeted screening. The specific process is as follows: ①Utilizing a peptide folding network server ( The three-dimensional structure of umami peptides (e.g.) Figure 1 (as shown) ② Pretreatment of the receptor structure: removal of all water molecules and ligands, and addition of all hydrogen atoms; ③ Use AutoDock Tools (ADT) to convert the PDB format files of the receptor and ligand to PDBQT format and add Gasteiger charge to the ligand; during docking, the receptor remains rigid while the umami peptide is set to be flexible, allowing its chemical bonds to rotate freely in order to explore the optimal binding mode. ④ Use AutoGrid4 to generate grid information. The grid boxes cover the entire structural domain of the receptor to comprehensively analyze the docking sites. The docking parameters are set to a genetic algorithm to generate 100 conformations. Finally, the docking is run through AutoDock4 to obtain a docking record file in .dlg format. ⑤ After docking, the binding affinity of umami peptides to T1R1 / T1R3 is quantified by the Wiener score (unit: kcal / mol) of AutoDock.

[0024] Table 2. Molecular docking results of umami peptides with T1R1 / T1R3 receptors

[0025] As shown in Table 2, the binding energies of the three potential umami peptides to T1R1 / T1R3 are −10.17, −9.1 and −10.04 kcal / mol, respectively. These negative binding energies indicate that these umami peptides can theoretically bind spontaneously to the receptor. Among them, peptide WHDCHR shows a stronger binding ability, which suggests that it may be the peptide with the greatest umami potential.

[0026] Figure 2In the two-dimensional structural diagram, amino acid residues are green and ligands are yellow; in the two-dimensional LigPlot molecular interaction visualization, hydrogen bonds are represented by green dashed lines, hydrophobic interactions by red flashing arcs, and salt bridges by red dashed lines; as shown in Table 2, Figure 2 and Figure 3 It is known that the binding of three potential umami peptides to 17 amino acid residues on the T1R1 receptor includes Lys193, Gln226, Asn230, Glu229, Ala227, Leu223, Gln181, Thr175, Ser173, Tyr182, Asp219, Glu174, Gln222, Lys242, Gln231, Leu228, and Val178. Most of these residues have been reported in studies of various umami peptides. Three potential umami peptides can also bind to 19 amino acid residues on the T1R3 receptor, including Arg220, Lys155, Len223, Ser224, Gln221, Leu242, Gln222, Leu173, Met171, Leu174, Glu217, Glu148, Val152, Ala228, Met151, IIe225, Ser147, Tyr218, and Glu172. These residues are also derived from a variety of previously reported umami peptides.

[0027] Depend on Figure 2 As shown in C1, the interaction between peptide CDDDF and T1R1 / T1R3 mainly occurs through hydrogen bonds with residues Lys193, Gln226, Asn230, Thr175, Arg220, and Lys155, and through hydrophobic interactions with residues Glu229, Gln221, Ala227, Leu223, Ser224, Gln222, Leu173, Met171, Met151, Gln217, Leu174, Leu242, and Glu217; additionally, residue Arg220 forms a salt bridge with CDDDF. Figure 2 As shown in C2, the peptide CWDVYR binds to residues Gln181, Thr175, Ser173, Gln226, and Gln221 via hydrogen bonds, and to residues Tyr182, Asp219, Glu174, Gln222, Leu223, Ala227, Glu229, Asn230, Leu228, Gln231, Lys242, Glu148, Val152, Gln217, Met151, Glu172, Met171, Leu173, Arg220, Ile225, Ser224, and Ala228 via hydrophobic interactions. Figure 2As shown in C3, the peptide WHDCHR binds to residues Thr175 and Ser147 via hydrogen bonds, and to residues Asp219, Leu223, Gln226, Glu174, Lys193, Val178, Gln222, Gln181, Gln231, Ala227, Met171, Leu174, Tyr218, Leu173, Met151, Glu148, Gln221, Glu217, and Arg220 via hydrophobic interactions. In summary, all three umami peptides interact with the key binding sites of the T1R1 / T1R3 receptors: Leu223, Arg220, Gln226, Thr175, Gln222, Leu173, Met171, Met151, Gln221, and Ala217, indicating that these sites are crucial regions for the umami peptides to exert their umami effects. Furthermore, the hydrogen bond lengths between the three potential umami peptides and the T1R1 / T1R3 receptors range from 2.81 to 3.34 Å, demonstrating the important role of hydrogen bonds in the binding of umami peptides to the receptors; simultaneously, hydrophobic interactions are also a key force in the binding of the three potential umami peptides to T1R1 / T1R3. Hydrogen bonding, hydrophobic interactions, and salt bridges are key factors in the interaction between umami peptides and the T1R1 / T1R3 receptors, and these forces contribute to the umami peptides' potential umami properties. However, the specific flavor characteristics of the umami peptides still need to be determined through further synthesis and sensory verification. Example 3

