ACE (Angiotensin Converting Enzyme) inhibitory octapeptide YI8 derived from cattle cheese protein hydrolysate and application

Abstract

The invention discloses ACE (Angiotensin Converting Enzyme) inhibitory octapeptide YI8 derived from bovine cheese protein hydrolysate and application, and relates to the technical field of bioactive peptides, and the technical scheme is characterized in that the ACE inhibitory octapeptide YI8 comprises an amino acid sequence of YKVPQLEI. The ACE inhibitory octapeptide YI8 derived from the cattle cheese protein hydrolysate is obtained by screening from the cattle cheese protein hydrolysate, and is an octapeptide YI8 with ACE inhibitory activity, the in-vitro ACE inhibition rate of the ACE inhibitory octapeptide YI8 derived from the cattle cheese protein hydrolysate is tested to be 70.86% under the condition of the concentration of 1mg / ml, and the ACE inhibitory octapeptide YI8 is suitable for functional food for assisting in lowering blood pressure.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and more specifically, it relates to an octapeptide YI8 with ACE inhibitory activity derived from milk casein hydrolysate and its application. Background Art

[0002] Angiotensin converting enzyme (ACE) has the effects of constricting blood vessels and raising blood pressure. Inhibiting the activity of ACE can play a role in lowering blood pressure. Food proteins such as dairy products, grains, eggs, and seafood can be used as raw materials to prepare ACE inhibitory peptides by enzymatic hydrolysis. Different protein raw materials and enzymatic hydrolysis processes will obtain different enzymatic hydrolysates with ACE inhibitory activity, but the compositions of the active peptides therein are not the same. The amino acid compositions and structures of ACE inhibitory peptides vary greatly, without a fixed or unified amino acid composition, and the components of the protease hydrolysate are complex, greatly increasing the difficulty of separation and extraction.

[0003] Chinese invention patent with the patent number L200480022213.9 discloses a casein hydrolyzate, its production process and application. The casein hydrolyzate, its production process and application disclose the use of extracellular enzyme groups of Aspergillus oryzae to hydrolyze casein to prepare a protease hydrolysate with an ACE inhibition rate; there are also research reports on using Moringa oleifera seed rennet to hydrolyze buffalo milk cheese (Zhong Yuwang, Yu Mengyi, Zhang Dan, et al. Application of Moringa oleifera seed rennet in the preparation of buffalo milk cheese and analysis of angiotensin-converting enzyme inhibitory peptides. Food Science, 2024, 45(18): 87-98.) and using alkaline protease to hydrolyze sheep milk casein (Tang Haixia, Zhang Yan, Ge Wupeng, et al. Process optimization of preparing ACE inhibitory peptides from sheep milk casein by enzymatic hydrolysis and its inhibitory mechanism. Journal of Chinese Institute of Food Science and Technology, 2022, 22(06): 220-231). However, the article does not further screen and isolate the peptide monomers with ACE inhibitory activity therein and further evaluate their ACE inhibitory activity through experiments.

[0004] SAITO Y et al. reported two ACE inhibitory peptides isolated from sake proteins in the article "Structure and activity of angiotensin I converting enzyme inhibitory peptides from sake and sake lees", namely IYPRY (in vitro IC50 = 8.5 mM) and YGGY (in vitro IC50 = 3.4 mM). Huan Xiang et al. reported the peptide GPPGPPGL with ACE inhibitory activity isolated from the enzymatic hydrolysate of eel (Anguilla japonica) bone collagen in the article "Angiotensin-I-converting enzyme inhibitory peptides from eel (Anguilla japonica) bone collagen: preparation, identification, molecular docking, and protective function on HUVECs", and its IC50 value was 535.84 μM. Guanmian Wei et al. reported the peptides VE, FEF, and WEEF with ACE inhibitory activity isolated from fermented tofu in the article "Identification and characterization of umami-ACE inhibitory peptides from traditional fermented soybean curds", and their IC50 values were WEEF (85 ± 2 μM) < FEF (170 ± 10 μM) < VE (205 ± 5 μM). Shenghao Xing et al. reported the ACE inhibitory peptides SLPQ and PYVRYL isolated from the enzymatic hydrolysate of goat milk in the article "Goat Milk Protein-Derived ACE Inhibitory Peptide SLPQ Exerts Hypertension Alleviation Effects Partially by Regulating the Inflammatory Stress of Endothelial Cells", and PYVRYL performed better than SLPQ.Meanwhile, Martin M et al. further listed in the article "Effects of natural peptides from food proteins on angiotensin converting enzyme activity and hypertension" the structures of active peptides derived from protease hydrolysates with ACE inhibitory function discovered in recent years.

