Pea protein source salty peptide and preparation method thereof

Abstract

The invention provides a pea protein source salty peptide and a preparation method thereof.The method comprises the steps that pea protein is subjected to enzymolysis, ultrafiltration, separation and purification to obtain a pea protein salty peptide primary extract, then the primary extract is subjected to LC-MS / MS analysis and computer simulation technologies to screen non-toxic polypeptide sequences capable of being combined with a TMC4 protein receptor, and finally the pea protein source salty peptide is synthesized through a solid-phase synthesis method. And carrying out sensory analysis on salty taste and salty strength to obtain the pea protein salty peptide. The peptide RA7, the peptide AR8 and the peptide FR6 screened by the invention have good salty taste scores, the salty taste intensity of the peptide RA7, the peptide AR8 and the peptide FR6 is equivalent to that of a salt solution with the concentration of 3g / L, meanwhile, the peptide RF8, the peptide FF5 and the peptide DS5 have relatively high salty taste increasing effects, and particularly, the salty taste increasing rate of the peptide RF8 is 136%. The method not only can promote high-valued application of the pea protein salty peptide, but also can be used for development of low-salt food to achieve the effect of reducing salt but not reducing salty.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and particularly relates to a pea protein-derived salty peptide and a preparation method thereof. Background Art

[0002] With the global population growing and resource constraints, finding sustainable protein sources has become an important topic in food science. Plant protein has gradually become a research hotspot due to its renewable, environmentally friendly and non-animal origin characteristics.

[0003] Pea protein, a high-quality plant protein resource, is valued for its high protein content, favorable amino acid profile, and low allergenicity. In recent years, the extraction and preparation of salty peptides from plant proteins through biotechnology has become a new trend in the food industry. Salty peptides have the potential to reduce sodium content in foods, improve food flavor, and promote healthy eating. The development of salty peptides can reduce salt intake and has important implications for improving public health. Pea protein contains abundant peptides that can be released under specific enzymatic hydrolysis conditions and exhibit salty properties. However, the enormous potential of pea-derived salty peptides remains to be fully realized. Summary of the Invention

[0004] Technical Problem to Be Solved: To address the aforementioned technical issues, the present invention aims to develop a pea protein salty peptide and its preparation method by enzymatically hydrolyzing pea protein, isolating and purifying the pea protein hydrolysate, and systematically evaluating its flavor properties. The resulting salty peptide exhibits excellent saltiness and salt-enhancing properties, enabling its use in the development of low-salt foods, reducing salt without reducing saltiness. This method not only provides a healthy salty alternative for the food industry but also expands the application areas of pea protein, promoting the high-value utilization of plant protein resources.

[0005] Technical solution: Pea protein-derived salty peptide, the pea protein salty peptide is one or more of peptide AR8, peptide RF8, peptide RA7, peptide FR6, peptide FF5, and peptide DS5, the amino acid sequence of the peptide AR8 is shown in SEQ ID NO.1, the amino acid sequence of the peptide RF8 is shown in SEQ ID NO.2, the amino acid sequence of the peptide RA7 is shown in SEQ ID NO.3, the amino acid sequence of the peptide FR6 is shown in SEQ ID NO.4, the amino acid sequence of the peptide FF5 is shown in SEQ ID NO.5, and the amino acid sequence of the peptide DS5 is shown in SEQ ID NO.6.

[0006] The preparation method of pea protein-derived salty peptide comprises the following steps: S1. Pea protein is added to water to prepare a pea protein solution and the pH is adjusted to 6.8-7.2. After enzymatic hydrolysis, the enzyme is inactivated, cooled, centrifuged, and the supernatant is obtained to obtain a pea protein enzymatic hydrolyzate; further, the pH is adjusted to 7.0; S2. The pea protein hydrolysate was subjected to ultrafiltration, gel filtration chromatography, and freeze-drying to obtain a primary extract of pea protein salty peptides. S3. The pea protein salty peptide primary extract was sequenced using liquid chromatography-mass spectrometry to obtain multiple polypeptide sequences; S4. Using computer simulation technology to screen the multiple polypeptide sequences obtained in S3 for non-toxic polypeptide sequences that bind to the TMC4 protein receptor; S5. The polypeptide sequence obtained in S4 was synthesized by solid-phase synthesis, and the saltiness and saltiness enhancement intensity were analyzed by sensory analysis to obtain pea protein salty peptide.

