Silver carp skin bone ACE inhibitory peptide and its preparation method and application

Abstract

The present invention discloses a silver carp skin and bone ACE inhibitory peptide, a preparation method and application thereof, and relates to the technical field of ACE inhibitory peptides. The preparation method of the silver carp skin and bone ACE inhibitory peptide provided by the present invention is as follows: steam explosion treatment is performed on a mixture of silver carp fish skin and fish bones, and the intermediate layer is centrifuged to obtain a steam explosion liquid; the pH of the steam explosion liquid is adjusted, and alkaline protease is added to the mass of the steam explosion liquid for enzymatic hydrolysis until the pH is neutral; then, flavor protease is added to the mass of the steam explosion liquid for enzymatic hydrolysis to obtain a primary enzymatic hydrolysis product containing the silver carp skin and bone ACE inhibitory peptide. Five ACE inhibitory peptides are obtained by ultrafiltration and screening analysis of the primary enzymatic hydrolysis products. These primary enzymatic hydrolysis products, ultrafiltration products and ACE inhibitory peptides all have good ACE activity inhibition rates and can be widely used in the preparation of blood pressure lowering drugs.

Description

Technical Field

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

[0002] Hypertension is categorized into primary hypertension and secondary hypertension based on etiology and demographic characteristics. Primary hypertension, which accounts for 90%-95% of all hypertensive patients, has no single clear cause and is associated with a combination of factors, including genetics, age, obesity, and a high-salt diet. It is primarily managed with long-term lifestyle interventions and medications (such as diuretics, ACE inhibitors / ARBs, and CCBs).

[0003] Angiotensin-converting enzyme (ACE) converts angiotensin I to angiotensin II, inactivating the vasodilator bradykinin and promoting aldosterone secretion, leading to elevated blood pressure. Therefore, inhibiting ACE activity can be effective in treating hypertension. Currently, commonly used ACE inhibitors include enalapril, captopril, benazepril, ramipril, and lixinpril. While they are effective in treating hypertension, long-term use can lead to kidney damage, hyperkalemia, allergic reactions, and fetal developmental impairment.

[0004] Compared to chemically synthesized ACE inhibitors, food-derived peptide ACE inhibitors offer advantages such as increased safety, ease of absorption, and minimal side effects, providing a new approach for the prevention and treatment of hypertension. Currently, a variety of bioactive collagen peptide products are available in China, but these generally suffer from high molecular weights, resulting in low levels of active ingredients and low product purity.

[0005] my country is rich in aquatic resources and has a high production of freshwater fish. The processing of fish produces large amounts of fish skin, fish bones, and fish scales, which are rich in collagen and minerals such as calcium and phosphorus, and are a source of high-quality bioactive peptides and calcium. However, in the industrial production of surimi, the byproducts fish skin and fish bones usually exist in the form of a mixture, making them difficult to separate. They are mainly used to produce feed and have low added value. Currently, the main way to high-value-added utilization of fish skin, fish scales, and fish bones is to use them separately to extract collagen or collagen peptides. If the fish skin and bone mixture can be fully recycled and utilized to develop polypeptide ACE inhibitors, it will provide important support for the development of functional products derived from aquatic product byproducts and food-borne polypeptide antihypertensive drugs. Summary of the Invention

[0006] The present invention provides a silver carp skin and bone ACE-inhibiting peptide, its preparation method, and application. The preparation method comprises first steam-exploding a mixture of silver carp skin and bones, then adding alkaline protease and flavor protease for enzymatic hydrolysis to obtain a primary enzymatic hydrolysis product containing the silver carp skin and bone ACE-inhibiting peptide. Nanofiltration and ultrafiltration can also be performed to obtain ultrafiltration products of varying molecular weights, and finally, five peptide segments with ACE-inhibiting activity are screened from the ultrafiltration products. This is achieved specifically through the following techniques.

[0007] The first aspect of the present invention provides a method for preparing an ACE inhibitory peptide from silver carp skin and bone, comprising the following steps:

[0008] The mixture of silver carp fish skin and fish bones is subjected to steam explosion treatment, and the middle layer is taken out by centrifugation to obtain steam explosion liquid;

[0009] The pH of the steam explosion liquid is adjusted to 8.0-10.0, and 1000-5000 U / g of alkaline protease is added based on the mass of the steam explosion liquid for enzymatic hydrolysis until the pH is neutral; then, 500-2500 U / g of flavor protease is added based on the mass of the steam explosion liquid for enzymatic hydrolysis to inactivate the enzyme, thereby obtaining a primary enzymatic hydrolysis product containing the silver carp skin 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 steam explosion treatment method is: maintaining a blasting pressure of 1.0-2.0 MPa for 2-4 minutes, a nitrogen concentration of 5%-10%, and collecting the product after instantaneous pressure relief.

