ACE inhibitory peptide monomer from goat milk as well as preparation method and application of ACE inhibitory peptide monomer

Abstract

The embodiment of the invention discloses a goat milk-derived ACE (angiotensin converting enzyme) inhibitory peptide monomer as well as a preparation method and application thereof, relates to the technical field of protein separation, and aims to improve a preparation source and preparation efficiency of a food-borne protein-derived ACE inhibitory peptide. The preparation method comprises the following steps: dissolving sheep milk casein in water, and carrying out heating treatment, enzymolysis treatment and freeze drying treatment to obtain a sheep milk source zymolyte; preparing the goat milk source zymolyte into a solution with the concentration of 145mg / mL-155mg / mL, passing through a 0.45 mu m filter membrane, and carrying out primary separation by adopting preparative high performance liquid chromatography to obtain a product Y; preparing the Y product into a solution with the concentration of 20 mg / mL-30 mg / mL, passing through a 0.45 mu m filter membrane, and carrying out secondary separation by adopting preparative high performance liquid chromatography to obtain a Y-X product; preparing the Y-X product into a solution with the concentration of 5mg / mL-15mg / mL, passing through a 0.45 mu m filter membrane, and carrying out re-separation preparation by using a C18 hydrophilic preparative column and preparative high performance liquid chromatography to obtain a product M; the product M is prepared into a solution with the concentration of 5 mg / mL-15 mg / mL, the solution passes through a 0.22-micron filter membrane, after re-separation preparation is conducted through RP-HPLC, rotary evaporation concentration treatment and freeze drying treatment are conducted, and a product F is obtained.

Description

Technical Field

[0001] This application relates to the technical field of protein separation, and particularly relates to an ACE inhibitory peptide monomer derived from goat milk, and its preparation method and application. Background Art

[0002] The renin-angiotensin system (RAS) is the main system regulating human blood pressure, and the key components in RAS have always been the focus of research on controlling hypertension and its complications. Traditional antihypertensive drugs have adverse side effects and limitations, while food-derived protein angiotensin-converting enzyme (ACE) inhibitory peptides have the characteristics of natural non-toxic side effects and good blood pressure-lowering effects, and have good application value. However, at present, the preparation sources and preparation efficiency of food-derived protein angiotensin-converting enzyme (ACE) inhibitory peptides are relatively low, making it difficult to meet market demands. Summary of the Invention

[0003] The main purpose of this application is to provide an ACE inhibitory peptide monomer derived from goat milk, and its preparation method and application, aiming to improve the preparation sources and preparation efficiency of food-derived protein angiotensin-converting enzyme (ACE) inhibitory peptides.

[0004] To achieve the above object, the technical solution adopted in the embodiments of this application is as follows: A preparation method of an ACE inhibitory peptide monomer derived from goat milk, comprising the following steps:

[0005] Dissolve goat milk casein in water, and then perform heat treatment, enzymatic hydrolysis treatment and freeze-drying treatment to obtain a goat milk-derived enzymatic hydrolysate;

[0006] Prepare the goat milk-derived enzymatic hydrolysate into a solution with a concentration of 145 mg / mL - 155 mg / mL, filter it through a 0.45 μm filter membrane, and then perform preliminary separation by preparative high performance liquid chromatography to obtain a Y product;

[0007] Prepare the Y product into a solution with a concentration of 20 mg / mL - 30 mg / mL, filter it through a 0.45 μm filter membrane, and then perform secondary separation by preparative high performance liquid chromatography to obtain a Y-X product;

[0008] Prepare the Y-X product into a solution with a concentration of 5 mg / mL - 15 mg / mL, filter it through a 0.45 μm filter membrane, use a C18 hydrophilic preparation column, and perform re-separation preparation by preparative high performance liquid chromatography to obtain an M product;

[0009] Prepare the M product into a solution with a concentration of 5 mg / mL - 15 mg / mL, filter it through a 0.22 μm filter membrane, perform re-separation preparation by RP-HPLC, and then perform rotary evaporation concentration treatment and freeze-drying treatment to obtain an F product.

