Umami peptide derived from euphausia superba and application thereof

Abstract

The application discloses umami peptides derived from Antarctic krill and application thereof, and belongs to the technical field of bioactive peptides. The umami peptides derived from Antarctic krill are five in number, and the amino acid sequences are shown as SEQ ID NO. 1-5. The application discloses the application of the umami peptides in being or preparing umami agents. The application takes Antarctic krill protein as raw material, carries out virtual hydrolysis, carries out joint screening by using umami peptide prediction model, and finally obtains five potential umami peptides which are non-toxic and have good water solubility by physical and chemical property prediction. Molecular interaction analysis shows that the umami peptides can form stable binding with T1R3 key sites, and the interaction mainly includes hydrogen bond and hydrophobic interaction and other non-covalent forces. The application constructs a kind of Antarctic krill umami peptide screening and mechanism analysis strategy based on virtual enzymolysis, multi-model prediction and molecular docking, and provides a theoretical basis for directional development of natural umami peptides and high-value utilization of Antarctic krill marine protein resources.

Description

Technical Field

[0001] This invention relates to umami peptides derived from Antarctic krill and their applications, belonging to the field of bioactive peptide technology. Background Technology

[0002] Umami, as one of the basic tastes, is a core component of food flavor. Natural umami enhancers, due to their safety and nutritional value, have become a research hotspot in the food industry. Umami peptides, as a class of small molecule peptides with flavor-enhancing activity, not only impart a rich umami flavor to food but also possess nutritional functions, making them more valuable than traditional umami enhancers (such as monosodium glutamate). However, the traditional screening of umami peptides relies on the conventional "enzymatic hydrolysis-separation-identification" route, which suffers from low screening efficiency, long experimental cycles, and high costs. Furthermore, the understanding of the flavor-enhancing mechanism is insufficient, severely hindering the development and industrialization of natural umami peptides.

[0003] Antarctic krill ( Euphausia superba Antarctic krill is the world's most abundant marine crustacean resource, with a protein content as high as 60%–70%, and a balanced amino acid composition, rich in umami-related amino acids (such as glutamic acid and aspartic acid). Upon hydrolysis, it readily produces short-chain bioactive peptides. Currently, the development of Antarctic krill resources is mainly focused on oil extraction and feed processing, with insufficient utilization of its protein resources for high-value applications. Research on developing natural umami peptides using Antarctic krill protein as a raw material has not yet formed a systematic framework. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides five umami peptides derived from Antarctic krill and their applications, belonging to the field of bioactive peptide technology.

[0005] This invention is achieved through the following technical solution: There are 5 umami peptides derived from Antarctic krill, with the following amino acid sequences: (1) The amino acid sequence is RHSRF, as shown in SEQ ID NO.1.

[0006] (2) The amino acid sequence is GAYR, as shown in SEQ ID NO.2.

[0007] (3) The amino acid sequence is DMGL, as shown in SEQ ID NO.3.

[0008] (4) The amino acid sequence is NRYHQF, as shown in SEQ ID NO.4.

[0009] (5) The amino acid sequence is SRAGL, as shown in SEQ ID NO.5.

[0010] The above-mentioned umami peptides derived from Antarctic krill are used as or in the preparation of umami agents.

[0011] This invention uses Antarctic krill protein (NCBI reference sequence: AUI80375.1) as raw material, and performs virtual hydrolysis using the BIOPEP-UWM platform. Subsequently, it employs two umami peptide prediction models, TastePeptidesDM and UMPred-FRL, for joint screening. Further physicochemical property prediction yielded five non-toxic, water-soluble potential umami peptides (DMGL, RHSRF, NRYHQF, SRAGL, and GAYR). To clarify the mechanism of action of these umami peptides, this invention uses AlphaFold to construct a 3D structural model of the human umami receptor T1R1 / T1R3 heterodimer, and uses the AutoDock Vina algorithm for molecular docking of the umami peptides and the umami receptor. Molecular interaction analysis shows that the umami peptides can form stable binding to key sites on T1R3, and their interactions mainly include non-covalent forces such as hydrogen bonds and hydrophobic interactions.