[0005] However, the enzymatic hydrolysis strategies adopted in the above-mentioned prior arts are based on different enzymatic hydrolysis strategies, which will have a huge impact on the functional activity of the enzymatic hydrolysate and the sequence composition and abundance of active peptides in the enzymatic hydrolysate, and none of them can obtain functional foods applied to assist in reducing blood pressure, so there is room for improvement. Summary of the Invention

[0006] In view of this, the first object of the present application is to provide an ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate. The specific scheme is as follows:

[0007] An ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, whose amino acid sequence is YKVPQLEI.

[0008] Preferably: Its molecular weight is 989.16 Da.

[0009] Preferably: It is screened from milk casein hydrolysate.

[0010] Preferably: The preparation method of the milk casein hydrolysate includes the following steps:

[0011] Step 1: Take milk casein, the addition amount of casein is 1-5%, pH 8-9, add 2-6% protease, carry out enzymatic hydrolysis, control the enzymatic hydrolysis temperature at 37-55°C, and the enzymatic hydrolysis time is 4-6 h to obtain an enzymatic hydrolysis product;

[0012] Step 2: Preliminarily filter the enzymatic hydrolysis product to remove residues to obtain a clarified polypeptide enzymatic hydrolysate, and perform freeze-drying treatment to obtain a polypeptide freeze-dried powder;

[0013] Step 3: Perform mass spectrometry determination on the polypeptide freeze-dried powder by LC-MS / MS, analyze the results of the mass spectrometry determination using mass spectrometry analysis software, and compare with the milk casein sequence to obtain a polypeptide sequence;

[0014] Step 4: Perform molecular docking on the polypeptide sequence with ACE protein. Before docking, convert the 2D structure of the polypeptide into a 3D structure by energy minimization, and screen to obtain a polypeptide sequence with strong binding ability to ACE protein;

[0015] Step 5: Solid-phase synthesize the screened polypeptide sequences, and use an in vitro ACE enzyme activity inhibition assay to obtain the octapeptide YI8 with ACE inhibitory activity.

[0016] Preferably: In step 1, the addition amount of milk casein is 2%; the addition amount of the mixed protease is 5%, and the protease is composed of trypsin and enzyme group X with a mixing ratio of 3:1. The enzymatic hydrolysis temperature is controlled at 37°C, the pH value is 8.0, and the enzymatic hydrolysis time is 6 h.

[0017] Preferably: In step 3, the conditions of the mass spectrometry determination are controlled as follows: in the liquid phase method, the chromatographic column is C18, 3 μm, 250 mm X 75 μm, Eksigent, phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300 nl / min, the injection volume is 4 μl, the chromatographic gradient is 60 min, and the elution gradient is: 0 - 48 min: phase A decreases uniformly from 95% to 60%; 48 - 55 min: phase A decreases uniformly from 60% to 30%; 55 - 56 min: phase A decreases uniformly from 30% to 0; 56 - 60 min: maintain 0% phase A.

[0018] Preferably: In the mass spectrometry determination, the equipment model used is: Orbitrap Exploris 480 - Thermo Fisher, based on the positive ion detection mode, and the following parameters are controlled for the determination: the primary resolution is 120,000, the AGC is set to 300, the scanning range is 200 - 1600 m / z, the MIPS mode is peptide, the selected valence states are 1 - 5, the secondary resolution is 15,000, and the separation window is 1.6 m / z.

[0019] Preferably: In step 5, the in vitro ACE enzyme activity inhibition assay is to test the in vitro ACE activity inhibition effect of the active peptide based on a concentration of 1 mg / ml.

[0020] The second object of the present invention is to provide an application of an ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, including using the ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate as described above and applying it to functional foods for assisting in reducing blood pressure.

[0021] Preferably: Under the concentration condition of 1 mg / ml, the in vitro ACE inhibition rate obtained by testing the functional food for assisting in reducing blood pressure is 70.86%.

