A porcine actomyosin flavor peptide PA1 and application thereof

Abstract

The application discloses a pork actin flavor peptide PA1, and the amino acid sequence of the pork actin flavor peptide PA1 is GNERFR, and the molecular weight of the flavor peptide is 778 Da. The flavor peptide belongs to a salty peptide, the salty taste threshold of the flavor peptide is less than the salty taste threshold of common salt, the flavor peptide has strong salty taste, can be used as a food flavoring agent to significantly improve the salty taste characteristics of food, and can be used as a new type of seasoning to replace common salt, and has a good market application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a pork actin flavor peptide PA1 and its applications. Background Technology

[0002] Saltiness is an indispensable and fundamental taste in food, forming one of the important bases of food flavor. Excessive salt intake can lead to many diseases, such as high blood pressure. Therefore, the government is strongly advocating for nationwide salt reduction. Flavor peptides are oligopeptides, usually extracted from food or synthesized from amino acids, with a molecular weight of 500–1500 Da. They possess excellent processing characteristics and flavor-enhancing functions; some flavor peptides also have physiological activity, exhibiting very high nutritional value and health benefits.

[0003] Pork is one of the most consumed meats in my country, accounting for about 60% of total meat consumption. Due to its high consumption, the number of pigs raised is also very large, making my country the world's largest pig-producing country, with an annual output of 600-700 million head. However, in practice, pork processing mainly involves basic cooking, and research on using pork as a raw material for flavorings and aromatics is still relatively limited.

[0004] Therefore, providing a novel condiment made from pork that has a salty taste and can replace table salt has good market application prospects. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pork actin flavor peptide PA1 and its application, thus solving the problems mentioned in the background art.

[0006] One of the technical solutions adopted by the present invention to solve its technical problem is: providing pork actin flavor peptide PA1. Its amino acid sequence is GNERFR, as shown in SEQ ID NO: 1.

[0007] Actin is a highly abundant intracellular protein present in every eukaryotic cell, existing in two forms: monomeric G-actin and multimeric F-actin. It is a key component of the cytoskeleton. Actin was chosen as the research material due to its high abundance in organisms.

[0008] Studies have shown that flavor peptides typically have molecular weights between 200 and 1500 Da, and their sequences must contain ionizable amino acid residues capable of removing cations, with a high degree of amino acid dissociation. The presence of arginine residues significantly enhances the salty taste. Therefore, this provides a theoretical basis for the search for flavor peptides. We first screened and calculated pork protein sequences, discovering a salty sequence GNERFR, named PA1, with a molecular weight of 778 Da.

[0009] The application of the flavor peptide PA1 in the abalone soup is proved to be able to replace salt well through actual application, and the flavor peptide PA1 can be used as a new salty flavoring.

[0010] The second technical solution of the application is to provide the biological material related to the pork actin flavor peptide PA1, which is any one of A1) to A8) as follows:

[0011] A1) a nucleic acid molecule encoding the recombinant protein in claim 1;

[0012] A2) an expression cassette containing the nucleic acid molecule in A1);

[0013] A3) a recombinant vector containing the nucleic acid molecule in A1),

[0014] A4) a recombinant vector containing the expression cassette in A2);

[0015] A5) a recombinant microorganism containing the nucleic acid molecule in A1);

[0016] A6) a recombinant microorganism containing the expression cassette in A2);

[0017] A7) a recombinant microorganism containing the recombinant vector in A3);

[0018] A8) a recombinant microorganism containing the recombinant vector in A4).

[0019] The third technical solution of the application is to provide the application of the pork actin flavor peptide PA1 in the preparation of food flavoring agents or health products.

[0020] The fourth technical solution of the application is to provide the application of the biological material related to the pork actin flavor peptide PA1 in the preparation of food flavoring agents or health products.

[0021] The fifth technical solution of the application is to provide a food flavoring agent, and the effective component of the food flavoring agent includes the pork actin flavor peptide PA1, and the amino acid sequence of the pork actin flavor peptide PA1 is SEQ ID NO: 1.

[0022] Preferably, the effective component of the food flavoring agent is the pork actin flavor peptide PA1, and the amino acid sequence of the pork actin flavor peptide PA1 is SEQ ID NO: 1.

[0023] The sixth technical solution of the application is to provide a health product, and the effective component of the health product includes the pork actin flavor peptide PA1, and the amino acid sequence of the pork actin flavor peptide PA1 is SEQ ID NO: 1.

