Silver carp skin bone ACE (angiotensin converting enzyme) inhibitory peptide as well as preparation method and application thereof

Abstract

The invention discloses a silver carp skin bone ACE (angiotensin converting enzyme) inhibitory peptide as well as a preparation method and application thereof, and relates to the technical field of ACE inhibitory peptides. The preparation method of the silver carp skin bone ACE inhibitory peptide provided by the invention comprises the following steps: performing steam explosion treatment on a mixture of skin and bone of silver carp, and centrifuging to take a middle layer to obtain steam explosion liquid; adjusting the pH value of the steam explosion liquid, and adding alkaline protease according to the mass of the steam explosion liquid for enzymolysis until the pH value is neutral; and adding flavourzyme according to the mass of the steam explosion liquid for enzymolysis to obtain a primary enzymolysis product containing the silver carp skin bone ACE inhibitory peptide. And carrying out ultrafiltration and screening analysis on the primary enzymolysis product to obtain five ACE inhibitory peptides. The primary enzymatic hydrolysate, the ultrafiltration product and the ACE inhibitory peptide all have better ACE activity inhibition rate, and can be widely applied to preparation of drugs for lowering blood pressure or functional foods for assisting in lowering blood pressure.

Description

Technical Field

[0001] The present invention relates to the technical field of ACE inhibitory peptides, and particularly relates to a silver carp skin and bone ACE inhibitory peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Hypertension is classified into primary hypertension and secondary hypertension according to etiology or population characteristics. Primary hypertension has no obvious single etiology and is related to comprehensive factors such as genetics, age, obesity, and high-salt diet, accounting for 90%-95% of the total hypertensive population. It is mainly treated by long-term lifestyle intervention + drug control (such as diuretics, ACEI / ARB, CCB, etc.).

[0003] Angiotensin-I converting enzyme (ACE) causes blood pressure to rise by converting angiotensin I into angiotensin II, inactivating bradykinin with vasodilatory effects, and promoting aldosterone secretion. Therefore, the effect of treating hypertension can be achieved by inhibiting ACE activity. Currently, commonly used clinical ACE inhibitors include enalapril, captopril, benazepril, ramipril, lisinopril, etc., which have obvious therapeutic effects on treating hypertension. However, long-term use is likely to cause problems such as kidney function damage, hyperkalemia, allergic reactions, and fetal dysplasia.

[0004] Compared with chemically synthesized ACE inhibitors, food-derived polypeptide ACE inhibitors have the advantages of high safety, easy absorption, and low side effects, providing a new way for the prevention and treatment of hypertension. Currently, there are various bioactive collagen peptide products in China, but they generally have a relatively large molecular weight, resulting in a low content of effective active ingredients and low product purity.

[0005] China is rich in aquatic resources, with a high output of freshwater fish. A large amount of fish skin, fish bone, and fish scale are produced during the processing. They contain rich collagen, mineral calcium, phosphorus, etc., and are high-quality sources of bioactive peptides and calcium. However, in industrialized surimi production, by-products such as fish skin and fish bone usually exist in a mixture form and are difficult to separate. They are mainly used for producing feed, with low added value. Currently, the high-value utilization methods of fish skin, fish scale, and fish bone are mainly for separately extracting collagen or collagen peptides. If the fish skin and bone mixture can be fully recycled to develop polypeptide ACE inhibitors, it can provide important support for the research and development of functional products from aquatic product by-products and food-derived polypeptide antihypertensive drugs. Summary of the Invention

[0006] The present invention provides a silver carp skin and bone ACE inhibitory peptide, a preparation method thereof and an application. The preparation method of the silver carp skin and bone ACE inhibitory peptide is to first perform steam explosion treatment on the mixture of the fish skin and fish bones of silver carp, and then add alkaline protease and flavor protease for enzymatic hydrolysis to obtain a primary enzymatic hydrolysis product containing the silver carp skin and bone ACE inhibitory peptide; it can also be subjected to nanofiltration and ultrafiltration to obtain ultrafiltration products with different molecular weights, and finally 5 peptide segments with ACE inhibitory activity are screened from the ultrafiltration products. Specifically, it is achieved through the following techniques.

