Milk-derived polypeptide mixture with anti-hypertension activity as well as preparation method and application of milk-derived polypeptide mixture

Abstract

The invention relates to the field of protein, in particular to a milk-derived polypeptide mixture with anti-hypertension activity as well as a preparation method and application of the milk-derived polypeptide mixture. The preparation method of the milk-derived polypeptide mixture comprises the following steps: preparing a mixed aqueous solution of whey protein and milk protein, carrying out high-temperature denaturation, adding neutral protease to carry out primary enzymolysis, and after the primary enzymolysis is finished, carrying out high-temperature enzyme deactivation; adding flavourzyme to carry out secondary enzymolysis, and after the secondary enzymolysis is finished, carrying out high-temperature enzyme deactivation to obtain enzymatic hydrolysate of whey protein and milk protein; and carrying out freeze drying or spray drying treatment on the enzymatic hydrolysate to obtain the milk-derived polypeptide mixture. Two-stage step-by-step enzymolysis is adopted, the milk-derived polypeptide mixture with small molecular weight and high hydrolysis degree is obtained, and in-vitro enzyme activity experiments prove that the prepared milk-derived polypeptide mixture has a good antihypertensive function. The milk-derived polypeptide mixture disclosed by the invention can directly inhibit the activity of angiotensin converting enzyme (ACE), and is of great significance to development of health care products and medicines with antihypertensive performance.

Description

Technical Field

[0001] This invention relates to the field of proteins, and in particular to a mixture of milk-derived polypeptides with antihypertensive activity, its preparation method, and its application. Background Technology

[0002] Milk and dairy products are among the oldest foods in human dietary history. Their rich protein content not only provides abundant nutrition but is also an important source of bioactive peptides. In recent years, the physiologically active peptides contained in milk and dairy products have attracted increasing attention. Small molecule peptides exhibit superior transport, absorption, and utilization rates in the intestine compared to monomeric amino acids. Furthermore, their different amino acid arrangements can result in various physiological activities, such as participating in immune regulation, scavenging free radicals, antihypertensive, antithrombotic, and antidiabetic effects.

[0003] Hypertension is a systemic disease characterized by elevated arterial blood pressure, accompanied by functional or organic changes in organs such as the heart, blood vessels, brain, and kidneys. It is one of the major threats to human health. Modern medicine has proven that persistent hypertension can induce a series of cardiovascular diseases, such as atherosclerosis, stroke, coronary heart disease, myocardial infarction, and kidney failure. Recent epidemiological studies show that hypertension is increasingly affecting younger people, making it a pressing global health problem that needs to be addressed.

[0004] Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that catalyzes the conversion of the plasma peptide angiotensin I (Ang I) into the potent vasoconstrictor angiotensin II (Ang II) in vivo. Ang II has a strong vasoconstrictive effect and can stimulate the body to release aldosterone, increasing the reabsorption of sodium by the kidneys, leading to increased blood volume and thus elevated blood pressure. ACE inhibitory peptides can inhibit the conversion of Ang I to Ang II and simultaneously degrade kinins, inactivating them, thereby inhibiting ACE activity and exerting an antihypertensive effect.

[0005] There is a lack of research and preparation methods for peptides with antihypertensive activity in the current technology. Summary of the Invention

[0006] The purpose of this invention is to provide a mixture of milk-derived polypeptides with antihypertensive activity, its preparation method, and its application. The milk-derived polypeptide mixture prepared by this method not only has a low molecular weight, high degree of hydrolysis, and good flavor, but also exhibits excellent antihypertensive activity, making it promising for application in the food, health product, and pharmaceutical fields.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a mixture of milk-derived polypeptides with antihypertensive activity, the mixture comprising one or more of the following amino acid sequences:

[0009] Trp-Glu-Asn-Pro-Gly-Val-Phe (peptide 1, as shown in SEQ ID NO.1), Asp-Ser-Ser-Tyr-Phe-Leu-Tyr (peptide 2, as shown in SEQ ID NO.2), Thr-Phe-Gly-Phe-Ala-Leu-Thr-Phe (peptide 3, as shown in SEQ ID NO.3), Gly-Ile-Asn-Tyr-Trp-Leu-Ala (peptide 4, as shown in SEQ ID NO.4), Phe-Pro-Glu-Ser-Gly-Thr-Phe (peptide 5, as shown in SEQ ID NO.5), Ser-Leu-Phe-Ser-His-Ala-Phe (peptide 6, as shown in SEQ ID NO.6), Ser-Leu-Arg-Glu-Phe-Pro-Gly-Pro-Leu-Pro (peptide 7, as shown in SEQ ID NO.1). (as shown in SEQ ID NO.7), Ile-Pro-Pro-Tyr-Gln-Gly-Tyr (peptide 8, as shown in SEQ ID NO.8), Ser-Gly-Pro-Phe-Pro-Gly-Pro-Ser-Gln-Pro (peptide 9, as shown in SEQ ID NO.9), Tyr-Pro-Gln-Arg-Asp-Met-Pro (peptide 10, as shown in SEQ ID NO.10), Ala-Gln-Ser-Ala-Pro-Leu-Arg-Val-Tyr (peptide 11, as shown in SEQ ID NO.11), Leu-Asp-Lys-Val-Gly-Ile-Asn-Tyr-Trp-Leu-Ala (peptide 12, as shown in SEQ ID NO.12), Gln-Asp-Trp-Thr-Gly-Gly-Lys-Glu-Phe (peptide 13, as shown in SEQ ID NO.7), Ile-Pro-Pro-Tyr-Gln-Gly-Tyr (peptide 8, as shown in SEQ ID NO.8), Ser-Gly-Pro-Phe-Pro-Gly-Pro-Ser-Gln-Pro (peptide 9, as shown in SEQ ID NO.9), Tyr-Pro-Gln-Arg-Asp-Met-Pro (peptide 10, as shown in SEQ ID NO.10), Ala-Gln-Ser-Ala-Pro-Leu-Arg-Val-Tyr (peptide 11, as shown in SEQ ID NO.11), Leu-Asp-Lys-Val-Gly-Ile-Asn-Tyr-Trp-Leu-Ala (peptide 12, as shown in SEQ ID NO.12), Gln-Asp-Trp-Thr-Gly-Gly-Ly As shown in SEQ ID NO. 13), Asp-Leu-Pro-Phe-Pro-Glu-Lys-Asn (peptide 14, as shown in SEQ ID NO. 14) and Tr p-Gly-Gln-Gly-Leu-Leu-Val-Thr (peptide 15, as shown in SEQ ID NO. 15).

[0010] In a second aspect, the present invention provides a method for preparing a mixture of milk-derived polypeptides with antihypertensive activity, the method comprising the following steps:

[0011] (1) Prepare a mixed aqueous solution of whey protein and milk protein, sterilize it at high temperature, add neutral protease for one enzymatic hydrolysis, after the first enzymatic hydrolysis is completed, inactivate the enzyme at high temperature; then add flavor protease for a second enzymatic hydrolysis, after the second enzymatic hydrolysis is completed, inactivate the enzyme at high temperature to obtain whey protein and milk protein hydrolysate.

[0012] (2) After freeze-drying or spray-drying whey protein and milk protein hydrolysate, a mixture of milk-derived polypeptides is obtained.

[0013] In one embodiment of the present invention, the whey protein and milk protein mixed aqueous solution contains whey protein with a mass fraction of 5-9% and milk protein with a mass fraction of 1-7%.

[0014] In one embodiment of the present invention, the amount of neutral protease added is 1000-5000 U / g.

[0015] In one embodiment of the present invention, the high-temperature sterilization temperature is 90-100°C and the time is 5-10 minutes.

[0016] In one embodiment of the present invention, the temperature of the first enzymatic hydrolysis is 45-55°C and the time is 1-3 hours.