[0012] Furthermore, the enzymatic hydrolysis temperature after adding the alkaline protease is 50-60° C.; and 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 was subjected to ultrafiltration treatment to obtain three ultrafiltration products containing the silver carp skin and bone ACE inhibitory peptide with molecular weight <1 kDa, 1kDa≤molecular weight≤3kDa, or molecular weight >3kDa.

[0014] The present invention verified the in vitro ACE inhibitory activity of three ultrafiltration products with different molecular weights and found that among these ultrafiltration products, the ACE activity inhibition rates of those with molecular weight <1kDa, 1kDa≤molecular weight≤3kDa, and molecular weight >3kDa were 89%, 81%, and 70%, respectively, which were all better than those of the primary enzymatic hydrolysate. After treatment with simulated gastrointestinal digestion, the ACE activity inhibition rates of the primary enzymatic hydrolysate and the three ultrafiltration products decreased significantly, but the inhibition rates of the three ultrafiltration products were all higher than those of the primary enzymatic hydrolysate, with the highest inhibition rate of <1kDa (70%), followed by 1-3kDa (62%), >3kDa (55%), and the primary enzymatic hydrolysate (49%). This shows that the digested digestive products still have a high ACE activity inhibition rate.

[0015] The second aspect of the present invention provides a product containing silver carp skin and bone ACE inhibitory peptide, including the primary enzymatic hydrolysis product obtained by the above preparation method, or the ultrafiltration product obtained by the above preparation method.

[0016] The third aspect of the present invention provides a silver carp skin bone ACE inhibitory peptide, which is any one of the amino acid sequences shown in SEQ ID NO.1-5. The specific amino acid sequence is:

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

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

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

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

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

[0022] The five peptides synthesized using solid-phase synthesis and assayed for their ACE inhibitory activity in vitro confirmed their high ACE inhibitory capacity, reaching 88%-93%, significantly higher than the 62% of the primary enzymatic hydrolysate, potentially providing a new treatment option for patients with hypertension. Among them, the silver carp skin and bone ACE inhibitory peptide represented by SEQ ID NO.1 exhibited the highest ACE inhibitory capacity (93%), significantly higher than the ultrafiltration product with a molecular weight of <1 kDa (89%).

[0023] In a fourth aspect, the present invention further provides any one of the following substances:

[0024] (1) A nucleic acid molecule encoding any one of the amino acid sequences shown in SEQ ID NOs. 1-5;

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

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

[0027] It will be understood by those skilled in the art that the term "nucleic acid molecule" herein actually includes either or both of the complementary double strands. The nucleotide sequences in the present invention include either DNA or RNA, and disclosure of one implies disclosure of the other.

[0028] Those skilled in the art will understand that the term "plasmid vector" in this application refers to a recombinant expression vector capable of effectively expressing any of the peptide segments of SEQ ID NOs. 1-5. The plasmid vector may also include optional control sequences. These control sequences are operably linked to the nucleic acid molecule. A control sequence is one or more control sequences that direct the expression of the nucleic acid molecule in a host. The resulting vector plasmid (e.g., vector or transformant) can effectively express the aforementioned peptide segment.

[0029] When the nucleic acid molecule is linked to a vector plasmid, the nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or exogenous, i.e., not derived from the vector itself. The nucleic acid molecule is operably linked to the control elements.

[0030] According to embodiments of the present invention, a vector plasmid can refer to either a cloning vector or an expression vector, and can be obtained by operably linking a nucleic acid molecule to a commercially available vector (e.g., a plasmid or viral vector). The vectors used in the present invention are not particularly limited, and commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0031] Those skilled in the art will understand that the term "recombinant cell" herein refers to a cell containing the aforementioned nucleic acid molecule or vector plasmid. The cell may be a prokaryotic cell, a eukaryotic cell, or a bacteriophage. Furthermore, prokaryotic cells include Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis; and eukaryotic cells include fungi, insect cells, plant cells, or mammalian cells.

[0032] The fifth aspect of the present invention also provides a product containing the silver carp skin and bone ACE inhibitory peptide, or the use of the silver carp skin and bone ACE inhibitory peptide in the preparation of a blood pressure lowering drug.