[0010] In some optional embodiments of the present application, after dissolving the caprine cheese protein in water, heat treatment, enzymatic hydrolysis treatment and freeze-drying treatment are carried out to obtain a caprine milk-derived enzymolysate, including:

[0011] Dissolve the caprine cheese protein in distilled water to a concentration of 5%-15%. When the solution is heated to 35°C - 45°C, add NaOH to adjust the pH to 7.5 - 8.5; then add trypsin to the reaction mixture at a w / w ratio of 0.3% - 0.5%. After enzymatic hydrolysis for 2h - 3h, heat in a water bath to 85°C - 95°C to inactivate the enzyme for 25min - 35min, and then obtain the caprine milk-derived enzymolysate by freeze-drying.

[0012] In some optional embodiments of the present application, the caprine milk-derived enzymolysate is formulated into a solution with a concentration of 145mg / mL - 155mg / mL. After passing through a 0.45μm filter membrane, preparative high performance liquid chromatography is used for preliminary separation to obtain the Y product, including:

[0013] The caprine milk-derived enzymolysate is formulated into a solution with a concentration of 145mg / mL - 155mg / mL using ultrapure water. After passing through a 0.45μm filter membrane, the caprine milk antihypertensive peptide is preliminarily separated by preparative high performance liquid chromatography; according to the peak elution time, it is divided into Y1 - Y5, and a total of 5 sections are sampled. The ACE inhibition rates of the 5 sections of samples are measured respectively, and the component with the highest ACE inhibition rate is selected as the Y product.

[0014] In some optional embodiments of the present application, the Y product is formulated into a solution with a concentration of 20mg / mL - 30mg / mL. After passing through a 0.45μm filter membrane, preparative high performance liquid chromatography is used for secondary separation to obtain the Y-X product, including:

[0015] The Y product is formulated into a solution with a concentration of 20mg / mL - 30mg / mL, passes through a 0.45μm filter membrane, and preparative high performance liquid chromatography is used for re-separation; according to the peak elution time, it is divided into Y3-1, Y3-2, Y3-3, Y3-4, and a total of 4 sections are sampled; the ACE inhibition rates of the 4 sections of samples are measured respectively, and the component with the highest ACE inhibition rate is selected as the Y-X product.

[0016] In some optional embodiments of the present application, the Y-X product is formulated into a solution with a concentration of 5mg / mL - 15mg / mL, passes through a 0.45μm filter membrane, and a C18 hydrophilic preparative column is used. Preparative high performance liquid chromatography is used for re-separation and preparation to obtain the M product, including:

[0017] Prepare the Y-X product into a solution with a concentration of 5 mg / mL - 15 mg / mL, filter it through a 0.45 μm filter membrane, use a C18 hydrophilic preparation column, and perform re-separation and preparation on it by preparative high-performance liquid chromatography; divide it into M1 - M4 according to the peak emergence time, and collect samples in 4 segments; measure the ACE inhibition rate of the 4 segments of samples respectively, and select the component with the highest ACE inhibition rate as the M product.

[0018] As some alternative embodiments of the present application, after preparing the M product into a solution with a concentration of 5 mg / mL - 15 mg / mL, filtering it through a 0.22 μm filter membrane, and performing re-separation and preparation by RP-HPLC, perform rotary evaporation concentration treatment and freeze-drying treatment to obtain the F product, including:

[0019] Prepare the M product into a solution with a concentration of 5 mg / mL - 15 mg / mL, filter it through a 0.22 μm filter membrane, and perform re-separation and preparation by RP-HPLC; according to the peak emergence situation, it is divided into F1 - F7, a total of 7 peaks for sample collection; measure the ACE inhibition rate of each component after collecting the samples by rotary evaporation concentration and freeze-drying, and select the component with the highest ACE inhibition rate as the M product.

[0020] The embodiments of the present application also provide the following technical solution: an ACE inhibitory peptide monomer derived from goat milk, which is obtained by the method described above.

[0021] As some alternative embodiments of the present application, the amino acid sequence of the ACE inhibitory peptide monomer is as shown in SEQ ID NO: 1.

[0022] The embodiments of the present application also provide the following technical solution: an application of an ACE inhibitory peptide monomer derived from goat milk as described above, and the ACE inhibitory peptide monomer is used to prepare a drug for treating hypertension.