[0022] As can be seen from the above solution, the present application provides an ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate and its application. The ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate is screened from milk casein hydrolysate and is the octapeptide YI8 with ACE inhibitory activity. Under the condition of a concentration of 1 mg / ml, the in vitro ACE inhibitory rate of the ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate is tested to be 70.86%, and it is applicable to functional foods for assisting in reducing blood pressure, thereby obtaining a significant blood pressure reduction effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the 3D molecular docking map of YKVPQLEI of the present application and ACE;

[0024] Figure 2 is Figure 1 the partial enlarged schematic diagram in

[0025] Figure 3 is the 2D molecular docking map of YKVPQLEI of the present application and ACE;

[0026] Figure 4 is the comparison chart of the ACE inhibitory rates of YI8 of the present application and AmealPeptide. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] To make the technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the scope of protection of the present application.

[0028] The following will specifically describe an ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate and its application of the present application.

[0029] An ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, its amino acid sequence is YKVPQLEI, and its molecular weight is 989.16 Da.

[0030] In order to obtain the ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, it will be screened from milk casein hydrolysate.

[0031] It should be noted that the preparation method of the milk casein hydrolysate in the embodiments of the present application includes the following steps:

[0032] Step 1: Take milk casein with a casein addition amount of 1-5%, pH 8-9, add 2-6% protease, and carry out enzymatic hydrolysis. The enzymatic hydrolysis temperature is controlled at 37-55°C, and the enzymatic hydrolysis time is 4-6 h to obtain an enzymatic hydrolysis product.

[0033] Step 2: Preliminarily filter the enzymatic hydrolysis product to remove residues to obtain a clear polypeptide enzymatic hydrolysate, and perform freeze-drying treatment to obtain polypeptide lyophilized powder.

[0034] Step 3: Perform mass spectrometry determination on the polypeptide lyophilized powder by LC-MS / MS. The results of the mass spectrometry determination are analyzed using mass spectrometry analysis software and compared with the milk casein sequence to obtain a polypeptide sequence. Among them, the conditions for mass spectrometry determination are controlled as follows: in the liquid phase method, the chromatographic column is C18, 3μm, 250mmX75μm, Eksigent, the mobile phase A is water, 0.1% formic acid; the mobile phase B is acetonitrile, 0.1% formic acid, the flow rate is 300 nl / min, the injection volume is 4 μl, the chromatographic gradient is 60 min, and the elution gradient is: 0-48 min: the mobile phase A decreases uniformly from 95% to 60%; 48-55 min: the mobile phase A decreases uniformly from 60% to 30%; 55-56 min: the mobile phase A decreases uniformly from 30% to 0; 56-60 min: maintain 0% mobile phase A. In the mass spectrometry determination, the equipment model used is: Orbitrap Exploris 480-Thermofisher, based on the positive ion detection mode, and the following parameters are controlled for determination: the first-order resolution is 120,000, the AGC is set to 300, the scanning range is 200-1600 m / z, the MIPS mode is peptide, the selected valence state is 1-5, the second-order resolution is 15,000, and the separation window is 1.6 m / z.

[0035] Step 4: Perform molecular docking of the polypeptide sequence with ACE protein. Before docking, convert the 2D structure of the polypeptide into a 3D structure by energy minimization, and screen to obtain a polypeptide sequence with strong binding ability to ACE protein.

[0036] Step 5: Solid-phase synthesize the screened polypeptide sequence, and use an in vitro ACE enzyme activity inhibition test to test the in vitro ACE activity inhibition effect of the active peptide based on a concentration of 1 mg / ml to obtain the octapeptide YI8 with ACE inhibitory activity.

[0037] In order to further increase the content of the ACE inhibitory octapeptide YI8 derived from the milk casein hydrolysate in the milk casein hydrolysate to reduce the screening difficulty, in Step 1 of the embodiment of the present application, the addition amount of milk casein is 2%; the addition amount of the mixed protease is 5%, and the protease is composed of trypsin and enzyme group X with a mixing ratio of 3:1. The enzymatic hydrolysis temperature is controlled at 37°C, the pH value is 8.0, and the enzymatic hydrolysis time is 6 h.

[0038] Application of an ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, including using the above-mentioned ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate and applying it to functional foods for assisting in reducing blood pressure. And under the concentration condition of 1 mg / ml, the in vitro ACE inhibitory rate of this functional food for assisting in reducing blood pressure was tested to be 70.86%.

