Bioactive polypeptide IVVLPTGVAH as well as preparation method and application thereof

Abstract

The invention relates to a bioactive polypeptide IVVLPTGVAH as well as a preparation method and application of the bioactive polypeptide IVVLPTGVAH. The bioactive polypeptide IVVLPTGVAH provided by the invention has bitter taste, the crystal of the bioactive polypeptide IVVLPTGVAH is powdery, and the bioactive polypeptide IVVLPTGVAH can be combined with a bitter taste receptor TAS2R14. The bioactive polypeptide can be used as a bitter additive and an additive for foods and beverages, and the beverages comprise alcoholic beverages and non-alcoholic beverages. The bioactive peptide IVVLPTGVAH provided by the invention is a small-molecular-weight peptide screened from yellow wine, sensory evaluation and electronic tongue results show that the extracted bitter peptide has good bitter taste and richness, on one hand, the bioactive peptide IVVLPTGVAH has high bitter taste strength, the bitter taste of wine can be properly improved, and the flavor quality can be possibly improved under certain conditions; on the other hand, the bitter peptide IVVLPTGVAH in the yellow rice wine is properly removed, so that the evaluation experience of the yellow rice wine can be improved.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to a bioactive polypeptide IVVLPTGVAH, its preparation method, and its applications. Background Technology

[0002] The bitterness of Shaoxing wine largely originates from derivatives produced during fermentation, including amino acids, polypeptides, and higher alcohols. The mechanism of bitterness in Shaoxing wine is closely related to the specific binding of bitter peptides to T2Rs family taste receptors. Bitter peptides are a diverse class of oligopeptides that frequently produce bitterness during the aging process of fermented products and during enzymatic processes that generate protein hydrolysates. The properties and quantity of these short-chain peptides determine the intensity of bitterness in food. Studies have shown that the specific binding of bitter peptides to T2Rs family taste receptors requires strict stereoconformity matching: the active group of the bitter peptide ligand must maintain a critical distance of approximately 4.1 Å from the stimulating unit in the receptor-binding domain, and its molecular size must be adapted to the geometric constraints of the receptor-binding pocket. Existing research indicates that bitter peptides, which contribute to bitterness, generally have a relative molecular mass below 6.0 kDa and an average hydrophobicity exceeding 1400 cal / mol (Cho M, Unklesbay N, Hsieh F, et al. Hydrophobicity of bitter peptides from soy protein hydrolysates. Journal of Agricultural & Food Chemistry, 2004, 52(19): 5895-5901.). These bitter peptides produce specific taste sensations by binding to specific receptors on the taste buds of the human tongue. Moderate bitterness can stimulate consumers' appetite and highlight the characteristics and style of rice wine, while persistent bitterness will affect the overall taste of rice wine. Therefore, it is necessary to properly adjust the bitterness of rice wine.

[0003] Chinese patent CN113390994B discloses a method for extracting, separating, identifying, and verifying bitter peptides in rice wine. The method includes: separating rice wine into components with different molecular weights using ultrafiltration membranes and determining the most bitter component through descriptive sensory evaluation; collecting the corresponding components from ultraviolet spectra using gel filtration chromatography and obtaining the dilution factor of each component using taste dilution analysis; determining the purity of the peptide components using high-performance liquid chromatography; performing amino acid sequence analysis on the peptide components using ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry; and verifying the effect of the identified key bitter peptides on the bitterness of the recombinant model using taste addition experiments. This method reveals the structure and bitter characteristics of bitter peptides in rice wine, and for the first time discovers a bitter peptide with the amino acid sequence Leu-Pro-Thr-Leu in rice wine.

[0004] The master's thesis, "Sensory-Oriented Analysis of Bitter Substances in Shaoxing Rice Wine," separates important bitter substances in Shaoxing rice wine using HPLC, ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), and taste dilution analysis (TDA). The thesis preliminarily deduces that their molecular formula is C0. 37 H 68 O8 and C 24 H 44 N4O4 was used to preliminarily predict that the important bitter substances in Shaoxing rice wine may be pentaerythritol tetraoctanoate (tetradecanoate) and 1,8,15,22-tetraazacyclooctadecane-2,9,16,23-tetraone. Eleven volatile substances that contribute to the bitterness of Shaoxing rice wine were identified, namely 3-methylbutanol, benzaldehyde, ethyl octanoate, phenethyl alcohol, octanoic acid, isobutanol, phenol, furfural, ethyl nonanoate, ethyl palmitate, and acetophenone.