[0007] Furthermore, the mass ratio of pea protein to water in S1 is 1:15-1:25; the amount of enzyme added in S1 is 3000-4000 U / g; and the enzyme is prepared by mixing flavor protease and composite protease in a ratio of 1:3-1:7; Furthermore, the mass ratio of pea protein to water in the S1 is 1:20; the added amount of enzyme in the S1 is 3500 U / g; and the enzyme is prepared by mixing flavor protease and composite protease in a ratio of 1:5.

[0008] Furthermore, the enzymatic hydrolysis conditions in S1 are enzymatic hydrolysis temperature 50-55°C and enzymatic hydrolysis time 4.0-4.5h; the enzyme inactivation conditions in S1 are enzyme inactivation temperature 90-100°C and enzyme inactivation time 10-20min; Furthermore, the enzymatic hydrolysis conditions in S1 are an enzymatic hydrolysis temperature of 51° C. and an enzymatic hydrolysis time of 4.2 h; and the enzyme inactivation conditions in S1 are an enzyme inactivation temperature of 95° C. and an enzyme inactivation time of 15 min.

[0009] Furthermore, the specific parameters of the liquid chromatography-mass spectrometry technology in S3 are: NanoViper C18 reversed-phase chromatography column (specifications: 75 μm id × 50 cm, 2 μm, 100Å); chromatographic instrument: Thermo Fisher Easy-nLC1200; mass spectrometer: Thermo Fisher Orbitrap Exploris 240; chromatographic separation time: 60 min; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 80% ACN / 0.1% formic acid aqueous solution; flow rate: 250 nL / min.

[0010] Furthermore, the specific operation of S4 is to use the ToxinPred database to select non-toxic polypeptide sequences from the multiple polypeptide sequences obtained in S3, and then screen out polypeptide sequences that can bind to the TMC4 receptor protein through molecular docking technology, and finally obtain non-toxic polypeptide sequences that can bind to the TMC4 receptor protein.

[0011] The application of the pea protein salty peptide in the preparation of low-salt foods and food seasonings.

[0012] 1. The present invention screened peptides AR8 (AVFDDKVR: Ala-Val-Phe-Asp-Asp-Lys-Val-Arg), RF8 (RSDPQNPF: Arg-Ser-Asp-Pro-Gln-Asn-Pro-Phe), RA7 (RDFLEDA: Arg-Asp-Phe-Leu-Glu-Asp-Ala), and FR6 (FDDKVR: Phe-Asp- Asp-Lys-Val-Arg), peptide FF5 (FDSRF: Phe-Asp-Ser-Arg-Phe) and peptide DS5 (DYWPS: Asp-Tyr-Trp-Pro-Ser); among them, peptide RA7, peptide AR8 and peptide FR6 have good salty sensory properties, and their saltiness intensity is equivalent to that of 3g / L salt solution. At the same time, peptide RF8, peptide FF5 and peptide DS5 have good salt-enhancing effects, especially peptide RF8, whose salt-enhancing rate is 136%.