[0007] In the first aspect of the present invention, a preparation method of a silver carp skin and bone ACE inhibitory peptide is provided, comprising the following steps:

[0008] Perform steam explosion treatment on the mixture of the fish skin and fish bones of silver carp, and centrifuge to take the middle layer to obtain a steam-exploded liquid;

[0009] Adjust the pH of the steam-exploded liquid to 8.0 - 10.0, add 1000 - 5000 U / g of alkaline protease based on the mass of the steam-exploded liquid for enzymatic hydrolysis until the pH is neutral; then add 500 - 2500 U / g of flavor protease based on the mass of the steam-exploded liquid for enzymatic hydrolysis, inactivate the enzyme activity, and obtain a primary enzymatic hydrolysis product containing the silver carp skin and bone ACE inhibitory peptide.

[0010] Furthermore, the mass ratio of the fish skin to the fish bones is 1:(2.5 - 3.5).

[0011] Furthermore, the method of the steam explosion treatment is: maintain at a blasting pressure of 1.0 - 2.0 MPa for 2 - 4 min, with a nitrogen concentration of 5% - 10%, and collect the product after instantaneous pressure relief.

[0012] Furthermore, the enzymatic hydrolysis temperature after adding the alkaline protease is 50 - 60°C; the enzymatic hydrolysis time after adding the flavor protease is 1 - 4 h.

[0013] Furthermore, the primary enzymatic hydrolysis product containing the silver carp skin and bone ACE inhibitory peptide is also subjected to ultrafiltration treatment to obtain three kinds of ultrafiltration products containing the silver carp skin and bone ACE inhibitory peptide with a molecular weight < 1 kDa, 1 kDa ≤ molecular weight ≤ 3 kDa, or molecular weight > 3 kDa.

[0014] Through the experimental verification of the in vitro ACE inhibitory activity of the ultrafiltration products with three different molecular weights, it was found that among these ultrafiltration products, the ACE activity inhibition rates of the molecular weights < 1 kDa, 1 kDa ≤ molecular weight ≤ 3 kDa, and molecular weight > 3 kDa were 89%, 81%, and 70% in sequence, and the effects were all better than those of the primary hydrolysate. After the treatment of simulated gastrointestinal digestion, the ACE activity inhibition rates of the primary hydrolysate and the three ultrafiltration products all decreased significantly, but the inhibition rates of the three ultrafiltration products were all higher than those of the primary hydrolysate, among which < 1 kDa was the highest (70%), followed by 1 - 3 kDa (62%), > 3 kDa (55%), and the primary hydrolysate (49%). Thus, it can be seen that the digested products still have a relatively high ACE activity inhibition rate.

[0015] In the second aspect of the present invention, there is provided a product containing the ACE inhibitory peptide from silver carp skin and bone, including the primary hydrolysate prepared by the above - mentioned preparation method, or including the ultrafiltration product prepared by the above - mentioned preparation method.

[0016] In the third aspect of the present invention, there is provided an ACE inhibitory peptide from silver carp skin and bone, which is any one of the amino acid sequences shown in SEQ ID NO.1 - 5. The specific amino acid sequences are as follows:

[0017] (1) Leu Asp Gly Ala Glu Glu Leu Gly Leu Ala, with a molecular mass of 986.492 Da, as shown in SEQ ID NO.1;

[0018] (2) Ala Glu Asp Val Ala Leu Val Arg Asp, with a molecular mass of 329.841, as shown in SEQ ID NO.2;

[0019] (3) Asp Asp Leu Gly Val Asp Val Leu Ala, with a molecular mass of 458.735, as shown in SEQ ID NO.3;

[0020] (4) Lys Asn Glu His Met Val Leu Thr Leu Gly, with a molecular mass of 571.302, as shown in SEQ ID NO.4;

[0021] (5) Gly Phe Asp Gly Leu Glu Gln Val Gly, with a molecular mass of 461.220, as shown in SEQ ID NO.5.