[0017] In one embodiment of the present invention, the amount of flavor protease added is 12.5 to 62.5 LAPU / g.

[0018] In one embodiment of the present invention, the temperature of the secondary enzymatic hydrolysis is 40-60°C and the time is 1-5 hours.

[0019] In one embodiment of the present invention, the freeze-drying temperature is -72 to -68°C and the vacuum degree is 2 Pa.

[0020] In a third aspect, the present invention provides the use of the milk-derived polypeptide mixture in the preparation of health products or medicines with antihypertensive functions.

[0021] In a fourth aspect, the present invention provides an antihypertensive product comprising a mixture of milk-derived polypeptides or derivatives thereof; the antihypertensive product includes antihypertensive health products or antihypertensive drugs; the derivative of the polypeptide mixture refers to a polypeptide derivative that still possesses antihypertensive biological activity obtained by modifying the amino acid side chain groups, amino terminus, or carboxyl terminus of a bioactive polypeptide with hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification, or glycosylation.

[0022] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0023] This invention employs a two-stage stepwise enzymatic hydrolysis to obtain a mixture of milk-derived polypeptides with small molecular weight and high degree of hydrolysis. In vitro enzyme activity experiments have demonstrated that the obtained milk-derived polypeptide mixture possesses excellent antihypertensive function. The milk-derived polypeptide mixture of this invention can directly inhibit angiotensin-converting enzyme (ACE) activity, which is of great significance for the development of health products and drugs with antihypertensive properties. Attached Figure Description

[0024] Figure 1 Chromatogram of a mixture of milk-derived peptides identified by nano-liquid chromatography-quadrupole orbital trap mass spectrometry;

[0025] Figure 2 The scavenging effect of a mixture of milk-derived polypeptides on ABTS free radicals;

[0026] Figure 3 Inhibitory effect of milk-derived polypeptide mixture on α-glucosidase;

[0027] Figure 4 Inhibitory effect of milk-derived polypeptide mixture on α-amylase;

[0028] Figure 5 The inhibitory effect of a mixture of milk-derived polypeptides on angiotensin-converting enzyme (ACE). Detailed Implementation

[0029] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0030] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0031] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; these series *METHODS INENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATINSTRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS INENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0033] Example 1: Preparation method of milk-derived polypeptide mixture

[0034] 1. Types of proteases and raw materials

[0035] The neutral protease used in the following examples of the present invention was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., derived from Bacillus subtilis, is an exonuclease, and has a protease activity of 110,000 U / g; the flavor protease used was purchased from Novozymes (China) Biotechnology Co., Ltd., derived from Aspergillus oryzae, is an endopeptidase, and has a protease activity of 1,000 LAPU / g.

[0036] The whey protein used in the following embodiments of the present invention is whey protein concentrate (WPC) with a whey protein content greater than 80%, purchased from Fonterra Trading (Shanghai) Co., Ltd.; the milk protein used in the following embodiments of the present invention is milk protein concentrate (MPC) with a protein content greater than 80%, purchased from Fonterra Trading (Shanghai) Co., Ltd.

[0037] 2. Preparation steps

[0038] 1) Dissolve 7% whey protein powder and 3% milk protein powder by mass in RO water, sterilize at 95°C for 8 minutes, and cool to 50°C in an ice water bath to obtain unhydrolyzed milk protein stock solution.

[0039] 2) Add neutral protease at a concentration of 3000 U / g, mix well, and then place in a shaker at 50°C for 1500 rpm for 2 hours to obtain a one-step enzymatic hydrolysate.

[0040] 3) The first-step enzymatic hydrolysate was inactivated by heating in a 95°C water bath for 8 minutes, cooled to 50°C in an ice-water bath, and flavor protease was added at a concentration of 35 LAPU / g. After mixing, the mixture was placed in a shaker at 50°C for 1500 rpm for 3 hours to obtain the second-step enzymatic hydrolysate.

