Method for modifying umami peptides

Abstract

The application discloses a method for modifying umami peptides, and belongs to the technical field of food. The method is used for modifying and modifying the existing beef octapeptide terminal in order to meet the demand of recombinant expression long peptide multiple copies, and the method is used for modifying umami peptides according to the umami taste rule and improving the umami of single copy peptide. The method comprises the following steps: using homologous modeling and molecular dynamics to construct the structure of umami receptor T1R1 / T1R3; creating a modified peptide sequence, and performing molecular simulation on the sequence to dock with the receptor, and comparing the scores of new umami peptides. Then, the eight copy form long chain peptide of some high score peptides is simulated to be digested. The effectiveness of the introduced enzyme cutting site is verified, and the molecular docking result meets the expectation. The modification of the umami peptide is completed, and a new umami peptide is obtained.

Description

Technical Field

[0001] This invention relates to a method for modifying umami peptides, belonging to the field of food technology. Specifically, it relates to a method for obtaining umami peptides with better flavor by introducing trypsin cleavage sites at specific positions and then modifying the amino acid sequence and types of peptides according to the rules of umami peptide formation. Background Technology

[0002] Sweet, bitter, salty, sour, and umami are recognized as the five basic tastes of humans. Umami was first discovered by Professor Ikeda in 1912 and mainly originates from monosodium glutamate (MSG). Umami peptides are another class of small molecule peptides with their own flavor, giving food a subtle and delicate taste. They not only suppress certain unpleasant tastes but also contain some essential amino acids, increasing the nutritional value of food. Since the discovery of umami peptides, researchers have devoted considerable effort to finding new umami peptides by hydrolyzing proteins from different food sources using proteases. Japanese scholar Yamazaki isolated, extracted, and purified a delicious peptide, beef peptide (BMP), from the hydrolysis of papain in beef. Its amino acid sequence is Lys-Gly-Asp-Glu-Glu-Ser-Leu-alanine 3. The threshold of BMP is 1.14 mmol / L, slightly lower than the recognized umami peptide MSG (1.56 mmol / L).

[0003] Given that BMP has the potential to replace MSG as a novel umami enhancer, researchers have explored methods such as enzymatic hydrolysis, chemical synthesis, and recombinant expression fermentation for large-scale production. Among these, large-scale expression of the BMP gene in engineered microbial cells through genetic engineering recombination technology is considered an effective approach for industrial-scale BMP production. Simultaneously, researchers have attempted to express BMP in engineered bacteria through genetic engineering to obtain large quantities of this peptide. For example, multiple copies of BMP in Bacillus subtilis were successfully expressed using a target gene tandem strategy. A food-safe Bacillus subtilis expression system was obtained, and a high-density fermentation process for BMP was explored, ultimately increasing BMP yield. However, a drawback of these methods is the difficulty in recombinant expression of short-chain peptides. Therefore, researchers have had to increase the copy number of the peptide chain to reduce the difficulty of recombinant expression. The optimal molecular weight range for umami peptides is considered to be 1000-5000 U, with optimal umami flavor in 3-9 amino acids. Therefore, although long-chain peptides expressed through genetic engineering have high yield and purity, their umami value is far lower than that of single-copy peptides. These issues limit the application of recombinantly expressed umami peptides. Summary of the Invention

[0004] To effectively address the problems encountered in the aforementioned microbial recombinant expression fermentation methods, a trypsin-specific cleavage site was introduced at the end of BMP, enabling it to be hydrolyzed by trypsin after recombinant expression, yielding a large number of single-replicated peptides. First, the amino acid sequence of BMP was modified by introducing trypsin-specific cleavage amino acids at the peptide ends. Subsequently, molecular docking of the modified BMP was performed using the umami receptor T1R1 / T1R3 homology model. After identifying and synthesizing the modified BMPs, their umami characteristics will be studied through taste evaluation and electronic tongue analysis. This work provides guidance for the industrial production of novel umami peptides and the future modification of other functional peptides.

[0005] This invention relates to a method for modifying existing umami peptides, specifically a method for obtaining umami peptides with better flavor by introducing trypsin cleavage sites at specific positions and then modifying the amino acid sequence and types of peptides according to the rules of umami peptide formation.

[0006] This invention provides a method for modifying umami peptides, the method comprising:

[0007] (1) Move the trypsin cleavage site Lys of BMP to the C-terminus to obtain a polypeptide with the amino acid sequence SLADEEGK;

[0008] (2) Change the connection order of the negatively charged acidic group, hydrophobic group and positively charged basic group in the umami peptide BMP in step (1) to obtain a series of polypeptides.