[0022] The above five peptide segments were synthesized by solid-phase synthesis, and their ACE inhibitory activities were measured in vitro. It was confirmed that all of the peptide segments had high ACE inhibitory ability, with the ACE inhibition rate reaching 88% - 93%, significantly higher than 62% of the primary enzymatic hydrolysate, which could provide a new treatment option for hypertensive patients. Among them, the silver carp skin and bone ACE inhibitory peptide shown in SEQ ID NO.1 had the highest ACE inhibitory ability (ACE inhibition rate reaching 93%), significantly higher than that of the ultrafiltration product with a molecular weight <1 kDa (ACE inhibition rate 89%).

[0023] In the fourth aspect of the present invention, there is also provided any one of the following substances:

[0024] (1) A nucleic acid molecule encoding any one of the amino acid sequences shown in SEQ ID NO.1 - 5;

[0025] (2) A plasmid vector containing the nucleic acid molecule;

[0026] (3) A recombinant cell containing the nucleic acid molecule or the plasmid vector.

[0027] Those skilled in the art should understand that the term "nucleic acid molecule" in this application actually includes either strand of the complementary double-strand, or both strands. The nucleotide sequences in the present invention include the DNA form or the corresponding RNA form. Disclosing one means the other is also disclosed.

[0028] Those skilled in the art should understand that the term "plasmid vector" in this application refers to a recombinant expression vector capable of effectively expressing any one of the peptide segments of SEQ ID NO.1 - 5. The plasmid vector may also include optional control sequences. The control sequences are operably linked to the nucleic acid molecule. Among them, the control sequences are one or more control sequences that can direct the expression of the nucleic acid molecule in the host. The vector plasmid constructed thereby (such as a vector or a transformant) can effectively express the above peptide segments.

[0029] When connecting the above nucleic acid molecule to the vector plasmid, the nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can be directly from the vector itself or exogenous, that is, not from the vector itself. The nucleic acid molecule is operably linked to the control elements.

[0030] According to an embodiment of the present invention, the vector plasmid may refer to a cloning vector or an expression vector, and can be obtained by operably connecting a nucleic acid molecule to a commercially available vector (such as a plasmid or a viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0031] Those skilled in the art should understand that the term "recombinant cell" in this application refers to a cell containing the above nucleic acid molecule or the above vector plasmid. The cell can be a prokaryotic cell, a eukaryotic cell or a phage. Further, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc.; the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell, etc.

[0032] In the fifth aspect of the present invention, there is also provided an application of the above product containing silver carp skin and bone ACE inhibitory peptide, or silver carp skin and bone ACE inhibitory peptide, in the preparation of antihypertensive drugs or functional foods for assisting in lowering blood pressure.

[0033] Compared with the prior art, the advantages of the present invention are as follows:

[0034] The present invention provides a method for preparing a primary hydrolysate of silver carp skin and bone containing silver carp skin and bone ACE inhibitory peptide by synergistically combining steam explosion and stepwise enzymatic hydrolysis, and further screening obtains 3 kinds of ultrafiltration products with different molecular weights and 5 kinds of ACE inhibitory peptide segments. The primary hydrolysate, ultrafiltration product and ACE inhibitory peptide obtained by the present invention all have good ACE inhibitory activity, and can provide a new treatment option for hypertensive patients. Description of the Drawings

[0035] Figure 1 Contents of total calcium and chelated calcium in the ultrafiltration product with a molecular weight <1 kDa prepared in Example 2.

[0036] Figure 2 Mass spectrometry identification diagram of the peptide segment in Example 4.

[0037] Figure 3 ACE activity inhibition rates of the primary hydrolysate and three ultrafiltration products.