[0033] Compared with the prior art, the present invention is beneficial in that:

[0034] The present invention provides a method for producing a primary enzymatic hydrolysate of silver carp skin and bone containing ACE-inhibiting peptides from silver carp skin and bone by synergistically combining steam explosion and stepwise enzymatic hydrolysis. Furthermore, three ultrafiltration products with different molecular weights and five ACE-inhibiting peptide segments were obtained through screening. The primary enzymatic hydrolysate, ultrafiltration product, and ACE-inhibiting peptides obtained by the present invention all exhibited excellent ACE-inhibiting activity, providing a new treatment option for patients with hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The total calcium and chelated calcium contents in the ultrafiltration product with a molecular weight less than 1 kDa prepared in Example 2.

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

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

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

[0039] Figure 5 is the ACE activity inhibition rate of the synthetic peptide segment in Example 4. DETAILED DESCRIPTION

[0040] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

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

[0042] The mixture of silver carp skin and fish bones is subjected to steam explosion treatment, and the middle layer is taken out by centrifugation to obtain steam explosion liquid;

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

[0044] In addition to the necessary processing steps mentioned above, the preparation method of silver carp skin and bone ACE inhibitory peptide can also include auxiliary processing steps in each step to facilitate subsequent processing and improve the yield of silver carp skin and bone ACE inhibitory peptide.

[0045] For example, the skin and bone mixture of silver carp is washed and drained before steam explosion.

[0046] For example, the product after steam explosion treatment can be sieved (e.g., 80-mesh sieve) to remove impurities before centrifugation.

[0047] For example, after the hydrolysis and inactivation of alkaline protease and flavor protease, centrifugation can be performed to remove sediment and oil.

[0048] For example, the hydrolyzed products of alkaline protease and flavor protease are also subjected to decolorization, deodorization, desalination and concentration treatments.

[0049] Decolorization, deodorization, desalination and concentration can be carried out using methods commonly used in the industry.

[0050] Specifically, an adsorbent such as activated carbon may be added, and vacuum filtration may be performed to complete the decolorization and deodorization treatment.

[0051] Specifically, desalination and concentration treatment can be carried out through nanofiltration (membrane filtration) and the like.

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

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

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

[0055] Optionally, in the above preparation method, the steam explosion treatment method is: maintaining a blasting pressure of 1.0-2.0 MPa for 2-4 min, 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.; and 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 is further subjected to ultrafiltration treatment to obtain three ultrafiltration products containing the silver carp skin and bone ACE inhibitory peptide with molecular weight <1 kDa, 1kDa≤molecular weight≤3kDa, or molecular weight >3kDa.

[0059] Example 1: Preparation of primary enzymatic hydrolysate of silver carp skin and bones

[0060] This embodiment provides a method for preparing a primary enzymatic hydrolysate of silver carp skin and bones, comprising the following steps:

[0061] (1) Steam explosion pretreatment: The fish skin and bone by-products (the mass ratio of fish skin to fish bone is 1:3) were cleaned, drained and placed in a preheated steam explosion device. 3 kg of sample was injected each time, the nitrogen concentration was 7%, and the pressure was maintained at 1.5 MPa for 3 min. The pressure was released instantly and the steam-exploded samples were collected.

[0062] (2) Centrifugation: Centrifuge the above sample at 3000 rpm for 10 min to remove the precipitate and floating oil, and add an appropriate amount of clean water to wash the residue. Collect the middle layer of clear liquid to obtain the steam explosion liquid as the enzymatic substrate.

[0063] (3) Adjusting pH value and enzymatic hydrolysis: dilute and adjust the protein content in the steam explosion liquid to 2.5%. After preheating, use 2 mol / L sodium hydroxide solution to slowly adjust the pH of the steam explosion liquid to 10.0.

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

[0065] Based on the mass of the steam explosion liquid, flavor protease was added at a concentration of 500 U / g and the enzymatic hydrolysis was continued for 1 h.

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

[0067] The enzyme was inactivated at 98°C for 20 min, cooled to room temperature, centrifuged to remove the precipitate and oil, and the supernatant was collected.

[0068] (4) Decolorization and deodorization: Add 1.0% activated carbon powder to the supernatant obtained in step (3) based on the mass of the supernatant, adjust the pH value of the supernatant to 5.0-6.0, decolorize at 50°C for 60 min, and filter to obtain the decolorized and deodorized liquid.

[0069] (5) Nanofiltration concentration: The decolorized and deodorized liquid obtained in step (4) is desalted and concentrated using a nanofiltration membrane with a cutoff of 150 Da to obtain a concentrated liquid.

