Goat milk bioactive polypeptide with anti-oxidation and angiotensin converting enzyme inhibition activity as well as preparation and application of goat milk bioactive polypeptide

Abstract

The invention discloses a goat milk bioactive polypeptide with antioxidation and angiotensin converting enzyme inhibition activity, and preparation and application thereof, and belongs to the field of biological polypeptides, and a preparation method and application thereof. In-vitro tests prove that the polypeptide has an excellent antioxidant effect, can remarkably reduce blood pressure and is beneficial to prevention and treatment of cardiovascular diseases, the interaction mechanism of the polypeptide and ACE is defined by using a molecular docking technology, scientific basis and targets are provided for research and development of new drugs, and the polypeptide has potential physiological functions such as antioxidant activity and ACE inhibitory activity and has a broad application prospect. The method is suitable for functional food and medicine development and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biological peptides and preparation methods and applications thereof, and specifically relates to a goat milk biologically active polypeptide with antioxidant and angiotensin converting enzyme inhibitory activity and preparation and application thereof. Background Art

[0002] As a nutritious food, goat milk contains a variety of bioactive ingredients, such as protein, fat, minerals, vitamins, and a variety of bioactive peptides. These ingredients give goat milk a variety of physiological functions, such as anti-oxidation, immunomodulation, hypoglycemia, and hypotension. Among them, bioactive peptides are important functional ingredients in goat milk and have broad application prospects. Peptides are a class of small molecules connected by multiple amino acids through peptide bonds, which have a variety of biological activities, such as antioxidant, anti-diabetic and hypotensive effects. Traditionally, the sources of peptides are mainly protein hydrolysates such as soybeans, fish, and dairy products. However, there are few studies on functional peptides from goat milk. Goat milk is not only rich in highly bioavailable proteins, but also has fewer allergens, making it an ideal source for peptide extraction.

[0003] Antioxidant activity refers to the ability of a substance to scavenge free radicals or inhibit the production of free radicals. Free radicals are harmful substances produced during human metabolism, which can damage cells and tissues, leading to aging, disease and other problems. Therefore, antioxidant activity is of great significance for maintaining human health. Certain bioactive peptides in goat milk have antioxidant activity, which can scavenge free radicals and protect cells and tissues from damage. Angiotensin converting enzyme (ACE) is an enzyme involved in the regulation of human blood pressure, which can catalyze the conversion of angiotensin I to angiotensin II, thereby increasing blood pressure levels. Inhibiting the activity of ACE can lower blood pressure levels, which is of great significance for the treatment of cardiovascular diseases such as hypertension. ACE inhibitors have become one of the important categories of drugs for the treatment of hypertension. Certain bioactive peptides in goat milk have the ability to inhibit the activity of ACE, and therefore have a potential blood pressure lowering effect.

[0004] In terms of activity, the existing technology for extracting bioactive peptides from goat milk makes it difficult to obtain multifunctional peptides with both antioxidant and angiotensin-converting enzyme (ACE) inhibition, and there is a lack of in-depth research on their mechanism of action. Summary of the invention

[0005] In view of the current technical status that it is difficult to extract bioactive polypeptides from goat milk that have both antioxidant activity and angiotensin converting enzyme inhibitory activity, the present invention aims to provide a goat milk bioactive polypeptide with antioxidant and angiotensin converting enzyme inhibitory activity and its preparation and application.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides a goat milk bioactive polypeptide with antioxidant and angiotensin converting enzyme inhibitory activities. The sequence of the goat milk bioactive polypeptide QEALELIR with antioxidant and angiotensin converting enzyme inhibitory activities is shown as SEQ ID NO.1.

[0008] The goat milk bioactive polypeptides having antioxidant and angiotensin converting enzyme inhibitory activities also include any one of the goat milk bioactive polypeptides QAQNVQHY (as shown in SEQ ID NO.2), NIPVGILR (as shown in SEQ ID NO.3) and LSMTDTR (as shown in SEQ ID NO.4).

[0009] The goat milk biologically active polypeptide with the activity of resisting oxidation and inhibiting angiotensin converting enzyme is artificially synthesized.

[0010] The goat milk bioactive polypeptide with antioxidant and angiotensin converting enzyme inhibitory activity is obtained by sequentially enzymatically hydrolyzing, separating and extracting fresh goat milk using two enzymes.