[0028] Solid-phase synthesis of peptides CDDDF, CWDVYR, and WHDCHR The umami peptide sequences (CDDDF, WHDCHR, and CWDVYR) were synthesized by Hefei Guotai Biotechnology Co., Ltd. (Hefei, China) using the Fmoc solid-phase synthesis method on 2-Cl Resin. Their purity and molecular weight were verified by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS). The synthesized peptides were stored at -20°C for further use.

[0029] Depend on Figure 4 and Figure 5 It can be seen that the purities of peptides CDDDF, WHDCHR, and CWDVYR are 93.904%, 90.458%, and 96.055%, respectively; their relative molecular weights are 613.30 g / mol, 852.60 g / mol, and 840.60 g / mol, respectively, which are basically consistent with the theoretical relative molecular masses of 613.60 g / mol, 852.92 g / mol, and 840.95 g / mol, further verifying the accuracy and reliability of the synthesis method.

[0030] Example 4 Sensory evaluation verification of peptides CDDDF, CWDVYR, and WHDCHR The sensory evaluation team consisted of six rigorously selected laboratory members (3 women and 3 men, aged 22-28). Prior to the formal experiments, all evaluators underwent two weeks of professional training, including: 1) basic taste recognition testing, requiring accurate identification of five basic tastes; 2) training on consistency of scoring criteria; and 3) training on standardized sample evaluation procedures. The reference standard solutions used included: sweetness (1% sucrose solution, w / v), saltiness (0.35% sodium chloride solution, w / v), umami (0.35% monosodium glutamate solution, w / v), sourness (0.08% citric acid solution, w / v), and bitterness (1% L-leucine solution, w / v). The experiments were conducted in a standard sensory evaluation room, with the ambient temperature controlled at 23±2℃ and the relative humidity maintained at 50-60%. Fasting and smoking are required for one hour prior to the evaluation. During the evaluation process, a mandatory 10-minute break is required every 30 minutes. Between evaluations of different samples, the patient must rinse their mouth thoroughly with 50 mL of purified water to minimize the impact of sensory fatigue on the evaluation results. Results are presented as a radar chart.

[0031] The synthetic peptide was prepared into a 50 mL sample solution with a concentration of 1 mg / mL (pH 6.5) using purified water. These solutions were randomly coded and placed in plastic cups for evaluation. During evaluation, team members gargled with 3 mL of the sample for 10 seconds and assessed its five taste characteristics: sweet, salty, bitter, sour, and umami. A 10-point intensity scale was used for scoring, with the reference standard solution (e.g., 0.35% monosodium glutamate) set at a baseline of 5 points. 0 points represented no taste, and 10 points represented an extremely strong taste.

[0032] (3) The umami threshold and flavor-enhancing effect of the synthetic peptide were determined using the triangular test method. The peptide solution was serially diluted with deionized water at a 1:1 ratio, and samples were provided in ascending order of concentration. When the evaluator could distinguish the previous sample from the blank control (pure water) but could not distinguish the next dilution gradient, the concentration was the threshold. The flavor-enhancing effect was evaluated by dissolving the peptide in a 0.35% monosodium glutamate solution and comparing its umami intensity with that of pure monosodium glutamate solution.

[0033] Depend on Figure 6 It was found that all synthetic peptides exhibited umami and bitterness, along with distinct sweet and salty flavors, while the sourness was relatively weak. Among the three eggshell synthetic peptides, the WHDCHR sequence peptide had the strongest umami flavor, scoring 4.33 points, which is close to that of the standard umami peptide solution; the CDDDF and CWDVYR sequence peptides had umami scores of 3.92 and 3.90 points, respectively, with similar umami intensity and little difference. The bitterness of all three synthetic peptides is due to the presence of hydrophobic amino acids (such as Trp, Val, and Phe). In addition, basic amino acids (such as Arg) can enhance the perception of bitterness.