[0012] This invention constructs a strategy for screening and analyzing the mechanism of Antarctic krill umami peptides based on a combination of virtual enzymatic hydrolysis, multi-model prediction, and molecular docking, providing a theoretical basis for the targeted development of natural umami peptides and the high-value utilization of Antarctic krill marine protein resources.

[0013] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0014] Figure 1 Flavor radar chart of 5 synthetic peptides.

[0015] Figure 2 : Taste PCA diagram of 5 synthetic peptides.

[0016] Figure 3 Molecular docking results of peptide NRYHQF and T1R3.

[0017] Figure 4 Results of docking of peptide RHSRF with T1R3 molecules.

[0018] Figure 5 Results of docking of peptide SRAGL with T1R3 molecules.

[0019] Figure 6 Results of docking of peptide GAYR with T1R3 molecules.

[0020] Figure 7 Results of docking of peptide DMGL with T1R3 molecules.

[0021] Figure 8 Statistical analysis of the active amino acid binding sites of 5 synthetic peptides to the receptor T1R3.

[0022] Figure 9Statistics on the binding force types of 5 synthetic peptides to the receptor T1R3. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0024] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0025] Virtual screening, molecular docking, and identification of umami peptides derived from Antarctic krill. 1. Project Introduction Umami peptides are important flavor-enhancing active substances in food. They enhance umami perception by interacting with umami receptors and have significant application value in the development of natural seasonings and functional foods.

[0026] In recent years, the rapid development of bioinformatics and computer simulation technologies has propelled umami peptide research into a precise stage of "virtual screening + experimental verification." This technology, based on protein sequences, utilizes virtual enzymatic digestion, activity prediction, and molecular docking to rapidly screen high-potential candidate peptides from a vast pool of peptides, significantly reducing experimental workload. Simultaneously, combined with molecular interaction analysis, it can elucidate the binding mechanism of umami peptides to taste receptors at the atomic level, providing theoretical support for peptide structure optimization. Compared to traditional methods, this strategy offers advantages such as high efficiency, low cost, and simultaneous mechanism research, and has become a novel pathway for discovering naturally bioactive peptides.

[0027] This project aims to integrate virtual screening, molecular docking, and experimental verification technologies to establish an integrated technical system encompassing "virtual enzymatic digestion - multi-index screening - receptor binding simulation - in vitro verification," enabling the efficient screening and identification of highly active umami peptides from Antarctic krill proteins. By elucidating the binding mode and key action sites of umami peptides to the T1R3 umami receptor, the project elucidates their structure-activity relationship, providing technical support for the targeted development of natural umami agents derived from marine organisms, promoting the high-value utilization of Antarctic krill protein resources, and expanding the development pathways for marine functional foods.

[0028] 2 Materials and Methods 2.1 Materials and Reagents The main materials and reagents used were: monosodium glutamate, potassium chloride, ethanol, hydrochloric acid, and tartaric acid; all of which were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0029] 2.2 Instruments and Equipment The main instruments and equipment used were: PL202-S electronic balance, purchased from Metteler Toledo; BC / BD-218SHT refrigerator, purchased from Qingdao Haier Co., Ltd.; and SA402B electronic tongue, purchased from Insent Co., Ltd., Japan.