[0005] However, current research reports on bioactive peptides that can exert bitterness are still very limited, and the types of bitter peptides disclosed are also very few. Existing technologies need to provide more active peptides that can serve as bitter peptides to enrich food flavor. Summary of the Invention

[0006] To enrich the variety of bitter peptides, this invention provides a bioactive polypeptide IVVLPTGVAH, its preparation method, and its applications.

[0007] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides a bioactive polypeptide IVVLPTGVAH, the amino acid sequence of which is Ile-Val-Val-Leu-Pro-Thr-Gly-Val-Ala-His, as shown in SEQ ID NO.1.

[0008] The bioactive polypeptide IVVLPTGVAH provided by this invention has a bitter taste, and its crystals are in powder form, capable of binding to the bitter taste receptor TAS2R14.

[0009] Preferably, the bioactive polypeptide is a polypeptide derived from rice wine.

[0010] Preferably, the bioactive polypeptide has a bitter taste and can be used as a bittering additive in food and beverages, including alcoholic and non-alcoholic beverages.

[0011] Preferably, the bioactive polypeptide is water-soluble.

[0012] In a second aspect, the present invention provides a method for preparing the bioactive polypeptide IVVLPTGVAH, which can be artificially synthesized by genetic engineering, obtained by screening from rice wine, or prepared directly by chemical synthesis.

[0013] The artificial synthesis of the bioactive peptide IVVLPTGVAH through genetic engineering is a technical solution that can be achieved by those skilled in the art. For example, it can be based on DNA recombination technology, using a suitable DNA template to control the sequence synthesis of the peptide.

[0014] Preferably, the bioactive peptide IVVLPTGVAH can be prepared by solid-phase synthesis. The specific method is as follows: using the Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as a solid-phase carrier, and sequentially introducing histidine, alanine, valine, glycine, threonine, proline, leucine, valine, valine, and isoleucine residues to extend the peptide chain from the C-terminus to the N-terminus, thereby synthesizing the yellow rice wine bitter peptide IVVLPTGVAH in a solid phase.

[0015] The method for screening bitter peptides from rice wine is as follows: Peptidomics was used to analyze the differentially expressed peptides in various rice wines produced using the same raw materials and processes but with varying bitterness intensities due to differences in environmental temperature and other conditions. Bioinformatics was used to predict bitter peptides, and potential bitter peptides were further screened based on their hydrophobicity and peptide chain length. Using TAS2R14 as a bitter receptor, molecular docking technology was used to screen for the bioactive peptide IVVLPTGVAH, a bitter peptide found in this application, which has the specific amino acid sequence: Ile-Val-Val-Leu-Pro-Thr-Gly-Val-Ala-His.

[0016] Preferably, peptides smaller than 10 kDa are purified by centrifugation using a 10 kDa ultrafiltration tube, and the data obtained by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) are analyzed using PEAKS Studio 10.6 software to identify different peptide fragments among various rice wines.

[0017] Preferably, a bioinformatics tool (BIOPEP-UWM) is used for preliminary screening of the bitterness potential of differentially expressed peptides.

[0018] Preferably, potential bitter peptides are further screened using a quantitative structure-activity relationship (QSAR) model. The mathematical expression is as follows: log1 / T = 1.87 + 0.08 × n1z1 + 0.07 × n1z2 - 0.04 × n1z3 - 0.02 × c1z1 + 0.03 × c1z2 + 0.01 × logM + 0.11 × HP + 0.09 × total number of amino acids. The bitterness value is described as log1 / T (where T is the molar concentration of the bitterness threshold). The first amino acid at the n-terminus of the peptide is designated as n1, and its three z-scale properties are described as n1z1, n1z2, and n1z3, while c1z1 and c1z2 are the two z-scale properties of the first C-terminal amino acid. HP represents the total hydrophobicity of all amino acids in the peptide chain, and M represents the total molecular mass.