[0011] The method for extracting the above-mentioned goat milk bioactive polypeptides with antioxidant and angiotensin converting enzyme inhibitory activity comprises the following steps: enzymolysis of the pretreated fresh goat milk with pepsin and trypsin in turn, filtration separation, purification to obtain goat milk bioactive polypeptides, in vitro screening, using Keap1 and ACE enzymes as receptors respectively, and obtaining goat milk bioactive polypeptides with antioxidant and angiotensin converting enzyme inhibitory activity through molecular docking virtual screening technology.

[0012] The added amount of pepsin is 0.1% to 0.3% of the mass volume ratio of fresh goat milk.

[0013] Furthermore, the added amount of pepsin is 0.1%.

[0014] The pepsin hydrolysis conditions are: pH=2-3, temperature 35°C-39°C, and hydrolysis for 3h-5h.

[0015] Furthermore, the enzymatic hydrolysis conditions are: pH=3.0, temperature of 37°C, and enzymatic hydrolysis for 4 hours.

[0016] The added amount of the trypsin is 0.1% to 0.3% of the mass volume ratio of fresh goat milk, and the trypsin enzymolysis conditions are: pH = 7 to 8, temperature 35°C to 39°C, enzymolysis 6 to 9h, heating to 90 to 100°C and keeping warm for 10min to 20min to terminate the enzymolysis reaction.

[0017] Furthermore, the addition amount of trypsin is 0.1%, the heating temperature is 95° C., and the heating time is 10 min.

[0018] The filtration is carried out using an ultrafiltration membrane, and the ultrafiltration membrane has a molecular weight cutoff of ≤3 kDa.

[0019] The in vitro screening adopts a solid phase chemical synthesis method to synthesize and verify the antioxidant and ACE inhibitory abilities.

[0020] The present invention provides the use of the goat milk bioactive polypeptide having antioxidant and angiotensin converting enzyme inhibitory activities in the preparation of antioxidant and blood pressure lowering drugs.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The present invention provides a goat milk bioactive polypeptide with antioxidant and angiotensin converting enzyme inhibitory activity, which has excellent antioxidant activity, can scavenge free radicals, and protect cells from oxidative damage. This ability of the polypeptide is verified by an in vitro antioxidant activity test, indicating that it has a significant effect in protecting cells from oxidative damage; the polypeptide has a significant inhibitory effect on angiotensin converting enzyme (ACE), and by inhibiting ACE, the polypeptide can reduce vascular inflammation and improve vascular elasticity, thereby helping to maintain the overall health of the cardiovascular system; the goat milk bioactive polypeptide with antioxidant and angiotensin converting enzyme inhibitory activity of the present invention not only has excellent antioxidant and antihypertensive effects, but also clarifies the interaction mechanism with ACE through molecular docking technology, providing a theoretical basis and scientific basis for the development of new drugs, and can be used to develop new drugs with antioxidant, antihypertensive and other activities, and the clear interaction mechanism with the target protein provides a scientific basis and target for the research and development of new drugs.

[0023] The extraction method provided by the present invention adopts two enzymes (pepsin and trypsin) to enzymolyze fresh goat milk in sequence. Compared with the traditional process, this method simplifies the extraction process and improves the extraction efficiency; after enzymolysis, ultrafiltration membrane filtration is adopted to effectively remove macromolecular impurities, improve the purity of polypeptides, ensure the integrity of polypeptides, and avoid degradation caused by excessive enzymolysis; by optimizing enzymolysis conditions and purification steps, polypeptide degradation caused by excessive enzymolysis is avoided, the integrity of polypeptides is ensured, and the extracted polypeptides maintain good biological activity, including antioxidant activity and ACE inhibitory activity; the extraction method is experimentally verified, has stable process parameters and controllable extraction process, has high stability and reliability in industrial production, and is conducive to large-scale production; through molecular docking technology, the interaction mechanism between polypeptides and ACE is clarified, which provides a theoretical basis for the application of polypeptides in drug development; the materials such as enzymes and ultrafiltration membranes used are relatively low in cost and easy to obtain, and the production cost can be further reduced and the economic benefit can be improved by optimizing process parameters and improving extraction efficiency.