[0030] 2.3 Experimental Methods 2.3.1 Virtual enzymatic hydrolysis of Antarctic krill-derived proteins and prediction of their polypeptide properties The amino acid sequences of Antarctic krill-derived proteins were retrieved from the UniProt database (https: / / www.uniprot.org / ). Myosin Timeless Protein 2 (NCBI reference sequence: AUI80375.1), with a length of 1364 amino acids, was selected as the virtual enzymatic digestion target. Virtual enzymatic digestion simulation of the template protein was performed using the BIOPEP-UWM database. Three common proteases recorded in the database were used to perform combined enzymatic digestion of Timeless Protein 2 to obtain theoretical peptide profiles. Subsequently, a Python script was used to extract peptides of 3–8 amino acids in length from the peptide profiles for subsequent screening and analysis. The peptides obtained from the virtual enzymatic digestion were screened using the TastePeptidesDM and UMPred-FRL umami prediction models. Peptides predicted to have umami characteristics by both models were identified as potential umami peptides and used in the next step of analysis. The bioactivity of potential umami peptides was scored using the PeptideRanker online tool, toxicity prediction of candidate peptides was performed using ToxinPred, and water solubility was evaluated using the Innovagen peptide property prediction platform. Peptides that were non-toxic and well-water-soluble were retained. Peptides meeting the above requirements were synthesized using the Fmoc solid-phase method developed by Shanghai Sango Biotechnology Co., Ltd., China.

[0031] 2.3.2 Verification of the taste characteristics of Antarctic krill umami peptides The taste characteristics of the synthetic peptides were determined using an SA-402B taste analysis system (Insent SA402B, Tokyo, Japan). Peptide solutions were prepared at a concentration of 0.3 mg / mL in a tasteless solution (30 mM KCl solution containing 0.3 mM tartaric acid). MSG solutions of the same concentration were used as controls. Each sample was measured four times, with the first measurement automatically discarded.

[0032] 2.3.3 Study on the umami-enhancing mechanism of Antarctic krill umami peptides 2.3.3.1 Construction of a 3D Model of Umami Receptors Since there are no available crystal structures for the human umami receptors T1R1 / T1R3, their three-dimensional structures were constructed using computer modeling. The amino acid sequences of T1R1 (UniProt ID: Q7RTX1) and T1R3 (UniProt ID: Q7RTX0) were obtained from the UniProt database. AlphaFold 2.3.0 was used to predict the structure of the T1R3 heterodimer complex, and the conformational rationality of the model was evaluated using Ramachandran plot analysis on the SAVES v6.0 server. The validated model was used for subsequent molecular docking studies.

[0033] 2.3.3.2 Molecular docking Two-dimensional structures of candidate peptides were drawn using KingDraw, and MM2 force field optimization was performed using KingDraw. The optimized structures were saved in mol2 format. The constructed receptor model was dehydrated and hydrogenated using PyMOL. Subsequently, the AutoDock Vina algorithm in the PyRx platform was used to simulate molecular docking between the receptor and peptide ligand. Eight docking calculations were performed for each group, and the conformation with the lowest Vina score was selected as the optimal binding mode. Finally, Discovery Studio 4.5 was used to analyze the interaction type and key binding sites between the peptide ligand and receptor.

[0034] 3 Results and Discussion 3.1 Virtual enzymatic hydrolysis of Antarctic krill-derived proteins and prediction of their polypeptide properties Based on the calculation of the relevant properties of the obtained peptides, the five peptides with the highest Ranker scores were selected for synthesis verification, and their information is detailed in Table 1.

[0035] Table 1. Ranker scores and related properties of each synthetic peptide.

[0036] 3.2 Results of Taste Test of Antarctic Krill Umami Peptides Flavor radar charts of 5 synthetic peptides are shown below. Figure 1 As shown, the results of the determination of the flavor profile of synthetic umami peptides are presented. The results indicate that three synthetic peptides (RHSRF, DMGL, GAYR) exhibited umami flavor, and all five synthetic peptides exhibited a certain degree of saltiness. In addition, the synthetic peptides also possessed certain sour and bitter tastes. The sourness of the synthetic peptides may be related to the presence of acidic amino acid residues (Asp, Glu) and residual chemical reagents (such as sodium acetate) during the synthesis process. Furthermore, the synthetic peptides also had a slightly bitter taste, which may be due to the presence of hydrophobic amino acids such as alanine (Ala) and glycine (Gly) in the peptides.