[0034] Table 3 Umami threshold and umami-enhancing effect of peptide CDDDF, peptide CWDVYR and peptide WHDCHR

[0035] From Table 3 and Figure 7 It can be seen that the umami threshold of the three synthetic peptides is in the order of peptide WHDCHR (0.04 mmol / L), peptide CWDVYR (0.08 mmol / L) and peptide CDDDF (0.10 mmol / L), all of which are significantly lower than that of traditional umami substance sodium glutamate (MSG, threshold is 1.635 mmol / L), which indicates that the synthetic peptides have strong umami and belong to high-sensitivity umami peptides. Further experiments show that compared with 0.35% standard sodium glutamate solution, the umami of the three synthetic peptides is significantly enhanced, among which the umami-enhancing effect of peptide CWDVYR and peptide WHDCHR is the most significant, and their umami-enhancing scores are (7.75 ± 0.5) and (7.75 ± 0.5), respectively, which are significantly higher than that of peptide CDDDF (6.75 ± 0.5). This stronger umami-enhancing effect may be due to the taste synergy between the synthetic peptides and sodium glutamate, thereby further highlighting the umami characteristics. Example 5

[0036] Electronic tongue analysis verification of peptide CDDDF, peptide CWDVYR and peptide WHDCHR The taste characteristics of the synthetic peptides were evaluated using a TS-5000Z electronic tongue system (INSENT, Japan). The three synthetic peptides were dissolved in deionized water at a concentration of 1 mg / mL, and the pH was adjusted to 6.5 with a 0.1 M NaOH solution to ensure that the samples were in the best state for taste detection. The treated samples were placed in the TS-5000Z electronic tongue system for analysis. The instrument was equipped with five specific taste sensors, including AAE (umami), CT0 (salty), CA0 (sour), C00 (bitter) and AE1 (astringent), which can accurately determine the intensity of different taste components. To ensure data reliability, each sample was measured independently for 3 times, and the average value was taken to eliminate system error.

[0037] It can be seen from Figure 8 that the taste characteristics of peptide CDDDF, peptide CWDVYR and peptide WHDCHR are similar, with umami being the most significant, followed by bitterness, and accompanied by slight astringency; among them, the umami score of peptide WHDCHR is the highest (16.17 ± 0.08), which is significantly higher than that of peptide CWDVYR (15.61 ± 0.27) and peptide CDDDF (15.48 ± 0.01) (P<0.05). In contrast, the umami characteristics of peptide CWDVYR and peptide CDDDF are not significantly different (P>0.05). Example 6

[0038] Determination of antioxidant activity of peptide CDDDF, peptide CWDVYR, and peptide WHDCHR (1) DPPH free radical scavenging rate Dissolve 10 mg of the freeze-dried peptide in water to prepare a solution with a mass concentration of 1 mg / mL; take 2 mL of the sample solution to be tested, mix with 2 mL of a 0.1 mmol / L DPPH anhydrous ethanol solution, and then place in the dark at room temperature for 30 min of reaction, and then measure the absorbance A1 at a wavelength of 517 nm; at the same time, take 2 mL of a 0.1 mmol / L DPPH anhydrous ethanol solution and add 2 mL of distilled water as a blank group, and then measure the absorbance A0 at a wavelength of 517 nm; additionally, take 2 mL of the sample solution to be tested and mix with 2 mL of anhydrous ethanol as a control group, and then place in the dark at room temperature for 30 min of reaction, and then measure the absorbance A2 at a wavelength of 517 nm; the calculation formula for the DPPH free radical inhibition rate is: DPPH free radical inhibition rate (%) = [1 - (A1 - A2) / A0] x 100%.

[0039] (2) ABTS free radical scavenging rate First, dilute the ABTS solution with a phosphate buffered saline (PBS) buffer until its absorbance at 734 nm reaches 0.7 ± 0.05; then add 10 μL of the sample (with a concentration of 1 mg / mL) to the ABTS solution, and then keep each mixture at 25°C for 5 min, and then evaluate the antioxidant activity of the sample by detecting the change in the absorbance of the system at a wavelength of 734 nm, with distilled water as a blank control. The calculation formula for the ABTS+ free radical scavenging capacity is: ABTS+ free radical scavenging rate (%) = (A control − A sample ) / A control x 100%, where A control is the absorbance of the blank control, and A sample is the absorbance of the sample.