[0024] The application provided by the present invention, the goat milk bioactive polypeptide provided by the present invention has the effects of lowering blood pressure and anti-oxidation, can simultaneously meet the needs of hypertensive patients for blood pressure control and anti-oxidation protection, is derived from goat milk, is a natural product, and therefore has a lower safety risk; has significant ACE inhibitory activity, and therefore can effectively reduce the generation of angiotensin II, thereby playing a role in lowering blood pressure; contains a large amount of amino acids that are beneficial to blood vessels, can quickly improve vascular elasticity, increase the flexibility of vascular smooth muscle, and repair damage to the inner wall of blood vessels, thereby further lowering blood pressure; has excellent antioxidant properties, can interact with free radicals, and protect the body from the influence of diseases by delaying oxidation, helps to reduce the damage of oxidative stress to cells, thereby maintaining cell health and stability; can not only be used in antihypertensive and antioxidant drugs, but can also be further expanded to the fields of health care products, cosmetics, etc., and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the computer simulation of enzymatic cleavage of β-lactoglobulin of the present invention;

[0026] Figure 2 It is a Venn diagram of the reconstituted milk enzymatic polypeptides of fresh goat milk and skimmed goat milk powder of the present invention;

[0027] Figure 3 The molecular weight distribution of two groups of enzymatic peptides identified by the mass spectrometry technology of the present invention;

[0028] Figure 4 The length distribution of two groups of enzymatic peptides identified by the mass spectrometry technology of the present invention;

[0029] Figure 5 IC of the four peptides of the present invention to ACE, DPPH and ABTS 50 Inhibitory concentration curve, where a is LSMTDTR, b is NIPVGILR, c is QAQNVQHY, and d is QEALELIR;

[0030] Figure 6The present invention is a schematic diagram of the docking of four polypeptides with the oxidative stress receptor (1O8A), wherein A1 is a three-dimensional stereogram of the docking of LSMTDTR with the oxidative stress receptor (1O8A), B1 is a three-dimensional stereogram of the docking of NIPVGILR with the oxidative stress receptor (1O8A), C1 is a three-dimensional stereogram of the docking of QAQNVQHY with the oxidative stress receptor (1O8A), and D1 is a three-dimensional stereogram of the docking of QEALELIR with the oxidative stress receptor (1O8A); A2 is a three-dimensional stereogram of the docking of LSMTDTR with the oxidative stress receptor (1O8A), B2 is a two-dimensional stereogram of the docking of NIPVGILR with the oxidative stress receptor (1O8A), C2 is a two-dimensional stereogram of the docking of QAQNVQHY with the oxidative stress receptor (1O8A), and D2 is a two-dimensional schematic diagram of the docking of QEALELIR with the oxidative stress receptor (1O8A);

[0031] Figure 7 The present invention is a schematic diagram of the docking of four polypeptides with the receptor ACE enzyme (2FLU), wherein A1 is a three-dimensional stereogram of the docking of LSMTDTR with the receptor ACE enzyme (2FLU), B1 is a three-dimensional stereogram of the docking of NIPVGILR with the receptor ACE enzyme (2FLU), C1 is a three-dimensional stereogram of the docking of QAQNVQHY with the receptor ACE enzyme (2FLU), and D1 is a three-dimensional stereogram of the docking of QEALELIR with the receptor ACE enzyme (2FLU); A2 is a three-dimensional stereogram of the docking of LSMTDTR with the receptor ACE enzyme (2FLU), B2 is a two-dimensional stereogram of the docking of NIPVGILR with the receptor ACE enzyme (2FLU), C2 is a two-dimensional stereogram of the docking of QAQNVQHY with the receptor ACE enzyme (2FLU), and D2 is a two-dimensional schematic diagram of the docking of QEALELIR with the receptor ACE enzyme (2FLU). DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0033] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0034] Example 1

[0035] This embodiment provides a goat milk bioactive polypeptide with antioxidant and angiotensin converting enzyme inhibitory activity, and the specific steps are as follows:

[0036] 1. Extraction of goat milk bioactive peptides with antioxidant and angiotensin converting enzyme inhibitory activity

[0037] (1) Computer simulation of enzymatic hydrolysis

[0038] The complete amino acid sequence of goat milk protein (Caprahircus) was obtained from the UniProtKB protein database (https: / / www.uniprot.org / ), which is mainly composed of casein (β-casein: P33048) and whey protein (β-lactoglobulin: P02756); the “enzyme(s)action” function of the “Analysis” module in the BIOPEP-UWN database was selected to simulate in vivo hydrolysis.