[0009] (3) In step (2), the amino acids with negatively charged acidic groups of the polypeptide are replaced by Asp and Glu, and the amino acids with hydrophobic groups are replaced by hydrophobic amino acids to obtain a novel umami peptide.

[0010] (4) Establish a homology model of taste receptors T1R1 / T1R3;

[0011] (5) The novel umami peptide from step (3) is molecularly docked with the homology model from step (4) to screen and obtain the target peptide.

[0012] In one embodiment, in step (2), the negatively charged acidic group is -Asp-Glu-Glu-, the hydrophobic group is -Ser-Leu-Ala-, and the positively charged basic group is Lys-Gly-.

[0013] In one embodiment, in step (2), the connection sequence is, in order, a negatively charged acidic group - a hydrophobic group - a positively charged basic group, or a hydrophobic group - a negatively charged acidic group - a positively charged basic group.

[0014] In one embodiment, in step (3), the hydrophobic amino acid is Trp, Phe, Val, Ser, Leu, or Ala.

[0015] In one implementation, in step (4), the login number of T1R1 is NP_619642.2 and the login number of T1R3 is NP_689414.2.

[0016] In one implementation, in step (4), a homologous model of T1R1 / T1R3 is established through the DS / Modeler protocol.

[0017] In one implementation, in step (5), molecular docking is performed using the DS / CDOCKER program, and the target peptide is screened using the I-score value as an indicator.

[0018] In one implementation, the I-score value is greater than -126.83 kcal / mol.

[0019] This invention provides umami peptides obtained by modifying according to the above method.

[0020] The present invention also provides a method for producing the above-mentioned umami peptides, the method being as follows:

[0021] (1) The above-mentioned umami peptides were prepared by chemical synthesis or biosynthesis.

[0022] (2) Use trypsin to hydrolyze the umami peptides in step (1).

[0023] In one embodiment, the biosynthesis method involves producing umami peptides through fermentation using microbial cells containing umami peptides.

[0024] In one embodiment, the microbial cells include, but are not limited to, Bacillus subtilis, Saccharomyces cerevisiae, and Escherichia coli.

[0025] The present invention provides a flavor enhancer comprising the flavor peptide.

[0026] The present invention also provides the application of the method for modifying the umami peptide, or the method for producing the umami peptide, in the food field.

[0027] The present invention also provides the application of the method for modifying the umami peptide or the method for producing the umami peptide in the preparation of seasonings, food additives or fermented foods.

[0028] In one embodiment, the seasonings include soy sauce, light soy sauce, shrimp oil, dark soy sauce, oyster sauce, chicken bouillon, sugar, salt, monosodium glutamate, fermented black beans, fermented bean curd, and concentrated soup cubes.

[0029] Beneficial effects:

[0030] (1) This invention modifies umami beef peptides (BMPs) with trypsin hydrolysis sites, creating a total of 1286 modified peptide sequences. Molecular simulation and homology modeling were used to construct umami receptors (T1R1 / T1R3). Among them, the I-score of 837 structurally modified BMPs exceeded that of BMPs, indicating that the structurally modified BMPs provided by this invention can dock better than beef peptides.

[0031] (2) Increasing the copy number of peptides is an effective method for recombinant expression in genetic engineering and for obtaining large quantities of umami peptides. However, the umami value of multi-copy umami peptides produced is lower than that of single-copy umami peptides, thus limiting the industrial application of recombinantly expressed umami peptides. This invention meets the needs of recombinant expression of multi-copy long peptides, improves the umami of single-copy peptides, and provides a method for obtaining umami peptides with better flavor performance. Attached Figure Description

[0032] Figure 1 Sequence alignment of T1R1 and metabotropic glutamate receptors;

[0033] Figure 2 Sequence alignment of T1R3 and metabotropic glutamate receptors;

[0034] Figure 3 The final homology model of taste receptors T1R1 / T1R3;

[0035] Figure 4 : Ramachandran diagram of homology modeling.

[0036] Figure 5 Electronic tongue is used to evaluate umami flavor. Detailed Implementation

[0037] The invention will be further illustrated below through specific implementation examples.

[0038] Molecular docking is the process by which two or more molecules recognize each other through geometric and energy matching, with the aim of finding the optimal binding mode between ligand molecules and acceptor molecules.

[0039] Discovery Studio Client v17.2.0.16349 software: Discovery Studio TM (DS for short) is a new generation of molecular modeling and simulation environment based on Windows / Linux systems and personal computers, targeting the life sciences field.

[0040] DS / Modeler: Given an unknown structural sequence and another protein with a known three-dimensional structure and a certain degree of homology, this industrial-standard rapid homology modeling method can automatically and quickly predict the protein structure. Predictable protein structure types include enzymes, antibodies, and transmembrane proteins.