[0019] The results of electronic tongue and sensory evaluation showed that the screened bitter peptides had high bitterness values ​​and good abundance.

[0020] When prepared directly by chemical synthesis, the synthesis is based on the structure of the bioactive peptide IVVLPTGVAH.

[0021] In a third aspect, the present invention provides the application of the bioactive polypeptide IVVLPTGVAH in the preparation of food or beverages, wherein the bioactive polypeptide IVVLPTGVAH is used as a bittering agent.

[0022] In some embodiments of the present invention, the food is a seasoning, and the beverage includes alcoholic and non-alcoholic beverages.

[0023] In some embodiments of the present invention, the food is a seasoning, and the beverage is selected as rice wine. By controlling the content of bitter peptides, the bitterness of rice wine can be effectively reduced, and its flavor quality can be improved.

[0024] In a fourth aspect, the present invention provides a food or beverage containing a bittering agent, wherein the bittering agent is a bioactive polypeptide IVVLPTGVAH. By controlling the content of the bioactive polypeptide IVVLPTGVAH, the bitterness of the food or beverage can be effectively adjusted.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects: The bioactive peptide IVVLPTGVAH provided by this invention is a low molecular weight peptide screened from rice wine. Sensory evaluation and electronic tongue results show that the extracted bitter peptide has good bitterness and richness. On the one hand, the high bitterness intensity of this bioactive peptide IVVLPTGVAH can appropriately increase the bitterness of wine and may increase the flavor quality under certain conditions. On the other hand, appropriately removing the bitter peptide IVVLPTGVAH from rice wine can improve the tasting experience of rice wine. Attached Figure Description

[0026] Figure 1 Mass spectrometry results of an artificially synthesized sample of the bitter peptide IVVLPTGVAH in Shaoxing wine; Figure 2 Mass spectrometry results of an artificially synthesized sample of the bitter peptide IVVLPTGVAH in Shaoxing wine; Figure 3 This is a schematic diagram of the 3D spatial interaction between IVVLPTGVAH and the bitter taste receptor TAS2R14. Detailed Implementation

[0027] The following description, in conjunction with the accompanying drawings and specific embodiments, illustrates exemplary implementations of the present invention to help those skilled in the art understand other objectives, features, advantages, and aspects of this application. It should be understood that while preferred embodiments of the present application are shown, the following descriptions and specific embodiments are for illustrative purposes only and do not constitute a limitation on the scope of the invention. The scope of the invention is defined by the appended claims. Unless otherwise stated, the specific experiments in the following embodiments were conducted according to conventional methods and conditions in the art, or in accordance with the product specification.

[0028] Example 1 Screening and identification of bitter bioactive peptides in rice wine (hereinafter referred to as rice wine bitter peptides) Four types of Shaoxing wine with varying bitterness intensities, brewed using traditional Shaoxing wine brewing techniques with glutinous rice, wheat koji, and yeast as raw materials and at average ambient temperatures of 10.56°C, 9.32°C, 9.05°C, and 11.63°C, were used as research subjects. Differential peptides in the four types of Shaoxing wine were identified through peptidomics analysis. Then, virtual screening using molecular docking and bitterness scoring was employed to obtain the Shaoxing wine flavor peptides (bitter peptides). The specific methods are as follows: (1) Take 500 μL of rice wine sample and place it in a 10 kD ultrafiltration tube. Centrifuge at 8000 r / min for 10 minutes at 4°C. Then collect the portion less than 10 kD and transfer it to a vacuum centrifuge at 45°C to evaporate the solvent to dryness.

[0029] (2) Add dithiothreitol solution to the sample to achieve a final concentration of 10 mmol / L, and then place the sample in a water bath at 56°C for a reduction reaction for 1 hour. After the reaction, add iodoacetamide solution to achieve a final concentration of 55 mmol / L, and carry out the reaction under light-protected conditions for 40 minutes.

[0030] (3) The desalting was performed again using a self-packed desalting column, and the solvent was evaporated in a vacuum centrifuge at 45℃. A 75 μm, d. × 25 cm Nano Viper C18 1.9 μm, 100A analytical column was used. The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was 80% acetonitrile (ACN) / 0.1% formic acid aqueous solution. The flow rate was set to 600 nL / min, and the analysis time for each component was 66 minutes. The detailed composition of mobile phase B is shown in Table 1.