[0070] (6) The concentrated solution is freeze-dried to obtain primary enzymatic hydrolysis product powder.

[0071] Example 2: Preparation of ultrafiltration product

[0072] 1. Preparation method of ultrafiltration product

[0073] The three ultrafiltration products of this embodiment were prepared by the following methods:

[0074] (1) The primary enzymatic hydrolysate concentrate prepared in step (5) of Example 1 was passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain a first filtrate with a molecular weight of less than 1 kDa, which was prepared into a freeze-dried powder for later use.

[0075] (2) The primary enzymatic hydrolysate concentrate prepared in step (5) of Example 1 was first passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain the first retentate; then passed through an ultrafiltration membrane with a pore size of 3 kDa to obtain the second filtrate, with a molecular weight of 1 kDa ≤ ≤ 3 kDa, and prepared into a freeze-dried powder for later use.

[0076] (3) The primary enzymatic hydrolysate concentrate prepared in step (5) of Example 1 was passed through an ultrafiltration membrane with a pore size of 3 kDa, and the second retentate with a molecular weight > 3 kDa was obtained and prepared into a freeze-dried powder for later use.

[0077] 2. Determination of total calcium and chelated calcium in ultrafiltration products with molecular weight less than 1 kDa

[0078] Weigh the peptide powder to prepare a 20 mg / ml peptide solution, add 8 times the volume of anhydrous ethanol, mix thoroughly, and refrigerate at 4°C for 4 h. Use a refrigerated centrifuge at 4°C and 8000 rpm / min for 20 min to obtain a precipitate and a supernatant.

[0079] The free calcium (Ca) in the supernatant was determined by atomic absorption spectrometry. 游离 ) content. Total calcium (Ca 总 ) content is determined by flame atomic absorption spectrometry in accordance with the national standard GB 5009.92-2016 "National Food Safety Standard - Determination of Calcium in Foods".

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

[0081] .

[0082] from Figure 1 The test results show that the total calcium content in the ultrafiltration product with a molecular weight of less than 1 kDa prepared in this example is 1276.28 mg / 100 g, and the chelated calcium content is 1222.56 mg / 100 g.

[0083] Example 3: Preparation of simulated gastrointestinal digestion products

[0084] In this embodiment, simulated gastric fluid was selected to prepare the simulated gastrointestinal digestion process.

[0085] Simulated gastric fluid (pH = 1.4) contained 5.51 mg / mL NaCl, 1.65 mg / mL KCl, 0.53 mg / mL NaH2PO4, 0.60 mg / mL CaCl2, 0.61 mg / mL NH4Cl, and 2000 U / mL porcine pepsin. The pH was adjusted with 1 M hydrochloric acid.

[0086] Simulated intestinal fluid (pH 7.8) contained: 14.02 mg / mL NaCl, 1.13 mg / mL KCl, 6.78 mg / mL NaHCO3, 0.16 mg / mL KH2PO4, 0.10 mg / mL MgCl2, 100 U / mL trypsin, and 25 U / mL chymotrypsin.

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

[0088] (1) 56 mL of simulated gastric fluid was added to the primary enzymatic hydrolysis product powder prepared in Example 1 and the three ultrafiltration product powders prepared in Example 2, and the mixture was shaken and digested in a constant temperature water bath at 37°C to obtain primary digestion products.

[0089] (2) The pH value of the primary digestion product was adjusted to 7.5 using saturated NaHCO3 solution and 1 M NaOH solution, and then 72 mL of simulated intestinal fluid was added. The product was digested in a water bath at 37°C for 4 h, and heated in a boiling water bath for 10 min to terminate the digestion. The product was freeze-dried to obtain the final four 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:

[0093] The ultrafiltration product powder sample with a molecular weight of less than 1 kDa prepared in Example 2 was dissolved (2 mL, 0.1% formic acid aqueous solution), centrifuged, and the supernatant was collected for detection.

[0094] The mobile phases used in liquid chromatography were: liquid A was 0.1% (v / v) formic acid in water, and liquid B was formic acid-acetonitrile in water (0.1 mL formic acid was diluted to 100 mL with 80% (volume fraction) acetonitrile in water).

[0095] A liquid chromatography column (50 μm×150 mm, Acclaim PepMapTM RSLC, Thermo Scientific Technology Inc.) was equilibrated with 92% solution A, and a 1 μL injection volume was used for column separation. The relevant liquid phase gradient settings were as follows: 0-98 min, solution B linear gradient from 8% to 28%; 98-113 min, solution B linear gradient from 28% to 37%; 113-117 min, solution B linear gradient from 37% to 100%; 117-120 min, solution B was maintained at 100%.