[0023] Compared with the prior art, the beneficial effects of the present application are: the present application uses goat milk casein as a raw material, and obtains a hypotensive oligopeptide through a variety of separation and purification means. The amino acid sequence of the oligopeptide is INNQFLPYPY, which has the characteristics of high purity, good activity, being able to be artificially synthesized, and being safe and non-toxic. The results of ACE inhibitory activity determination show that the hypotensive peptide has a significant inhibitory effect on ACE in vitro, can be applied to the field of hypotensive treatment, and is beneficial to the comprehensive utilization of goat milk casein. Description of the Drawings

[0024] Figure 1 It is the ACE inhibition rate before and after the hydrolysis of goat milk casein involved in the embodiments of the present application;

[0025] Figure 2 It is the preparative liquid phase primary separation chromatogram involved in the embodiments of the present application;

[0026] Figure 3 ACE inhibition rate of each component sample separated by preparative liquid chromatography for the first time in the embodiments of the present application;

[0027] Figure 4 Preparative liquid chromatography secondary separation chromatogram for the embodiments of the present application;

[0028] Figure 5 ACE inhibition rate of each component sample separated by preparative liquid chromatography for the second time in the embodiments of the present application;

[0029] Figure 6 Hydrophilic preparative column tertiary separation chromatogram for the embodiments of the present application;

[0030] Figure 7 ACE inhibition rate of each component sample separated by hydrophilic preparative column for the third time in the embodiments of the present application;

[0031] Figure 8 Reverse high performance liquid chromatography separation chromatogram for the embodiments of the present application;

[0032] Figure 9 ACE inhibition rate of each component sample separated by reverse high performance liquid chromatography for the embodiments of the present application;

[0033] Figure 10 Secondary mass spectrum of highly active monomer F2 for the embodiments of the present application;

[0034] Figure 11 N-terminal amino acid sequence of F2 measured by PPSQ for the embodiments of the present application. Detailed implementation manners

[0035] It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0036] As the main by-product of producing whey protein industrially, feta cheese protein is very wasteful of resources when sold at a low price or discarded as waste. Using it as a good source of bioactive peptides to prepare ACE inhibitory peptides by enzymatic hydrolysis can improve its application value. The present application takes the ovine milk-derived polypeptide obtained by enzymatic hydrolysis of feta cheese protein as the research object, uses the ACE inhibition model for activity tracking, and performs separation and purification by preparative liquid chromatography and reverse phase high performance liquid chromatography to obtain highly active ACE inhibitory peptides and identify their structures.

[0037] Specifically, the preparation method of the ACE inhibitory peptide monomer derived from ovine milk in the present application includes steps 1-step 5, that is:

[0038] Step 1: Dissolve feta cheese protein in water, then perform heat treatment, enzymatic hydrolysis treatment and freeze-drying treatment to obtain ovine milk-derived enzymolysate.

[0039] In a preferred embodiment, step 1 further includes: dissolving the caprine cheese protein in distilled water to a concentration of 5%-15%, adding NaOH to adjust the pH to 7.5-8.5 when the solution is heated to 35°C-45°C; then adding trypsin to the reaction mixture at a w / w ratio of 0.3%-0.5%, enzymatically hydrolyzing for 2h-3h, heating in a water bath to 85°C-95°C to inactivate the enzyme for 25min-35min, and then freeze-drying to obtain the caprine milk-derived hydrolysate.

[0040] Step 2: Prepare a solution of the caprine milk-derived hydrolysate with a concentration of 145mg / mL-155mg / mL, filter it through a 0.45μm filter membrane, and then perform preliminary separation using preparative high-performance liquid chromatography to obtain product Y.

[0041] In a preferred embodiment, step 2 further includes: preparing a solution of the caprine milk-derived hydrolysate with a concentration of 145mg / mL-155mg / mL using ultrapure water, filtering it through a 0.45μm filter membrane, and then performing preliminary separation of the caprine milk antihypertensive peptide using preparative high-performance liquid chromatography; dividing it into 5 segments of Y1-Y5 according to the peak elution time, collecting samples for each segment, respectively measuring the ACE inhibitory rate of the 5 segment samples, and selecting the component with the highest ACE inhibitory rate as product Y.