[0031] Table 1: Elution time and percentage of phase B The BIOPEP-UWM database was then used to predict the bitterness of peptides, and a total of 144 peptides that may have a bitter taste were obtained. Then, based on the criteria of a relative molecular mass of less than 6.0 kDa and a hydrophobicity of more than 1400 cal / mol, 9 potential bitter peptides were further screened out, as shown in Table 2.

[0032] The bitterness values ​​of potential bitter peptides were predicted using the QSAR model. The mathematical expression is as follows: log1 / T=1.87+0.08×n1z1+0.07×n1z2-0.04×n1z3-0.02×c1z1+0.03×c1z2+0.01×log M +0.11×HP+0.09×total number of amino acids. The bitterness value is described as log1 / T (where T is the molar concentration of the bitterness threshold). The first amino acid at the n-terminus of the peptide is designated n1, and its three z-scale properties are described as n1z1, n1z2, and n1z3, while c1z1 and c1z2 are the two z-scale properties of the first C-terminal amino acid. HP represents the total hydrophobicity of all amino acids in the peptide chain, and M represents the total molecular weight. The bitterness values ​​of the nine potential bitter peptides are shown in Table 2.

[0033] Table 2: Potential bitter peptides and their bitterness values The TAS2R14 crystal structure was downloaded from the PDB database, and water molecules, co-crystallization ligands, and irrelevant ions were removed to serve as the acceptor. The acceptor was hydrogenated using AutoDock Tools, the Gasteiger charge was calculated, and the structure was saved in PDBQT format. Chem3D was used to predict its 3D structure. Semi-flexible docking was performed using AutoDock, and the center coordinates of the docking box were determined based on the acceptor's active site. Twenty docking attempts were made. The docking results of nine peptides were visualized using Pymol. Finally, a bitter peptide that could stably bind to the bitter taste acceptor TAS2R14 was selected, with the following amino acid sequence: Ile-Val-Val-Leu-Pro-Thr-Gly-Val-Ala-His.

[0034] The 3D spatial interaction between IVVLPTGVAH and its receptor binding is as follows: Figure 3 As shown, by Figure 3 It is known that the optimal docking posture of IVVLPTGVAH with the bitter taste receptor TAS2R14 is: IVVLPTGVAH binds to the Ile-1 residue and the Ser-219 site, and the Pro-5 residue and the Thr-224 site, respectively.

[0035] Example 2 Synthesis of bioactive peptide IVVLPTGVAH (yellow wine bitter peptide) The bitter peptide Ile-Val-Val-Leu-Pro-Thr-Gly-Val-Ala-His (IVVLPTGVAH) was synthesized by Nanjing Yuanpeptide Biotechnology Co., Ltd. using a peptide solid-phase synthesis method. The purity of the synthesized peptide was verified to be greater than 95% by high-performance liquid chromatography and mass spectrometry. Specific results are as follows: Figure 1 and Figure 2 As shown.

[0036] Example 3 Sensory evaluation of bitter peptides in rice wine The sensory evaluation team was formed as follows: First, in accordance with the GB / T 16291.1-2012 standard, 20 candidates were selected. The first stage involved basic taste discrimination ability assessment (sweet, sour, bitter, salty) and typical yellow wine flavor characteristic description test. The second stage involved enhanced aroma identification assessment for those who passed the test. Finally, 12 sensory evaluators (4 men and 8 women) were selected to participate in the experiment.

[0037] Selected personnel must complete a systematic sensory training program: (1) Standardize the training of tasting operation, including standardized procedures such as temperature control of wine samples, oral rinsing and swallowing methods; (2) Based on gradient concentration sample recognition training, the ability to quantitatively perceive bitterness intensity is established by using the nine-level scaling method; (3) Establish standardized scoring criteria through blind sample testing and consistency training to eliminate individual sensory bias.

[0038] A 0.005 mg / mL bitter peptide solution was prepared, thoroughly stirred, and its bitterness intensity was tasted. The bitterness of the rice wine bitter peptide was scored, and the average score was calculated to be 7.583 (see Table 3). Sensory evaluation results showed that the rice wine bitter peptide can enhance the bitterness characteristics of food.

