Novel umami peptide with ACE inhibitory activity as well as preparation method and application of novel umami peptide

Abstract

The invention discloses a novel umami peptide with ACE (angiotensin converting enzyme) inhibitory activity as well as a preparation method and application thereof, and belongs to the technical field of bioactive peptide biology. The umami peptide with ACE inhibitory activity is prepared and identified from viscera of sea cucumbers, can enhance umami perception in the presence of salt, plays a role in reducing salt and increasing freshness, also has ACE inhibitory activity, plays a role in relieving cardiovascular diseases such as hypertension and the like, and has application value in preparation of products with the effect of reducing blood pressure. The umami peptide provided by the invention enhances umami perception in the presence of salt, plays a role in reducing salt and increasing the umami, has ACE inhibitory activity, has the characteristics of simple structure, safety, strong activity and the like, can play nutritional and health-care roles, is expected to provide effective active ingredients for developing foods, food seasonings, pressure-reducing products without side effects and the like, and has broad market prospects. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of active peptide biotechnology, and in particular to a novel umami peptide with ACE inhibitory activity and a preparation method and application thereof. Background Art

[0002] Umami peptides, as a class of short peptides generated by the hydrolysis of natural proteins, have attracted widespread attention due to their umami taste and diverse functionality. Umami peptides are mainly rich in glutamic acid, aspartic acid and other amino acids with umami properties. They are widely used in the food industry to enhance flavor and reduce salt addition. The significance of umami peptides not only lies in enhancing food flavor, but also in their potential multifunctionality, including specific physiological regulatory functions such as antioxidant, anti-inflammatory, lowering blood pressure and lowering blood sugar. Umami peptides can reduce salt and increase freshness while also having certain functionality. This is currently an important direction for the development of umami peptides from natural raw materials.

[0003] Hypertension is one of the main risk factors for cardiovascular disease, and angiotensin-converting enzyme (ACE) plays an important role in its pathogenesis. ACE promotes blood pressure by catalyzing the conversion of angiotensin I to angiotensin II (a potent vasoconstrictor) and degrading bradykinin (a vasodilator). Therefore, inhibiting ACE activity is considered an important therapeutic approach to control blood pressure. Although chemically synthesized ACE inhibitors (such as captopril and enalapril) have shown good antihypertensive effects in clinical practice, their potential side effects limit long-term use by patients. In contrast, functional peptides with ACE inhibitory activity derived from natural proteins have become a more ideal choice due to their high safety and low side effects.

[0004] As a high-protein, low-fat marine organism, sea cucumber is rich in a variety of nutrients, including collagen, sulfated polysaccharides, sea cucumber saponins and abundant trace elements. In particular, the amino acid composition of the protein in sea cucumber is unique, containing a high proportion of hydrophobic amino acids (such as alanine, leucine) and aromatic amino acids (such as phenylalanine, tyrosine). These characteristics provide the possibility for its protein to generate functional peptides with ACE inhibitory activity after hydrolysis. In addition, as a traditional food, sea cucumber has long been considered safe and reliable, and its processing byproducts (such as viscera and skin) have also been shown to contain a large amount of protein resources. These characteristics make sea cucumber an important potential source for the development of umami peptides with ACE inhibitory activity. Existing studies have shown that through appropriate enzymatic treatment, multifunctional peptides that have both umami characteristics and can inhibit ACE activity can be isolated from sea cucumber proteins. This not only helps to develop high-value-added functional foods, but also provides a new direction for natural intervention methods for hypertension. Therefore, providing new sea cucumber umami peptides has become a research hotspot. Summary of the invention

[0005] The purpose of the present invention is to provide a novel umami peptide with ACE inhibitory activity and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions: One of the technical solutions of the present invention is an umami peptide with ACE inhibitory activity, whose amino acid sequence is IDGAVFP.

[0007] The second technical solution of the present invention is a method for preparing the umami peptide, comprising the following steps: (1) using aminopeptidase and flavor protease to enzymatically hydrolyze the sea cucumber viscera to obtain a sea cucumber viscera enzymatic hydrolyzate; (2) After the sea cucumber viscera hydrolysate is subjected to ultrafiltration and desalting treatment, the peptide sequences in the sea cucumber viscera hydrolysate are identified by LC-MS / MS liquid chromatography-mass spectrometry to construct a sea cucumber viscera hydrolysate peptide database, and an enzymatic hydrolysate containing the umami peptide with ACE inhibitory activity is obtained; (3) Screening out umami peptides with potential ACE inhibitory activity.