[0041] 4) After the enzyme hydrolysate is heated in a 95°C water bath for 8 minutes to inactivate the enzyme, it is placed in a vacuum freeze dryer for vacuum freeze drying. The parameters are: temperature -70°C, time 48 hours, and vacuum degree 2 Pa. The resulting powder is a mixture of milk-derived polypeptides with antihypertensive activity.

[0042] Example 2: Isolation and screening of antihypertensive peptides from a mixture of milk-derived peptides

[0043] 1) UPLC analysis

[0044] The UPLC conditions are as follows:

[0045] Instruments: Waters ACQUITY UPLC (Ultra-High Performance Liquid Chromatography), Electrospray Chromatography, Quadrupole, Time-of-Flight Mass Spectrometry

[0046] Column specifications: BEH C18 column

[0047] Flow rate: 0.4 mL / min

[0048] Temperature: 50℃

[0049] UV detection wavelength: 210nm

[0050] Injection volume: 2 μL

[0051] Gradient conditions: Solution A: water containing 0.1% formic acid (v / v), Solution B: acetonitrile containing 0.1% formic acid (v / v)

[0052]

[0053] 2) Mass spectrometry analysis

[0054] The mass spectrometry conditions are as follows:

[0055] Ionization method: ES+

[0056] Mass range (m / z): 100, 1000

[0057] Capillary voltage (kV): 3.0

[0058] Sampling cone (V): 35.0

[0059] Ion source temperature (°C): 115

[0060] Desolventization temperature (°C): 350

[0061] Solvent removal gas flow (L / hr): 700.0

[0062] Collision energy (eV): 4.0

[0063] Scan time (sec): 0.25

[0064] Internal scan time (sec): 0.02

[0065] Based on the above analytical methods, the milk-derived polypeptide mixture obtained in Example 1 was subjected to chromatographic separation and mass spectrometric analysis using ultra-high performance liquid chromatography, electrospray ionization, quadrupole chromatography, and time-of-flight mass spectrometry. The amino acid sequences of the polypeptides in the milk-derived polypeptide mixture were obtained. Specific results are shown in [link to results]. Figure 1 .

[0066] The same amino acid sequences were selected from the parallel control group. Then, using the peptide database Peptide Ranker, amino acid sequences with lower bioactivity probability scores and publicly available amino acid sequences were excluded. The remaining amino acid sequences were the milk-derived peptides with antihypertensive activity.

[0067] 3) Results

[0068] The amino acid sequences of the peptides with antihypertensive activity are shown in Table 1. A total of 15 milk-derived peptides with antihypertensive activity were identified.

[0069] Table 1 Information on milk-derived peptides with antihypertensive activity

[0070]

[0071] Example 3: The regulatory effect of a mixture of milk-derived polypeptides on blood pressure

[0072] I. ABTS Free Radical Scavenging Rate Experiment of Milk-Derived Peptide Mixture (Spectrophotometry)

[0073] 1. Experimental reagents and instruments:

[0074] Reagents: Milk protein stock solution and milk-derived peptide mixture obtained in Example 1; ABTS test kit, Shanghai Beyotime Biotechnology Co., Ltd.

[0075] Instruments and equipment: Electronic balance, Sartorius, Germany; Constant temperature water bath, Shanghai Yiheng Technology Co., Ltd.; 96-well cell culture plate, Millipore, USA; RO15 pure water system, Likang Biomedical Technology Holding Co., Ltd.; Freeze dryer, Ningbo Xinzhi Biotechnology Co., Ltd.; SpectraMax M2 microplate reader, Molecular Devices.

[0076] 2. Experimental Methods:

[0077] Prepare an ABTS working stock solution using equal volumes of ABTS solution and oxidant solution, and store at room temperature in the dark for 12 hours before use. Dilute the ABTS working stock solution 40 times with PBS solution before use.