[0096] After capillary high-performance liquid chromatography separation, mass spectrometry analysis was performed on a Thermo QE HF mass spectrometer (Thermo Fisher) for 120 minutes. Positive ion detection was used. Mass-to-charge ratios of peptides and peptide fragments were acquired using the following method: 20 fragmentation spectra (MS2 scans) were acquired after each full scan. The scan range was 100–1500, with a primary resolution of 60,000, a secondary resolution of 15,000, and a collision energy of 28 eV.

[0097] Mass spectrometry identification results Figure 2 shown.

[0098] 2. Screening, identification and synthesis

[0099] Bioinformatics analysis was performed on the ultrafiltration product with a molecular weight of less than 1 kDa prepared in Example 2. Specifically, peptide function prediction was performed using a peptide property calculator and the ToxinPred online tool. Subsequently, molecular docking was used to identify the amino acid sequences of single peptides with high ACE inhibitory activity, as shown in SEQ ID NOs. 1 to 5. Specifically, the following:

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

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

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

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

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

[0105] The five peptides were synthesized by solid phase synthesis, and their purity was analyzed by HPLC. They were then made into freeze-dried powders for later use.

[0106] Based on the results of mass spectrometry, several other peptides <1 kDa were synthesized using the same method in this example as control peptides. These control peptides were based on the repeated amino acid sequences of the above five peptides as much as possible. Their amino acid sequences are shown in SEQ ID NOs. 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 rate of the samples was determined using an ACE inhibition screening kit. The samples tested included 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. Captopril (1 µM, 20 µL) was used as a positive control.

[0113] The determination method is:

[0114] (1) Add HHL (maleurylhistidylleucine, 100 μL) and peptide solution 40 μL to a 5 mL centrifuge tube in sequence 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 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 layer, 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] Where: A-absorbance value of sample group at 228 nm, B-absorbance value of control group at 228 nm, C-absorbance value of blank group at 228 nm.

[0122] The ACE activity inhibition rates of captopril, primary enzymatic hydrolysis products and three ultrafiltration products are as follows Figure 3 The results showed that, with the ACE activity inhibition rate of the positive control (captopril) as 100%, the inhibition rate of the primary hydrolysate at 3 mg / mL was 62%. At the same concentration, the ACE activity inhibition rates of the three ultrafiltration products with molecular weights <1 kDa, 1 kDa ≤ ≤ 3 kDa, and >3 kDa were 89%, 81%, and 70%, respectively, demonstrating superior effects to the primary hydrolysate.

[0123] The ACE activity inhibition rates of the primary enzymatic hydrolysate and three ultrafiltration products after simulated gastrointestinal digestion in Example 3 are as follows: Figure 4 The results showed that after simulated gastrointestinal digestion, the ACE activity inhibition rates of all four products decreased significantly, with the inhibition rates of the three ultrafiltration products still higher than those of the primary hydrolysate. The ultrafiltration product with a molecular weight of less than 1 kDa had the highest ACE activity inhibition rate, reaching 70%, followed by the ultrafiltration product with a molecular weight of 1 kDa ≤ ≤ 3 kDa, with an ACE activity inhibition rate of 62%; the ultrafiltration product with a molecular weight of >3 kDa had an ACE activity inhibition rate of 55%. The primary hydrolysate showed the greatest decrease in ACE activity inhibition, with an ACE activity inhibition rate of 49%. These digestion products still have 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 follows: Figure 5 The results showed that the ACE activity inhibition rate of the five synthetic peptides reached 88%-93%, significantly higher than the primary enzymatic hydrolysate prepared in Example 1 (62%). This shows that the five peptides screened in Example 4 all have very high ACE activity inhibition effects and can be effectively used to treat hypertension. Among them, the peptide shown in SEQ ID NO. 1 has the highest ACE activity inhibition effect, with an ACE activity inhibition rate of 93%.

[0125] This test example also uses the same test method to determine the ACE activity inhibition rate of the four control peptides 1-4 synthesized in Example 4. Figure 5 As shown in FIG, the inhibition rates of the four control peptides were 69%, 74%, 72% and 71%, respectively, which were significantly lower than those of the five ACE inhibitory peptides synthesized in Example 4.

[0126] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of 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 solution of the present invention, and these simple modifications all fall within the scope of protection 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 containing the nucleic acid molecule or plasmid vector.

4. Use of the silver carp skin and bone ACE inhibitory peptide according to claim 1 in the preparation of a blood pressure lowering drug.

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