[0041] DS 4.0 / CDOCKER is a flexible docking program based on CHARMm, employing soft-core potentials and optional grid representation to dock ligand molecules with receptor active sites. First, a kinetic method is used to randomly search for small molecule conformations, followed by simulated annealing to optimize each conformation within the receptor active site region, thus resulting in more accurate docking results.

[0042] Example 1: Modification of BMP Sequence

[0043] A method for modifying umami peptides, comprising the following steps:

[0044] S1, BMP sequence modification: The trypsin cleavage site Lys of the BMP amino acid sequence (KGDEESLA) was moved to the C-terminus to obtain a polypeptide with the amino acid sequence SLADEEGK. Subsequently, the order of the negatively charged acidic group (-Asp-Glu-Glu-) and the hydrophobic group (-Ser-Leu-Ala-) was changed so that the final sequence order was (1) negatively charged acidic group-hydrophobic group-positively charged basic group; (2) hydrophobic group-negatively charged acidic group-positively charged basic group. In the next step, the amino acids in these groups were modified. The amino acids with negatively charged groups were replaced by Asp and Glu, and the amino acids with hydrophobic groups were replaced by Trp, Phe, Val, Ser, Leu, and Ala, resulting in a series of polypeptides (Table 1).

[0045] Homology modeling of S2 and T1R1 / T1R3: The heterodimers of the taste receptors T1R1 / T1R3 were modeled using DS (DiscoveryStudio Client v17.2.0.16349). The amino acid sequences of T1R1 (accession number: NP_619642.2) and T1R3 (accession number: NP_689414.2) were obtained online at https: / / www.ncbi.nlm.nih.gov / . Before constructing the receptor model, the amino acid sequences of various receptor templates were compared with the amino acid sequences of T1R1 / T1R3 using ESPript 3.0. Based on the 3D structure and sequence blast results of the metabolite glutamate receptor, a homology model of T1R1 / T1R3 was established using the DS / Modeler protocol.

[0046] S3. Molecular docking of novel umami peptides with T1R1 / T1R3: Dock ligands were used to generate pockets from the 3D structures of the T1R1 / T1R3 receptors. All water molecules and foreign components were removed. Hydrogen atoms were added to the CHARMM force field. Virtual screening was performed using modified BMPs with DS 4.0 / CDOCKER based on the T1R1 receptor and T1R3 structures. CDOCKER is a semi-flexible docking method based on energy minimization for molecular docking. Minisinate and CHARMM force fields were used to minimize the energy of the peptides. Preferred conformations were analyzed to determine the binding modes with the receptors, and potential binding modes were identified through comparative analysis. Pockets were selected based on the volume of the receptor cavity to ensure that the modified BMPs could smoothly enter the active pocket and achieve substantial interactions with the umami receptors. Molecular docking was evaluated based on docking energies, and the optimal docking conformation was retained to display a two-dimensional diagram. The docking interaction energy score of CDOCKER was used to evaluate the composite conformation of the receptor and ligand to obtain the optimal conformation for the ligand binding site.

[0047] Table 1. Energy of docking between modified umami peptide segments and T1R1 / T1R3 molecules.

[0048]

[0049]

[0050] Currently, homology modeling and computational molecular docking are novel methods for determining potential binding sites of umami peptides to taste receptors and elucidating the taste mechanism of umami peptides. In this invention, ENDscript 3.0 was first used to find suitable receptor construction templates. After comparison and scoring, the metabotropic glutamate receptor (PDB ID: 1EWK) was selected as the receptor construction template. The metabotropic glutamate receptor is a protein that has been previously used as a template. The sequence similarity between T1R1 / T1R3 and the metabotropic glutamate receptor is as follows: Figure 1 and Figure 2 As shown in the figure. The results indicate that their sequences highly overlap, suggesting that the metabolite glutamate receptor can serve as a template for the T1R1 / T1R3 receptor construction. This model is similar to the model obtained by Dang et al. using the protein prediction software DS17.

[0051] This invention uses the DS homology modeling program to construct the T1R1 / T1R3 heterodimer and optimizes the model using DS software. The final homology model of the taste receptors T1R1 / T1R3 is as follows: Figure 3 As shown. Figure 3 The subunit on the left is the T1R1 portion of the umami receptor, while the subunit on the right is the T1R3 portion. The umami receptor T1R1 / T1R3 is a heterodimer in the first class of taste receptors in the G protein-coupled receptor family. Figure 3 The receptor-binding region of its T1R1 / T1R3 heterodimer and polypeptide exhibits a "Venus flytrap" structure, consisting of a heterodimer linked by cysteine-rich regions. The two segments are hinged together, forming a cavity. This cavity is the primary region where the ligand interacts with the taste receptor. The taste receptor consists of a complex of 1042 amino acids in its extracellular region, containing both T1R1 and T1R3.