[0042] Step 3: Prepare a solution of product Y with a concentration of 20mg / mL-30mg / mL, filter it through a 0.45μm filter membrane, and then perform secondary separation using preparative high-performance liquid chromatography to obtain product Y-X.

[0043] In a preferred embodiment, step 3 further includes: preparing a solution of product Y with a concentration of 20mg / mL-30mg / mL, filtering it through a 0.45μm filter membrane, and performing re-separation using preparative high-performance liquid chromatography; dividing it into 4 segments of Y3-1, Y3-2, Y3-3, and Y3-4 according to the peak elution time, collecting samples for each segment; respectively measuring the ACE inhibitory rate of the 4 segment samples, and selecting the component with the highest ACE inhibitory rate as product Y-X.

[0044] Step 4: Prepare a solution of product Y-X with a concentration of 5mg / mL-15mg / mL, filter it through a 0.45μm filter membrane, use a C18 hydrophilic preparative column, and perform re-separation and preparation using preparative high-performance liquid chromatography to obtain product M.

[0045] In a preferred embodiment, step 4 further includes: formulating the Y-X product into a solution with a concentration of 5 mg / mL - 15 mg / mL, passing it through a 0.45 μm filter membrane, using a C18 hydrophilic preparation column, and performing re-separation and preparation on it by preparative high-performance liquid chromatography; dividing it into M1 - M4 according to the elution time, and collecting samples in 4 segments; respectively measuring the ACE inhibitory rates of the 4 segments of samples, and selecting the component with the highest ACE inhibitory rate as the M product.

[0046] Step 5: Formulate the M product into a solution with a concentration of 5 mg / mL - 15 mg / mL, pass it through a 0.22 μm filter membrane, perform re-separation and preparation using RP-HPLC, and then perform rotary evaporation concentration and freeze-drying treatments to obtain the F product.

[0047] In a preferred embodiment, step 5 further includes: formulating the M product into a solution with a concentration of 5 mg / mL - 15 mg / mL, passing it through a 0.22 μm filter membrane, and performing re-separation and preparation using RP-HPLC; according to the elution conditions, it is divided into F1 - F7, a total of 7 peaks for sample collection; after the collected samples are concentrated by rotary evaporation and freeze-dried, measure the ACE inhibitory rates of each component, and select the component with the highest ACE inhibitory rate as the M product.

[0048] For the ACE inhibitory peptide monomer prepared through the above steps, use a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF-MS / MS) to determine its amino acid sequence. Take 1 μL of the peptide solution on the target plate; after drying at room temperature, in-situ add 0.1 μL of the matrix (α-cyano-4-hydroxycinnamic acid, HCCA). Set the instrument parameters, calculate the mass-to-charge ratio by detecting the flight time of the sample ions, and obtain the spectrum. And use an amino acid sequencer (PPSQ) to identify the structure of the ACE inhibitory peptide monomer prepared through the above steps. The analysis principle of the amino acid sequencer used is the Edman degradation reaction. Before analyzing the sample, first perform pretreatment on the glass fiber membrane. First inject 15 μL of polybrene solution into the glass fiber membrane, and after drying, perform pretreatment in the reaction chamber. After the pretreatment is completed, inject 15 μL of the sample to be tested with a concentration of 0.2 mg / mL into the glass fiber membrane, and after drying, perform amino acid sequence analysis to obtain the amino acid sequence of the sample, as shown in SEQ ID NO: 1, which is INNQFLPYPY.

[0049] The above technical solutions will be further described in detail with specific embodiments as follows:

[0050] Unless otherwise specified, the kit materials involved in the embodiments are all commercially available.

[0051] Wherein:

[0052] The AL104 one in ten-thousandth electronic balance was purchased from Mettler-Toledo Instruments Co., Ltd.

[0053] The TDL-5 centrifuge was purchased from Shanghai Anting Instruments Co., Ltd.

[0054] The R204B3 rotary evaporator was purchased from Shanghai Shen Science and Technology Co., Ltd.

[0055] The Thermo Modulyo freeze dryer was purchased from Thermo Fisher Scientific Co., Ltd.