[0038] Figure 4 ACE activity inhibition rates of the primary hydrolysate and three ultrafiltration products digested in Example 3.

[0039] Figure 5 ACE activity inhibition rate of the synthetic peptide segment in Example 4. Detailed Embodiments

[0040] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0041] In some embodiments of the present invention, a method for preparing silver carp skin and bone ACE inhibitory peptide comprises the following steps:

[0042] Perform steam explosion treatment on the mixture of silver carp skin and bone, and centrifuge to obtain the middle layer to get the steam explosion liquid;

[0043] Adjust the pH of the steam explosion liquid to 8.0 - 10.0, add alkaline protease at 1000 - 5000 U / g based on the mass of the steam explosion liquid and enzymatically hydrolyze until the pH is neutral; then add flavor protease at 500 - 2500 U / g based on the mass of the steam explosion liquid and enzymatically hydrolyze, inactivate the enzyme, to obtain a primary enzymatic hydrolysis product containing the silver carp skin and bone ACE inhibitory peptide.

[0044] In addition to the above necessary treatment steps for the method of preparing silver carp skin and bone ACE inhibitory peptide, in order to facilitate subsequent processing and improve the yield of silver carp skin and bone ACE inhibitory peptide, etc., auxiliary treatment steps can also be added in each step.

[0045] For example, before steam explosion treatment, wash the mixture of silver carp skin and bone and drain the water.

[0046] For example, for the product after steam explosion treatment, it can also be sieved (such as through an 80 - mesh sieve) to remove impurities first and then centrifuged.

[0047] For example, after the hydrolysis and enzyme inactivation treatment with alkaline protease and flavor protease, centrifugation treatment can also be carried out to remove precipitation and grease.

[0048] For example, the hydrolysis products of alkaline protease and flavor protease also undergo decolorization, deodorization, desalting, and concentration treatment.

[0049] Decolorization, deodorization, desalting, and concentration can be operated by commonly used methods in the industry.

[0050] Specifically, adsorbents such as activated carbon can be added and vacuum filtration is carried out to complete decolorization and deodorization treatment.

[0051] Specifically, desalting and concentration treatment can be carried out by methods such as nanofiltration (membrane filtration).

[0052] For example, for the obtained primary enzymatic hydrolysis product containing the silver carp skin and bone ACE inhibitory peptide, it can be lyophilized at low temperature into a powder for storage.

[0053] Optionally, in the above preparation method, the mass ratio of fish skin to fish bone is 1:(2.5 - 3.5).

[0054] Optionally, the sample injection amount during steam explosion is 1 - 3 kg / time.

[0055] Optionally, in the above preparation method, the method of steam explosion treatment is: maintaining for 2 - 4 min at a blasting pressure of 1.0 - 2.0 MPa, with a nitrogen concentration of 5% - 10%, and collecting the product after instantaneous pressure relief.

[0056] Optionally, the mass fraction of protein in the steam explosion liquid is 15% - 20%.

[0057] Optionally, in the above preparation method, the enzymatic hydrolysis temperature after adding the alkaline protease is 50 - 60 °C; the enzymatic hydrolysis time after adding the flavor protease is 1 - 4 h.

[0058] Optionally, the product containing the silver carp skin and bone ACE inhibitory peptide was also subjected to ultrafiltration treatment to obtain three ultrafiltration products containing the silver carp skin and bone ACE inhibitory peptide with a molecular weight < 1 kDa, 1 kDa ≤ molecular weight ≤ 3 kDa, or molecular weight > 3 kDa.

[0059] Example 1: Preparation of the primary enzymatic hydrolysis product of silver carp skin and bone

[0060] This example provides a preparation method for the primary enzymatic hydrolysis product of silver carp skin and bone, including the following steps:

[0061] (1) Steam explosion pretreatment: Clean the fish skin and bone by - product (the mass ratio of fish skin to fish bone is 1:3), drain the water, and place it in a pre - heated steam explosion device. Inject 3 kg each time, with a nitrogen concentration of 7%, maintain the pressure at 1.5 MPa for 3 min, and instantaneously relieve the pressure and collect the sample after steam explosion.

