A hydrolyzed casein oligopeptide with auxiliary blood pressure lowering function and its preparation method and application

Abstract

The present invention belongs to the technical field of functional oligopeptides, and discloses a hydrolyzed casein oligopeptide with auxiliary blood pressure lowering function and its preparation method and application. The present invention uses casein as raw material, collects casein hydrolyzate containing active peptides through directional and controllable enzymolysis, and obtains the above-mentioned hydrolyzed casein oligopeptide with significant blood pressure lowering function. It also has the effect of lowering blood lipids, improving endothelial function and vascular elasticity. In the present invention, the main sequence of the hydrolyzed casein oligopeptide with blood pressure lowering function containing the hydrolyzed casein oligopeptide with blood pressure lowering function is VNEISK, LQSW, VLPVPQK, MIMKEP, GPFPIIV, FFVAPFPEVFGK, wherein VNEISK is the hydrolyzed casein oligopeptide with the auxiliary blood pressure lowering function newly discovered in this study with the largest activity and the highest content, which has the effect of assisting in lowering blood pressure, softening blood vessels, improving vascular elasticity, and improving headaches, dizziness, palpitations, tinnitus, insomnia and the like caused by hypertension. The blood pressure lowering peptide prepared by the present invention can be used in various forms of products such as milk powder, beverages, and tablet candies.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional oligopeptides, and more specifically, relates to a hydrolyzed casein oligopeptide with auxiliary blood pressure lowering function, and a preparation method and application thereof. Background Art

[0002] Antihypertensive peptides derived from food are a general term for a class of small molecule polypeptides that can lower human blood pressure. Generally, the molecular weight of active antihypertensive peptides is below 1000Da, and they are usually obtained by hydrolyzing proteins with proteases under relatively mild conditions. Due to its obvious antihypertensive effect and no effect on normal blood pressure and no side effects, it has become a hot topic of research.

[0003] The mechanism of action of antihypertensive peptides is to achieve the function of lowering blood pressure by inhibiting the activity of angiotensin converting enzyme (ACE). ACE has an important physiological function in regulating blood pressure in the renal-angiotensin system. Renin acts on angiotensinogen to release inactive angiotensin I, which is then generated through the action of ACE to produce angiotensin II with vascular smooth muscle activity, thereby causing blood pressure to rise. Antihypertensive peptides inhibit the activity of angiotensin converting enzyme, preventing angiotensin type I (inactive) from being converted to type II (active), thereby inhibiting the contraction of peripheral blood vessels and preventing blood pressure from rising.

[0004] Most of the existing antihypertensive peptides only have the effect of lowering blood pressure, but their effects on changes in blood lipids, endothelial function, and vascular elasticity are uncertain. At the same time, there are relatively few milk-derived antihypertensive peptides. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the above problems existing in the prior art. Firstly, a milk-derived blood pressure-lowering peptide is provided.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned blood pressure lowering peptide.

[0007] The third object of the present invention is to provide a functional product containing the above-mentioned blood pressure lowering peptide.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A hydrolyzed casein oligopeptide with a blood pressure-lowering assisting function, wherein the amino acid sequence of the hydrolyzed casein oligopeptide is VNEISK.

[0010] The present invention uses casein as a raw material, collects casein hydrolysate containing active peptides through directional and controllable enzymatic hydrolysis, and obtains the hydrolyzed casein oligopeptides having a significant blood pressure lowering function.

[0011] The present invention provides the use of the hydrolyzed casein oligopeptide in preparing a product with auxiliary blood pressure lowering function.

[0012] Preferably, the dosage of the hydrolyzed casein oligopeptide is 600-1000 mg / day.

[0013] Preferably, the product is a health product or a medicine.

[0014] Preferably, the hydrolyzed casein oligopeptide has at least one of the following functions:

[0015] (1) Lowering blood pressure;

[0016] (2) Reduce the activity of lipoprotein phospholipase A2;

[0017] (3) Enhance vasodilation function.

