A umami peptide derived from apostichopus japonicus, and a preparation method, identification method and application thereof
By combining stepwise enzymatic hydrolysis with multiple techniques, umami peptides from sea cucumber imitations were successfully prepared and identified. This solved the problem of low protein resource utilization in sea cucumber imitations, provided an efficient method for preparing and identifying umami peptides, and enhanced the added value of sea cucumber imitations and the application potential of umami peptides.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WEIHAI FOOD & DRUG INSPECTION & TESTING RES INST
- Filing Date
- 2026-02-06
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, the protein resource utilization rate of sea cucumber imitations is low, and there are no systematic reports on the development and preparation methods of umami peptides, resulting in low added value and a limited variety of seasonings.
A stepwise enzymatic hydrolysis method was adopted. First, the sea cucumber protein was hydrolyzed with Alcalase, then further hydrolyzed with flavor protease, and purified by ultrafiltration and gel chromatography. Finally, peptide sequences with umami activity were identified by electronic tongue, LC-MS/MS and virtual screening tools.
This method enables the efficient and controllable preparation of small molecule umami peptides, enriches the marine-derived umami peptide database, improves the discovery efficiency and accuracy of umami peptides, and enhances the umami effect of food.
Smart Images
Figure CN122103252A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of food science and bioactive peptide technology, and in particular to a umami peptide derived from sea cucumber, its preparation method, identification method and application. Background Technology
[0002] Umami, as the fifth basic taste, is one of the key dimensions for measuring the deliciousness of food. Umami peptides are a class of small molecule peptides with umami characteristics. Because they can impart a rich and full-bodied flavor to food and may offer health benefits such as reducing salt intake and enhancing umami, they have become a research hotspot in the development of novel natural seasonings. Currently, the sources of umami peptides have expanded to include terrestrial animal proteins, plant proteins, and microorganisms, while the exploration of novel umami peptides from marine organisms, especially high-protein seafood, is showing great potential.
[0003] The sea cucumber, also known as the spiny sea cucumber, is an important edible sea cucumber in my country. Its body wall is rich in protein, with the dried product containing more than 70% protein. It also has a balanced amino acid composition, containing a high proportion of umami amino acids such as glutamic acid and aspartic acid, making it an ideal raw material for developing marine-derived umami peptides.
[0004] However, current deep processing of sea cucumber mainly focuses on the extraction of active ingredients such as polysaccharides and saponins. Research on its vast protein resources, especially the preparation of flavor peptides through controlled enzymatic hydrolysis, is insufficient. The sequences, flavor characteristics, and preparation processes of related flavor peptides have not been systematically reported. Therefore, developing a small molecule peptide derived from sea cucumber with clearly defined flavor characteristics, and establishing efficient and controllable preparation and identification methods, is of significant practical importance for increasing the added value of sea cucumber processing and enriching the variety of natural marine condiments. Summary of the Invention
[0005] The technical solution adopted in this invention is to provide a flavor peptide derived from sea cucumber, its preparation method, identification method and application, so as to solve the technical problems of low utilization rate, low added value and limited seasoning categories of sea cucumber.
[0006] To solve the above-mentioned technical problems, the technical solution adopted in this application is to provide a umami peptide derived from sea cucumber, wherein the amino acid sequence of the umami peptide is any one of Glu-Gly-Ala-Thr (EGAT), Gly-Ala-Asp-Ala (GADA), Asp-Ser-Ala-Thr (DSAT), Asp-Ser-Thr-Thr (DSTT) or Ser-Ser-Ala-Glu (SSAE).
[0007] A second aspect of this application provides a method for preparing the above-described umami peptide derived from sea cucumber, comprising the following steps: S1. The imitation sea cucumber is washed, homogenized, degreased, dried, and pulverized to obtain imitation sea cucumber protein powder; S2. Dissolve the sea cucumber protein powder in water, adjust the pH, and obtain the first enzymatic hydrolysate through the first enzymatic hydrolysis step; S3. Adjust the pH of the first step enzymatic hydrolysate, and then proceed with the second enzymatic hydrolysate to obtain the second step enzymatic hydrolysate; S4. Inactivate the enzyme in the second step enzyme hydrolysate, centrifuge, and obtain the supernatant; S5. Use an ultrafiltration membrane to separate the supernatant by ultrafiltration, collect the permeate, and obtain a mixture of small molecule peptides; S6. The mixture of small molecule peptides was separated and purified using a gel chromatography column. The eluent was collected based on the ultraviolet absorption peak and then freeze-dried to obtain umami peptides.