[0078] Experimental Group 1: 10 μL of different concentrations of milk-derived polypeptide mixture solutions were pipetted into 96-well plates, with three replicates. 200 μL of ABTS working solution was added to each well, the plates were shaken thoroughly, and the reaction was carried out at room temperature in the dark for 4 min. The absorbance OD1 was measured at 734 nm.

[0079] Experimental Group 2: 10 μL of different concentrations of milk protein stock solution were pipetted into 96-well plates, with three replicates. The remaining procedures were the same as in Experimental Group 1.

[0080] Blank control group: Add 10 μL of deionized water (ddH2O) to each well as a blank control instead of the sample. Shake well and react at room temperature in the dark for 4 min. Measure the absorbance (OD0) at 734 nm. Calculate the absorbance using the formula:

[0081]

[0082] Wherein, OD0 is the absorbance of the blank control solution, and OD1 is the absorbance of the sample after the reaction is completed.

[0083] 3. Experimental Results and Analysis:

[0084] according to Figure 2 The in vitro total antioxidant activity of the milk-derived peptide mixture was determined by measuring the ABTS free radical scavenging rate. It was found that the absorbance of the experimental group with the added milk-derived peptide mixture was lower than that of the control group, indicating better reducing and oxidizing capabilities. Figure 2 It was found that the milk-derived peptide mixture could significantly scavenge ABTS free radicals starting from 0.35 mg / mL, and this scavenging effect increased with increasing concentration. The milk-derived peptide mixture at 0.92 mg / mL could scavenge 50% of ABTS free radicals, while milk protein at 9.90 mg / mL could scavenge 50% of ABTS free radicals. Therefore, it can be concluded that the prepared milk-derived active peptide mixture has a significantly greater antioxidant capacity than the unenzymatically hydrolyzed milk protein stock solution, and the total antioxidant level of the milk-derived peptide mixture was optimal at a concentration of 4.09 mg / mL.

[0085] II. Experiment on the inhibition of α-glucosidase activity by a mixture of milk-derived polypeptides (spectrophotometry)

[0086] 1. Experimental reagents and instruments:

[0087] Reagents: α-glucosidase, Shanghai Yuanye Biotechnology Co., Ltd.; sucrose, Shanghai Aladdin Biochemical Technology Co., Ltd.; milk protein stock solution and milk-derived polypeptide mixture obtained in Example 1; glucose detection kit, Beijing Solarbio Technology Co., Ltd.

[0088] Instruments and equipment: Electronic balance, Sartorius, Germany; Constant temperature water bath, Shanghai Yiheng Technology Co., Ltd.; 96-well cell culture plate, Millipore, USA; RO15 pure water system, Likang Biomedical Technology Holding Co., Ltd.; Freeze dryer, Ningbo Xinzhi Biotechnology Co., Ltd.; SpectraMax M2 microplate reader, Molecular Devices.

[0089] 2. Experimental Methods:

[0090] Based on the number of samples, add 1 volume of glucose assay kit reagent B (1:1) to 1 volume of glucose assay kit reagent A to prepare an appropriate amount of glucose detection working solution, and then mix thoroughly with a pipette tip. The working solution should be prepared and used immediately. Table 2 shows the addition system for the assay group and control group in this experiment.

[0091] Test group 1: Added milk-derived polypeptide mixture;

[0092] Test group 2: Added milk protein stock solution;

[0093] Blank control group: RO water was added.

[0094] Table 2. Experimental additive system for inhibiting α-glucosidase activity using milk-derived polypeptide mixtures.

[0095]

[0096] The absorbance change was measured at 405 nm using an ELISA reader at 37°C for 30 minutes.

[0097]

[0098] Wherein, ΔA0 is the change in absorbance of the blank control solution, and ΔA1 is the change in absorbance of the sample before and after the reaction is completed.