[0037] Principal component analysis (PCA) was used to obtain the taste characteristic distribution of each synthetic peptide in the electronic tongue test. The taste PCA diagrams of the five synthetic peptides are shown below. Figure 2 As shown in the figure, PCA results indicate that PC1 (80.2%) and PC2 (10.45%) together explain most of the taste differences. Different peptides showed significant separation along the PC1 axis, indicating substantial differences in their overall taste characteristics. Peptides such as DMGL and RHSRF exhibited some aggregation along the PC1 axis, suggesting that their characteristics are relatively similar across multiple taste dimensions, presenting a complex taste profile, especially the balance between umami and saltiness. GAYR, SRAGL, and NRYHQF, on the other hand, showed strong separation along the PC2 axis, revealing significant differences in sensory dimensions such as sourness and bitterness.

[0038] 3.3 Analysis of the umami peptide mechanism in Antarctic krill Molecular docking results showed that the Vina fractions of the five synthetic peptides were: DMGL (-7.2), RHSRF (-7.5), NRYHQF (-9.4), SRAGL (-7.2), and GAYR (-8.1). These results indicate that all five synthetic peptides can bind to T1R3 and exhibit good Vina fractions, suggesting that they possess potential for strong umami activity.

[0039] 3.3.1 Molecular docking analysis of peptide WIMRFF and T1R3 The molecular docking results of peptide NRYHQF and T1R3 are as follows: Figure 3 As shown. From Figure 3 The 3D interaction diagram shows that the peptide NRYHQF is completely embedded in the active pocket of the T1R3 receptor with its side chain structure, forming a tight binding. In the 2D interaction diagram, several key interactions were observed: amino acid residues such as 66SER, 45GLU, 68ASN, 302ALA, 387HIS, 278HIS, 147SER, 146SER, 148GLU, and 168GLY form conventional hydrogen bond interactions with the peptide; 277VAL forms a hydrophobic interaction with the peptide; and simultaneously, 301GLU forms a π-anion interaction with the benzene ring structure of the peptide.

[0040] 3.3.2 Analysis of peptide RHSRF-T1R3 molecular docking The docking results of peptide RHSRF with T1R3 molecules are as follows: Figure 4 As shown. From Figure 4The 3D interaction diagram shows that the peptide RHSRF is completely embedded in the active pocket of the T1R3 receptor, forming a tight binding conformation. In the 2D interaction diagram, several key interactions were observed: amino acid residues such as 216ASP, 148GLU, 68ASN, 302ALA, 44GLY, 42PRO, 104SER, 147SER, 145HIS, 170SER, and 66SER form conventional hydrogen bond interactions with the peptide; 277VAL forms a hydrophobic interaction with the peptide; 389GLN and 388HIS form polar interactions with the peptide; and simultaneously, 301GLU and 45GLU form π-anion interactions with the aromatic ring structure of the peptide.

[0041] 3.3.3 Molecular docking analysis of peptide SRAGL and T1R3 The docking results of peptide SRAGL with T1R3 molecules are as follows: Figure 5 As shown. From Figure 5 The 3D interaction diagram shows that the peptide SRAGL inserts its terminal structure into the active pocket entry region of the T1R3 receptor, forming a relatively loose binding conformation. In the 2D interaction diagram, it is observed that amino acid residues 147SER, 148GLU, 145HIS, 146SER, and 170SER form conventional hydrogen bond interactions with the peptide; 277VAL forms a hydrophobic interaction with the peptide; and 278HIS forms a polar interaction with the peptide.

[0042] 3.3.4 Molecular docking analysis of peptide GAYR and T1R3 The docking results of peptide GAYR and T1R3 molecules are as follows: Figure 6 As shown. From Figure 6 The 3D interaction diagram shows that the peptide GAYR attaches to the surface region of the T1R3 receptor with its side chain structure, and part of its structure is embedded in the shallow groove of the receptor. In the 2D interaction diagram, several key interactions were observed: amino acid residues such as 145HIS, 146SER, 302ALA, 170SER, 389GLN, and 388HIS form conventional hydrogen bond interactions with the peptide; 218TYR forms a π-π stacking interaction with the aromatic ring of the peptide; and simultaneously, 148GLU forms a π-anion interaction with the peptide.