[0039] Since various enzymes have different cleavage sites on proteins, the active peptides produced have different amino acid sequences, resulting in different activity spectra. Computer software (BIOPEP-UWN) can not only simulate the enzymatic digestion characteristics of proteases, but also predict the functional activity of the desired peptides, mainly based on the position and frequency of amino acid residues in the peptide sequence (Gangopadhyay et al., 2016). The purpose of this study was to understand and verify the active functions of peptides produced by digesting goat milk protein in the human gastrointestinal tract. We used the "Enzyme (S) ACTION" function in the BIOPEP-UWN database to select pepsin (EC 3.4.23.1) and trypsin (EC 3.4.21.4) to hydrolyze goat milk protein and obtain their cleavage sites, biological activities and theoretical hydrolysis degrees. In the prediction of biological activity functions, the frequencies of ACE inhibition, DPP-Ⅳ inhibition and antioxidant activity fragments accounted for a large proportion.

[0040] (2) Centrifuge 1 L of fresh goat milk at 7500 rpm for 10 min to remove fat and impurities.

[0041] (3) After centrifugation, add pepsin accounting for 0.1% of the mass volume ratio of fresh goat milk, and enzymatically hydrolyze at pH = 3.0 and 37°C for 4 hours; then quickly inactivate the enzyme for 10 minutes to fully lose its enzyme activity, add trypsin accounting for 0.1% of the mass volume ratio of fresh goat milk, and continue enzymatic hydrolysis at pH = 8.0 and 37°C for 8 hours, and heat to 95°C for 10 minutes to inactivate the enzyme; filter and separate the supernatant, freeze-dry, and then dissolve the freeze-dried sample in ultrapure water, using a molecular weight cutoff of ≤3kD. a is ultrafiltered and separated at 4°C with an ultrafiltration membrane, and components with a molecular weight of less than 3KDa are collected. The crude bioactive polypeptide from goat milk is obtained by vacuum freeze-drying technology; the crude bioactive polypeptide from goat milk is re-dissolved in ultrapure water, passed through a gel chromatography column, and eluted with ultrapure water. The elution conditions are: a sample volume of 1 mL, a flow rate of 0.4 mL / min, a UV detector with a sensitivity of 1.0, a detection wavelength of 220 nm, the collection tube is updated every 6 minutes, the collection is repeated multiple times, and then concentrated and freeze-dried again to obtain the bioactive polypeptide from goat milk.

[0042] 390 and 389 peptides were identified in fresh skim goat milk hydrolysate (FGMH) and reconstituted skim goat milk hydrolysate (GMPH), respectively, of which 387 peptides overlapped with each other, accounting for 98.7% of the total number of peptides. Figure 2 As shown, this indicates that FGMH and GMPH hydrolysates have a high degree of similarity in peptide composition, and the vast majority of peptides are shared.

[0043] See attached Figure 3 In the FGMH hydrolysate, 61.28% of the polypeptides have a molecular weight between 0KDa-1.0KDa, 36.92% of the polypeptides have a molecular weight between 1.0KDa-1.5KDa, and only 1.80% of the polypeptides have a molecular weight between 1.5KDa-3.0KDa; the polypeptide molecular weight distribution of GMPH hydrolysate is very similar to that of FGMH, and is also mainly concentrated in the range of 0-1.5KDa. This molecular weight distribution characteristic indicates that most of the polypeptides in the two hydrolysates are concentrated in the range of 0-1.5KDa. The polypeptides in the two hydrolysates are mainly small molecular peptides, which is due to the action mode of proteases. Small molecular peptides usually have better solubility, stability and biological activity, and therefore have broad application prospects in food, health products and medicine.

[0044] 2. LC-MS / MS mass spectrometry identification of goat milk bioactive peptides

[0045] The sequence of the goat milk bioactive peptides obtained by step enzymatic hydrolysis was identified by mass spectrometry, and then the goat milk bioactive peptides with antioxidant and angiotensin converting enzyme inhibitory activities were obtained by virtual screening.

[0046] The online prediction tools Peptide Ranker and ToxinPred were used to predict the toxicity and allergenicity of the active peptides identified and screened by mass spectrometry. Figure 4 It can be observed that the peptides formed by hydrolysis of goat milk protein are mainly composed of 7 to 10 amino acids, and the most common hydrophobic amino acids in the polypeptide sequence are Leu and Pro.