[0039] Example 1

[0040] An ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, its amino acid sequence is YKVPQLEI, and its molecular weight is 989.16 Da.

[0041] In order to obtain this ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, it will be screened from milk casein hydrolysate.

[0042] It should be noted that the preparation method of the milk casein hydrolysate in the embodiments of this application includes the following steps:

[0043] Step 1: Take milk casein, with the addition amount of casein being 2%, pH 8.0, add 5% protease (trypsin: enzyme group X = 3:1) for enzymatic hydrolysis, control the enzymatic hydrolysis temperature at 37°C, and the enzymatic hydrolysis time is 6 h;

[0044] Step 2: Conduct preliminary filtration on the enzymatic hydrolysis product, remove the residue to obtain a clear polypeptide enzymatic hydrolysate, and obtain a polypeptide lyophilized powder after lyophilization;

[0045] Step 3: Perform mass spectrometry determination on the polypeptide lyophilized powder using LC-MS / MS, analyze the results of the mass spectrometry determination using mass spectrometry analysis software, and compare with the milk casein sequence to obtain several polypeptide sequences; The LC-MS / MS determination conditions are as follows: In the liquid phase method, the chromatographic column is C18, 3 μm, 250 mm × 75 μm (Eksigent), phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300 nl / min, the injection volume is 4 μl, and the 60 min chromatographic gradient is as follows: 0 - 48 min: phase A decreases uniformly from 95% to 60%; 48 - 55 min: phase A decreases uniformly from 60% to 30%; 55 - 56 min: phase A decreases uniformly from 30% to 0; 56 - 60 min: maintain 0% phase A. Mass spectrometry method: Orbitrap Exploris 480 - Thermofisher, positive ion detection mode, the first - level resolution is 120000, the AGC is set to 300, the scanning range is 200 - 1600 m / z. The MIPS mode is peptide, select valence states 1 - 5, the second - level resolution is 15000, and the separation window is 1.6 m / z;

[0046] As shown in Table 1, 20 high-abundance active heptapeptide and octapeptide sequences were finally obtained.

[0047] Table 1. High-abundance active peptide sequences in milk casein hydrolysate

[0048]

[0049]

[0050] Step 4: Use molecular docking software to perform molecular docking of the polypeptide sequences with ACE protein. Before docking, convert the 2D structure of the polypeptide to a 3D structure by energy minimization, and screen out the polypeptide sequences with strong binding ability to ACE protein. The 3D structure of ACE can be downloaded from the RCSB Protein Data Bank (PDB ID: 1O8A). The results of molecular docking are shown in Table 2 below. The docking results are represented by -CiE. The larger the -CiE value, the stronger the binding ability of the polypeptide to ACE, and the more likely it is to inhibit ACE activity.

[0051] Table 2. Prediction results of the interaction between milk casein components and ACE

[0052]

[0053]

[0054] S5: Among all the tested active peptides, the docking score of YKVPQLEI (YI8) is the largest. Therefore, we used YI8 for further molecular docking analysis, and the binding mode of the ACE inhibitory peptide YI8 of the present invention to ACE is as Figure 1 , Figure 2 , Figure 3 shown. Among them, as shown in Table 3, there are 5 H-H bond interactions, 6 C-H bond interactions, 2 salt bridges between YI8 and ACE, and 35 amino acid residues participate in the interaction between YI8 and ACE.

[0055] Table 3. Specific situation of the molecular docking of YI8 and ACE

[0056]

[0057]

[0058] Step 6: Solid-phase synthesize YKVPQLEI (YI8) and evaluate its in vitro ACE inhibitory activity.

