Whey protein peptide with antihypertensive activity as well as preparation method and application thereof

Abstract

The invention provides a whey protein peptide with antihypertensive activity and a preparation method and application thereof, and belongs to the technical field of bioengineering. The preparation method at least has the following beneficial effects that the preparation method of the whey protein peptide with the antihypertensive activity, provided by the invention, comprises the following steps: carrying out first-step enzymolysis on whey protein by using nattokinase to obtain an initial zymolyte; and performing second-step enzymolysis on the initial zymolyte by using protease to obtain the whey protein peptide. Nattokinase is selected as a first-step hydrolase, the whey protein hydrolysis efficiency is high, and the content of active peptide fragments is high, and protease is selected for second-step enzymolysis, so that the bitter taste of whey protein peptide can be effectively removed, the flavor and taste are remarkably improved, the palatability is improved, the proportion of oligopeptides is further increased, the number of oligopeptides with the antihypertensive function is increased, and the antihypertensive effect is improved. Animal experiments prove that the whey protein peptide obtained by the two-step enzymolysis preparation method provided by the invention can effectively reduce blood pressure.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and specifically to a whey protein peptide having blood pressure lowering activity, and a preparation method and application thereof. Background Art

[0002] Whey protein is an important nutrient in whey. It is a single-molecule protein that is sensitive to environmental factors such as heat and acid, and is prone to protein denaturation and precipitation. Whey protein peptides, which are processed from whey protein, are mainly composed of short peptides and amino acids. They have stronger functional activity than whey protein and can also reduce sensitivity to environmental factors such as heat and acid. Whey protein peptides have broader development and application prospects, can significantly increase the added value of whey protein, and expand the field of functional food development.

[0003] There are several major technical difficulties in the industrial production of whey protein: on the one hand, the utilization rate of protein raw materials is low and the production cost is high; secondly, the target peptide content is low and the efficacy is not significant; finally, unpleasant flavors such as bitterness occur during the processing.

[0004] How to provide a process that can efficiently produce whey protein peptides with stable quality, high functional activity, and excellent flavor and taste is still a technical problem that needs to be solved in this field. Summary of the invention

[0005] In view of this, the present application provides a whey protein peptide with antihypertensive activity and a preparation method and application thereof, in order to solve the problems of low enzymatic hydrolysis efficiency, low functional activity, obvious bitterness and poor flavor in the existing whey protein peptide enzymatic hydrolysis process.

[0006] The embodiment of the present application is implemented by providing a method for preparing whey protein peptides with antihypertensive activity, comprising: performing a first step of enzymatic hydrolysis on whey protein using nattokinase to obtain an initial enzymatic hydrolysate; and performing a second step of enzymatic hydrolysis on the initial enzymatic hydrolysate using a protease to obtain whey protein peptides.

[0007] In some embodiments, the protease is selected from one or more of carboxypeptidase and flavor protease.

[0008] In some embodiments, the protease is a composite protease, which includes carboxypeptidase and flavor protease, and the mass ratio of the carboxypeptidase to the flavor protease is (0.8-1.2):1.

[0009] In some embodiments, the nattokinase is homemade, and the nattokinase is obtained by fermenting soybeans with Bacillus subtilis, with a purity of ≥95%.

[0010] In some embodiments, the first step of enzymatic hydrolysis of whey protein comprises:

[0011] Weigh the whey protein and pure water in proportion, put them into a hydrolysis tank and mix them to obtain a whey protein solution;

[0012] Weigh nattokinase according to proportion, add it to the whey protein solution, stir it thoroughly to dissolve, and perform the first enzymolysis.

[0013] In some embodiments, the whey protein content of the whey protein solution is 2%-8%; and / or,

[0014] The added amount of nattokinase is 4000-8000 IU / g; and / or,

[0015] The enzymatic hydrolysis time of the first step is 4-6 hours, and the enzymatic hydrolysis temperature is 50-60° C.; and / or,

[0016] After the first step of enzymatic hydrolysis, an enzyme inactivation treatment is further included, and the conditions of the enzyme inactivation treatment are: heating to above 95° C. and inactivating the enzyme for 10-15 minutes.