[0008] Preferably, in step S2, the sea cucumber protein powder is dissolved in water, the pH is adjusted to 8.5-9.5, and Alcalase enzyme is added for the first enzymatic hydrolysis. The Alcalase enzyme activity is ≥2.4AU / g, the amount of Alcalase enzyme added is 1-3% of the mass of the sea cucumber protein powder, the temperature of the first enzymatic hydrolysis is 50-60℃, and the time is 2-4h.
[0009] Preferably, in step S3, the pH of the first step enzymatic hydrolysate is adjusted to 6-7, and flavor protease is added for the second enzymatic hydrolysis. The enzyme activity of the flavor protease is ≥500 LAPU / g, and the amount of flavor protease added is 1-3% of the mass of the sea cucumber protein powder. The temperature of the second enzymatic hydrolysis is 45-55℃, and the time is 1-3h.
[0010] Preferably, in step S4, the sterilization temperature is 90-100℃ and the time is 5-20 minutes.
[0011] Preferably, in step S5, the ultrafiltration membrane is an ultrafiltration membrane with a molecular cutoff of less than 3 kDa, and the ultrafiltration separation is carried out at 4-10°C.
[0012] Preferably, in step S6, the gel chromatography column is a Sephadex G-15 column, the eluent is ultrapure water or a low-concentration salt solution, and the detection wavelength is 210-220 nm.
[0013] A third aspect of this application provides a method for identifying umami peptides derived from sea cucumber as described above, comprising the following steps: S1. Use an electronic tongue system to perform flavor profile analysis on the collected eluted components and screen out the components with the highest umami intensity; S2. The peptide sequence of the component with the highest umami intensity was identified by liquid chromatography-tandem mass spectrometry. S3. Use virtual screening tools to predict the umami potential of the identified peptide sequences; S4. Molecular docking simulation was performed between the peptide sequences predicted to have high umami potential and the umami receptors T1R1 / T1R3 to screen out peptides with binding energies lower than those of the control. S5. The umami characteristics of peptides with lower binding energies than the control were verified using an electronic tongue system, and the amino acid sequence of the umami peptides was finally determined.
[0014] Preferably, in step S3, the virtual screening tool includes the iUmami-SCM online prediction platform; in step S4, the reference standard includes monosodium glutamate.
[0015] The fourth aspect of this application provides the application of the above-described umami peptide derived from sea cucumber in the preparation of food seasonings, nutritional fortifiers, or flavor enhancers.
[0016] This invention provides a flavor peptide derived from sea cucumber, its preparation method, identification method, and application. Compared with existing technologies, its advantages are: (1) This invention provides a method for preparing umami peptides derived from sea cucumber. Using sea cucumber as raw material, a stepwise enzymatic hydrolysis method is employed, involving raw material pretreatment, first-step enzymatic hydrolysis with Alcalase enzyme, second-step enzymatic hydrolysis with flavor protease, sterilization and centrifugation, ultrafiltration, and gel chromatography purification to obtain umami peptides. This invention utilizes the synergistic effect of two proteases with different characteristics. First, Alcalase enzyme efficiently hydrolyzes large molecular proteins into polypeptide fragments, and then flavor protease further cleaves the peptide chains from the ends, releasing small molecule umami peptides and reducing bitterness, thereby directionally enriching the target product. The stepwise enzymatic hydrolysis preparation method of this invention has mild conditions and a clear process. Through the rational combination of enzymes, it effectively promotes the generation of small molecule umami peptides and helps improve the flavor profile. The preparation method is stable, controllable, and suitable for large-scale preparation.