[0018] The present invention also provides a method for preparing the hydrolyzed casein oligopeptide, comprising the following steps:

[0019] (1) Add 3-4 times the mass of casein in water and stir to dissolve at 40°C-50°C;

[0020] (2) adding trypsin for enzymolysis, the amount of enzyme added is 0.1%-0.2% of the casein mass, the reaction temperature is 40°C-50°C, the reaction time is 1h-2h, and the pH is 7.0-9.0;

[0021] After the trypsin hydrolysis is completed, add pepsin with a casein mass of 0.05%-0.1% and hydrolyze for 1h-2h;

[0022] (3) Adjust the temperature to 80-90°C and keep warm for 20-30 minutes for sterilization;

[0023] (4) adjusting the pH value to 4.0-5.0 and filtering to remove the undigested macromolecular protein precipitate;

[0024] (5) Purification: adjust the pH to 3.0-5.0, the temperature to -20°C-4°C, the ethanol concentration to 40%-60%, stand for 4h-6h, centrifuge, and remove the precipitate;

[0025] (6) Anion exchange resin purification: The pH of the supernatant was adjusted to 7.0, and the supernatant was purified using 717 anion exchange resin for 2 h. The supernatant was first washed with water, the washing solution was discarded, and then eluted with 0.5% hydrochloric acid;

[0026] (7) Reverse phase silica gel purification: The eluate is further purified by reverse phase silica gel.

[0027] HPLC system: LC-8A Shimadzu preparative high performance liquid chromatograph; chromatographic column: HW-0121; chromatographic conditions: flow rate: 8 mL / min; detection wavelength: 214 nm; injection volume: 2 mL; mobile phase: A pump ultrapure water (containing 0.1% (v / v) TFA), B pump acetonitrile, B pump initial concentration is 25%; elution mode: binary gradient elution; elution gradient: 0.01 min ~ 60 min, 25% ~ 36%; 60.01 min ~ 80 min, 36%; 80.01 min ~ 90 min, 90% ~ 90%; 90.01 min ~ 100 min, 90% ~ 25%; repeat injection, collect T16 elution peak;

[0028] (8) Drying: The product is dried into powder by freeze drying, spray drying or vacuum drying to obtain hydrolyzed casein oligopeptide.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention uses casein as a raw material, collects casein hydrolysates containing active peptides through directional and controllable enzymolysis, and obtains the above-mentioned hydrolyzed casein oligopeptides with significant blood pressure lowering function. At the same time, it also has the effects of lowering blood lipids, improving endothelial function and vascular elasticity. The present invention can make full use of dairy product resources and provide a new way for the high-value utilization of milk-derived peptides. At the same time, the hydrolyzed casein oligopeptides prepared by the present invention are safe and non-toxic and have a wide range of application value. Studies have shown that the hydrolyzed casein oligopeptides have a variety of effects such as assisting in lowering blood pressure, softening blood vessels, improving vascular elasticity, and improving headaches, dizziness, palpitations, tinnitus, insomnia, etc. caused by hypertension. The blood pressure-lowering peptides prepared by the present invention can be used in various forms of products such as milk powder, solid beverages, beverages, and tablet candies. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The changes in blood pressure before and after the application of antihypertensive peptides;

[0032] Figure 2 The changes of lipoprotein phospholipase a2 before and after the application of antihypertensive peptides;

[0033] Figure 3 The changes of FMD of vasodilator function before and after the application of antihypertensive peptides;

[0034] Figure 4 The changes in symptom scores before and after the use of antihypertensive peptides;

[0035] Figure 5 This is a summary of the symptom score improvement rate before and after the application of antihypertensive peptides. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] Example 1 Preparation of blood pressure lowering peptide

[0038] Taking casein as raw material, casein hydrolysate containing active peptides is obtained through directional and controllable enzymatic hydrolysis.

[0039] A preparation method of a high-purity blood pressure-lowering peptide comprises the following steps:

[0040] (1) Add 400 mL of pure water to a beaker and stir at 200 rpm. Slowly add 100 g of casein to the beaker and stir at 40°C to dissolve.

[0041] (2) Add trypsin for enzymatic hydrolysis, and the enzymatic hydrolysis conditions are: the amount of enzyme added is 0.1% of the casein mass, the reaction temperature is 40°C, the reaction time is 1 hour, and the pH is 7.0. After the trypsin enzymatic hydrolysis, the pH is adjusted to 4.0, and pepsin with a casein mass of 0.05% is added, and the enzymatic hydrolysis is continued for another 1 hour.

[0042] (3) Adjust the temperature to 80°C and keep warm for 30 minutes for sterilization.

[0043] (4) Adjust the pH to 4.0 and centrifuge at 3000 r / min for 30 min to remove the undigested macromolecular protein precipitate.

[0044] (5) The supernatant was adjusted to pH 2.0, temperature -20°C, ethanol concentration 60%, allowed to stand for 4 h, centrifuged, the precipitate discarded, and the supernatant collected;

[0045] (6) Purification by anion exchange resin: The pH of the supernatant was adjusted to 7.0, and the supernatant was purified using 717 anion exchange resin for 2 h, first washed with water, discarded the washing solution, and then eluted with 0.5% hydrochloric acid.