[0017] In some embodiments, the amount of the protease added is 6000-9000 U / g; and / or,

[0018] The enzymatic hydrolysis time of the second step is 1-5h, and the enzymatic hydrolysis temperature is 40-50°C; and / or,

[0019] After the second step of enzymolysis, an enzyme inactivation treatment is further included, wherein the conditions of the enzyme inactivation treatment are: heating to above 95° C. and inactivating the enzyme for 10-15 minutes; and / or,

[0020] After the second step of enzymatic hydrolysis, an enzyme inactivation treatment is further included. After the enzyme inactivation is completed, the solution after the enzyme inactivation is subjected to solid-liquid separation, and the clear liquid is dried to obtain the whey protein peptide.

[0021] The present application also provides a whey protein peptide having blood pressure lowering activity obtained according to the preparation method described above.

[0022] In some embodiments, the whey protein peptides include:

[0023] The proportion of peptides with a molecular weight less than 2000 Da is greater than 30%; and / or,

[0024] The peptides with molecular weight less than 2000Da account for 60%-95%; and / or,

[0025] The proportion of peptides with a molecular weight of 2000-5000Da is less than 40%; and / or,

[0026] The peptides with molecular weight between 2000-5000Da account for 5%-30%; and / or,

[0027] The proportion of peptides with a molecular weight greater than 5000 Da is less than 20%; and / or,

[0028] Peptides with a molecular weight greater than 5000Da account for 2%-10%.

[0029] The present application also provides an application of the whey protein peptide described above, and the application of the whey protein peptide in food, health food and medicine for treating hypertension or maintaining a healthy blood pressure level.

[0030] The present application includes at least the following beneficial effects: the method for preparing whey protein peptides with antihypertensive activity provided in the present application comprises the following steps: using nattokinase to perform a first step of enzymolysis on whey protein to obtain an initial enzymolysate; and using a protease to perform a second step of enzymolysis on the initial enzymolysate to obtain whey protein peptides. Nattokinase is selected as the first step hydrolase, which has high efficiency in hydrolyzing whey protein and high content of active peptide segments, and the protease is selected for the second step of enzymolysis, which can effectively remove the bitterness of whey protein peptides, significantly improve the flavor and taste, increase palatability, and further increase the proportion of short peptides, increase the number of short peptides with antihypertensive function, and enhance the antihypertensive effect. The whey protein peptides obtained by the two-step enzymolysis preparation method provided in the present application have been verified by animal experiments to effectively lower blood pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 It is a schematic flow chart of an embodiment of a method for preparing a whey protein peptide with blood pressure lowering activity provided in the present application. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. In addition, it should be understood that the specific implementation methods described herein are only used to illustrate and explain the present application and are not used to limit the present application. In the present application, unless otherwise stated, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present application, the term "including" means "including but not limited to".

[0034] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0035] In this application, "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0036] In the present application, "at least one" means one or more, and "plurality" means two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0037] The present application provides a method for preparing whey protein peptides having blood pressure lowering activity, see Figure 1 , Figure 1 This is a flow chart of an embodiment of a method for preparing a whey protein peptide with blood pressure lowering activity provided by the present application, comprising the following steps:

[0038] Step S1: using nattokinase to perform a first enzymatic hydrolysis on whey protein to obtain an initial enzymatic hydrolysate;

[0039] Step S2: using a protease to perform a second step of enzymolysis on the initial enzymolysis product to obtain whey protein peptides.