[0017] (2) This invention provides an umami peptide derived from sea cucumber, the amino acid sequence of which is any one of Glu-Gly-Ala-Thr (EGAT), Gly-Ala-Asp-Ala (GADA), Asp-Ser-Ala-Thr (DSAT), Asp-Ser-Thr-Thr (DSTT), or Ser-Ser-Ala-Glu (SSAE). This invention is the first to identify five tetrapeptides with umami activity—EGAT, GADA, DSAT, DSTT, and SSAE—from sea cucumber protease hydrolysates. These peptides have small molecular weights, have been proven to have pure umami flavor, and have weak undesirable flavors such as bitterness and sourness, thus enriching the marine-derived umami peptide database.
[0018] (3) This invention provides a method for identifying umami peptides derived from sea cucumber. First, high-umami-active components are screened out using electronic tongue taste profile analysis. Then, peptide sequences are identified using LC-MS / MS. Subsequently, the iUmami-SCM virtual screening tool, molecular docking simulation with umami receptors T1R1 / T1R3, and electronic tongue taste verification are comprehensively used to screen layer by layer, finally determining the peptide sequences with clear umami characteristics. The identification method of this invention combines bioinformatics prediction with experimental verification, which can efficiently and accurately screen out target umami peptides from complex enzymatic hydrolysis products, improving the efficiency and accuracy of umami peptide discovery. The integrated identification strategy of "electronic tongue initial screening - peptide sequence identification - virtual screening - molecular docking simulation - electronic tongue taste verification" provides a reliable technical path for the discovery of target-oriented umami peptides.
[0019] (4) This invention provides an application of umami peptides derived from sea cucumber. Umami peptides have a pure umami flavor and a low umami threshold, and can be used to prepare food seasonings (such as chicken essence, soy sauce, compound seasoning sauce, soup base), nutritional fortifiers, or flavor enhancers. They can also be used as natural and healthy umami ingredients in various foods, providing new raw material options and technical support for the development of high-end marine seasonings. Umami peptides can be used directly or compounded with other umami substances (such as monosodium glutamate and flavor nucleotides) to produce a synergistic umami-enhancing effect. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1 Elution curves of a mixture of small molecule peptides separated by Sephadex G-15 gel chromatography; Figure 2 Radar plot of electronic tongue taste profile analysis of the three components after separation by Sephadex G-15 gel chromatography; Figure 3 The image shows the PCA analysis chromatogram of the three components separated by Sephadex G-15 gel chromatography using electronic tongue detection. Figure 4 To identify the total ion chromatogram of all peptides in the G2 fraction; Figure 5 Mass spectra of the five umami peptides identified; Figure 6 A homology model diagram of the taste receptor T1R1 / T1R3-VFD; Figure 7 To evaluate the Raphtogram for the T1R1 / T1R3 protein model; Figure 8 The diagram shows the docking of EGAT with T1R1 / T1R3 and the interaction diagram of the active amino acid residues. Figure 9 The diagram shows the docking of GADA with T1R1 / T1R3 and the interaction diagram of the active amino acid residues. Figure 10 The diagram shows the docking of DSAT with T1R1 / T1R3 and the interaction diagram of the active amino acid residues. Figure 11 The diagram shows the docking of DSTT with T1R1 / T1R3 and the interaction diagram of the active amino acid residues. Figure 12 The diagram shows the docking of SSAE with T1R1 / T1R3 and the interaction diagram of the active amino acid residues. Figure 13 Radar chart of electronic tongue taste profile analysis for the five selected umami peptides; Figure 14 A bar chart comparing the umami-enhancing effects of EGAT and GADA in a simulated chicken broth system; Figure 15 The molecular weight distribution of umami peptides obtained by stepwise enzymatic hydrolysis (Example 1) and by single enzymatic hydrolysis (Comparative Example 1) is shown in the figure. Detailed Implementation
[0022] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application. Unless otherwise specified, the methods used in this invention are conventional methods; the raw materials and apparatus used, unless otherwise specified, are conventional commercially available products.