[0056] The KQ500-B ultrasonic cleaner was purchased from Kunshan Ultrasonic Instruments Co., Ltd.

[0057] The VM-300S vortex mixer was purchased from Qunan Experimental Instruments Co., Ltd.

[0058] The HT-400B electrothermal constant temperature incubator was purchased from Shanghai Hetian Scientific Instruments Co., Ltd.

[0059] The LC-8 preparative high performance liquid chromatography was purchased from Shimadzu Corporation of Japan.

[0060] The LC-10ATvp plus analytical high performance liquid chromatography was purchased from Shimadzu Corporation of Japan.

[0061] Example 1 Separation and purification of ACE inhibitory peptide

[0062] S1. Dissolve the sheep milk cheese protein in distilled water to a concentration of 10%. When the solution is heated to 40 °C, add NaOH to adjust the pH to 8.0. Then add trypsin to the reaction mixture at a ratio of 0.4% (w / w). After enzymatic hydrolysis for 2.5 h, heat in a water bath to 90 °C to inactivate the enzyme for 30 min, and then freeze-dry to obtain the enzymolysate from sheep milk. Measure the ACE inhibitory rate of its enzymolysate to be 28.03%. As Figure 1 shown.

[0063] S2. First, perform the first separation of the sheep milk-derived polypeptide by preparative high performance liquid chromatography, number according to the elution peak order, and collect them into 5 segments respectively, named Y1, Y2, Y3, Y4 and Y5, as Figure 2 shown. Then perform vacuum concentration and freeze-drying. Conduct ACE inhibitory activity measurement experiments on the six initially separated samples under the same conditions. The results are as Figure 3 shown. When the unified concentration of the samples is 1 mg / mL, the inhibitory rate of Y3 reaches 46.13%, which is better than the activity of other samples in inhibiting ACE. Therefore, Y3 is further separated and purified.

[0064] S3. Select Y3 with better ACE inhibitory activity in the previous separation step and perform the second separation using preparative liquid chromatography. According to the elution order, Y3 was further divided into 4 segments and collected separately, named Y3-1, Y3-2, Y3-3, and Y3-4, as Figure 4 shown. Then, perform vacuum concentration and freeze-drying, and measure the ACE inhibitory activity of each component. The results are as Figure 5 shown. After the second liquid-phase separation, the ACE inhibitory activity of component Y3-3 is better than that of other components, and the inhibition rate reaches 62.58%. Therefore, Y3-3 is further separated and purified.

[0065] S4. Select an ECOSIL C18 hydrophilic preparative column to further separate Y3-3. According to the elution order, Y3-3 was further divided into four segments, named M1, M2, M3, and M4, as Figure 6 shown. Then, perform vacuum concentration and freeze-drying, and measure the ACE inhibitory activity of each component. The results are as Figure 7 shown. Among them, the activity of M3 is the highest, and the inhibition rate reaches 70.17%. However, the polypeptide composition in this component is still relatively complex. In order to further clarify the polypeptide fragment with the strongest activity in the goat milk-derived polypeptide, component M3 is further purified and analyzed.

[0066] S5. Further analyze and purify the most active component M3 in the previous step by RP-HPLC. The liquid chromatogram of this component is as Figure 8 shown. The chromatogram shows that there are a total of 7 chromatographic peaks, named F1, F2, F3, F4, F5, F6, and F7. Collect each component in turn and then perform freeze-drying. Subsequently, measure the ACE inhibitory activity of each component under the same conditions. The results are as Figure 9 shown. Among them, the ACE inhibition rate of component F3 is the highest, and F2 is the second.

[0067] S6. Use MALDI-TOF-MS / MS to measure the F2 monomer. The M / Z of the parent ion is 1290.6005; from Figure 10 the secondary mass spectrum, it can be seen that the M / Z of the main fragment peaks are 1063, 949, 821, etc. It is thus speculated that this ACE inhibitory peptide is an oligopeptide composed of 9-12 amino acids. On this basis, set the operating parameters of the amino acid sequencer and perform the N-terminal amino acid sequence determination of F2.