[0008] The third technical solution of the present invention is the use of the umami peptide in the preparation of angiotensin converting enzyme inhibitors.

[0009] A fourth technical solution of the present invention is an angiotensin converting enzyme inhibitor, comprising the umami peptide.

[0010] A fifth technical solution of the present invention is the use of the umami peptide in the preparation of blood pressure lowering drugs.

[0011] The sixth technical solution of the present invention is a blood pressure lowering drug, comprising the umami peptide.

[0012] The seventh technical solution of the present invention is the use of the umami peptide in improving the umami taste of food or in the preparation of products for improving the umami taste of food.

[0013] An eighth technical solution of the present invention is a food seasoning comprising the umami peptide.

[0014] Based on the above technical solution, the present invention has the following technical effects: The present invention prepares and identifies an umami peptide with ACE inhibitory activity from sea cucumber viscera, which can not only enhance the umami perception in the presence of salt, thereby reducing salt and increasing the umami taste, but also has ACE inhibitory activity, thereby alleviating cardiovascular diseases such as hypertension, and has application value in the preparation of products with antihypertensive effect.

[0015] The umami peptide involved in the present invention enhances the perception of umami in the presence of salt, plays the role of reducing salt and increasing umami, and at the same time has ACE inhibitory activity, has the characteristics of simple structure, safety, strong activity, etc., can play a role in nutrition and health care, and is expected to provide effective active ingredients for the development of food, food condiments and blood pressure-lowering products without side effects, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is the secondary mass spectrometry identification diagram of the amino acid structure and sequence of umami peptide with ACE inhibitory activity.

[0018] Figure 2 The docking interaction diagram of the umami peptide IDGAVFP with ACE inhibitory activity and the umami receptor. A is a schematic diagram of the overall three-dimensional conformation of the umami peptide IDGAVFP docking with the umami receptor, B is a local enlarged view of the box in A, and C is a two-dimensional schematic diagram of the docking interaction force between the umami peptide IDGAVFP and the umami receptor.

[0019] Figure 3 The sensory score diagram of IDGAVFP, an umami peptide with ACE inhibitory activity, in the presence of different salt concentrations.

[0020] Figure 4 This is a graph showing the inhibition rate of ACE by the umami peptide IDGAVFP with ACE inhibitory activity at different concentrations.

[0021] Figure 5 The molecular simulation docking diagram of the umami peptide IDGAVFP with ACE inhibitory activity and ACE. A is a three-dimensional conformational diagram of the docking site amino acids and interaction forces between the umami peptide IDGAVFP and ACE, and B is a two-dimensional diagram of the docking interaction forces between the umami peptide IDGAVFP and the umami receptor. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0028] The embodiment of the present invention provides an umami peptide with ACE inhibitory activity, and the amino acid sequence thereof is IDGAVFP.

[0029] The present invention also provides a method for preparing the umami peptide, comprising the following steps: (1) enzymatically hydrolyzing the sea cucumber viscera using aminopeptidase and flavor protease to obtain a sea cucumber viscera enzymatic hydrolyzate; (2) After the sea cucumber viscera hydrolysate is subjected to ultrafiltration and desalting treatment, the peptide sequences in the sea cucumber viscera hydrolysate are identified by LC-MS / MS liquid chromatography-mass spectrometry to construct a sea cucumber viscera hydrolysate peptide database, and an enzymatic hydrolysate containing the umami peptide with ACE inhibitory activity is obtained; (3) Screening out umami peptides with potential ACE inhibitory activity.

[0030] In some specific embodiments, the enzymatic hydrolysis conditions are: the amount of aminopeptidase used is 1000 U / g, the amount of flavor protease used is 500 U / g, the amount of sea cucumber viscera substrate is 10%, the enzymatic hydrolysis is at 50°C for 18 h, and the enzymatic hydrolysis pH is 7.

[0031] In some specific embodiments, the screening method is: identifying the molecular weight and sequence of peptides in the sea cucumber viscera hydrolysate, and screening out 5 ppm peptides; further predicting the ACE inhibitory activity and umami taste of the peptides, and based on the umami taste score, screening out umami peptides with potential ACE inhibitory activity, and analyzing their secondary mass spectrometry structure to obtain the amino acid sequence and structure of the umami peptide.

[0032] The embodiment of the present invention also provides the use of the umami peptide in the preparation of angiotensin converting enzyme inhibitors.

[0033] The embodiment of the present invention also provides an angiotensin converting enzyme inhibitor, comprising the umami peptide.

[0034] The embodiment of the present invention also provides the use of the umami peptide in the preparation of a blood pressure lowering drug.