[0023] Example 1: Preparation method of umami peptides derived from sea cucumber. This embodiment provides a method for preparing umami peptides derived from sea cucumber, including the following steps: S1. Raw material pretreatment: After rehydrating and washing the dried sea cucumber imitation body wall, it was homogenized using a tissue homogenizer. Then, it was defatted twice with n-hexane at a material-to-liquid ratio of 1:3, 2 hours each time. After evaporating the solvent, it was freeze-dried and pulverized through a 60-mesh sieve to obtain sea cucumber imitation protein powder.
[0024] S2. Alcalase enzyme first step of enzymatic hydrolysis: Weigh 100g of sea cucumber protein powder and add it to deionized water at a material-to-liquid ratio of 1:8, then stir until homogeneous. Adjust the pH to 8.5 with 1mol / L NaOH solution, add 2g of Alcalase enzyme (enzyme activity 2.4AU / g), and stir in a 55℃ water bath for 2.5h to obtain the first step enzymatic hydrolysate.
[0025] S3. Second step of enzymatic hydrolysis of flavor protease: Adjust the pH of the first-step enzymatic hydrolysate to 7.0 with 1 mol / L HCl solution, add 1.5 g of flavor protease (enzyme activity 500 LAPU / g), and continue to stir and enzymatically hydrolyze in a 50℃ water bath for 1.5 h to obtain the second-step enzymatic hydrolysate.
[0026] S4. Sterilization and centrifugation: The second-step enzymatic hydrolysate was sterilized in a 95°C water bath for 10 minutes to terminate the reaction. After cooling to room temperature, it was centrifuged at 4°C and 8000 rpm for 20 minutes, and the supernatant was collected.
[0027] S5. Ultrafiltration separation: The supernatant was placed in a 3kDa ultrafiltration cup pre-cooled at 4℃ and ultrafiltration was performed at 4℃ and 0.3MPa nitrogen pressure. All permeate was collected, concentrated, and freeze-dried to obtain a mixture of small molecule peptides.
[0028] S6. Gel chromatography separation and purification: The mixture of small molecule peptides was dissolved in ultrapure water (concentration 10 mg / mL), filtered through a 0.22 μm filter membrane, and then loaded onto a Sephadex G-15 column (1.6 cm × 60 cm). Ultrapure water was used as the mobile phase at a flow rate of 0.5 mL / min, and the elution peaks were detected at 220 nm. Different eluent fractions were collected according to their elution times, and after freeze-drying, the umami peptides were obtained and stored for later use.
[0029] Please see Figure 1 The image shows the elution curves of a mixture of small molecule peptides separated by Sephadex G-15 gel chromatography. Figure 1 It can be seen that after separation by Sephadex G-15 gel chromatography, three components G1, G2 and G3 were separated, among which G2 is the target collection peak with the strongest umami flavor.
[0030] Example 2: Identification method of umami peptides derived from sea cucumber (Stichopus japonicus) This embodiment provides a method for identifying umami peptides derived from sea cucumber, including the following steps: S1. Electronic tongue screening: The gel chromatography groups collected in Example 1 were prepared into 1 mg / mL aqueous solutions. The flavor profiles of the above components were analyzed using an electronic tongue system. The components with the highest umami intensity were screened out using 0.3% sodium glutamate solution as the umami reference.
[0031] Please see Figure 2 This is a radar image showing the electronic tongue flavor profiles of the three components separated by Sephadex G-15 gel chromatography. Figure 2 It can be seen that G2 performs better in terms of umami flavor, which is related to Figure 1 This aligns with the conclusion that "G2 is the target collection peak with the strongest umami flavor".
[0032] Please see Figure 3 The image shows the PCA analysis chromatogram of the three components separated by Sephadex G-15 gel chromatography using electronic tongue detection. The main component PC1 accounted for 74.7%, and PC2 accounted for 21.5%. Figure 3 It can be seen that the three components G1, G2 and G3 are clearly distinguishable in terms of taste characteristics. G2 has unique taste characteristics in the principal component space, which further confirms the specificity of its umami activity.
[0033] S2. Peptide sequence identification: Peptide sequences of component G2 were identified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Chromatographic conditions: C18 column; mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile, gradient elution. Mass spectrometry conditions: electrospray ionization source, positive ion mode. The mass spectrometry data were then searched using Proteome Discoverer software in the sea cucumber proteome database, ultimately identifying multiple peptide sequences.