[0062] (2) Centrifugation: Centrifuge the above sample at 3000 rpm for 10 min, remove the precipitate and floating grease, add an appropriate amount of water to wash the residue, and collect the middle - layer supernatant, which is used as the enzymatic hydrolysis substrate, namely the steam explosion liquid.

[0063] (3) Adjust the pH value and perform enzymatic hydrolysis: Dilute and adjust the mass content of protein in the steam explosion liquid to 2.5%, pre - heat it, and slowly adjust the pH of the steam explosion liquid to 10.0 using 2 mol / L sodium hydroxide solution.

[0064] Based on the mass of the steam explosion liquid, add alkaline protease at 3000 U / g and perform enzymatic hydrolysis at 55 °C until the pH value of the steam explosion liquid reaches 7.3.

[0065] Based on the mass of the steam-exploded liquid, add flavor protease at 500 U / g and continue enzymatic hydrolysis for 1 h.

[0066] During the above enzymatic hydrolysis process, calcium ions Ca in the steam-exploded liquid 2+ chelated with the peptide segments of the enzymatic hydrolysis product to form a chelate of silver carp skin and bone ACE inhibitory peptide and calcium.

[0067] Inactivate the enzyme at 98 °C for 20 min. After cooling to room temperature, centrifuge to remove the precipitate and grease, and collect the supernatant.

[0068] (4) De-colorization and deodorization: In the supernatant obtained in step (3), add 1.0% activated carbon powder based on the mass of the supernatant, adjust the pH value of the supernatant to 5.0 - 6.0, shake and de-colorize at 50 °C for 60 min, and then filter by suction to obtain the de-colorized and deodorized liquid.

[0069] (5) Nanofiltration concentration: Use a nanofiltration membrane with a cut-off of 150 Da to perform desalination and concentration on the de-colorized and deodorized liquid obtained in step (4) to obtain the concentrated liquid.

[0070] (6) Perform low-temperature freeze-drying on the concentrated liquid to obtain the primary enzymatic hydrolysis product powder.

[0071] Example 2: Preparation of ultrafiltration products

[0072] 1. Preparation method of ultrafiltration products

[0073] The three ultrafiltration products in this example were prepared by the following methods respectively:

[0074] (1) Pass the primary enzymatic hydrolysate concentrate prepared in step (5) of Example 1 through a 1 kDa ultrafiltration membrane to obtain the first filtrate with a molecular weight < 1 kDa, and make it into a freeze-dried powder for standby.

[0075] (2) First pass the primary enzymatic hydrolysate concentrate prepared in step (5) of Example 1 through a 1 kDa ultrafiltration membrane, take the first retentate; then pass it through a 3 kDa ultrafiltration membrane, and take the second filtrate with 1 kDa ≤ molecular weight ≤ 3 kDa, and make it into a freeze-dried powder for standby.

[0076] (3) Pass the primary enzymatic hydrolysate concentrate prepared in step (5) of Example 1 through a 3 kDa ultrafiltration membrane, take the second retentate with a molecular weight > 3 kDa, and make it into a freeze-dried powder for standby.

[0077] 2. Determination of the total calcium and chelated calcium contents in the ultrafiltration product with a molecular weight < 1 kDa

[0078] Weigh the peptide powder and prepare a 20 mg / ml polypeptide solution. Add 8 times the volume of absolute ethanol, mix well, and let it stand refrigerated at 4°C for 4 h. Then, centrifuge at 8000 rpm / min for 20 min at 4°C using a refrigerated centrifuge to obtain a precipitate and supernatant.