[0099] 3. Experimental Results and Analysis:

[0100] from Figure 3 It was found that the milk-derived polypeptide mixture and milk protein stock solution significantly inhibited α-glucosidase activity starting from 0.80 mg / mL and 3.12 mg / mL, respectively, and this regulatory effect increased with increasing polypeptide concentration. The milk-derived polypeptide mixture inhibited 50% of α-glucosidase activity at 1.64 mg / mL, while milk protein at 9.85 mg / mL inhibited 50% of α-glucosidase activity. Therefore, it can be concluded that the prepared active milk-derived polypeptides have a significantly greater ability to inhibit α-glucosidase than the undigested milk protein stock solution, with the milk-derived polypeptide mixture exhibiting the best α-glucosidase inhibition level at a concentration of 5.17 mg / mL. This demonstrates that the milk-derived polypeptide mixture can inhibit α-glucosidase activity, reducing the ability of α-glucosidase to hydrolyze carbohydrates containing glucosidic bonds into easily absorbed monosaccharides (such as glucose) in the intestine, thereby regulating postprandial blood glucose levels.

[0101] III. Assay for the inhibition of α-amylase by a mixture of milk-derived polypeptides (spectrophotometry)

[0102] 1. Experimental reagents and instruments:

[0103] Reagents: α-amylase, Shanghai Aladdin Biochemical Technology Co., Ltd.; maltodextrin, Shanghai Aladdin Biochemical Technology Co., Ltd.; milk protein stock solution and milk-derived polypeptide mixture obtained in Example 1; p-dinitrosalicylic acid, Sangon Biotech (Shanghai) Co., Ltd.; glucose standard, Sangon Biotech (Shanghai) Co., Ltd.

[0104] Instruments and equipment: Electronic balance, Sartorius, Germany; Constant temperature water bath, Shanghai Yiheng Technology Co., Ltd.; 96-well cell culture plate, Millipore, USA; RO15 pure water system, Likang Biomedical Technology Holding Co., Ltd.; Freeze dryer, Ningbo Xinzhi Biotechnology Co., Ltd.; SpectraMax M2 microplate reader, Molecular Devices.

[0105] Experimental methods:

[0106] Prepare a 3% maltodextrin (w / v) solution based on the number of samples, and mix thoroughly with a pipette tip. Prepare an appropriate amount of amylase working solution by adding 2 volumes of deionized water (1:2) to 1 volume of amylase concentrate, and mix thoroughly with a pipette tip. The amylase working solution should be prepared fresh and stored at 4°C. Prepare an appropriate amount of reducing sugar detection working solution by adding 2 volumes of deionized water (1:2) to 1 volume of p-dinitrosalicylic acid concentrate, and mix thoroughly with a pipette tip. Table 3 shows the addition systems for the assay group and control group in the experiment of inhibiting α-amylase activity using this milk-derived polypeptide mixture.

[0107] Test group 1: Added milk-derived polypeptide mixture;

[0108] Test group 2: Added milk protein stock solution;

[0109] Blank control group: RO water was added.

[0110] Table 3. Experimental additive system for inhibiting α-amylase activity using milk-derived polypeptide mixtures.

[0111]

[0112]

[0113] Wherein, OD0 is the change in absorbance of the blank control solution, and OD1 is the change in absorbance of the sample before and after the reaction is completed.

[0114] 3. Experimental Results and Analysis:

[0115] from Figure 4 It was found that the milk-derived polypeptide mixture significantly inhibited α-amylase activity starting from 2.68 mg / mL, and this regulatory effect increased with increasing concentration. At 3.97 mg / mL, the milk-derived polypeptide mixture inhibited 50% of α-amylase activity. Milk protein inhibited 50% of α-amylase activity at 9.97 mg / mL. Therefore, it can be concluded that the prepared active milk-derived polypeptides have a significantly greater ability to inhibit α-amylase than undigested milk protein stock solution. The α-amylase inhibition level of the milk-derived polypeptide mixture was optimal at a concentration of 7.42 mg / mL. This demonstrates that the milk-derived polypeptide mixture can inhibit α-amylase activity from low to high concentrations, reducing the ability of α-amylase to hydrolyze carbohydrates containing α-1,4 glycosidic bonds into easily absorbed reducing monosaccharides in the intestine, thereby regulating postprandial blood glucose levels.