[0034] Please see Figure 4 This was done to identify the total ion chromatogram of all peptides in the G2 fraction. Figure 4 It is known that the G2 component contains multiple peptide sequences, including Glu-Gly-Ala-Thr (EGAT), Gly-Ala-Asp-Ala (GADA), Asp-Ser-Ala-Thr (DSAT), Asp-Ser-Thr-Thr (DSTT), Ser-Ser-Ala-Glu (SSAE), Val-Thr-Gly-Glu (VTGE), Ala-Thr-Gly-Asp (ATGD), Val-Asp-Gly-Glu (VDGE), and Asp-Ser-Gly-Val-Leu (DSGVL).
[0035] S3. Virtual Filtering: The identified peptide sequences were imported into the iUmami-SCM online prediction platform for umami scoring, and peptides with scores greater than 580 were selected.
[0036] Please see Figure 5 The image shows the mass spectra of the five identified umami peptides, where A is the peptide sequence EGAT, B is the peptide sequence GADA, C is the peptide sequence DSAT, D is the peptide sequence DSTT, and E is the peptide sequence SSAE. Figure 5 The composition of five peptides, EGAT, GADA, DSAT, DSTT, and SSAE, was determined, and it was verified that G2 contains these five peptides.
[0037] S4. Molecular docking simulation: Sequences of human umami receptors T1R1 (Q7RTX1) and T1R3 (Q7RTX0) were obtained from the UniProt database, and homology modeling was performed using Swiss-Model. The virtually selected peptides were then molecularly docked with the receptor proteins using AutoDock Vina software. Monosodium glutamate (MSG) was used as a positive control (binding energy approximately -5.7 kcal / mol), and peptides with binding energies below -6.0 kcal / mol were screened.
[0038] Please see Figure 6 Figure 1 shows a homology model of the taste receptors T1R1 / T1R3-VFD, where Figure A is a 3D representation of T1R1 / T1R3 and Figure B is a 3D surface map of T1R1 / T1R3. Please refer to Figure 2. Figure 7 This is used to evaluate the Laplace plot for the T1R1 / T1R3 protein model. Figure 7 It can be seen that most of the black dots (representing active amino acid residues) are concentrated in the red allowed region, and a small portion are concentrated in the yellow edge region. This indicates that the conformation of most residues in the model is reasonable, that is, the T1R1 / T1R3 protein model is reliable.
[0039] Please see Figures 8-12 The diagrams show the docking of EGAT, GADA, DSAT, DSTT, SSAE with T1R1 / T1R3 and the interaction diagram of the active amino acid residues. Figures 8-12 It is known that the five peptides EGAT, GADA, DSAT, DSTT, and SSAE have relatively low binding energies to the receptors T1R1 / T1R3 and exhibit stable binding, all of which contribute to umami flavor. These five peptides primarily rely on hydrogen bonds and van der Waals forces to connect with the receptors T1R1 / T1R3, with hydrogen bonds and van der Waals forces playing a crucial role in driving umami flavor formation.
[0040] S5. Electronic tongue taste verification: The flavor profiles of the five peptides (synthesized by the company with a purity >95%) were analyzed using an electronic tongue system, and the amino acid sequences of the umami peptides were finally determined.
[0041] Please see Figure 13 The image shows a radar chart of the electronic tongue taste profiles of the five selected umami peptides. Figure 13 It can be seen that the five peptides EGAT, GADA, DSAT, DSTT, and SSAE all exhibit a significant umami response, with EGAT having the highest umami intensity.
[0042] Example 3: Application of umami peptides derived from sea cucumber (Stichopus japonicus) This embodiment provides an application of umami peptides derived from sea cucumber, including the following steps: S1. Add 0.02% of umami peptides EGAT and GADA to the base chicken broth (containing 1% NaCl and 0.05% MSG).
[0043] S2. The umami flavor is evaluated by the sensory evaluation team.