[0079] Use atomic absorption spectrometry to determine the content of free calcium (Ca 游离 ) in the supernatant. The total calcium (Ca 总 ) content in the peptide powder was determined by the flame atomic absorption spectrometry method in the national standard GB 5009.92-2016 "National Food Safety Standard - Determination of Calcium in Foods".

[0080] The calculation formula for the calcium chelation rate in the peptide powder is as follows:

[0081] .

[0082] From Figure 1 the detection results, it can be seen that the total calcium content in the ultrafiltration product with a molecular weight < 1 kDa prepared in this example is 1276.28 mg / 100g, and the chelated calcium content is 1222.56 mg / 100g.

[0083] Example 3: Preparation of Simulated Gastrointestinal Digestion Products

[0084] In this example, simulated gastric juice was selected to simulate the gastrointestinal digestion process.

[0085] The simulated gastric juice (pH = 1.4) contains: 5.51 mg / mL NaCl, 1.65 mg / mL KCl, 0.53 mg / mL NaH 2 PO 4 , 0.60 mg / mL CaCl 2 , 0.61 mg / mL NH 4 Cl and 2000 U / mL porcine pepsin. The pH value was adjusted with 1 M hydrochloric acid.

[0086] The simulated intestinal juice (pH 7.8) contains: 14.02 mg / mL NaCl, 1.13 mg / mL KCl, 6.78 mg / mL NaHCO 3 , 0.16 mg / mL KH 2 PO 4 , 0.10 mg / mL MgCl 2 , 100 U / mL trypsin and 25 U / mL chymotrypsin.

[0087] The specific gastrointestinal digestion process is as follows:

[0088] (1)Add 56 mL of simulated gastric juice to the primary enzymatic hydrolysis product powder prepared in Example 1 and the three ultrafiltration product powders prepared in Example 2, and perform constant-temperature water bath shaking digestion at 37°C to obtain the primary digestion product.

[0089] (2)Adjust the pH value of the primary digestion product to 7.5 using saturated NaHCO 3 solution and 1 M NaOH solution, then add 72 mL of simulated intestinal juice, perform water bath shaking digestion at 37°C for 4 h, heat in a boiling water bath for 10 min to terminate digestion, and freeze-dry to obtain the final 4 kinds of simulated gastrointestinal digestion product powders.

[0090] Example 4: Screening, Identification and Synthesis of Target Peptides

[0091] 1. LC-MS / MS Detection.

[0092] The specific method is as follows:

[0093] Dissolve the ultrafiltration product powder sample with a molecular weight < 1 kDa prepared in Example 2 (2 mL, 0.1% formic acid aqueous solution), centrifuge, and take the supernatant for detection.

[0094] The mobile phase used in liquid chromatography, Solution A is 0.1% (v / v) formic acid aqueous solution, and Solution B is formic acid-acetonitrile aqueous solution (0.1 mL of formic acid is made up to 100 mL with 80% (v / v) acetonitrile aqueous solution).

[0095] Equilibrate the liquid chromatography column (50 μm × 150 mm, Acclaim PepMapTM RSLC, thermo scientific Technology Inc.) with 92% of Solution A, inject 1 μL of the sample, separate it through the chromatographic column, and set the relevant liquid phase gradient as follows: 0 - 98 min, the linear gradient of Solution B ranges from 8% to 28%; 98 - 113 min, the linear gradient of Solution B ranges from 28% to 37%; 113 - 117 min, the linear gradient of Solution B ranges from 37% to 100%; 117 - 120 min, Solution B is maintained at 100%.

[0096] After capillary high-performance liquid chromatography separation, perform mass spectrometry analysis using a Thermo QE HF mass spectrometer (Thermo Fisher). Analysis duration: 120 min. Detection mode: positive ion. The mass-to-charge ratios of polypeptides and polypeptide fragments are collected according to the following method: 20 fragment spectra (MS2 scan) are collected after each full scan. Scanning range 100 - 1500, primary resolution 60000, secondary resolution 15000, collision energy CE 28 eV.

