Novel kokumi polypeptide as well as screening preparation method and application thereof

Abstract

The invention discloses a novel kokumi polypeptide as well as a screening preparation method and application thereof, belongs to the technical field of food science and biology, and discloses a polypeptide with an amino acid sequence of Arg-Ser-Gln-Pro-Val-Gly-Val, the polypeptide is identified by high-throughput screening and molecular docking technologies, has a remarkable flavor enhancing effect, and can be used for preparing a food flavor enhancer. And particularly, the salty taste, the delicate taste and the kokumi attribute are enhanced. The preparation method of the polypeptide is simple and low in cost, and can be widely applied to development of low-salt, low-sodium and low-sugar foods.

Description

Technical Field

[0001] The present invention belongs to the fields of food science and biotechnology, and particularly relates to a novel kokumi polypeptide, a screening and preparation method thereof, and an application thereof. Background Art

[0002] Kokumi polypeptides are a class of bioactive substances that can enhance the richness, smoothness, and fullness of taste in foods. These polypeptides generally do not exhibit obvious taste characteristics in pure water, but when added to solutions containing basic taste substances (such as saltiness and umami), they can significantly enhance the original taste intensity and improve the taste. The concept of kokumi originally originated in Japan and has attracted extensive attention in the field of food science in recent years.

[0003] However, the number of currently known kokumi polypeptides is limited, and the quantification of their taste characteristics and biological mechanisms of action have not been fully elucidated. Therefore, developing novel kokumi polypeptides and deeply studying their taste mechanisms have important scientific significance and application value. Summary of the Invention

[0004] In view of this, the present invention discloses a novel kokumi polypeptide, a screening and preparation method thereof, and an application thereof.

[0005] The present invention adopts the following technical solutions:

[0006] A novel kokumi polypeptide, the amino acid sequence of the polypeptide being: Arg-Ser-Gln-Pro-Val-Gly-Val.

[0007] Further, the polypeptide is added to food as a food flavor enhancer.

[0008] Further, the food includes low-salt food or low-sugar food.

[0009] Further, the food is low-sodium food.

[0010] Further, the added weight percentage of the polypeptide in the food is 0.05 - 0.2%.

[0011] A screening and preparation method of the above polypeptide, the method comprising:

[0012] S1. Screening candidate polypeptides using a high-throughput modeling and molecular docking program to obtain the polypeptide; the standard for successful molecular docking in the molecular docking program is set as: docking binding energy ≤ -5 kcal / mol;

[0013] S2. Synthesizing the screened polypeptide using solid-phase synthesis technology.

[0014] Further, step S1 includes: taking the calcium-sensing receptor as the target protein, programming in Python language, and developing a high-throughput polypeptide modeling and molecular docking program in combination with software such as Pymol, Autodock Vina, Discovery Studio, PLIP or Openbabel, and performing molecular docking and molecular dynamics simulation on the candidate polypeptide; the docking binding energy of the polypeptide and the calcium-sensing receptor is -9.929 kcal / mol.

[0015] Further, step S2 includes: selecting a resin, installing a protecting group, adding amino acids one by one, coupling reaction, washing, deprotection and cleavage.

[0016] Advantages of the present invention:

[0017] The present invention discloses a novel kokumi polypeptide and its screening, preparation method and application. The polypeptide is identified by high-throughput screening and molecular docking technology, and has a significant flavor-enhancing effect, especially in enhancing salty, umami and kokumi attributes. The polypeptide preparation method disclosed by the present invention is simple and low-cost, and can be widely applied to the development of low-salt, low-sodium and low-sugar foods.

[0018] The present invention develops an efficient and high-throughput method for screening kokumi polypeptides, providing technical support for the discovery of more kokumi polypeptides in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 : Schematic diagram of the molecular structure of the polypeptide of the present invention;

[0021] Figure 2 : Schematic diagram of the detailed site and 3D of the molecular docking of the polypeptide of the present invention and the CaSR receptor protein;

[0022] Figure 3 : 3D Gibbs free energy topography diagram of the 100 ns molecular dynamics simulation results of the complex of the molecular docking result of the polypeptide of the present invention and the CaSR receptor molecule;

[0023] Figure 4 : Schematic diagram of the sensory evaluation results of the polypeptide in the monosodium glutamate salt solution of the present invention;

[0024] Figure 5:Schematic diagram of the original curve and the fitted curve of the interaction between the polypeptide of the present invention and the CaSR receptor protein;

[0025] Figure 6 :Verification results of animal experiments of the polypeptide of the present invention. Detailed implementation manners

[0026] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0027] It should be clear that the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0028] Embodiment 1:

[0029] A novel kokumi polypeptide, the amino acid sequence of the polypeptide is: Arg-Ser-Gln-Pro-Val-Gly-Val. Its molecular structure is as Figure 1 shown.