[0044] Please see Figure 14 The bar chart shows the comparison of the umami-enhancing effects of EGAT and GADA in a simulated chicken broth system. Figure 14 It is known that adding the umami peptide EGAT to chicken broth increased the umami intensity by approximately 40% compared to base chicken broth, resulting in a richer and fuller flavor. Adding the umami peptide GADA to chicken broth increased the umami intensity by approximately 35%, and the umami flavor lasted longer. This indicates that the umami peptides derived from sea cucumber biosynthetic, as described in this invention, have excellent umami-enhancing effects in complex food systems.
[0045] Comparative Example 1: Preparation of Umami Peptides by Single Enzymatic Hydrolysis The difference between this comparative example and Example 1 is that step S3 was omitted, and only Alcalase enzyme was used for single enzymatic hydrolysis for 3.5 hours, while other conditions remained unchanged, and finally, umami peptides were obtained by single enzymatic hydrolysis.
[0046] Sensory evaluation and molecular weight distribution of umami peptides obtained by single enzymatic hydrolysis were performed.
[0047] Please refer to Table 1 for a comparison of sensory evaluations of umami peptides obtained by stepwise enzymatic hydrolysis (Example 1) and umami peptides obtained by single enzymatic hydrolysis (Comparative Example 1).
[0048] Table 1. Comparison of sensory evaluation of umami peptides obtained by stepwise enzymatic hydrolysis and single enzymatic hydrolysis (n=10)
[0049] Note: Superscripts for different letters in the same line indicate significant differences (p<0.05). Scoring criteria: 1 point (very weak / none), 9 points (very strong).
[0050] As shown in Table 1, the umami peptides obtained by single enzymatic hydrolysis (Comparative Example 1) have a lower umami intensity than those obtained by stepwise enzymatic hydrolysis (Example 1), and also have a slight bitter and astringent taste.
[0051] Please see Figure 15 The graph shows the peptide molecular weight distribution of umami peptides obtained through stepwise enzymatic hydrolysis (Example 1) and single enzymatic hydrolysis (Comparative Example 1). Figure 15 It can be seen that the proportion of peptides larger than 1000 Da in the umami peptides obtained by single enzymatic hydrolysis (Comparative Example 1) is relatively high. This indicates that the second enzymatic hydrolysis of the flavor protease in Example 1 is crucial for generating umami peptides with smaller molecular weight and purer flavor. This further proves that stepwise enzymatic hydrolysis is superior to single enzymatic hydrolysis in terms of umami intensity and control of bitterness and astringency.
[0052] The present invention also provides the application of the umami peptides derived from sea cucumber prepared by the preparation method of Example 1 in the preparation of food seasonings, nutritional fortifiers or flavor enhancers.
[0053] In summary, this invention provides a umami peptide derived from sea cucumber, its preparation method, identification method, and applications, compared with existing technologies: (1) This invention provides a method for preparing umami peptides derived from sea cucumber. Using sea cucumber as raw material, a stepwise enzymatic hydrolysis method is employed, involving raw material pretreatment, first-step enzymatic hydrolysis with Alcalase enzyme, second-step enzymatic hydrolysis with flavor protease, sterilization and centrifugation, ultrafiltration, and gel chromatography purification to obtain umami peptides. This invention utilizes the synergistic effect of two proteases with different characteristics. First, Alcalase enzyme efficiently hydrolyzes large molecular proteins into polypeptide fragments, and then flavor protease further cleaves the peptide chains from the ends, releasing small molecule umami peptides and reducing bitterness, thereby directionally enriching the target product. The stepwise enzymatic hydrolysis preparation method of this invention has mild conditions and a clear process. Through the rational combination of enzymes, it effectively promotes the generation of small molecule umami peptides and helps improve the flavor profile. The preparation method is stable, controllable, and suitable for large-scale preparation.
[0054] (2) This invention provides an umami peptide derived from sea cucumber, the amino acid sequence of which is any one of Glu-Gly-Ala-Thr (EGAT), Gly-Ala-Asp-Ala (GADA), Asp-Ser-Ala-Thr (DSAT), Asp-Ser-Thr-Thr (DSTT), or Ser-Ser-Ala-Glu (SSAE). This invention is the first to identify five tetrapeptides with umami activity—EGAT, GADA, DSAT, DSTT, and SSAE—from sea cucumber protease hydrolysates. These peptides have small molecular weights, have been proven to have pure umami flavor, and have weak undesirable flavors such as bitterness and sourness, thus enriching the marine-derived umami peptide database.