[0059] The test method is as follows: Both the sample and the hydrolyzed sample are diluted 9 times with distilled water to make the final concentration 1 mg / ml. 0.1 mM boric acid-borax buffer: Weigh 12.37 g of boric acid and make up the volume to 1000 mL with distilled water. Weigh 19.07 g of sodium tetraborate and make up the volume to 1000 mL with distilled water. Take 175 mL of sodium tetraborate solution and 325 mL of boric acid solution, adjust the pH to 8.3 with hydrochloric acid or sodium hydroxide, and finally add 17.532 g of sodium chloride, dissolve and make up the volume to 1000 mL. 5 mM HHL solution: Weigh 2 mg of HHL and dissolve it in 920 μL of 0.1 mol / L borate buffer (pH 8.3) containing 0.3 mol / L sodium chloride. 0.1 U / mL ACE solution: The whole bottle of ACE is 0.25 U in total, dissolve it with 125 μL of 0.01 mol / L potassium phosphate buffer (pH 7.0) containing 0.5 M sodium chloride, store it at -20 °C, and dilute it 20 times with 0.1 mol / L borate buffer (pH 8.3) containing 0.3 M sodium chloride before use. 1 M hydrochloric acid solution: Slowly add 9 mL of hydrochloric acid along the wall into 100 mL of distilled water. 1 mg / mL hippuric acid standard solution: Weigh 10 mg of hippuric acid and make up the volume to 10 mL with distilled water. Dilute it with distilled water to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL standard solutions before use.

[0060] Reaction conditions: Take a 2 mL centrifuge tube, add 80 μL of HHL solution (5 mM) and 20 μL of the sample, shake well, incubate at 37 °C for 5 min, then add 20 μL of ACE solution (0.1 U / mL), mix well and incubate at 37 °C for 30 min. Add 200 μL of 1 M hydrochloric acid solution to terminate the reaction. Filter with a 0.22 μm filter membrane and determine the content of hippuric acid by HPLC. In the control experiment, use 20 μL of distilled water instead of the sample.

[0061] Liquid phase conditions: Chromatographic column: Agilent ZORBAX SB-C18 chromatographic column (4.6×250 mm, 5 μm). Mobile phase: The volume ratio of acetonitrile to water is 25:75, and 0.05% trifluoroacetic acid is added to the water phase. Detection parameters: Column oven temperature 30 °C, injection volume 20 μL, flow rate 0.5 mL / min, detection wavelength 228 nm.

[0062] ACE inhibition rate calculation formula: ACE inhibition rate (%) = (B - A) x 100 / B. Where A is the concentration of hippuric acid in the sample; B is the concentration of hippuric acid in the blank control.

[0063] Finally, as Figure 4 shown, the in vitro ACE inhibition rate of YKVPQLEI is 70.86% at a concentration of 1 mg / ml, and the in vitro ACE inhibition rate of Anylac polypeptide is 54.36% at the same concentration.

[0064] Example 2

[0065] An ACE-inhibitory octapeptide YI8 derived from milk casein hydrolysate, with the amino acid sequence YKVPQLEI and a molecular weight of 989.16 Da.

[0066] To obtain this ACE-inhibitory octapeptide YI8 derived from milk casein hydrolysate, it will be screened from milk casein hydrolysate.

[0067] It should be noted that the preparation method of the milk casein hydrolysate in the embodiments of this application includes the following steps:

[0068] Step 1: Take milk casein with a casein addition amount of 1%, pH 8.5, add 2% protease, and carry out enzymatic hydrolysis. The enzymatic hydrolysis temperature is controlled at 45°C and the enzymatic hydrolysis time is 4 h to obtain an enzymatic hydrolysis product;

[0069] Step 2: Preliminarily filter the enzymatic hydrolysis product to remove residues to obtain a clear polypeptide hydrolysate, and perform freeze-drying treatment to obtain a polypeptide freeze-dried powder;

[0070] Step 3: Perform mass spectrometry determination on the polypeptide freeze-dried powder using LC-MS / MS. The results of the mass spectrometry determination are analyzed using mass spectrometry analysis software and compared with the milk casein sequence to obtain a polypeptide sequence; among them, the conditions for mass spectrometry determination are controlled as follows: in the liquid phase method, the chromatographic column is C18, 3μm, 250mmX75μm, Eksigent, phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300 nl / min, the injection volume is 4 μl, the chromatographic gradient is 60 min, and the elution gradient is: 0 - 48 min: phase A decreases uniformly from 95% to 60%; 48 - 55 min: phase A decreases uniformly from 60% to 30%; 55 - 56 min: phase A decreases uniformly from 30% to 0; 56 - 60 min: maintain 0% phase A. In the mass spectrometry determination, the equipment model used is: Orbitrap Exploris 480 - Thermofisher, based on the positive ion detection mode, and the following parameters are controlled for determination: the first-level resolution is 120000, the AGC is set to 300, the scanning range is 200 - 1600 m / z, the MIPS mode is peptide, the selected valence states are 1 - 5, the second-level resolution is 15000, and the separation window is 1.6 m / z.