[0097] The mass spectrometry identification results are asFigure 2 as shown

[0098] 2. Screening, identification and synthesis

[0099] Bioinformatics analysis was performed on the ultrafiltration product with a molecular weight < 1 kDa prepared in Example 2. Specifically, the online tools peptide property calculator and ToxinPred were used to predict the functions of the polypeptides. Subsequently, molecular docking was used to identify the amino acid sequences of the single peptides with high ACE inhibitory activity as shown in SEQ ID NO.1 to SEQ ID NO.5. Specifically:

[0100] (1) Leu Asp Gly Ala Glu Glu Leu Gly Leu Ala, with a molecular mass of 986.492 Da, abbreviated as LDGAEELGLA;

[0101] (2) Ala Glu Asp Val Ala Leu Val Arg Asp, with a molecular mass of 329.841 Da, abbreviated as AEDVALVRD;

[0102] (3) Asp Asp Leu Gly Val Asp Val Leu Ala, with a molecular mass of 458.735 Da, abbreviated as DDLGVDVLA;

[0103] (4) Lys Asn Glu His Met Val Leu Thr Leu Gly, with a molecular mass of 571.302 Da, abbreviated as KNEHMVLTLG;

[0104] (5) Gly Phe Asp Gly Leu Glu Gln Val Gly, with a molecular mass of 461.220 Da, abbreviated as GFDGLEQVG.

[0105] The above five peptide segments were synthesized by solid-phase synthesis method, and the purity analysis by HPLC was carried out, and they were respectively made into freeze-dried powder for standby.

[0106] According to the results of mass spectrometry identification, several other peptide segments < 1KDa were synthesized as control peptides in this example by the same method. These control peptides referred to the repeated amino acid sequences in the above 5 peptide segments as much as possible, and their amino acid sequences are shown in SEQID NO.6-9, specifically:

[0107] (1) Control 1: Ser Gly Leu Glu Glu Leu, molecular weight 324.168, abbreviated as SGLEEL;

[0108] (2) Control 2: Glu Glu Leu Glu Glu Leu, molecular weight 761.363, abbreviated as EELEEL;

[0109] (3) Control 3: Glu Pro Leu Thr Leu, molecular weight 614.344, abbreviated as EPLTL;

[0110] (4) Control 4: Val Glu Pro Phe, molecular weight 491.248, abbreviated as VEPF.

[0111] Test Example 1: Verification of in vitro ACE inhibitory activity

[0112] The ACE inhibition screening kit was used to measure the ACE inhibition rate of the samples. The samples tested were the primary enzymatic hydrolysate prepared in Example 1, the three ultrafiltration products prepared in Example 2, the four simulated gastrointestinal digestion products prepared in Example 3, and the five peptides synthesized in Example 4, and Captopril (1 µM, 20 μL) was selected as a positive control.

[0113] The determination method is:

[0114] (1) Add HHL (maleuryl histidyl leucine, 100 μL) and peptide solution 40 μL to a 5 mL centrifuge tube and incubate in a 37°C water bath for 5 min.

[0115] Preparation method of polypeptide solution: The primary enzymatic hydrolysate lyophilized powder prepared in Example 1, the three ultrafiltration product lyophilized powders prepared in Example 2, the four simulated gastrointestinal digestion product lyophilized powders prepared in Example 3, and the five peptide segment lyophilized powders synthesized in Example 4 were respectively prepared into 3 mg / ml polypeptide solutions using 0.1 M boric acid buffer.

[0116] (2) Add ACE (20 μL) and incubate in a 37°C water bath for 30 min. After the reaction is complete, add 250 μL of 1 M hydrochloric acid solution to inactivate the enzyme.

[0117] (3) Add 1.5 mL of pre-cooled ethyl acetate, shake to extract hippuric acid, centrifuge at 4000 r / min at 4°C for 5 min, aspirate 1.0 mL of the upper ethyl acetate, and dry in an oven at 105°C;

[0118] (4) Add 4 mL of distilled water to the dried test tube to prepare the reconstituted extract and measure the absorbance at 228 nm.