[0055] (3) This invention provides a method for identifying umami peptides derived from sea cucumber. First, high-umami-active components are screened out using electronic tongue taste profile analysis. Then, peptide sequences are identified using LC-MS / MS. Subsequently, the iUmami-SCM virtual screening tool, molecular docking simulation with umami receptors T1R1 / T1R3, and electronic tongue taste verification are comprehensively used to screen layer by layer, finally determining the peptide sequences with clear umami characteristics. The identification method of this invention combines bioinformatics prediction with experimental verification, which can efficiently and accurately screen out target umami peptides from complex enzymatic hydrolysis products, improving the efficiency and accuracy of umami peptide discovery. The integrated identification strategy of "electronic tongue initial screening - peptide sequence identification - virtual screening - molecular docking simulation - electronic tongue taste verification" provides a reliable technical path for the discovery of target-oriented umami peptides.
[0056] (4) This invention provides an application of umami peptides derived from sea cucumber. Umami peptides have a pure umami flavor and a low umami threshold, and can be used to prepare food seasonings (such as chicken essence, soy sauce, compound seasoning sauce, soup base), nutritional fortifiers, or flavor enhancers. They can also be used as natural and healthy umami ingredients in various foods, providing new raw material options and technical support for the development of high-end marine seasonings. Umami peptides can be used directly or compounded with other umami substances (such as monosodium glutamate and flavor nucleotides) to produce a synergistic umami-enhancing effect.
[0057] It should be noted that: (1) In Example 1 above, in step S2, the pH is adjusted to 8.5 with 1 mol / L NaOH solution, the enzyme activity of Alcalase is 2.4 AU / g, the amount of Alcalase added is 2% of the mass of the sea cucumber protein powder, and the enzymatic hydrolysis is carried out in a 55℃ water bath for 2.5 h with stirring. This is only a preferred example. In actual production, the pH is adjusted to 8.5-9.5, the enzyme activity of Alcalase is ≥2.4 AU / g, the amount of Alcalase added is 1-3% of the mass of the sea cucumber protein powder, the enzymatic hydrolysis temperature is 50-60℃, and the time is 2-4 h. The specific values should be adjusted according to the actual situation.
[0058] (2) In Example 1 above, in step S3, the pH of the first-step enzymatic hydrolysate is adjusted to 7.0 with 1 mol / L HCl solution, the enzyme activity of the flavor protease is 500 LAPU / g, and the amount of flavor protease added is 1.5% of the mass of the sea cucumber protein powder. The enzymatic hydrolysis is continued for 1.5 h with stirring in a 50℃ water bath. This is only a preferred embodiment. In actual production, the pH of the first-step enzymatic hydrolysate is adjusted to 6-7, the enzyme activity of the flavor protease is ≥500 LAPU / g, the amount of flavor protease added is 1-3% of the mass of the sea cucumber protein powder, the enzymatic hydrolysis temperature is 45-55℃, and the time is 1-3 h. Adjustments should be made according to actual conditions.
[0059] (3) In the above Example 1, in step S4, sterilization is carried out in a water bath at 95°C for 10 minutes. This is only a preferred example. In actual production, the sterilization temperature is 90-100°C and the time is 5-20 minutes, which should be adjusted according to the actual situation.
[0060] (4) In the above Example 1, ultrafiltration is performed at 4°C in step S5. This is only a preferred embodiment. In actual production, ultrafiltration separation is performed at 4-10°C, and the specific temperature is adjusted according to the actual situation.
[0061] (5) In the above Example 1, in step S6, ultrapure water is used as the mobile phase, and the elution peak is detected at a wavelength of 220 nm. This is only a preferred embodiment. In actual production, the eluent is ultrapure water or a low-concentration salt solution, and the detection wavelength is 210-220 nm. The specific wavelength can be adjusted according to the actual situation.
[0062] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this application.