[0024] Preferably, the effective component of the above-mentioned pork actin flavor peptide PA1 is pork actin flavor peptide PA1, and the amino acid sequence of the pork actin flavor peptide PA1 is SEQ ID NO: 1.

[0025] The flavor peptide of the present application can be synthesized by a method known to those skilled in the art, such as solid-phase synthesis, and purified by a method known to those skilled in the art, such as high-performance liquid chromatography.

[0026] The present application has the following beneficial effects:

[0027] The present application takes pork actin as the research object, and through screening and calculation, a polypeptide PA1 with a novel amino acid sequence is found. The flavor peptide PA1 presents strong salty taste and can be used as a substitute for salt. The flavor peptide PA1 can replace salt in abalone soup and present good flavor in the abalone soup. The flavor peptide PA1 has good application prospect in the production of food condiments or health products. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Fig. 1 is a structural schematic diagram of the flavor peptide PA1.

[0029] Figure 2 Fig. 3 is a flavor radar chart of the flavor peptide PA1.

[0030] Figure 3 Fig. 5 is a contrast chart of the flavor peptide PA1 and three salty taste peptides in taste intensity.

[0031] Figure 4 Fig. 7 is a 3D space conformation chart of the molecular docking of the flavor peptide PA1 and the receptor TRPV1.

[0032] Figure 5 Fig. 9 is a difference contrast chart of the flavor peptide PA1 abalone soup and the traditional salt abalone soup. DETAILED DESCRIPTION

[0033] In order to better understand the present application, the present application will be further described in detail below in combination with the embodiments and drawings, but those skilled in the art understand that the following embodiments are not a limitation on the protection scope of the present application, and any changes and variations made on the basis of the present application are within the protection scope of the present application.

[0034] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0035] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0036] Example 1: Screening process and preparation method of flavor peptide PA1

[0037] The existing research found that salty peptides and salty taste enhancing peptides are oligopeptides composed of amino acids connected by peptide bonds, most of which are oligopeptides composed of 2-6 amino acids, with a molecular weight of less than 1 kDa, and most of which are related to acidic amino acids. Among them, arginine (Arg), glutamic acid (Glu) and aspartic acid (Asp) are usually present in salty peptides and salty taste enhancing peptides. Therefore, in this study, actin in pork was selected as the object, and the amino acid sequence meeting the above conditions was finally found to be GNERFR, which may be a potential salty peptide.

[0038] Construction of salty peptide expression vector

[0039] (1) Gene synthesis

[0040] With the amino acid sequence of GNERFR as the target product, B. subtilis 168 was selected as the expression host, and the coding gene of the target salty peptide was codon optimized to obtain the expression gene. The gene sequence is SEQ ID NO. 1, and finally the salty peptide gene sequence with Nde I and EcoR I enzyme cutting sites at the 5' and 3' ends, respectively, is synthesized.

[0041] (2) Double enzyme digestion to construct recombinant expression vector

[0042] The synthesized salty peptide gene and the vector plasmid were subjected to Nde I / EcoR I double enzyme digestion, and then the target enzyme cutting fragments were purified and recovered by 1% agarose gel electrophoresis to obtain the salty peptide target gene with sticky ends and the linear vector plasmid with sticky ends. Then the two were connected by high-efficiency ligase to obtain the salty peptide recombinant vector—GNERFR.

[0043] Among them, the enzyme digestion reaction system (100 μL in total) and the reaction program are as follows: salty peptide target gene or expression plasmid, 10*K buffer 10 μL, Nde I 5 μL, EcoR I 5 μL, enzyme digestion temperature is 37°C.

[0044] (3) Screening, identification and sequencing of recombinant expression vector

[0045] Bacterial liquid PCR screening was used, and the PCR system is shown in Table 2, and the PCR amplification program is shown in Table 2. The products of bacterial liquid PCR were detected by 1% agarose gel electrophoresis to screen positive transformants.

[0046] After sequencing verification of the positive transformants, the bacterial liquid of the transformants with correct sequencing was preserved in a glycerol tube.

[0047] Table 1 PCR reaction system

[0048] System components Reaction volume Bacterial solution 1 μL Upstream primer (10 μmol / L) 0.5 μL Downstream primer (10 μmol / L) 0.5 μL 2 x EasyTaq Mix 10 μL Sterile ultrapure water to make up the volume to 20 μL

[0049] Table 2 PCR amplification procedure

[0050]

[0051]

[0052] (4) Fermentation production of E. coli

[0053] The above positive transformants with correct expression were transformed into BL21 for fermentation production. The fermentation lasted for 7 days. After the fermentation, the supernatant and the precipitate after centrifugation of the bacterial cells before and after ultrasonic disruption were taken for SDS-PAGE analysis. The fermentation broth after ultrasonic disruption was subjected to Ni column affinity chromatography purification, and the desired flavor peptide PA1 was obtained.