[0039] Table 3: Sensory Rating Table Example 4 Sensory evaluation of bitter peptides in rice wine using electronic tongue The TS-5000Z electronic tongue system, equipped with its COO sensor, was used to quantitatively analyze the bitterness intensity of the bitter peptide IVVLPTGVAH in rice wine. After system startup, self-testing, diagnostics, and calibration procedures were completed sequentially. The sensor was then pretreated with a dedicated cleaning solution (positive electrode cleaning solution: 10mM potassium hydroxide + 100mM potassium chloride + 30% ethanol; negative electrode cleaning solution: 100mM hydrochloric acid + 30% ethanol) and a reference solution (30mM potassium chloride + 0.3mM tartaric acid). Sample detection was initiated after the signal baseline stabilized. Specific detection parameters were set as follows: 30 seconds for a single sample detection, followed by a 3-second pulse cleaning in the reference solution after each detection, before proceeding to the next sample. To ensure data reliability, strict sensor standardization management was implemented throughout the process, and all samples underwent three repeated measurements. This detection system effectively ensured the stability and reproducibility of the sensor response values ​​by cyclically executing the "determination-cleaning-equilibration" operation procedure.

[0040] A solution of the bitter peptide IVVLPTGVAH with a concentration of 0.005 mg / mL was prepared, and its bitterness value was scored using an electronic tongue. The score obtained was 18.11. The bitterness value of caffeine at the same concentration was 6.58. The results show that the bitter peptide has a strong bitter taste.

[0041] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A bioactive polypeptide IVVLPTGVAH, characterized in that, Its amino acid sequence is Ile-Val-Val-Leu-Pro-Thr-Gly-Val-Ala-His.

2. The bioactive polypeptide IVVLPTGVAH according to claim 1, characterized in that, The bioactive polypeptide IVVLPTGVAH has a bitter taste, and its crystals are in powder form. It can bind to the bitter taste receptor TAS2R14.

3. The bioactive polypeptide IVVLPTGVAH according to claim 1, characterized in that, The bioactive polypeptide IVVLPTGVAH is water-soluble.

4. A polynucleotide encoding the bioactive peptide IVVLPTGVAH of claim 1.

5. The method for preparing the bioactive peptide IVVLPTGVAH according to claim 1, characterized in that, It can be artificially synthesized through genetic engineering, obtained by screening from rice wine, or prepared directly through chemical synthesis.

6. The method for preparing the bioactive peptide IVVLPTGVAH according to claim 5, characterized in that, The peptide was prepared by solid-phase synthesis. The specific method is as follows: using Fmoc solid-phase synthesis strategy, Fmoc-protected amino acids are used as raw materials, Wang resin is selected as solid-phase carrier, and histidine, alanine, valine, glycine, threonine, proline, leucine, valine, valine, and isoleucine residues are introduced in sequence to extend the peptide chain from the C-terminus to the N-terminus, and the bitter peptide IVVLPTGVAH of rice wine is synthesized in solid phase.

7. The method for preparing the bioactive peptide IVVLPTGVAH according to claim 5, characterized in that, Peptidomics was used to analyze the differential peptides contained in various rice wines made from the same raw materials and using the same process, but with different bitterness intensities due to different environmental conditions such as temperature. Bioinformatics was used to predict bitter peptides, and potential bitter peptides were further screened based on the hydrophobicity and peptide chain length of the bitter peptides. Using TAS2R14 as a bitter taste receptor, the bioactive peptide IVVLPTGVAH described in claim 1 was obtained through molecular docking technology.

8. The application of the bioactive polypeptide IVVLPTGVAH according to claim 1 in the preparation of food or beverage, characterized in that, The bioactive polypeptide IVVLPTGVAH is used as a bittering agent.

9. A food or beverage containing a bittering agent, characterized in that, The bittering agent is selected as the bioactive polypeptide IVVLPTGVAH described in claim 1. By controlling the content of the bioactive polypeptide IVVLPTGVAH, the bitterness of food or beverage can be adjusted.

10. A food or beverage containing a bittering agent according to claim 9, characterized in that, The beverages include alcoholic beverages and non-alcoholic beverages.

Images