[0116] Example 4: The regulatory effect of a mixture of milk-derived polypeptides on blood pressure

[0117] I. Assay on the inhibition of angiotensin-converting enzyme (ACE) activity by a mixture of milk-derived polypeptides (spectrophotometry)

[0118] 1. Experimental reagents and instruments:

[0119] Reagents: Angiotensin-converting enzyme (ACE, 0.25 U), Sigma-Aldrich (Shanghai) Trading Co., Ltd.; N-[3-(2-furanyl)acryloyl]-Phe-bisglycine (FAPGG), Sigma-Aldrich (Shanghai) Trading Co., Ltd.; the milk protein stock solution and milk-derived peptide mixture obtained in Example 1;

[0120] Instruments and equipment: Electronic balance, Sartorius, Germany; Constant temperature water bath, Shanghai Yiheng Technology Co., Ltd.; 96-well cell culture plate, Millipore, USA; RO15 pure water system, Likang Biomedical Technology Holding Co., Ltd.; Freeze dryer, Ningbo Xinzhi Biotechnology Co., Ltd.; SpectraMax M2 microplate reader, Molecular Devices.

[0121] 2. Experimental Methods:

[0122] Prepare a 0.1 U / mL ACE enzyme working solution according to the number of samples, and then mix thoroughly with a pipette tip. Prepare a 0.1 mmol / L furanoacryl tripeptide solution according to the number of samples, and then mix thoroughly with a pipette tip. The ACE enzyme working solution and FAPGG working solution should be prepared fresh and stored at 4°C for later use. Table 4 shows the addition system for the assay group and blank control group in the experiment of inhibiting ACE activity of this milk-derived peptide mixture.

[0123] Test group 1: Added milk-derived polypeptide mixture;

[0124] Test group 2: Added milk protein stock solution;

[0125] Blank control group: Tris-HCl buffer was added.

[0126] Table 4. Experimental addition system for inhibiting ACE activity of milk-derived polypeptide mixture.

[0127]

[0128] The absorbance change before and after the reaction at 37℃ for 30 minutes was measured using an ELISA reader at 340 nm.

[0129]

[0130] Wherein, ΔA1 is the change in absorbance of the blank control solution, and ΔA2 is the change in absorbance of the sample before and after the reaction is completed.

[0131] 3. Experimental Results and Analysis:

[0132] from Figure 5It was found that the milk-derived polypeptide mixture significantly inhibited the activity of angiotensin-converting enzyme (ACE) starting from 0.39 mg / mL. This regulatory effect increased with increasing concentration of the milk-derived polypeptide mixture, reaching 1.46 mg / mL, where it inhibited 50% of ACE activity. In contrast, milk protein at 396.57 mg / mL inhibited ACE activity by 50%. Therefore, it can be concluded that the prepared milk-derived polypeptide mixture has a significantly greater ability to inhibit ACE than undigested milk protein stock solution. The optimal ACE inhibition level was observed at a concentration of 11.61 mg / mL. This demonstrates that the milk-derived polypeptide mixture can inhibit ACE activity from low to high concentrations, preventing the conversion of angiotensin I (Ang I) to angiotensin II (Ang II), thereby inhibiting peripheral vasoconstriction, preventing hypertension, and achieving a good antihypertensive effect.

[0133] In summary, milk-derived polypeptide mixtures can directly affect the activity of angiotensin-converting enzyme (ACE), or exert blood pressure-lowering effects by enhancing the body's antioxidant capacity or regulating blood sugar.