[0035] The embodiment of the present invention also provides a blood pressure lowering drug, comprising the umami peptide.

[0036] The embodiments of the present invention also provide the use of the umami peptide in improving the umami taste of food or in preparing a product that improves the umami taste of food.

[0037] The embodiment of the present invention also provides a food seasoning, comprising the umami peptide.

[0038] The umami peptide with ACE inhibitory activity provided by the present invention can not only clearly feel the umami taste in the presence of salt, increase the palatability, and achieve the effect of reducing salt and increasing the umami taste, but also can effectively inhibit the activity of ACE, play a role in preventing or even alleviating diseases such as hypertension, and has important significance for the actual production and theoretical research of functional umami peptides.

[0039] Example 1 Preparation of umami peptides from sea cucumber viscera After pretreatment such as homogenization, the sea cucumber viscera were hydrolyzed by aminopeptidase and flavor protease. The dosage of aminopeptidase was 1000 U / g, the dosage of flavor protease was 500 U / g, the dosage of sea cucumber viscera substrate was 10%, the hydrolysis condition was 50℃ for 18 h, and the pH value of the hydrolysis system was continuously monitored and adjusted to 7 during the hydrolysis process. The sea cucumber viscera hydrolysate was ultrafiltered and desalted using a solid phase extraction cartridge (SEP-PAK C18 solid phase extraction cartridge, Waters, USA) to prepare sea cucumber viscera hydrolysate containing umami peptides. LC-MS / MS liquid chromatography-mass spectrometry was further used to determine the molecular weight and sequence of each peptide prepared in the sea cucumber viscera hydrolysate to form a sea cucumber viscera hydrolysate peptide database.

[0040] Example 2 Screening and mining of umami peptide IDGAVFP with ACE inhibitory activity The molecular weight and sequence of peptides in the sea cucumber viscera hydrolysate were identified by LC-MS / MS liquid phase mass spectrometry, and 1222 peptides of 5 ppm were screened using the DeltaM tool. The ACE inhibitory activity and umami taste of the peptides were further predicted using the TastePeptidesDM and UMPred-FRL umami score predictors, the PeptideRanker functional activity predictor, and the Toixinpred safety and toxicity evaluation predictor. Based on the umami score, umami peptides with potential ACE inhibitory activity were screened, and their secondary mass spectrometry structures were analyzed to obtain the amino acid sequence and structure of the umami peptides. The results are as follows Figure 1 As shown, the secondary mass spectrum of the screened umami peptide shows that the amino acid sequence and arrangement of the umami peptide are IDGAVFP.

[0041] Example 3 Molecular simulation docking of ACE-inhibitory umami peptide IDGAVFP with umami taste receptor T1R1 / R1R3 The umami peptide with ACE activity prepared and mined from sea cucumber viscera was docked with the umami receptor T1R1 / R1R3 by molecular simulation to further determine the umami characteristics and taste mechanism of the umami peptide. The three-dimensional conformational map of the umami peptide IDGAVFP was drawn using Discovery studio software, and molecular simulation docking was performed with the active center of the constructed umami receptor T1R1 / R1R3, i.e., the cavity hairpin binding region. Based on the docking results, the interaction force between the umami peptide IDGAVFP and the key amino acid residues in the active center of the umami receptor T1R1 / R1R3 was analyzed.

[0042] The results are as follows Figure 2As shown, the umami peptide IDGAVFP forms 6 traditional hydrogen bonding forces, 1 pi-pi stacking force, 1 pi-alkyl force and multiple van der Waals forces with the receptor binding cavity region in the umami receptor T1R1 / R1R3. And the amino acids in the umami peptide IDGAVFP sequence bind to key amino acid residues in the umami receptor T1R1 / R1R3, such as Ala170, Asp147, His71, Ser276, Gln278 and Arg277, thus showing umami characteristics. Therefore, through the molecular simulation docking of the umami peptide IDGAVFP with the umami receptor T1R1 / R1R3, it is further determined that the umami peptide IDGAVFP prepared and identified from the sea cucumber viscera has umami characteristics.

[0043] Example 4 Sensory evaluation of umami taste of ACE-inhibitory peptide IDGAVFP at different salt concentrations Ten healthy sensory evaluation panel members (6 females and 4 males, aged 24-30 years old) with sensory evaluation experience were recruited from the laboratory. First, the evaluation panel members were trained in sensory evaluation for 2 weeks. The training content was to present aqueous solutions of six sour, sweet, bitter, salty, fresh and astringent taste standards to the sensory evaluators. The umami standard was sodium glutamate, which was the main sensory evaluation indicator of the present invention. Different concentrations of the umami peptide IDGAVFP described in the present invention (0.5, 1, 1.5 mg / mL) and different concentrations of NaCl (1, 1.5, 2, 3 mg / mL) were prepared into different solutions, which were presented to the sensory evaluators to taste and rank their umami intensity and record the scores.