[0052] To assess the reliability of the model, a Ramachandran diagram was implemented. For example... Figure 4 As shown, the calculated Ramachandran results indicate that nearly 96.8% of the residues are located in the permitted region (92% of the residues are located in the most favorable region, and 4.8% of the residues are located in the permitted region), while only 3.2% of the residues are located in the disallowed region. Based on the 90% critical evaluation principle, the model's conformation is reasonable.

[0053] Table 1 lists several peptides selected based on extensive docking results, corresponding to each fraction (80, 90, ..., 150 kcal / mol) from the highest to the lowest I-score peptides. Molecular docking involves the process by which two or more molecules recognize each other through assembly and energy matching to achieve an optimal binding mode between molecules. The stimulation of the T1R1 / T1R3 receptor by the umami peptide is due to insertion into the active site of T1R3, ultimately leading to an increase in T1R1 / T1R3 receptor activity. The strong binding of this peptide to T1R1 / T1R3 indicates that the peptide has a good ability to increase T1R1 / T1R3 receptor activity and enhance umami. A high I-score value indicates strong binding force and good affinity. The I-score value represents the overall score of the docking interaction energy between the umami peptide and the T1R1 / T1R3 receptor. The I-score of BMP is 126.83 kcal / mol. This invention created 1286 modified peptide sequences and performed molecular simulations and homology modeling to construct umami receptors (T1R1 / T1R3). Among these, 837 structurally modified BMPs had I-scores exceeding those of BMP, indicating that the structurally modified BMPs provided in this invention dock better than beef peptides. This finding also confirms the effectiveness of our modification strategy; referencing the flavor generation mechanism, the flavor presentation of the modified umami peptides is improved.

[0054] Example 2: Simulated hydrolysis of umami peptides

[0055] Simulated hydrolysis of novel umami peptides was performed using ExPASy PeptideCutter (http: / / web.expasy.org / peptide_cutter). Eight-copy BMP proteolysis was performed using trypsin. The cleavage site map was set to 60 to ensure sufficient enzymatic activity for each peptide. The results are shown in Table 2.

[0056] Table 2. Simulated enzymatic hydrolysis results of 8 copies of 5 different novel umami peptides

[0057]

[0058] Example 3 Umami Evaluation

[0059] Among 1286 modified BMPs, the peptide with the highest I score (MLSEDEGK) and the polypeptide with a medium I score (DEDSWLGK) were selected for chemical synthesis. BMPs were also chemically synthesized for flavor identification. Ultrapure water was added to prepare a 0.1 mg / mL solution of the synthesized peptides. The umami control group used the same concentration of MSG. Detection was performed using an electronic tongue and sensory evaluation; the test results are as follows: Figure 5 As shown, the results of the electronic tongue show that the umami of BMP is very close to that of MSG. Moreover, apart from MSG having a higher umami value and poor performance in other tastes, BMP's other performances are also excellent. Its bitterness is the lowest among the four samples, and it is far ahead of MSG in terms of body flavor.

[0060] The two novel umami peptides synthesized based on BMP modification inherit the advantages of BMP. Observation of the taste profile diagram of the electronic tongue shows that the umami of the two modified novel umami peptides is very similar to that of MSG, and the sour, bitter and astringent tastes are much less than those of MSG, while the body and salty tastes are much stronger.

[0061] Furthermore, the umami value of MLSEDEGK, which scored higher, was significantly higher than that of DEDSWLGK, which scored lower. This verifies that the docking results of Example 1 are consistent with and credible in real-world experiments.

[0062] Sensory evaluation results showed that BMP performed significantly better in all flavors compared to MSG. The peptides were more palatable and had a richer flavor profile than MSG alone. This suggests that BMP, as a novel umami peptide, overcomes the monotonous flavor and discomfort associated with traditional MSG. This is because peptides themselves possess nutritional value, making them more appealing to human senses.

[0063] Example 4: Preparation of Umami Peptides

[0064] The novel umami peptide from Example 2 was constructed into the pMA5 plasmid and transferred into Bacillus subtilis. The plasmid was then spread on LB medium, and single clones were picked and inoculated into LB medium. Fermentation was carried out at 37°C and 200 rpm to obtain a fermentation broth containing the novel umami peptide.

[0065] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A umami peptide, characterized in that, The amino acid sequence of the umami peptide is MLSEDEGK or DEDSWLGK.

2. A flavor enhancer, characterized in that, The umami agent comprises the umami peptide of claim 1.

3. The application of the umami peptide of claim 1 or the umami agent of claim 2 in the food industry.

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