[0047] More than 300 peptides identified using mass spectrometry were evaluated online by Peptide Ranker. The results are shown in Table 1. The higher the score, the higher the biological activity. Peptide sequences with a score of 0.5 or above were selected for subsequent toxicity, sensitivity, and solubility evaluation. Specifically, ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php), AllergenFP V.1.0 (https: / / www.ddg-pharmfac.net / AllergenFP / ) and Innovagen (https: / / pepcalc.com / peptide-solubility-calculator.php) online programs were used to screen peptide toxicity, sensitization, and solubility.

[0048] Table 1.The two groups ofsamples hadatotalnumber ofpeptides withascore of>0.5fromthe

[0049] online scoring program.

[0050]

[0051]

[0052]

[0053] In addition, the same online program prediction was performed for 2 peptides unique to the GMPH group and 3 peptides unique to the FGMH group.

[0054] 3. Use solid phase chemical synthesis method to synthesize the target peptide and verify its antioxidant and ACE inhibitory ability

[0055] The peptide, HHL and ACE were dispersed in a certain volume of 0.1M sodium borate buffer (pH 8.3). 50μL of hipuryl-histidyl-leucine (HHL) solution (5.0mM) was mixed with 25μL of peptide solution; after incubation at 37°C for 10min, 25μL of 0.1U / mL ACE was added; incubation was performed again at 37°C for 30min, and 400μL of 0.3M NaOH was added to stop the reaction. Then 25μL of 2% o-phthalaldehyde (OPA) was added and allowed to stand at room temperature for 10min. Finally, 50μL of 3.0M HCl was added to the solution system to terminate the reaction. The excitation wavelength was 340nm and the emission wavelength was 455nm for fluorescence intensity detection. Captopril was used as a positive control. The ACE inhibition rate (%) of the peptide was calculated according to formula (1).

[0056]

[0057] ABTS cationic solution is prepared by adding ABTS solution (7.0 mM) to an equal volume of potassium persulfate (2.45 mM) solution and incubating at room temperature in the dark for 20 h. Before use, the ABTS cationic solution is continuously diluted with phosphate buffer (5.0 mM, pH 7.4) until the absorbance at 734 nm is 0.70 ± 0.02. The diluted ABTS cationic solution is mixed with 0.2 mL of sample, incubated at room temperature for 10 min, and the absorbance at 734 nm is measured. Phosphate buffer is used as a control instead of the sample. The ABTS scavenging activity of the polypeptide sample is calculated according to formula (2):

[0058]

[0059] Dissolve 1.0 mL of sample in 1 mL of 0.1 M sodium phosphate buffer (pH 7.4), and dissolve DPPH in ethanol (0.1 mM, 95%). Incubate for 30 min in the dark, and measure the absorbance at 517 nm using an ELISA reader. Use ethanol instead of the sample as the control group. The scavenging activity of the peptide against DPPH free radicals is calculated according to formula (3):

[0060]

[0061] Figure 5 The IC values ​​of four different peptides for ACE inhibition, DPPH free radical scavenging and ABTS free radical scavenging activities are shown. 50 curve.

[0062] By the attached Figure 5 The data shows that

[0063] The inhibitory activity of the peptide LSMTDTRACE is relatively strong, and the inhibition rate increases with the concentration, close to 80%; the DPPH scavenging activity is relatively weak, and the curve rises slowly; the ABTS scavenging activity is between ACE and DPPH, and the curve is steeper, indicating that it has a significant inhibitory effect even at lower concentrations.

[0064] The inhibitory activity of peptide NIPVGILR on ACE is relatively low, and the scavenging activity curves of DPPH and ABTS are similar, indicating good antioxidant properties.

[0065] Peptide QAQNVQHYACE inhibitory activity: high, and with the increase of concentration, the inhibition rate increased significantly.

[0066] The ACE inhibitory effect of the peptide QEALELIR remained stable within the tested concentration range, but was slightly lower than that of other peptides. However, its DPPH scavenging activity was significantly enhanced at higher concentrations, and the ABTS curve was steeper, indicating that it had a strong inhibitory effect at low concentrations.

[0067] In summary, the target peptides synthesized by solid phase chemical synthesis method showed different activity characteristics after verification of antioxidant and ACE inhibitory abilities. Among them, the peptides LSMTDTR and QAQNVQHY showed strong activity in ACE inhibition, while the peptide NIPVGILR had good antioxidant properties. The ACE inhibitory effect of the peptide QEALELIR was relatively stable, which provided an important reference for the subsequent development and optimization of peptide drugs.