[0046] (7) Reverse-phase silica gel purification. The eluent was further purified by reverse-phase silica gel, HPLC system: LC-8A Shimadzu preparative high performance liquid chromatograph; chromatographic column: HW-0121; chromatographic conditions: flow rate: 8 mL / min; detection wavelength: 214 nm; injection volume: 2 mL; mobile phase: A pump ultrapure water (containing 0.1% (v / v) TFA), B pump acetonitrile, B pump initial concentration is 25%; elution mode: binary gradient elution; elution gradient: 0.01min~60min, 25%~36%; 60.01min~80min, 36%; 80.01min~90min, 90%~90%; 90.01min~100min, 90%~25%. Repeat the injection and collect the T16 elution peak.

[0047] (8) Concentration and drying: The eluted peak is collected and concentrated in vacuo, and freeze-dried to obtain the T16 antihypertensive peptide.

[0048] (9) ACE (angiotensin converting enzyme) inhibitory activity determination: hippuric acid method.

[0049] Under the above conditions, 2.55 g of T16 antihypertensive peptide was obtained with a molecular weight of 971.2 Da. At a concentration of 1 mg / mL, the inhibition rate of ACE enzyme was 51.8%. It had good solubility and a 20% ethanol solution of the antihypertensive peptide was clear and transparent.

[0050] The amino acid sequence of the T16 antihypertensive peptide was determined to be: VNEISK.

[0051] Example 2 ACE inhibitory activity

[0052] The T16 antihypertensive peptide prepared in Example 1 and the CEI12 peptide isolated in the previous study were used as the target to study the ACE inhibitory activity of the two and their combination, and the detection method was the hippuric acid method.

[0053] Experimental Grouping

[0054] Group 1: 500 μg / mL T16 antihypertensive peptide; Group 2: 500 μg / mL CEI12 peptide; Group 3: T6 antihypertensive peptide and CEI12 peptide were mixed so that the final concentrations of T6 antihypertensive peptide and CEI12 peptide in the mixed solution were both 500 μg / mL.

[0055] Results: The ACE inhibition rates of group 1, group 2 and group 3 were 91.34%, 86.67% and 68.19%, respectively.

[0056] The N-terminal sequence of the CEI12 peptide is FFVAPFPEVFGK

[0057] Example 3 Application of blood pressure lowering peptide

[0058] Sample: Green Extract Hydrolyzed Casein Oligopeptide Powder (T16 Antihypertensive Peptide), light yellow to white appearance, added with excipients to make tablets. Specification: 250 mg / tablet, the effective amount of hydrolyzed casein oligopeptide is 200 mg / tablet, 60 tablets / bottle. Batch number: 20210908. The placebo is the same as the sample in dosage form, taste, appearance and packaging, batch number: 20210908.

[0059] Experimental design and grouping requirements: Two control designs, self-control and inter-control, were used. According to the requirements of random blind method, 70 subjects were randomly divided into a trial group and a control group. The main factors affecting the results, such as the course of disease, were considered as much as possible, and a balance test was conducted to ensure the comparability between the groups. There were 35 subjects in each group. The trial group took the test sample, and the control group took a placebo. The trial period was 120 days.

[0060] Subjects: All subjects were adults aged 18 to 65 years old, and all signed informed consent forms; those with systolic blood pressure between 120 and 159 mmHg and diastolic blood pressure between 80 and 99 mmHg could be included if either condition was met (including hypertensive subjects with systolic blood pressure ≥140 mmHg and / or diastolic blood pressure ≥90 mmHg, and stable medication in the previous 3 months).

[0061] Dosage and time of consumption: The test group took the test product orally according to the recommended method and dosage, 4 tablets per day, for 120 consecutive days; the control group took the placebo orally, 4 tablets per day, for 120 consecutive days. Each indicator was tested once at the beginning and end of the trial, and blood pressure and heart rate were measured every 15 days.

[0062] Efficacy index determination standard: According to the "Health Food Function Evaluation Method (2020 Edition) (Draft for Comments)", when comparing the tasting group itself before and after the tasting, the diastolic or systolic blood pressure measurement values ​​decreased significantly, and the difference was significant. The diastolic blood pressure decreased by ≥10mmHg or dropped to normal, or the systolic blood pressure decreased by ≥20mmHg or dropped to normal. After the tasting, the diastolic or systolic blood pressure measurement values ​​or the percentage of decrease between the tasting group and the control group were significantly different. It can be determined that the test sample has the function of maintaining a healthy blood pressure level.