[0040] (3) Fe 3+ reducing capacity The potassium ferricyanide method was used for determination. The specific procedure was as follows: Take 2 mL of each 0.8 mg / mL sample solution, add 2 mL of 0.2 M phosphate buffer (pH 6.6) and 2 mL of 1% potassium ferricyanide solution respectively, mix well, and incubate in a 50℃ oven for 20 min. Then, add 2 mL of 10% trichloroacetic acid solution to terminate the reaction, mix well, centrifuge at 6500 r / min for 10 min, take 2 mL of the supernatant, add 2.5 mL of distilled water and 0.5 mL of ferric chloride hexahydrate solution, and incubate for 10 min. Use 1 mL of distilled water as a blank control, and measure the absorbance at 700 nm. A higher absorbance indicates a stronger reducing power of the sample; conversely, a lower absorbance indicates a weaker reducing power.

[0041] Depend on Figure 9 It can be seen that peptides CDDDF, CWDVYR, and WHDCHR all exhibit significant DPPH radical scavenging and ABTS+ radical scavenging abilities, with CDDDF showing the highest DPPH radical scavenging rate and CWDVYR showing the highest ABTS+ radical scavenging rate. Simultaneously, the Fe... 3+ The reducing power was ranked from strongest to weakest as follows: peptide CDDDF > peptide WHDCHR > peptide CWDVYR. The results indicate that peptide CDDDF has the strongest antioxidant potential compared to peptides WHDCHR and CWDVYR.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. An eggshell membrane-derived antioxidant umami peptide, characterized in that: The eggshell membrane-derived antioxidant umami peptides include peptide CDDDF, peptide CWDVYR, and peptide WHDCHR, wherein the amino acid sequence of peptide CDDDF is Cys-Asp-Asp-Asp-Phe, the amino acid sequence of peptide CWDVYR is Cys-Trp-Asp-Val-Tyr-Arg, and the amino acid sequence of peptide WHDCHR is Trp-His-Asp-Cys-His-Arg.

2. The eggshell membrane-derived antioxidant umami peptide according to claim 1, characterized in that: The umami threshold of the eggshell membrane-derived antioxidant umami peptide is 0.04-0.10 mmol / L.

3. The eggshell membrane-derived antioxidant umami peptide according to claim 1, characterized in that, The peptides CDDDF, CWDVYR, and WHDCHR all possess scavenging activity against DPPH free radicals, ABTS+ free radicals, and Fe. 3+ Reduction activity.

4. A method for preparing an eggshell membrane-derived antioxidant umami peptide according to any one of claims 1-3, comprising the following steps: S1. Using the BIOPEP-UWM tool, a specific protein was virtually digested to obtain a set of theoretical peptide sequences; S2. Initial screening of theoretical peptide sequence sets, followed by duplicate checking and PeptideRanker scoring for secondary screening; and evaluation of the water solubility, allergenicity, stability and toxicity of peptides, screening out potential umami peptides with good water solubility, no allergenicity, no toxicity and good stability. S3. Potential umami peptides were screened using the iUmami-SCM, Umami_YYDS, and Tastepeptides_DM models; S4. The antioxidant activity of potential umami peptides was evaluated using the UniDL4BioPep deep learning model, and bifunctional umami peptides were screened. S5. Molecular docking and molecular dynamics simulation techniques were used to simulate the binding of bifunctional umami peptides to umami receptors T1R1 / T1R3, and candidate umami peptides were screened. S6. Candidate umami peptides were synthesized using a biological solid-phase synthesis method; verification experiments were conducted to verify the umami characteristics and antioxidant activity of the candidate umami peptides, thus obtaining eggshell membrane-derived antioxidant umami peptides.

5. A hydrolysate of eggshell membrane, characterized in that: It includes one or more of the peptides CDDDF, CWDVYR, and WHDCHR as described in claim 1.

6. The application of an eggshell membrane-derived antioxidant umami peptide according to any one of claims 1-4 in the preparation of food, food additives and health products.

7. The application of an eggshell membrane-derived antioxidant umami peptide according to any one of claims 1-4 in enhancing the umami flavor and improving the taste of food.

8. The application of the eggshell membrane hydrolysate according to claim 5 in the preparation of food, food additives and health products.

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