[0054] The structure of the polypeptide PA1 is shown in Figure 1 .

[0055] Example 2 Sensory verification of flavor peptide PA1

[0056] The flavor peptide PA1 solution was used as the sample solution for sensory evaluation. The 0.35% salt solution, 1% sucrose solution, 0.08% citric acid solution, 0.35% monosodium glutamate solution, and 0.08% quinine solution were used as the basic solutions for salty, sweet, sour, fresh, and bitter tastes, respectively. The basic solution was rated as 5 points, and the target taste solution was evaluated using a 10-point system, so as to evaluate the taste intensity of the flavor peptide PA1. The sensory evaluation was performed by a trained professional sensory evaluation team (half male and half female). Each evaluation sample was randomly numbered and given to the evaluators in random order for taste evaluation. Each evaluator evaluated the same sample 3 times, and the average of the 3 measurements was taken as the final evaluation score. Each sample was evaluated at room temperature. Each sensory evaluator also evaluated the taste characteristics of each sample. The sensory evaluation data was analyzed using EXCEL, and the final sensory evaluation results are shown in Table 3.

[0057] Table 3 Flavor peptide PA1 taste characteristics

[0058] Evaluators 0 Fresh Sour Salty Bitter Sweet Notes A 6 Salty taste is more prominent B 8 Obvious salty taste C 7 Has obvious salty taste D 7 Salty taste is more prominent than fresh taste E 7 Salty taste is obvious

[0059] Example 3 Electronic tongue evaluation of flavor peptide PA1

[0060] Firstly, the flavor peptide PA1 was prepared into a 1 mg / mL solution. Next, 25 mL of the test solution was accurately transferred into the sample cup dedicated to the electronic tongue. Before measurement, the electronic tongue was self-tested, activated, calibrated, and diagnosed to ensure the reliability and stability of the collected data. The Ref Sol reference solution was prepared by mixing 30 mmol / L KCl solution with 0.3 mmol / L tartaric acid solution. The sensor was placed in the reference solution for 30 s to be zeroed, and then the salty taste determination was started. The test time was 30 s, and after the test, the reference solution was used for cleaning for 3 s, and then the aftertaste determination was performed again, with a test time of 30 s. Each sample was repeated 4 times, and the last 3 times were taken as the test results. The test results are shown in Table 4 and Figure 2 Figure 2 It can be seen from Table 4 and

[0061] Table 4 Electronic tongue identification results 6 kinds of taste response value table

[0062] Number Fresh taste Sour taste Bitter taste Astringent taste Salty taste Sweet taste Aftertaste Richness 1 3.07 0.48 0.05 0.80 9.01 1.01 2.66 2.53 1 3.07 0.48 0.05 0.80 9.01 1.01 2.66 2.53 1 3.07 0.48 0.05 0.80 9.01 1.01 2.66 2.53

[0063] Example 4 Comparison of flavor peptide PA1 taste intensity

[0064] In order to explore the salty taste intensity of the flavor peptide PA1, the synthesized flavor peptide PA1 was prepared into a 0.5 mg / mL solution. Then, the reported salty taste peptides Ala-Phe, Phe-Ile, and Ile-Phe were selected for synthesis of standard samples, and then prepared into solutions with the same concentration for sensory evaluation. The taste intensity of the flavor peptide PA1 and the three reported salty taste peptides was evaluated. The evaluation method referred to the sensory evaluation method in Example 1, and the scoring was based on a 10-point system, with 0 representing no taste and 10 representing significant taste. The final taste intensity results are shown in Table 5. Figure 3

[0065] Example 5 Molecular docking simulation of flavor peptide PA1 and receptor TRPV1

[0066] ​​The binding state of flavor peptide PA1 and the receptor was simulated by using molecular docking software AutoDock Vina to analyze the taste mechanism of the salty peptide. The structure of the receptor TRPV1 was downloaded from the AlphaFold database, the ligand, water molecules, and hydrogen were removed, and the receptor molecule was set. The two-dimensional (2D) structure of the peptide segment was predicted by ChemDraw 21.0.0 software, and then the three-dimensional (3D) structure thereof was generated by energy minimization through Chem3D 21.0.0, and polar hydrogen atoms were added, and set as the ligand molecule. The ligand molecule was docked into the set receptor molecule by AutoDock Vina software, and the docking result was visualized by PyMoL 2.5 (as shown in Figure 4 The binding energy of flavor peptide PA1 and receptor TRPV1 is-7.3 kcal / mol, and seven bonding amino acids are contained, and the hydrogen bond interaction is the main force between flavor peptide PA1 and receptor TRPV1 molecules.