[0043] 3.3.5 Molecular docking analysis of peptide DMGL and T1R3 The docking results of peptide DMGL and T1R3 molecules are as follows: Figure 7 As shown. From Figure 7The 3D interaction diagram shows that peptide DMGL inserts its C-terminus into a shallow groove on the surface of the T1R3 receptor, forming a curved binding conformation. In the 2D interaction diagram, 387HIS and 145HIS are observed to form conventional hydrogen bonds with the peptide; 277VAL, 302ALA, and 168GLY form hydrophobic interactions with the peptide; and 193GLN forms a polar interaction with the peptide.

[0044] 3.3.6 Analysis of peptide binding sites and binding interactions with T1R3 The binding sites of the five synthetic peptides to the receptor T1R3 are statistically analyzed as follows: Figure 8 As shown. By Figure 8 It is known that the binding sites of T1R3 are mainly distributed among multiple amino acid residues, such as HIS145, GLU148, and ALA302, with HIS145 being the most important amino acid residue. These amino acid residues play an important role in the study of ligand docking with T1R3 molecules.

[0045] Statistics on the binding forces of 5 synthetic peptides to receptor T1R3 are as follows: Figure 9 As shown. From Figure 9 As can be seen, the main binding forces include conventional hydrogen bonds, π-alkyl interactions, π-hydrophobic interactions, and alkyl interactions, with conventional hydrogen bonds being the most prevalent type of interaction. The figure also illustrates different binding mechanisms, such as π-hydrophobic interactions, which, as a relatively rare but highly selective binding mode, play a unique role in the binding of peptides to T1R3, significantly enhancing the stability of the binding.

[0046] 4. Conclusion In this study, using Antarctic krill protein resources as the experimental subject, a research strategy combining virtual screening, experimental verification, and molecular docking was constructed to explore potential umami peptides and analyze their mechanisms of action. Through virtual enzymatic hydrolysis, multi-model activity prediction, and safety screening, several small molecule peptides with umami potential were obtained from a large number of candidate peptides, and representative peptides were further screened for subsequent research. Electronic tongue and principal component analysis results showed that different peptides exhibited significant differences in umami, saltiness, and overall flavor balance, with some peptides showing better umami characteristics and flavor harmony.

[0047] Molecular docking results showed that the screened bioactive peptides could form stable binding sites with the umami receptor T1R3. These binding sites were mainly concentrated on key amino acid residues such as HIS145, GLU148, and ALA302. The binding forces were primarily traditional hydrogen bonds and hydrophobic interactions, supplemented by π-π stacking and π-anion interactions, which collectively enhanced the stability of the peptide binding to the receptor. These results reveal the possible mechanism by which umami peptides exert their flavor-enhancing effects at the molecular level, providing a theoretical basis for the structural optimization and functional design of umami peptides.

[0048] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A flavor peptide derived from Antarctic krill, characterized in that: The amino acid sequence is RHSRF, as shown in SEQ ID NO.

1.

2. The use of the umami peptide derived from Antarctic krill as described in claim 1 in the preparation of umami enhancers.

3. A flavor peptide derived from Antarctic krill, characterized in that: The amino acid sequence is GAYR, as shown in SEQ ID NO.

2.

4. The use of the umami peptide derived from Antarctic krill as described in claim 3 in the preparation of umami enhancers.

5. A flavor peptide derived from Antarctic krill, characterized in that: The amino acid sequence is DMGL, as shown in SEQ ID NO.

3.

6. The use of the umami peptide derived from Antarctic krill as described in claim 5 in the preparation of umami enhancers.

7. A flavor peptide derived from Antarctic krill, characterized in that: The amino acid sequence is NRYHQF, as shown in SEQ ID NO.

4.

8. The use of the umami peptide derived from Antarctic krill as described in claim 7 in the preparation of umami enhancers.

9. A flavor peptide derived from Antarctic krill, characterized in that: The amino acid sequence is SRAGL, as shown in SEQ ID NO.

5.

10. The use of the umami peptide derived from Antarctic krill as described in claim 9 in the preparation of umami enhancers.

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