[0013] 2. The pea protein salty peptide prepared by the present invention can be synthesized by a solid-phase synthesis method or extracted from pea protein hydrolysate. In addition, the present invention utilizes peptidomics and molecular docking methods to efficiently screen salty peptides with specific amino acid sequences from pea protein, simplifying the preparative liquid phase purification step and saving instrument costs. It is also conducive to the widespread application of pea protein. The salty peptide is prepared by controlled enzymatic hydrolysis of food-derived materials, which reduces costs and facilitates the large-scale production of salty peptides. The substance can be used as a food flavor regulator, and its application provides theoretical support for the development of new food condiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The MS spectra of peptide RF8, peptide DS5, and peptide YF6 are shown in Figure 1, where A represents peptide RF8, B represents peptide DS5, and C represents peptide YF6; Figure 2 These are the secondary mass spectra of peptide YS5, peptide FF5, and peptide YQ7, where D represents peptide YS5, E represents peptide FF5, and F represents peptide YQ7; Figure 3These are the secondary mass spectra of peptide RA7, peptide AR8, peptide FR6, and peptide ID5, where G represents peptide RA7, H represents peptide AR8, I represents peptide FR6, and J represents peptide ID5; Figure 4 The structural model diagram (A) and confidence interval diagram (B) of TMC4 receptor protein; Figure 5 Schematic diagrams of the complex conformations of peptide RF8 and peptide DS5 docked with TMC4 receptor molecules and their 3D and 2D interaction structures, where A represents peptide RF8 and B represents peptide DS5; Figure 6 Schematic diagram of the complex conformation and 3D and 2D interaction structure of peptide YF6 and peptide YS5 docked with TMC4 receptor molecules, respectively, where C represents peptide YF6 and D represents peptide YS5; Figure 7 Schematic diagram of the complex conformation and 3D and 2D interaction structure of peptide FF5 and peptide YQ7 docked with TMC4 receptor molecules, respectively, where E represents peptide FF5 and F represents peptide YQ7; Figure 8 Schematic diagram of the complex conformation and 3D and 2D interaction structure of peptide RA7 and peptide AR8 docked with TMC4 receptor molecules, respectively, where G represents peptide RA7 and H represents peptide AR8; Figure 9 Schematic diagram of the complex conformation and 3D and 2D interaction structure of peptide FR6 and peptide ID5 docked with TMC4 receptor molecules, respectively, where I represents peptide FR6 and J represents peptide ID5; Figure 10 This is the sensory radar chart of pea protein salty peptides; Figure 11 This is a diagram showing the saltiness intensity and salt-enhancing effect of pea protein salty peptides and salty peptides compounded with salt. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with examples, which are provided to explain the present invention and are not limited to the following examples: The flavor protease and compound protease used below were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0016] Example 1 A method for preparing a primary extract of pea protein salty peptides, comprising the following steps: Step 1: freeze-dry the pea protein to obtain pea protein powder; Step 2: Weigh 2g of pea protein powder, add 40mL of ultrapure water, adjust the pH to 7.0 with soda ash solution (the main component is sodium carbonate), add 7000U of flavor protease and compound protease (the addition ratio of flavor protease and compound protease is 1:5), place it in a magnetic stirring water bath at 51°C for enzymatic hydrolysis for 4.2h, then use a 95°C water bath to inactivate the enzyme for 15min, and then centrifuge at 10000rpm for 10min, collect the supernatant to obtain pea protein hydrolyzate; Step 3: The pea protein hydrolysate was ultrafiltered through an ultrafiltration system with a molecular cutoff of 1000 Da and a pressure not exceeding 0.15 MPa, and fractions <1 kDa were collected and freeze-dried in a vacuum (vacuum freeze-drying time was 72 h) to obtain fractions <1 kDa; Step 4: The fraction <1 kDa obtained from S3 was further separated and purified using an ÄKTA protein purifier. The parameters for separation and purification were injection volume 5 mL / time, ultrapure water as mobile phase, flow rate 0.5 mL / min, and UV detection wavelength 214 nm to obtain a fraction with a prominent salty taste. The fraction with a prominent salty taste was freeze-dried to obtain a primary extract of pea protein salty peptide.