[0067] Example 6 Practical application of flavor peptide PA1 in abalone soup

[0068] The abalone soup formula ingredients and proportions are as follows: pig big bone: 50 parts, granulated sugar: 1 part, cooking oil 2 parts, pig skin 10 parts, flavor peptide PA1: 0.05 parts.

[0069] The abalone soup preparation method is as follows:

[0070] Directly weigh the formula amount of each material and directly cook for 4 hours to obtain the salty peptide braised soup.

[0071] An equal amount of salt is used to replace the salty peptide in the above abalone soup to make a traditional salt abalone soup with the same ordinary process. The salty peptide abalone soup and the traditional salt abalone soup are subjected to sensory evaluation. The sensory evaluation table is shown in Table 5 and Table 6. The test method is to select five people to score from four aspects of color, aroma, taste, and mouthfeel, and calculate the final average score and total score. The abalone soup difference comparison is shown in Figure 5 .

[0072] Table 5 Sensory test table of two kinds of abalone soup

[0073]

[0074] Table 6 Sensory evaluation results

[0075] Item Fresh taste peptide abalone juice soup Traditional salt abalone juice soup Color 9.5 9.5 Flavor 9.6 9.5 Taste 9.4 9.3 Total score 28.5 28.3

[0076] Referring to Table 5 and Table 6, through data analysis of Example 6, it can be known that the salty peptide abalone soup is better than the traditional salt abalone soup in flavor and taste. The salty peptide abalone soup is only the same as the traditional salt abalone soup in color. Therefore, it can be determined that the technical scheme of the present application has obvious technical effect better than the traditional abalone soup.

[0077] In summary, the pork actin flavor peptide PA1 provided by the application has a saltiness threshold value less than that of salt, has strong saltiness, and can significantly improve the saltiness characteristics of food. The flavor peptide PA1 has a good application prospect in the production of food flavoring agents or health products.

[0078] Although the specific embodiments of the application are described above, those skilled in the art should understand that the specific examples described are only illustrative, and are not intended to limit the scope of the application, and equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the application should be covered within the scope of the claims of the application.

Claims

1. A porcine actomyosin flavor peptide PA1, characterized in that, The amino acid sequence of the pork actin flavor peptide PA1 is shown as SEQ ID NO:

1.

2. A biological material related to the pork actin flavor peptide PA1 according to claim 1, which is any one of the following A1) to A8): A1) a nucleic acid molecule encoding the recombinant protein according to claim 1; A2) an expression cassette containing the nucleic acid molecule according to A1); A3) a recombinant vector containing the nucleic acid molecule according to A1), A4) a recombinant vector containing the expression cassette according to A2); A5) a recombinant microorganism containing the nucleic acid molecule according to A1); A6) a recombinant microorganism containing the expression cassette according to A2); A7) a recombinant microorganism containing the recombinant vector according to A3); A8) a recombinant microorganism containing the recombinant vector according to A4).

3. Use of the pork actin flavor peptide PA1 according to claim 1 in the preparation of a food flavoring agent.

4. Use of the biological material related to the pork actin flavor peptide PA1 according to claim 2 in the preparation of a food flavoring agent.

5. A food flavoring, characterized by: The effective component thereof includes the pork actin flavor peptide PA1, and the amino acid sequence of the pork actin flavor peptide PA1 is SEQ ID NO:

1.

6. The food flavoring of claim 5 wherein: The effective component thereof is the pork actin flavor peptide PA1, and the amino acid sequence of the pork actin flavor peptide PA1 is SEQ ID NO:

1.

7. A health product, characterized by: The effective component thereof includes the pork actin flavor peptide PA1, and the amino acid sequence of the pork actin flavor peptide PA1 is SEQ ID NO:

1.

8. The health care product according to claim 7, wherein: The effective component thereof is the pork actin flavor peptide PA1, and the amino acid sequence of the pork actin flavor peptide PA1 is SEQ ID NO: 1.

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