[0068] 4. Molecular docking of the screened peptides with the antioxidant receptor Keap1 and ACE enzyme after ligand removal

[0069] The three-dimensional crystal structures of the receptor proteins human ACE-Lisinopril complex and keap1 were downloaded from the RCSB PDB database (https: / / www.rcsb.org / ), with PDB IDs 108a (resolution 2.00A) and 2FLU (resolution 1.50A), respectively. The three-dimensional structures of peptides LSMTDTR, NIPVGILR, QAQNVQHY and QEALELIR were generated through an online website (https: / / cloud.yinfotek.com / console / tools), and the structures were prepared and pdbqt files were generated using AutoDock Tools 1.5.6; ACE peptide molecule-specific docking pathway: Before docking analysis, the receptor protein was structurally processed using AutoDock Tools 1.5.6, the inhibitor lisinoprilide and water molecules were deleted, hydrogen atoms were added, Gasteiger charges were assigned, and it was converted to a pdbqt structure, retaining the zinc ion in 108a. Before docking analysis, PyMOL (1.7.6) software was used to build the ring, minimize the energy, remove water molecules, remove the Nrf-216-mer peptide, and perform protonation.

[0070] Figure 6The three-dimensional structural model (A1-D1) and the corresponding two-dimensional interaction map (A2-D2) of each peptide-receptor complex are shown. In terms of 3D structure, the peptide LMSTDTR binds to the active site of the receptor, and the surrounding amino acids include Glu, Ser, Asp and other residues, indicating the participation of hydrogen bonds and electrostatic interactions. In the corresponding 2D map, multiple red arcs mark the hydrophobic interactions formed with the receptor, and there are also several hydrogen bonds (dashed lines) and possible electrostatic interactions (such as interactions with Glu-411). The peptide NIPVGILR showed multi-point binding with Val, Lys, Tyr and other residues, indicating the existence of hydrophobic and polar interactions. The binding mode of the complex showed that this peptide had more contact points with the active site of the receptor, especially with key sites such as Lys and Asp, which enhanced the stability. The peptide QAQNVQHY interacted with Ser, Tyr, Gln and other residues. These polar amino acids were stably bound to the peptide through hydrogen bonds. The binding region of this complex was similar to that of other groups, indicating that they may be optimized for the same target region. Special attention was paid to the interaction between Ser-355 and Tyr-352, which was crucial to the activity of the complex. The binding of the peptide QEALELIR to the receptor was more complex, involving multiple amino acid residues, including Leu, His and Gln. Structurally, there were dense hydrogen bonds and hydrophobic interactions, indicating that the peptide had a strong binding ability. The participation of Leu-361 and His-319 may have enhanced the stability of the complex. Table 2 summarizes their binding energies and hydrogen bond numbers. QEALELIR has the strongest binding ability to the receptor (-7.7 Kcal / mol) because it shows the most complex interaction network and tight binding in structure; QAQNVQHY has the weakest binding ability (-5.4 Kcal / mol), which is consistent with its structural characteristics of relying on polar interactions and insufficient hydrophobicity. QEALELIR is a very potential candidate peptide because of its strong binding ability to the ACE enzyme; NIPVGILR and LSMTDTR also have good binding to the ACE enzyme and need to be optimized in terms of hydrophobicity to improve stability.

[0071] Figure 7 The molecular docking results of four peptides and protein receptor 2FLU (Keap1) are shown. Figure 7 The data shows that

[0072] LSMTDTR: mainly binds to key sites of the receptor through hydrogen bonds (Tyr-325, Gln-310, and Arg-311) and hydrophobic interactions (Ile-461, Phe-377), which has a positive impact on its activity;

[0073] NIPVGILR: forms a large number of hydrogen bonds with the receptor (Tyr-372, Gln-530, etc.), and multiple polar and charged residues participate, which stabilizes the ligand-receptor complex and enhances its biological activity;

[0074] QAQNVQHY: The binding mode includes hydrogen bonding (Tyr-334, Gln-520) and hydrophobic interaction (Phe-478), indicating that it has advantages in antioxidant or inhibitory activity;

[0075] QEALELIR: Relies on a small number of hydrogen bonds (Gln-530, Tyr-325) and limited polar interactions, with moderate binding stability.

[0076] The specific binding energies and hydrogen bond numbers between the four peptides and the receptor protein are shown in Table 2.