[0068] S7. Use an amino acid sequencer to determine the N-terminal amino acid sequence of the polypeptide ( Figure 11) The N-terminal sequence of F2 was obtained as: INNQFLPYPY. At the same time, it was compared with the amino acid sequence deduced from the secondary mass spectrometry of the precursor ion in the MALDI-TOF-MS / MS first-level mass spectrometry, and the results of the two were consistent. It was determined that the sequence of this highly active ACE inhibitory peptide was INNQFLPYPY. By searching the UniProt protein database, it was determined that the peptide of this sequence was derived from κ-casein in goat milk, and the inhibitory peptide with the sequence INNQFLPYPY was reported for the first time.

[0069] The ACE inhibitory activity detection method was carried out according to the following method:

[0070] 10 μL of ACE solution (0.2 U / mL) was added to the blank group, the control group and the sample group respectively. 10 μL of ACEI was added to the sample group, and 10 μL of buffer was added to the blank group and the control group. After standing at 37 °C for 5 min, 30 μL of HHL solution (6.5 mmol / mL, HHL was dissolved in 0.1 mol / L boric acid buffer at pH 8.3 containing 0.3 mol / L NaCl) was added to all groups. After reacting at 37 °C for 1 h, 80 μL of 1.0 mol / L HCl was added to the control group and the sample group to terminate the reaction.

[0071]

[0072] Liquid chromatography conditions: ① Chromatographic column: ECOSIL C18 (260 mm × 4.6 mm, 5 μm); ② Mobile phase: acetonitrile: ultrapure water = 25:75 (containing 0.1% (v / v) TFA); ③ Flow rate: 1 mL / min; ④ Detection wavelength: 228 nm; ⑤ Column temperature: 30 °C; ⑥ Injection volume: 20 μL.

[0073] Result calculation:

[0074] Principle: HHL is rapidly decomposed under the catalysis of ACE to produce hippuric acid (Hip) and dipeptide (His-Leu, HL). Hippuric acid has the maximum absorption at 228 nm. When an ACEI sample is added, the ACE enzyme activity is inhibited, and the production amount of hippuric acid decreases. Therefore, the inhibitory rate of ACEI on ACE activity can be evaluated by measuring the production amount of hippuric acid by high performance liquid chromatography.

[0075] The calculation formula is:

[0076] In the formula: R: The inhibitory rate of the ACEI sample on ACE (%);

[0077] A: The peak area of hippuric acid in the control group;

[0078] B: The peak area of hippuric acid in the group added with ACEI;

[0079] A0: The peak area of hippuric acid in the blank tube.

[0080] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A preparation method of an ACE inhibitory peptide monomer derived from goat milk, characterized in that, It includes the following steps: Dissolve the caprine cheese protein in water, then perform heat treatment, enzymatic hydrolysis treatment and freeze-drying treatment to obtain a caprine milk-derived enzymolysate; Prepare a solution of the caprine milk-derived enzymolysate with a concentration of 145 mg / mL - 155 mg / mL, filter it through a 0.45 μm filter membrane, and then perform preliminary separation by preparative high performance liquid chromatography to obtain a Y product; Prepare a solution of the Y product with a concentration of 20 mg / mL - 30 mg / mL, filter it through a 0.45 μm filter membrane, and then perform secondary separation by preparative high performance liquid chromatography to obtain a Y-X product; Prepare a solution of the Y-X product with a concentration of 5 mg / mL - 15 mg / mL, filter it through a 0.45 μm filter membrane, use a C18 hydrophilic preparative column, and perform re-separation and preparation by preparative high performance liquid chromatography to obtain an M product; Prepare a solution of the M product with a concentration of 5 mg / mL - 15 mg / mL, filter it through a 0.22 μm filter membrane, perform re-separation and preparation by RP-HPLC, and then perform rotary evaporation concentration treatment and freeze-drying treatment to obtain an F product.

2. The preparation method of the ACE inhibitory peptide monomer derived from goat milk according to claim 1, characterized in that, The step of dissolving the caprine cheese protein in water, then performing heat treatment, enzymatic hydrolysis treatment and freeze-drying treatment to obtain a caprine milk-derived enzymolysate includes: Dissolve the caprine cheese protein in distilled water to a concentration of 5% - 15%. When the solution is heated to 35°C - 45°C, add NaOH to adjust the pH to 7.5 - 8.5; then add trypsin to the reaction mixture at a w / w ratio of 0.3% - 0.5%, after enzymatic hydrolysis for 2 h - 3 h, heat it in a water bath to 85°C - 95°C to inactivate the enzyme for 25 min - 35 min, and then freeze-dry to obtain a caprine milk-derived enzymolysate.