[0134] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A milk-derived polypeptide mixture having antihypertensive activity, characterized in that, The milk-derived polypeptide mixture comprises one or more of the polypeptides corresponding to the following amino acid sequences: Trp-Glu-Asn-Pro-Gly-Val-Phe, Asp-Ser-Ser-Tyr-Phe-Leu-Tyr, Thr-Phe-Gly-Phe-Ala-Leu-Thr-Phe, Gly-Ile-Asn-Tyr-Trp-Leu-Ala, Phe-Pro-Glu-Ser-Gly-Thr-Phe, Ser-Leu-Phe-Ser-His-Ala-Phe, Ser-Leu-Arg-Glu-Phe-Pro-Gly-Pro-Leu-Pro, Ile-Pro-Pro-Tyr-Gln-Gly-Tyr, Ser-Gly-Pro-Phe-Pro-Gly-Pro-Ser-Gln-Pro, Tyr-Pro-Gln-Arg-Asp-Met-Pro, Ala-Gln-Ser-Ala-Pro-Leu-Arg-Val-Tyr, Leu-Asp-Lys-Val-Gly-Ile-Asn-Tyr-Trp-Leu-Ala, Gln-Asp-Trp-Thr-Gly-Gly-Lys-Glu-Phe, Asp-Leu-Pro-Phe-Pro-Glu-Lys-Asn, and Trp-Gly-Gln-Gly-Leu-Leu-Val-Thr.

2. A method for the preparation of a milk-derived polypeptide mixture as claimed in claim 1, characterized in that, The method comprises the following steps: Step (1): preparing a mixed aqueous solution of whey protein and milk protein, high-temperature sterilizing, adding neutral protease for primary enzymolysis, high-temperature inactivating the enzyme after the primary enzymolysis, adding flavor protease for secondary enzymolysis, high-temperature inactivating the enzyme after the secondary enzymolysis, and obtaining a whey protein and milk protein enzymolysis solution; Step (2): freeze-drying or spray-drying the whey protein and milk protein enzymolysis solution to obtain a milk-derived polypeptide mixture.

3. A method for the preparation of a milk-derived polypeptide mixture according to claim 2, characterized in that, In the mixed aqueous solution of whey protein and milk protein, the mass fraction of whey protein is 5-9%, and the mass fraction of milk protein is 1-7%.

4. A method of producing a milk-derived polypeptide mixture according to claim 2, c h a r a c t e r i s e d in that, In step (1), the addition amount of the neutral protease is 1000-5000 U / g, and the addition amount of the flavor protease is 12.5-62.5 LAPU / g.

5. A method of producing a milk-derived polypeptide mixture according to claim 2, c h a r a c t e r i s e d in that, In step (1), the high-temperature inactivation temperature of the enzyme is 90-100°C, and the time is 5-10 min.

6. A method of producing a milk-derived polypeptide mixture according to claim 2, c h a r a c t e r i s e d in that, In step (1), the temperature of the primary enzymolysis is 45-55°C, and the time is 1-3 h.

7. A method for the preparation of a milk-derived polypeptide mixture according to claim 2, characterized in that, In step (1), the temperature of the secondary enzymolysis is 40-60°C, and the time is 1-5 h.

8. A method of producing a milk-derived polypeptide mixture according to claim 2, c h a r a c t e r i z e d in that, In step (2), the freeze-drying temperature is -72 to -68°C.

9. Use of a milk-derived polypeptide mixture according to claim 1, characterized in that The milk-derived polypeptide mixture is used for preparing health-care products or drugs with anti-hypertensive function.

10. An antihypertensive product, characterized in that, The anti-hypertensive product comprises an anti-hypertensive health product or an anti-hypertensive drug; and the derivative of the polypeptide mixture refers to a polypeptide derivative obtained by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, the amino terminal or the carboxyl terminal of the bioactive polypeptide, and the polypeptide derivative still has the anti-hypertensive biological activity. The anti-hypertensive product comprises an anti-hypertensive health product or an anti-hypertensive drug; and the derivative of the polypeptide mixture refers to a polypeptide derivative obtained by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, the amino terminal or the carboxyl terminal of the bioactive polypeptide, and the polypeptide derivative still has the anti-hypertensive biological activity.

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