Claims
1. A umami peptide derived from sea cucumber, characterized in that, The amino acid sequence of the umami peptide is any one of Glu-Gly-Ala-Thr (EGAT), Gly-Ala-Asp-Ala (GADA), Asp-Ser-Ala-Thr (DSAT), Asp-Ser-Thr-Thr (DSTT) or Ser-Ser-Ala-Glu (SSAE).
2. A method for preparing umami peptides derived from sea cucumber as described in claim 1, characterized in that, Includes the following steps: S1. The imitation sea cucumber is washed, homogenized, degreased, dried, and pulverized to obtain imitation sea cucumber protein powder; S2. Dissolve the sea cucumber protein powder in water, adjust the pH, and obtain the first enzymatic hydrolysate through the first enzymatic hydrolysis step; S3. Adjust the pH of the first step enzymatic hydrolysate, and then proceed with the second enzymatic hydrolysate to obtain the second step enzymatic hydrolysate; S4. Inactivate the enzyme in the second step enzyme hydrolysate, centrifuge, and obtain the supernatant; S5. Use an ultrafiltration membrane to separate the supernatant by ultrafiltration, collect the permeate, and obtain a mixture of small molecule peptides; S6. The mixture of small molecule peptides was separated and purified using a gel chromatography column. The eluent was collected based on the ultraviolet absorption peak and then freeze-dried to obtain umami peptides.
3. The method for preparing umami peptides derived from sea cucumber as described in claim 2, characterized in that, In step S2, the sea cucumber protein powder is dissolved in water, the pH is adjusted to 8.5-9.5, and Alcalase enzyme is added for the first enzymatic hydrolysis. The enzyme activity of Alcalase is ≥2.4AU / g, and the amount of Alcalase added is 1-3% of the mass of the sea cucumber protein powder. The temperature of the first enzymatic hydrolysis is 50-60℃, and the time is 2-4h.
4. The method for preparing umami peptides derived from sea cucumber as described in claim 2, characterized in that, In step S3, the pH of the first step enzymatic hydrolysate is adjusted to 6-7, and flavor protease is added for the second enzymatic hydrolysis. The enzyme activity of the flavor protease is ≥500 LAPU / g, and the amount of flavor protease added is 1-3% of the mass of the sea cucumber protein powder. The temperature of the second enzymatic hydrolysis is 45-55℃, and the time is 1-3h.
5. The method for preparing umami peptides derived from sea cucumber as described in claim 2, characterized in that, In step S4, the sterilization temperature is 90-100℃ and the time is 5-20 minutes.
6. The method for preparing umami peptides derived from sea cucumber as described in claim 2, characterized in that, In step S5, the ultrafiltration membrane is an ultrafiltration membrane with a molecular cutoff of less than 3 kDa, and the ultrafiltration separation is carried out at 4-10℃.
7. The method for preparing umami peptides derived from sea cucumber as described in claim 2, characterized in that, In step S6, the gel chromatography column is a Sephadex G-15 column, the eluent is ultrapure water or a low-concentration salt solution, and the detection wavelength is 210-220 nm.
8. A method for identifying umami peptides derived from sea cucumber as described in claim 1, characterized in that, Includes the following steps: S1. Use an electronic tongue system to perform flavor profile analysis on the collected eluted components and screen out the components with the highest umami intensity; S2. The peptide sequence of the component with the highest umami intensity was identified by liquid chromatography-tandem mass spectrometry. S3. Use virtual screening tools to predict the umami potential of the identified peptide sequences; S4. Molecular docking simulation was performed between the peptide sequences predicted to have high umami potential and the umami receptors T1R1 / T1R3 to screen out peptides with binding energies lower than those of the control. S5. The umami characteristics of peptides with lower binding energies than the control were verified using an electronic tongue system, and the amino acid sequence of the umami peptides was finally determined.
9. The method for identifying umami peptides derived from sea cucumber as described in claim 8, characterized in that, In step S3, the virtual screening tool includes the iUmami-SCM online prediction platform; in step S4, the control standard includes monosodium glutamate.
10. The use of a umami peptide derived from sea cucumber as described in claim 1 in the preparation of food seasonings, nutritional fortifiers or flavor enhancers.