3. The preparation method of the ACE inhibitory peptide monomer derived from goat milk according to claim 1, characterized in that, The step of preparing a solution of the caprine milk-derived enzymolysate with a concentration of 145 mg / mL - 155 mg / mL, filtering it through a 0.45 μm filter membrane, and then performing preliminary separation by preparative high performance liquid chromatography to obtain a Y product includes: Prepare a solution of the caprine milk-derived enzymolysate with a concentration of 145 mg / mL - 155 mg / mL in ultrapure water, filter it through a 0.45 μm filter membrane, and then perform preliminary separation of caprine milk antihypertensive peptides by preparative high performance liquid chromatography; divide it into 5 segments of Y1 - Y5 according to the peak elution time, collect samples in 5 segments respectively, measure the ACE inhibition rate of the 5 segments of samples, and select the component with the highest ACE inhibition rate as the Y product.

4. The preparation method of the ACE inhibitory peptide monomer derived from goat milk according to claim 1, characterized in that, The step of preparing a solution of the Y product with a concentration of 20 mg / mL - 30 mg / mL, filtering it through a 0.45 μm filter membrane, and then performing secondary separation by preparative high performance liquid chromatography to obtain a Y-X product includes: Prepare a solution of the Y product with a concentration of 20 mg / mL - 30 mg / mL, filter it through a 0.45 μm filter membrane, and perform re-separation on it by preparative high performance liquid chromatography; divide it into 4 segments of Y3-1, Y3-2, Y3-3, Y3-4 according to the peak elution time; collect samples in 4 segments respectively; measure the ACE inhibition rate of the 4 segments of samples, and select the component with the highest ACE inhibition rate as the Y-X product.

5. The preparation method of the ACE inhibitory peptide monomer derived from goat milk according to claim 1, characterized in that, The Y-X product is formulated into a solution with a concentration of 5 mg / mL - 15 mg / mL, filtered through a 0.45 μm filter membrane, and then re-separated and prepared using a C18 hydrophilic preparation column by preparative high performance liquid chromatography to obtain the M product, including: The Y-X product is formulated into a solution with a concentration of 5 mg / mL - 15 mg / mL, filtered through a 0.45 μm filter membrane, and then re-separated and prepared using a C18 hydrophilic preparation column by preparative high performance liquid chromatography. It is divided into 4 sections for sample collection according to the peak elution time, namely M1 - M4. The ACE inhibitory rates of the 4 sections of samples are measured respectively, and the component with the highest ACE inhibitory rate is selected as the M product.

6. The preparation method of the ACE inhibitory peptide monomer derived from goat milk according to claim 1, characterized in that, The M product is formulated into a solution with a concentration of 5 mg / mL - 15 mg / mL, filtered through a 0.22 μm filter membrane, re-separated and prepared by RP-HPLC, and then subjected to rotary evaporation concentration and freeze-drying treatments to obtain the F product, including: The M product is formulated into a solution with a concentration of 5 mg / mL - 15 mg / mL, filtered through a 0.22 μm filter membrane, and re-separated and prepared by RP-HPLC. According to the peak elution situation, it is divided into 7 peaks for sample collection, namely F1 - F7. After the collected samples are concentrated by rotary evaporation and freeze-dried, the ACE inhibitory rates of each component are measured, and the component with the highest ACE inhibitory rate is selected as the M product.

7. An ACE inhibitory peptide monomer derived from goat milk, characterized in that, Obtained by the method according to any one of claims 1 - 6.

8. The ACE inhibitory peptide monomer derived from goat milk according to claim 7, characterized in that, The amino acid sequence of the ACE inhibitory peptide monomer is as shown in SEQ ID NO:

1.

9. Use of the ACE inhibitory peptide monomer derived from goat milk according to any one of claims 7-8, characterized in that, The ACE inhibitory peptide monomer is used for preparing a drug for treating hypertension.

Images