[0044] The results are as follows Figure 3 As shown, different concentrations of the umami peptide IDGAVFP can show a certain umami enhancement effect in the presence of different concentrations of NaCl, that is, when the concentration of the umami peptide IDGAVFP is 0.5-1 mg / mL, only a low salt concentration (1-1.5 mg / mL) is required to provide a higher level of umami; and at a higher concentration of the umami peptide IDGAVFP, a comparable umami level can be produced under low salt (1-1.5 mg / mL) conditions compared to high salt (2-3 mg / mL). The above results indicate that the umami peptide IDGAVFP has a certain effect of reducing salt and increasing umami.

[0045] Example 5 Verification of ACE inhibitory activity of umami peptide IDGAVFP The ACE inhibitory activity of the umami peptide IDGAVFP was determined using a 96-well plate using a visible spectrophotometer. The ACE inhibition rate was determined using 1 mM N-[3-(2-furanyl)acryloyl]-L-phenylalanylglycylglycine (FAPPG) as a substrate; 0.1 U / mL angiotensin converting enzyme (ACE); and HEPES as a buffer matrix (1.901 g HEPES reagent and 1.755 g NaCl reagent were weighed and dissolved in an appropriate amount of deionized water, and then the buffer pH was adjusted to 8.3 with NaOH solution, and the volume was made up to 100 mL for standby use). The assay steps are as follows: different concentrations of umami peptide IDGAVFP samples (0.5, 1, 1.5, 2 mg / mL), as well as FAPPG substrate, ACE and buffer were added to a 96-well plate in sequence; the absorbance of the sample system at 340 nm was measured using an ELISA reader and recorded; the sample system was placed in a 37°C shaker for 30 min, and the absorbance of the sample system after the reaction was measured again at 340 nm using an ELISA reader; the ACE inhibition rate of the sample was determined by calculating the change in absorbance before and after the reaction.

[0046] The calculation formula is: ACE inhibition rate of sample %=1-change in sample absorbance value / change in blank absorbance value.

[0047] The results are as follows Figure 4 As shown, when the concentration of the umami peptide IDGAVFP is 0.5 mg / mL, the inhibition rate of the umami peptide IDGAVFP is 92.5%; when the concentration of the umami peptide IDGAVFP is 1 mg / mL, the inhibition rate of ACE is 102.7%; when the concentration of the umami peptide IDGAVFP is 1.5 mg / mL, the inhibition rate of ACE is 100.6%; when the concentration of the umami peptide IDGAVFP is 2 mg / mL, the inhibition rate of the umami peptide IDGAVFP is 99.8% (see Figure 4 ). According to the results of ACE inhibitory activity assay, when the concentration of the umami peptide IDGAVFP is greater than 0.5 mg / mL, it can completely inhibit ACE. Combined with the sensory evaluation results in Example 4, when the concentration of the umami peptide IDGAVFP is 0.5-1 mg / mL, it can provide a considerable level of umami taste for solutions with high and low salt concentrations, and within this concentration range, the umami peptide IDGAVFP can also completely inhibit ACE, indicating that the umami peptide IDGAVFP can also have a certain ACE inhibitory effect at a concentration that can reduce salt and increase freshness.

[0048] In summary, the umami peptide IDGAVFP described in the present invention has a certain ACE inhibitory activity, and at a concentration that can reduce salt, increase freshness and present umami characteristics, it can also effectively inhibit the activity of ACE.

[0049] Example 6 Molecular simulation docking of ACE-inhibitory peptide IDGAVFP with ACE The umami peptide with ACE activity prepared and mined from sea cucumber viscera was docked with ACE by molecular simulation to further determine the mechanism of action of the umami peptide when it exerts its ACE inhibitory activity. The three-dimensional conformational map of the umami peptide IDGAVFP was drawn using Discovery studio software, and molecular simulation docking was performed with the catalytic active center of ACE (PDB: 1o8a), and the interaction force between the umami peptide IDGAVFP and the key amino acid residues in the active center of ACE was analyzed based on the docking results.