[0071] Step 4: Perform molecular docking on the polypeptide sequence and the ACE protein. Before docking, convert the 2D structure of the polypeptide into a 3D structure through energy minimization, and screen to obtain a polypeptide sequence with strong binding ability to the ACE protein;

[0072] Step 5: Solid-phase synthesize the screened polypeptide sequences, and use an in vitro ACE enzyme activity inhibition assay to test the in vitro ACE activity inhibition effect of the bioactive peptides based on a concentration of 1 mg / ml, thereby obtaining the octapeptide YI8 with ACE inhibitory activity.

[0073] Application of an ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, including using the ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate as described above and applying it to functional foods for assisting in reducing blood pressure.

[0074] Example 3

[0075] An ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, with an amino acid sequence of YKVPQLEI and a molecular weight of 989.16 Da.

[0076] In order to obtain the ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, it is screened from milk casein hydrolysate.

[0077] It should be noted that the preparation method of the milk casein hydrolysate in the embodiments of the present application includes the following steps:

[0078] Step 1: Take milk casein, with a casein addition amount of 5%, pH 9, add 6% protease, and carry out enzymatic hydrolysis. The enzymatic hydrolysis temperature is controlled at 55°C, and the enzymatic hydrolysis time is 6 h to obtain an enzymatic hydrolysis product;

[0079] Step 2: Preliminarily filter the enzymatic hydrolysis product to remove residues to obtain a clarified polypeptide enzymatic hydrolysate, and perform freeze-drying treatment to obtain a polypeptide lyophilized powder;

[0080] Step 3: Perform mass spectrometry determination on the freeze-dried polypeptide using LC-MS / MS. Analyze the results of the mass spectrometry determination with mass spectrometry analysis software and compare them with the milk casein sequence to obtain the polypeptide sequence. Among them, the conditions for mass spectrometry determination are controlled as follows: in the liquid phase method, the chromatographic column is C18, 3μm, 250mmX75μm, Eksigent, mobile phase A is water with 0.1% formic acid; mobile phase B is acetonitrile with 0.1% formic acid, the flow rate is 300 nl / min, the injection volume is 4 μl, and the chromatographic gradient is 60 min. The elution gradient is: 0 - 48 min: mobile phase A decreases uniformly from 95% to 60%; 48 - 55 min: mobile phase A decreases uniformly from 60% to 30%; 55 - 56 min: mobile phase A decreases uniformly from 30% to 0; 56 - 60 min: maintain 0% mobile phase A. In the mass spectrometry determination, the equipment model used is: Orbitrap Exploris 480 - Thermo Fisher, based on the positive ion detection mode, and the following parameters are controlled for determination: the primary resolution is 120,000, the AGC is set to 300, the scanning range is 200 - 1600 m / z, the MIPS mode is peptide, the selected valence states are 1 - 5, the secondary resolution is 15,000, and the isolation window is 1.6 m / z.

[0081] Step 4: Perform molecular docking of the polypeptide sequence with the ACE protein. Before docking, convert the 2D structure of the polypeptide into a 3D structure through energy minimization, and screen to obtain the polypeptide sequence with strong binding ability to the ACE protein.

[0082] Step 5: Solid-phase synthesize the screened polypeptide sequence, and use an in vitro ACE enzyme activity inhibition test to test the in vitro ACE activity inhibition effect of the active peptide based on a concentration of 1 mg / ml to obtain the octapeptide YI8 with ACE inhibitory activity.

[0083] An application of an ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate, including using the ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate as described above and applying it to functional foods for assisting in reducing blood pressure.

[0084] In summary, the present application provides an ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate and its application. The ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate is screened from milk casein hydrolysate and is the octapeptide YI8 with ACE inhibitory activity. Under the condition of a concentration of 1 mg / ml, the in vitro ACE inhibition rate of the ACE inhibitory octapeptide YI8 derived from milk casein hydrolysate is tested to be 70.86%, and it is applicable to functional foods for assisting in reducing blood pressure, thereby obtaining a significant blood pressure reduction effect.