[0030] Furthermore, the polypeptide is added to food as a food flavor enhancer.

[0031] Furthermore, the food includes low-salt food or low-sugar food.

[0032] Furthermore, the food is low-sodium food.

[0033] Furthermore, the added weight percentage of the polypeptide in the food is 0.05-0.2%.

[0034] A screening and preparation method of the above polypeptide, the method includes:

[0035] S1. Screening candidate polypeptides by using high-throughput modeling and molecular docking programs to obtain the polypeptide; the standard for successful molecular docking in the molecular docking program is set as: docking binding energy ≤ -5 kcal / mol;

[0036] S2. Synthesizing the screened polypeptide by using solid-phase synthesis technology.

[0037] Furthermore, step S1 includes: taking the calcium-sensing receptor as the target protein, programming with the Python language, and developing a high-throughput polypeptide modeling and molecular docking program in combination with software such as Pymol, Autodock Vina, Discovery Studio, PLIP or Openbabel, and performing molecular docking and molecular dynamics simulation on the candidate polypeptide; the docking binding energy of the polypeptide and the calcium-sensing receptor is -9.929 kcal / mol.

[0038] Furthermore, step S2 includes: selecting a resin, installing a protecting group, adding amino acids one by one, coupling reaction, washing, deprotection, and cleavage.

[0039] Example 2

[0040] (1) Screening and identification of the kokumi polypeptide RSQPVGV.

[0041] Using the calcium-sensing receptor (CaSR) as the target protein, programming with the Python language, and combining software such as Pymol, AutodockVina, Discovery Studio, PLIP, and Openbabel to develop a high-throughput polypeptide modeling and molecular docking program, molecular docking and molecular dynamics simulations were performed on the candidate polypeptides. As Figure 2 shown.

[0042] During the screening process, LC-MS / MS was used to identify polypeptides in yeast. A total of 5917 pure peptide segments were found, with the amino acid sequence length ranging from 5 to 24. Considering that the longest reported kokumi peptide amino acid sequence length is currently 14, a total of 4772 pure peptide segments with a length of 5 to 14 were selected from the identification results. Each polypeptide was individually docked with CaSR, and the docking binding energy between them was statistically analyzed. Taking ≤ -5 kcal / mol as the standard, the docking success rate and interaction sites were statistically analyzed. It was found that the 7-peptide (RSQPVGV) with the highest docking success rate and the strongest binding to CaSR had a docking binding energy of -9.929 kcal / mol with CaSR. The results of molecular dynamics simulations showed that the RMSD value of the complex was stable within the range of 0.4 ± 0.05 nm, and the 3D Gibbs free energy topography showed a specific and smooth energy column, indicating that RSQPVGV binds stably to the CaSR receptor. As Figure 3 shown, Figure 3 A: RMSD value of the RSQPVGV-CaSR complex, Figure 3 B: 3D Gibbs free energy topography of the RSQPVGV-CaSR complex.

[0043] (2) Synthesis and sensory evaluation of the kokumi polypeptide RSQPVGV.

[0044] The polypeptide RSQPVGV was synthesized using solid-phase synthesis technology. The synthesis steps included: selecting a suitable resin, installing protecting groups, adding amino acids one by one, coupling reactions, washing, deprotection, and cleavage, etc. Specifically: Selecting a resin, choosing a suitable resin such as Wang resin or Rink amide resin; Installing protecting groups, installing protecting groups on the α-amino group of amino acids, and commonly used protecting groups are tert-butoxycarbonyl (BOC) and 9-fluorenylmethoxycarbonyl (FMOC); Adding amino acids one by one, adding amino acids to the resin one by one and carrying out coupling reactions; Washing, washing after each addition of amino acids to remove unreacted amino acids and other impurities; Deprotection, using appropriate chemical reagents to remove the protecting groups to expose the α-amino group of the next amino acid; Cleavage, after all amino acids are added, using appropriate reagents to cleave the resin to obtain the polypeptide product. The synthesized polypeptide RSQPVGV was used with a monosodium glutamate-salt solution containing 1% monosodium glutamate and 0.3% salt as the medium, and glutathione (GSH) as the positive control for sensory analysis and electronic tongue detection.