[0119] The control group used 0.1 M boric acid buffer instead of the sample. The blank group added 80 μL of 1 M hydrochloric acid solution before adding ACE solution.

[0120] ;

[0121] In the formula: A is the absorbance value at 228 nm of the sample group, B is the absorbance value at 228 nm of the control group, and C is the absorbance value at 228 nm of the blank group.

[0122] The ACE activity inhibition rates of captopril, the primary enzymatic hydrolysate, and the three ultrafiltration products are as Figure 3 shown. The results show that based on the ACE activity inhibition rate of the positive control (captopril) being 100%, the inhibition rate of the 3 mg / mL primary enzymatic hydrolysate on ACE activity is 62%. At the same concentration, among the three ultrafiltration products, the ACE activity inhibition rates of those with a molecular weight < 1 kDa, 1 kDa ≤ molecular weight ≤ 3 kDa, and molecular weight > 3 kDa are 89%, 81%, and 70% respectively, and the effects are all better than that of the primary enzymatic hydrolysate.

[0123] The ACE activity inhibition rates of the primary enzymatic hydrolysate and the three ultrafiltration products after simulated gastrointestinal digestion treatment in Example 3 are as Figure 4 shown. The results show that after simulated gastrointestinal digestion treatment, the ACE activity inhibition rates of all 4 products have decreased significantly, and the inhibition rates of the three ultrafiltration products are still higher than that of the primary enzymatic hydrolysate. Among them, the ACE activity inhibition rate of the ultrafiltration product with a molecular weight < 1 kDa is the highest, reaching 70%, followed by the ultrafiltration product with 1 kDa ≤ molecular weight ≤ 3 kDa, with an ACE activity inhibition rate of 62%; the ACE inhibition rate of the ultrafiltration product with a molecular weight > 3 kDa is 55%, and the one with the largest decrease is the primary enzymatic hydrolysate, with an ACE activity inhibition rate of 49%. Thus, it can be seen that these digestion products still have relatively high ACE activity inhibition rates and have broad application prospects.

[0124] The ACE activity inhibition rates of the five peptide segments synthesized in Example 4 are as Figure 5 shown. The results show that the ACE activity inhibition rates of the 5 synthetic peptide segments reach 88% - 93%, which is significantly higher than that of the primary enzymatic hydrolysate prepared in Example 1 (62%). Thus, it can be seen that the five peptide segments screened in Example 4 all have very high ACE activity inhibition effects and can be effectively applied to the treatment of hypertension; among them, the peptide segment shown in SEQ ID NO.1 has the highest ACE activity inhibition effect, with an ACE activity inhibition rate reaching 93%.

[0125] In this test example, the same test method was also used to measure the ACE activity inhibition rates of 4 control peptide segments, namely Control 1 - 4, synthesized in Example 4. As Figure 5 shown, the inhibition rates of these 4 control peptide segments are 69%, 74%, 72%, and 71% respectively. They are all significantly inferior to the 5 ACE activity inhibitory peptides synthesized in Example 4.

[0126] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solutions of the present invention, and these simple variations all fall within the protection scope of the present invention.

Claims

1. A silver carp skin bone ACE inhibitory peptide, characterized in that: It is any one of the amino acid sequences shown in SEQ ID NO.1-5.

2. The silver carp skin-bone ACE inhibitory peptide according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

3. A substance, characterized in that The substance is any of the following: (1) A nucleic acid molecule encoding any one of the amino acid sequences shown in SEQ ID NOs. 1-5; (2) a plasmid vector containing the nucleic acid molecule; (3) A recombinant cell comprising the nucleic acid molecule or plasmid vector.

4. Use of the silver carp skin-bone ACE inhibitory peptide according to claim 1 in the preparation of a blood pressure lowering drug or a functional food that assists in lowering blood pressure.

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