[0063] Observation indicators: safety, blood pressure and heart rate, symptom score, blood lipids (total cholesterol TC, total triglycerides TG, low-density cholesterol LDL-C), blood rheology (whole blood viscosity, plasma viscosity, fibrin concentration, hematocrit, red blood cell aggregation index, red blood cell deformation index, red blood cell rigidity index, red blood cell electrophoresis time), endothelial function (endothelin ET-1, cell adhesion molecule ICAM-1, lipoprotein phospholipase a2), and vascular elasticity (vascular thickness, vasodilator function FMD).

[0064] 1. Balance comparison

[0065] A total of 89 subjects were included, including 44 in the trial group and 45 in the control group. After excluding 9 subjects who dropped out of the trial group and 5 subjects who dropped out of the control group, there were 75 subjects, including 35 in the trial group and 40 in the control group. The two groups were balanced and comparable (Table 1).

[0066] Table 1

[0067]

[0068]

[0069] 2. Safety index analysis 1

[0070] The participants were asked about their mental state, sleep, diet, and bowel movements, and the results were divided into good, average, and poor categories. Most participants were in good general condition, and the trial had no adverse effects on the participants (Table 2).

[0071] Table 2

[0072]

[0073] 3. Safety index analysis 2

[0074] After the test, the results of the subjects' white blood cell, red blood cell, hemoglobin, platelet, liver and kidney function tests were basically within the normal range (Table 3).

[0075] Table 3

[0076]

[0077]

[0078] 4. Blood pressure and heart rate

[0079] Table 4

[0080]

[0081]

[0082] Note: The difference is the difference between the end of the tasting and the beginning of the tasting; self-comparison ** P<0.01; comparison between groups # P < 0.05; ## P<0.01.

[0083] Blood pressure changes such as Figure 1 It can be seen that the systolic and diastolic blood pressures of the trial group decreased compared with those before the trial, and the difference was significant. As can be seen in Table 4, the systolic blood pressure of the trial group decreased by an average of 14.54 mmHg, and the diastolic blood pressure decreased by an average of 10.17 mmHg.

[0084] 5. Blood rheology

[0085] Comparison between the trial group before and after the trial showed no significant changes in the four blood lipids (P>0.05) (Table 5).

[0086] Table 5

[0087]

[0088] 6. Endothelial function

[0089] As shown in Table 6, the endothelin level in the trial group showed a downward trend when compared with that in the trial group before and after the trial, but the difference was not significant (P>0.05).

[0090] Table 6

[0091]

[0092] 7. Lipoprotein phospholipase A2

[0093] Lipoprotein phospholipase A2 is a human vascular-specific inflammatory marker and an independent risk factor for coronary heart disease and ischemic stroke. The reference range for adult serum is 131-376 (male). If the result is very high, consider the possibility of cardiovascular disease. It is recommended to go to the hospital for regular review of this indicator, pay attention to a low-salt, low-fat diet, ensure balanced nutrition, normal life and rest, and adequate sleep.

[0094] The lipoprotein phospholipase a2 level in the trial group showed a downward trend when compared with that in the control group before and after the trial, and the difference was significant (P<0.01).

[0095] like Figure 2 As shown in Table 7, after the trial diet, the lipoprotein phospholipase a2 in the trial diet group was lower than that in the control group, and the difference was significant (P < 0.01).

[0096] Table 7

[0097]

[0098] Note: Self-comparison ** P<0.01; comparison between groups ## P<0.01.

[0099] 8. Vascular elasticity

[0100] In conjunction with non-invasive brachial artery ultrasound, which measures brachial artery flow-mediated vasodilation (FMD) to assess endothelial function, the results are shown in Table 8.

[0101] Table 8

[0102]

[0103] Note: Self-comparison *P<0.05**P<0.01 Inter-group comparison #P<0.05.

[0104] FMD (%) = expanded inner diameter (inner diameter after decompression and release - basic inner diameter) / basic inner diameter × 100.

[0105] A decrease in FMD indicates peripheral vascular endothelial dysfunction. Meta-analysis shows that for every 1% decrease in brachial artery FMD, the risk of future cardiovascular disease increases by 8%. It can be used to evaluate coronary endothelial dysfunction with an accuracy comparable to that of angiography.

[0106] 9. Symptom score

[0107] As shown in Tables 9 and 10.