[0040] In this embodiment, whey protein is subjected to two-step enzymatic hydrolysis. Nattokinase is first used to efficiently hydrolyze the whey protein to obtain an initial enzymatic hydrolyzate with a high content of active peptides. Then, protease is used for enzymatic hydrolysis to remove the bitterness and astringency of the whey protein peptides, and the proportion of short peptides is further increased, thereby increasing the number of short peptides with antihypertensive function and improving the antihypertensive effect.

[0041] Furthermore, the prepared whey protein peptide has been verified by sensory evaluation and animal experiments to have the advantages of good taste and strong activity, and has a significant blood pressure lowering effect.

[0042] In step S1:

[0043] Whey protein is a protein mixture separated from whey. Its main components contain a variety of amino acids, including all essential amino acids, as well as some immunoglobulins, growth factors, etc. It can be purchased commercially.

[0044] In one embodiment, the first step of enzymatic hydrolysis of whey protein specifically comprises the following steps:

[0045] Step S11: weighing whey protein and pure water in proportion, putting them into a hydrolysis tank and mixing them to obtain a whey protein solution;

[0046] Step S12: Weigh enzyme powder according to proportion, add it to the whey protein solution, stir it thoroughly to dissolve, and perform the first enzymolysis.

[0047] The whey protein content in the whey protein solution is 2%-8%, specifically 2%-4%, 4%-6%, 6%-8%, etc.

[0048] In one embodiment, the added amount of nattokinase is 4000-8000 IU / g, the enzymolysis time is 4-6h, and the enzymolysis temperature is 50-60°C.

[0049] Nattokinase is homemade and is obtained by fermenting soybeans with Bacillus subtilis, with a purity of ≥95%.

[0050] In a specific embodiment, the preparation method of nattokinase is as follows: soybeans and nutrient solution are mixed according to a mass volume ratio of 1:2 to 1:4, soaked at 2 to 30 ° C for 6 to 12 hours and then drained; wherein the nutrient solution contains: glucose 15 to 45 g / L, disodium hydrogen phosphate dodecahydrate 5 to 10 g / L, sodium dihydrogen phosphate monohydrate 0.5 to 2 g / L, magnesium sulfate 0.1 to 1 g / L, pH 7.0 to 7.6; then steamed at 105 ° C to 115 ° C, 0.02 MPa for 30 to 60 minutes; after steaming and cooling, inoculated with Bacillus subtilis seed liquid at 1 to 10% ml / 100g, fermented under fermentation conditions of 37 to 41 ° C and humidity 75 to 85% for 24 to 48 hours to obtain fermented fresh natto; the fresh natto is dried to obtain solid fermentation nattokinase powder; purified to obtain nattokinase with a purity of ≥95%.

[0051] In one embodiment, after the first enzymatic hydrolysis is performed in step S12, an enzyme inactivation step may be further included. The enzyme inactivation may be performed using an enzyme inactivation method known in the art. In a specific embodiment, the enzyme inactivation method is: heating to above 95° C., inactivating the enzyme for 10-15 minutes, and cooling to 40-50° C. after enzyme inactivation.

[0052] In step S2:

[0053] In one embodiment, the protease may include one or more of carboxypeptidases, flavor enzymes, and the like.

[0054] In a specific embodiment, the protease is a composite protease, which may include carboxypeptidase and flavor protease. The second step of enzymatic hydrolysis by the composite protease can remove bitterness and astringency, increase the proportion of short peptides, and increase the number of short peptides with blood pressure lowering function.

[0055] In a specific embodiment, the composite protease is composed of carboxypeptidase and flavor protease. Further, the mass ratio of carboxypeptidase to flavor protease is (0.8-1.2):1, such as 0.8:1, 1:1, 1.2:1, etc.

[0056] In one embodiment, the added amount of the protease is 6000-9000 U / g, the enzymolysis time is 1-5 h, and the enzymolysis temperature is 40-50°C.

[0057] In one embodiment, after the second step of enzymolysis, an enzyme inactivation step may be further included. The enzyme inactivation may be performed using an enzyme inactivation method known in the art. In a specific embodiment, the enzyme inactivation method is: heating to 90° C. to inactivate the enzyme for 10 minutes.