[0045] The results showed that the taste threshold of the polypeptide RSQPVGV was 0.37 mmol / L. At a concentration of 0.1%, it could significantly enhance the saltiness, umami, and kokumi properties of the monosodium glutamate-salt solution. Electronic tongue detection also showed that it could significantly increase the Richness value (an index reflecting the kokumi property). As Figure 4 shown, Figure 4 in A: The radar chart of the 5-point sensory evaluation method, Figure 4 in B: The detailed difference bar chart of the saltiness, umami, and kokumi indexes.

[0046] (3) Research on the biological mechanism of the kokumi polypeptide RSQPVGV.

[0047] The binding ability of the polypeptide RSQPVGV to the human CaSR protein was detected using surface plasmon resonance technology (SPR). The results showed that the binding energy of the polypeptide RSQPVGV to CaSR increased with the increase in concentration, and the calculated KD value was 1.97×10^ -5 , suggesting a tight binding between the two. As Figure 5 shown.

[0048] (4) Verification of the kokumi polypeptide RSQPVGV through animal experiments.

[0049] Through the mouse two-bottle preference experiment, it was found that the polypeptide RSQPVGV could significantly enhance the taste intensity of 0.6% monosodium glutamate. After adding the CaSR receptor inhibitor NPS-2143, the flavor-enhancing effect of the polypeptide RSQPVGV decreased significantly, indicating that its mechanism of action was closely related to the CaSR receptor. As Figure 6 shown.

[0050] The present invention first discovered and identified a novel kokumi polypeptide RSQPVGV, which has a significant flavor-enhancing effect, especially showing excellent performance in enhancing salty, umami, and kokumi attributes. The binding mechanism between the polypeptide RSQPVGV and the CaSR receptor was elucidated through molecular docking, molecular dynamics simulation, and SPR technology, providing a theoretical basis for its application in food.

[0051] The present invention developed an efficient and high-throughput screening method for kokumi polypeptides, providing technical support for the discovery of more kokumi polypeptides in the future. The preparation method of the polypeptide RSQPVGV is simple and low-cost, and can be widely applied to the development of low-salt, low-sodium, and low-sugar foods.

[0052] The above has introduced the embodiments of the present invention in detail. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation manner and application scope. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A novel kokumi polypeptide, characterized in that, The amino acid sequence of the polypeptide is: Arg-Ser-Gln-Pro-Val-Gly-Val.

2. Use of a polypeptide as described in claim 1, characterized in that, The polypeptide is added to food as a food flavor enhancer.

3. Use of the polypeptide according to claim 2, characterized in that, The food includes low-salt food or low-sugar food.

4. Use of the polypeptide according to claim 3, characterized in that, The food is low-sodium food.

5. Use of the polypeptide according to claim 4, characterized in that, The added weight percentage of the polypeptide in the food is 0.05-0.2%.

6. A method for screening and preparing the polypeptide according to claim 1, characterized in that, The method includes: S1. Screening candidate polypeptides using high-throughput modeling and molecular docking programs to obtain the polypeptide; the standard for successful molecular docking in the molecular docking program is set as: docking binding energy ≤ -5 kcal / mol; S2. Synthesizing the screened polypeptide using solid-phase synthesis technology.

7. The method according to claim 6, characterized in that, Step S1 includes: using the calcium-sensing receptor as the target protein, programming with the Python language, and developing a high-throughput polypeptide modeling and molecular docking program in combination with software such as Pymol, Autodock Vina, Discovery Studio, PLIP, or Openbabel, and performing molecular docking and molecular dynamics simulations on the candidate polypeptide; the docking binding energy of the polypeptide and the calcium-sensing receptor is -9.929 kcal / mol.

8. The method according to claim 7, wherein Step S2 includes: selecting resin, installing protecting groups, adding amino acids one by one, coupling reactions, washing, deprotection, and cleavage.

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