[0077] Table 2: Specific binding energy and number of hydrogen bonds between four peptides and receptor proteins

[0078]

[0079] From the data in Table 2, we can see that the lower the binding energy (the more negative), the more stable the ligand-receptor complex. For humanACE-Lisinopril complex, QEALELIR has the lowest binding energy (-7.7Kcal / mol) and QAQNVQHY has the highest binding energy (-5.4Kcal / mol); for keap1(2FLU), QAQNVQHY has the lowest binding energy (-7.4Kcal / mol) and LSMTDTR has the highest binding energy (-6.6Kcal / mol).

[0080] In summary, QAQNVQHY has the lowest binding energy (-7.4Kcal / mol), indicating that it binds most firmly to the receptor and has more advantages in terms of biological activity. LSMTDTR has the highest binding energy (-6.6Kcal / mol), indicating that its binding is relatively weak and its activity needs to be improved by increasing hydrogen bonds or optimizing the sequence. The binding of these peptides to proteins related to the oxidative stress pathway affects their activity, and thus exhibits different biological activities in terms of anti-oxidation or anti-diabetes.

[0081] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A goat milk bioactive polypeptide having antioxidant and angiotensin converting enzyme inhibitory activity, characterized in that: The sequence of the goat milk bioactive polypeptide having antioxidant and angiotensin converting enzyme inhibitory activities is shown in SEQ ID NO.

1.

2. The goat milk bioactive polypeptide having antioxidant and angiotensin converting enzyme inhibitory activity according to claim 1, characterized in that: The goat milk bioactive polypeptide having antioxidant and angiotensin converting enzyme inhibitory activities also includes any one of the goat milk bioactive polypeptides whose sequences are shown in SEQ ID NOs. 2 to 4.

3. The goat milk bioactive polypeptide having antioxidant and angiotensin converting enzyme inhibitory activity according to claim 1 or 2, characterized in that: The goat milk biologically active polypeptide with the activity of resisting oxidation and inhibiting angiotensin converting enzyme is artificially synthesized.

4. The goat milk bioactive polypeptide having antioxidant and angiotensin converting enzyme inhibitory activity according to any one of claims 1 to 3, characterized in that: The goat milk bioactive polypeptide with antioxidant and angiotensin converting enzyme inhibitory activity is obtained by sequentially enzymatically hydrolyzing, separating and extracting fresh goat milk using two enzymes.

5. The method for extracting the goat milk bioactive polypeptide having antioxidant and angiotensin converting enzyme inhibitory activity according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: enzymolyzing the pretreated fresh goat milk with pepsin and trypsin in turn, filtering and separating, purifying to obtain goat milk bioactive polypeptides, and screening in vitro, using Keap1 and ACE enzymes as receptors respectively, and obtaining goat milk bioactive polypeptides with antioxidant and angiotensin converting enzyme inhibitory activities through molecular docking virtual screening technology.

6. The method for extracting goat milk bioactive polypeptides having antioxidant and angiotensin converting enzyme inhibitory activities according to claim 5, characterized in that: The added amount of pepsin is 0.1% to 0.3% of the mass volume ratio of fresh goat milk.

7. The method for extracting goat milk bioactive polypeptides having antioxidant and angiotensin converting enzyme inhibitory activities according to claim 5, characterized in that: The pepsin hydrolysis conditions are: pH=2-3, temperature 35-39° C., and hydrolysis for 3-5 hours.

8. The method for extracting goat milk bioactive polypeptides having antioxidant and angiotensin converting enzyme inhibitory activities according to claim 5, characterized in that: The added amount of the trypsin is 0.1% to 0.3% of the mass volume ratio of fresh goat milk, and the trypsin enzymolysis conditions are: pH = 7 to 8, temperature 35°C to 39°C, enzymolysis 6h to 9h, heating to 90°C to 100°C and keeping warm for 10min to 20min to terminate the enzymolysis reaction.

9. The method for extracting goat milk bioactive polypeptides having antioxidant and angiotensin converting enzyme inhibitory activities according to claim 1, characterized in that: The filtration separation adopts ultrafiltration membrane filtration, and the ultrafiltration membrane has a molecular weight cutoff of ≤3kDa. The in vitro screening adopts a solid phase chemical synthesis method to synthesize and verify the antioxidant and ACE inhibitory abilities.

10. Use of the goat milk bioactive polypeptide having antioxidant and angiotensin converting enzyme inhibitory activity according to any one of claims 1 to 4 in the preparation of antioxidant and blood pressure lowering drugs.

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