[0085] The "first", "second", "third", "fourth", etc. (if any) involved in this application are used to distinguish similar objects and do not necessarily describe a specific order or sequence. It should be understood that the data used in this way can be interchanged under appropriate circumstances so that the embodiments described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method or device comprising a series of steps or units need not be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods or devices.

[0086] It should be noted that the descriptions involving "first", "second", etc. in this application are only for descriptive purposes and should not be construed as indicating or implying their relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one such feature. Additionally, the technical solutions between various embodiments can be combined with each other, but it must be based on the ability of those of ordinary skill in the art to implement. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0087] Specific examples are used herein to illustrate the principles and implementation manners of this application. The descriptions of the above embodiments are only for helping to understand the method and its core idea of this application; at the same time, for those of ordinary skill in the art, based on the idea of this application, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to this application.

Claims

1. An ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate, characterized in that: Its amino acid sequence is YKVPQLEI.

2. The ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate according to claim 1, characterized in that: Its molecular weight is 989.16 Da.

3. The ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate according to claim 1, characterized in that: It is screened from cow cheese protein hydrolysate.

4. The ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate according to claim 3, characterized in that: The method for preparing the cow cheese protein hydrolysate comprises the following steps: Step 1, taking cow casein, adding 1-5% casein, pH 8-9, adding 2-6% protease, performing enzymolysis, controlling the enzymolysis temperature at 37-55°C, and the enzymolysis time at 4-6h, to obtain an enzymolysis product; Step 2, preliminarily filtering the enzymatic hydrolysate to remove the residue to obtain a clarified polypeptide enzymatic hydrolysate, and freeze-drying to obtain polypeptide freeze-dried powder; Step 3, the polypeptide freeze-dried powder is subjected to mass spectrometry analysis by LC-MS / MS, and the results of the mass spectrometry analysis are analyzed by mass spectrometry analysis software, and compared with the milk casein sequence to obtain the polypeptide sequence; Step 4: Molecular docking of the polypeptide sequence with the ACE protein. Before docking, the 2D structure of the polypeptide is converted into a 3D structure by energy minimization, and a polypeptide sequence with strong binding ability to the ACE protein is screened; Step 5: Solid phase synthesize the screened polypeptide sequence, and use an in vitro ACE enzyme activity inhibition test to obtain the octapeptide YI8 with ACE inhibitory activity.

5. The ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate according to claim 4, characterized in that: In step 1, the added amount of milk casein is 2%; the added amount of the mixed protease is 5%, and the protease is composed of trypsin and enzyme group X in a mixing ratio of 3:1, the enzymatic hydrolysis temperature is controlled at 37°C, the pH value is 8.0, and the enzymatic hydrolysis time is 6h.

6. The ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate according to claim 4, characterized in that: In step 3, the mass spectrometry conditions are controlled as follows: in the liquid phase method, the chromatographic column is C18, 3μm, 250mmX75μm, Eksigent, phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300nl / min, the injection volume is 4μl, the chromatographic gradient is 60min, and the elution gradient is: 0-48min: phase A is uniformly reduced from 95% to 60%; 48-55min: phase A is uniformly reduced from 60% to 30%; 55-56min: phase A is uniformly reduced from 30% to 0; 56-60min: maintain 0% phase A.

7. The ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate according to claim 6, characterized in that: In the mass spectrometry, the device model used is: Orbitrap Exploris480-Thermofisher, based on the positive ion detection mode, and the following parameters are controlled for measurement: the primary resolution is 120000, the AGC is set to 300, the scanning range is 200-1600m / z, the MIPS mode is peptide, the valence state 1-5 is selected, the secondary resolution is 15000, and the separation window is 1.6m / z.

8. The ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate according to claim 4, characterized in that: In step 5, the in vitro ACE enzyme activity inhibition test is based on testing the in vitro ACE activity inhibition effect of the active peptide at a concentration of 1 mg / ml.

9. An application of ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate, characterized in that: The method comprises using the ACE inhibiting octapeptide YI8 derived from cow cheese protein hydrolysate as described in any one of claims 1 to 8 and applying it to functional food for assisting in lowering blood pressure.

10. The use of the ACE-inhibiting octapeptide YI8 derived from cow cheese protease hydrolysate according to claim 9, characterized in that: The functional food for assisting in lowering blood pressure has an in vitro ACE inhibition rate of 70.86% under the condition of a concentration of 1 mg / ml.

Images