[0050] The results are as follows Figure 5 As shown in the figure, the umami peptide IDGAVFP with ACE inhibitory activity can bind tightly to the ACE active center and can be simulated and docked within the normal analysis time. The umami peptide IDGAVFP with ACE inhibitory activity interacts with multiple amino acid residues in the ACE active center, and the main amino acid residues are Phe527, Try520, His383, Glu384, Gln281, His513, Tyr523, Glu403, His410, Pro407, Val379, His353, Lys511 and Zn 2+ .

[0051] The results of the interaction analysis between the ACE inhibitory peptide IDGAVFP and the amino acid residues in the active center of ACE are shown in Figure 2. Figure 5 The umami peptide IDGAVFP with ACE inhibitory activity forms 4 hydrogen bond forces with ACE, 5 interactions between alkyl groups and π-alkyl groups, 1 salt bridge and 1 mutual attraction force, 2 carbon-hydrogen forces, and Zn 2+ Combination effect.

[0052] In summary, the umami peptide IDGAVFP with ACE inhibitory activity can bind to ACE mainly through hydrogen bonds, hydrophobic interactions and Zn 2+ The binding effect interacts with the active center residues of ACE and inhibits the activity of ACE. That is, from the perspective of molecular simulation docking, it is further proved that the umami peptide IDGAVFP has significant ACE inhibitory activity.

[0053] In summary, the present invention prepares and identifies an umami peptide IDGAVFP with ACE inhibitory activity from sea cucumber viscera by a composite enzyme hydrolysis method. The present invention obtains its amino acid sequence and structure by performing secondary mass spectrometry analysis on the umami peptide, and explains its umami characteristics and taste mechanism by molecular simulation docking with umami receptor T1R1 / T1R3 and umami sensory evaluation under different salt concentrations. The ACE inhibitory activity of the umami peptide IDGAVFP is further verified, and the umami peptide is docked with the ACE active center by molecular simulation docking to explain its inhibitory mechanism on ACE activity.

[0054] The umami peptide IDGAVFP with ACE inhibitory activity provided by the present invention can present a high level of umami at a certain concentration, and the umami levels presented at low salt and high salt concentrations are comparable, having a certain salt-reducing and umami-enhancing effect; and at this flavor concentration, the umami peptide IDGAVFP can also significantly inhibit the activity of ACE, having a certain blood pressure-lowering effect.

[0055] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A novel umami peptide with ACE inhibitory activity, characterized in that: Its amino acid sequence is IDGAVFP.

2. The method for preparing the novel umami peptide according to claim 1, characterized in that: The following steps are involved: (1) enzymatically hydrolyzing the sea cucumber viscera using aminopeptidase and flavor protease to obtain a sea cucumber viscera enzymatic hydrolyzate; (2) After the sea cucumber viscera hydrolysate is subjected to ultrafiltration and desalting treatment, the peptide sequences in the sea cucumber viscera hydrolysate are identified by LC-MS / MS liquid chromatography-mass spectrometry to construct a sea cucumber viscera hydrolysate peptide database, and an enzymatic hydrolysate containing the novel umami peptide with ACE inhibitory activity is obtained; (3) Screening of novel umami peptides with potential ACE inhibitory activity.

3. The preparation method according to claim 2, characterized in that: The enzymatic hydrolysis conditions are as follows: the use amount of aminopeptidase is 1000 U / g, the use amount of flavor protease is 500 U / g, the use amount of sea cucumber viscera substrate is 10%, the enzymatic hydrolysis is carried out at 50° C. for 18 hours, and the enzymatic hydrolysis pH is 7.

4. The preparation method according to claim 2, characterized in that: The screening method comprises: identifying the molecular weight and sequence of peptides in the sea cucumber viscera enzymatic hydrolysate, and screening out 5 ppm peptides; further predicting the ACE inhibitory activity and umami taste of the peptides, and screening out novel umami peptides with potential ACE inhibitory activity based on the umami taste score, and analyzing the secondary mass spectrometry structure thereof to obtain the amino acid sequence and structure of the novel umami peptide.

5. Use of the novel umami peptide according to claim 1 in the preparation of angiotensin converting enzyme inhibitors.

6. An angiotensin converting enzyme inhibitor, characterized in that The invention comprises the novel umami peptide according to claim 1.

7. Use of the novel umami peptide according to claim 1 in the preparation of antihypertensive drugs.

8. A blood pressure lowering drug, characterized in that: The invention comprises the novel umami peptide according to claim 1.

9. Use of the novel umami peptide according to claim 1 in improving the umami taste of food or in preparing products for improving the umami taste of food.

10. A food seasoning, characterized in that: The invention comprises the novel umami peptide according to claim 1.

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