[0017] Example 2

[0018] This example provides a method for screening pea protein salty peptides, the steps of which are as follows: S1: The salty pea protein salty peptide extract obtained in Example 1 was identified using LC-MS / MS. The specific technical parameters were: reversed-phase column: 75 μm ID × 50 cm, NanoViper C18 2 μm, 100Å; chromatographic instrument: ThermoFisher Easy-nLC 1200; mass spectrometer: Thermo Fisher Orbitrap Exploris 240; chromatographic separation time: 60 min; mobile phase A: 0.1% formic acid in water; mobile phase B: 80% ACN / 0.1% formic acid in water; flow rate: 250 nL / min. Multiple peptide sequences were obtained. S2: The peptide sequences obtained by S1 screening were used to generate peptide models using Chem3D Pro software. The peptide models were then ligand minimized using Auto Dock software to obtain the optimal peptide model. S3: The amino acid sequence of TMC4 receptor (NP_001138775) was retrieved from the NCBI database, and a homology model was constructed on the SWISS-MODEL website (https: / / swissmodel.expasy.org / ). The receptor model with the highest QMEANDisCoGlobal score was selected and used as the docking receptor model (e.g. Figure 4); S4: The peptides obtained in S2 were docked with the receptor model obtained in S3 using the molecular docking software Auto Dock to obtain peptide sequences that successfully docked with the TMC4 receptor. Peptides were screened based on binding energy, frequency of occurrence of salty segments, and ratio of hydrophobic amino acids. S5: The peptides screened in S4 were screened using the ToxinPred database (http: / / crdd.osdd.net / raghava / toxinpred / ) to obtain 10 non-toxic and non-allergenic peptide sequences, as shown in Table 1; the secondary mass spectra of the 10 peptides are shown in Figure 1 、 Figure 2 and Figure 3 The docking structures of 10 peptides and TMC4 receptor are shown in Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 and Figure 9 .

[0019] Table 1 Amino acid sequence information of 10 pea protein salty flavors

[0020] Example 3

[0021] Sensory evaluation and saltiness-enhancing effect evaluation test of pea protein salty peptide The 10 pea protein salty peptides obtained in Example 2 were subjected to solid phase synthesis, and the purity was greater than 95%. The salty properties and salt-enhancing effects of the 10 pea protein salty peptides were evaluated as follows: ① Sensory evaluation: Peptide RF8, peptide DS5, peptide YF6, peptide YS5, peptide FF5, peptide YQ7, peptide RA7, peptide AR8, peptide FR6, and peptide ID5 were prepared into 2 mg / mL peptide solutions in deionized water, and 3 mg / mL salt solution was used as the standard saltiness intensity reference solution for sensory analysis; ② Evaluation of saltiness-enhancing effect: Peptide RF8, peptide DS5, peptide YF6, peptide YS5, peptide FF5, peptide YQ7, peptide RA7, peptide AR8, peptide FR6, peptide ID5 and NaCl were respectively mixed with ultrapure water to prepare a mixed solution with a peptide concentration of 2 mg / mL and a NaCl concentration of 3 mg / mL, and the saltiness sensory evaluation was performed.

[0022] Depend on Figure 10It can be seen that the sensory properties of peptides RF8, DS5, YF6, YS5, FF5, YQ7, RA7, AR8, FR6 and ID5 are mainly sour, salty and umami. Among them, the highest salty characteristic is peptide FR6 (4.25), followed by peptide AR8 (4.125) and peptide RA7 (4), while the lowest salty characteristic is peptide RF8 (2.5); the peptides with higher umami and sour sensory scores are ID5 (umami 4.75, sour 7.0), peptide FF5 (umami The sensory properties of the 10 pea protein salty peptides, in addition to saltiness, sourness, and umami, are primarily attributed to the presence of the sour amino acids aspartic acid (Asp) and glutamic acid (Glu), which are also umami amino acids, and the sourness caused by the low pH buffer solution. The other 10 pea protein salty peptides had relatively balanced bitterness and sweetness scores, while peptide FR6 had the highest astringency score of 4.75. In summary, with the exception of peptide YS5, the remaining peptides were highly acceptable.