[0108] Table 9

[0109] Grouping Number of cases Before tasting Food tasting 45 days After tasting Food tasting group 35 2.51±0.89 <![CDATA[1.94±1.06 ** ]]> <![CDATA[1.26±0.95** ## ]]> Control group 40 2.17±0.71 2.25±0.87 2.28±0.91

[0110] Note: **P<0.01 for self-comparison, **P<0.01 for inter-group comparison ## P<0.01.

[0111] Table 10

[0112] Main symptoms Number of cases Number of effective cases Invalid cases Improvement rate (%) Headache 16(18) 9(1) 7(17) 56.3(5.6) dizziness 13(13) 6(0) 7(13) 46.2(0) Palpitations 10(9) 6(0) 4(9) 60.0(0) tinnitus 13(12) 8(0) 5(12) 61.5(0) Insomnia 8(9) 5(0) 3(9) 62.5(0) Irritability 14(13) 7(1) 7(12) 50.0(7.7) Weakness in waist and knees 12(12) 8(0) 4(12) 66.7(0)

[0113] The data in parentheses are for the control group. Figure 4 As shown in Figure 2, the improvement rate of symptom scores is as follows: Figure 5 shown.

[0114] 10. Observation of dropout rate and adverse reactions

[0115] A total of 89 subjects were included, 14 of whom did not undergo follow-up examinations at the prescribed time and met the exclusion criteria. There were 75 valid subjects, with a dropout rate of 15.7%.

[0116] During the experiment, no allergic or other adverse reactions were observed (Table 11). The experimental summary, as shown in Table 12, according to the judgment criteria of this scheme, can be explained that the test sample has a positive result in the human food test in maintaining a healthy blood pressure level.

[0117] Table 11

[0118]

[0119] Table 12

[0120]

[0121] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. The use of hydrolyzed casein oligopeptide in the preparation of a product with auxiliary blood pressure lowering function, characterized in that: The amino acid sequence of the hydrolyzed casein oligopeptide is VNEISK; the product is a health product or a medicine.

2. The use according to claim 1, characterized in that: The dosage of the hydrolyzed casein oligopeptide is 600-1000 mg / day.

3. The use according to claim 1, characterized in that: The preparation method of the hydrolyzed casein oligopeptide comprises the following steps: (1) Add 3-4 times the mass of casein in water and stir to dissolve at 40℃-50℃; (2) Add trypsin for enzymatic hydrolysis, the amount of enzyme added is 0.1%-0.2% of the casein mass, the reaction temperature is 40℃-50℃, the reaction time is 1 h-2h, and the pH is 7.0-9.0; After the trypsin hydrolysis is completed, add pepsin with a casein mass of 0.05%-0.1% and hydrolyze for 1h-2h; (3) Adjust the temperature to 80-90°C and keep warm for 20 min-30 min for sterilization; (4) Adjust the pH value to 4.0-5.0 and filter out the undigested macromolecular protein precipitate; (5) Purification: adjust the pH to 3.0-5.0, the temperature to -20℃-4℃, the ethanol concentration to 40%-60%, let stand for 4h-6h, centrifuge and remove the precipitate; (6) Anion exchange resin purification: The pH of the supernatant was adjusted to 7.0, and purified using 717 anion exchange resin for 2 h. The resin was first washed with water, the washing solution was discarded, and then eluted with 0.5% hydrochloric acid. (7) Reverse phase silica gel purification: The eluent is then purified by reverse phase silica gel. HPLC system: LC-8A Shimadzu preparative high performance liquid chromatograph; chromatographic column: HW-0121; chromatographic conditions: flow rate: 8 mL / min; detection wavelength: 214 nm; injection volume: 2 mL; mobile phase: ultrapure water containing 0.1% (v / v) TFA in pump A, acetonitrile in pump B, initial concentration of pump B is 25%; elution mode: binary gradient elution; elution gradient: 0.01 min~60 min, 25%~36%; 60.01 min~80 min, 36%; 80.01 min~90 min, 90%~90%; 90.01 min~100 min, 90%~25%; repeat injection, collect T16 elution peak; (8) Drying: After freeze drying, spray drying or vacuum drying, the obtained product is dried into powder to obtain hydrolyzed casein oligopeptide.

4. The use according to claim 3, characterized in that: The hydrolyzed casein oligopeptide has at least one of the following functions: (1) Lowering blood pressure; (2) Reduce the activity of lipoprotein phospholipase A2; (3) Enhance vasodilation function.

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