[0023] Depend on Figure 11 It can be seen that after compounding with NaCl, the highest saltiness sensory scores were obtained by peptides RF8 (5.9) and DS5 (5.9), followed by RA7 (5.8), while peptides YF6 and YS5 had the lowest saltiness sensory scores of 4.5. The saltiness sensory scores of the remaining peptides were all higher than 5. The highest saltiness increase rate was obtained by RF8 (136%), followed by FF5 (69.23%), while YS5 had the lowest saltiness increase rate of 20%. Figure 10 and Figure 11 According to the results, peptide RF8, peptide DS5, peptide YF6, peptide YS5, peptide FF5, peptide YQ7, peptide RA7, peptide AR8, peptide FR6 and peptide ID5 all have salty taste and have a certain salt-increasing effect, and can be used in food salt-reducing products; while peptide RA7, peptide AR8 and peptide FR6 have the best saltiness scores, and peptide RF8, peptide FF5 and peptide DS5 have the best salt-increasing effect.

[0024] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A pea protein-derived salty peptide, characterized in that: The pea protein salty peptide is one or more of peptide AR8, peptide RF8, peptide RA7, peptide FR6, peptide FF5, and peptide DS5, the amino acid sequence of the peptide AR8 is shown in SEQ ID NO.1, the amino acid sequence of the peptide RF8 is shown in SEQ ID NO.2, the amino acid sequence of the peptide RA7 is shown in SEQ ID NO.3, the amino acid sequence of the peptide FR6 is shown in SEQ ID NO.4, the amino acid sequence of the peptide FF5 is shown in SEQ ID NO.5, and the amino acid sequence of the peptide DS5 is shown in SEQ ID NO.

6.

2. The method for preparing pea protein-derived salty peptide according to claim 1, characterized in that: The following steps are involved: S1. Add water to pea protein to prepare a pea protein solution and adjust the pH to 6.8-7.

2. After enzymatic hydrolysis, inactivate the enzyme, cool, centrifuge, and obtain the supernatant to obtain a pea protein enzymatic hydrolyzate. S2. The pea protein hydrolysate was subjected to ultrafiltration, gel filtration chromatography, and freeze-drying to obtain a primary extract of pea protein salty peptides. S3. The pea protein salty peptide primary extract was sequenced using liquid chromatography-mass spectrometry to obtain multiple polypeptide sequences; S4. Using computer simulation technology to screen the multiple polypeptide sequences obtained in S3 for non-toxic polypeptide sequences that bind to the TMC4 protein receptor; S5. The polypeptide sequence obtained in S4 was synthesized by solid-phase synthesis, and the saltiness and saltiness enhancement intensity were analyzed by sensory analysis to obtain pea protein salty peptide.

3. The method for preparing pea protein-derived salty peptide according to claim 2, characterized in that: The mass ratio of pea protein to water in the S1 is 1:15-1:25; the added amount of enzyme in the S1 is 3000-4000 U / g; and the enzyme is prepared by mixing flavor protease and composite protease in a ratio of 1:3-1:

7.

4. The method for preparing pea protein-derived salty peptide according to claim 2, characterized in that: The conditions for enzymatic hydrolysis in S1 are as follows: an enzymatic hydrolysis temperature of 50-55° C. and an enzymatic hydrolysis time of 4.0-4.5 h; and the conditions for enzyme inactivation in S1 are as follows: an enzyme inactivation temperature of 90-100° C. and an enzyme inactivation time of 10-20 min.

5. The method for preparing pea protein-derived salty peptide according to claim 2, characterized in that: The specific parameters of the liquid chromatography-mass spectrometry technology in S3 are: NanoViper C18 reversed-phase chromatography column, specifications 75μm id × 50cm, 2μm, 100Å; chromatographic instrument: Thermo Fisher Easy-nLC 1200; mass spectrometer: Thermo Fisher Orbitrap Exploris 240; chromatographic separation time: 60min; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 80% ACN / 0.1% formic acid aqueous solution; flow rate: 250nL / min.

6. The method for preparing pea protein-derived salty peptide according to claim 2, characterized in that: The specific operation of S4 is to use the ToxinPred database to select non-toxic polypeptide sequences from the multiple polypeptide sequences obtained in S3, and then screen out polypeptide sequences that can bind to the TMC4 receptor protein through molecular docking technology, and finally obtain non-toxic polypeptide sequences that can bind to the TMC4 receptor protein.

7. Use of the pea protein-derived salty peptide according to claim 1 in the preparation of low-salt foods and food seasonings.

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