[0058] In one embodiment, after the enzyme is inactivated, the solution after the enzyme is inactivated is subjected to solid-liquid separation, and the clear liquid is dried to obtain whey protein peptides. The solid-liquid separation and drying methods can be used in a manner known in the art. Specifically, the solid-liquid separation can be a superposition of one or more methods such as centrifugation, filtration, and sieving, and the drying can include one or more of vacuum drying, spray drying, and reduced pressure drying.

[0059] The present application provides a whey protein peptide with blood pressure lowering activity, which is prepared by the preparation method of the whey protein peptide with blood pressure lowering activity provided by the present application.

[0060] In one embodiment, the proportion of peptide segments with a molecular weight less than 2000 Da in the prepared whey protein peptides is greater than 30%, and can further be greater than 50%, specifically 60%-95%, such as 86.43%, 84.10%, 80.31%, 70.74%, etc.

[0061] The proportion of peptide segments with a molecular weight of 2000-5000 Da in whey protein peptides is less than 40%, specifically 5%-30%, such as 7.70%, 9.63%, 12.57%, 21.68%, etc.

[0062] The proportion of peptide segments with a molecular weight greater than 5000 Da in the prepared whey protein peptides is less than 20%, and can further be less than or equal to 10%. The specific proportion can be 2%-10%, such as 5.87%, 6.27%, 7.12%, 7.58%, etc.

[0063] In this embodiment, the whey protein peptide contains a high proportion of small molecular weight peptide segments, so the whey protein peptide has a high ACE inhibition rate activity and the highest blood pressure lowering activity. As the molecular weight of the peptide increases, the ACE inhibition rate activity decreases and the blood pressure lowering activity decreases.

[0064] The present application also provides an application of a whey protein peptide with blood pressure lowering activity in food, health food and medicine for treating hypertension or maintaining a healthy blood pressure level.

[0065] The technical scheme and technical effects of the present application are described in detail below through specific embodiments, comparative examples and experimental examples. The following embodiments are only some embodiments of the present application and do not specifically limit the present application. It can be understood that the reagents used in each embodiment, comparative example and experimental example are all commercially available reagents unless otherwise specified.

[0066] Example 1

[0067] S1. Weigh whey protein (commercially available from Milk Specialties Global, batch number FL2003A1) and pure water in proportion to make a 3% whey protein solution, put it into a hydrolysis tank for mixing, and heat it to 55° C. in a water bath;

[0068] S2, weigh nattokinase powder at a ratio of 6000 IU / g, add it to the enzymolysis tank S1, stir thoroughly to dissolve, and enzymolysis time 5h;

[0069] S3. After the enzymatic hydrolysis is completed, the temperature of the hydrolyzate is raised to 90°C for 10 minutes to inactivate the enzyme, and then the temperature is lowered to 45°C;

[0070] S4, adding a composite protease of carboxypeptidase (purchased from Jiangsu Enming Bioengineering Technology Co., Ltd.) and flavor protease (purchased from Novozymes Biotechnology Co., Ltd.) in a ratio of 1:1 at 6000 U / g, and performing two-step enzymolysis for 2 h;

[0071] S5. The hydrolyzate after enzymatic hydrolysis of S4 was heated to 90°C, and the enzyme was inactivated for 10 minutes. After the enzyme was inactivated, the hydrolyzate was centrifuged and filtered;

[0072] The supernatant after centrifugation in steps S6 and S5 is spray-dried, the spray-drying air inlet temperature is 155° C., and the air outlet temperature is 75° C., to obtain whey protein polypeptides.

[0073] Example 2

[0074] The implementation steps are the same as those in Example 1, except that the complex enzyme in step S4 is replaced by carboxypeptidase.

[0075] Example 3

[0076] The implementation steps are the same as those in Example 1, except that in step S4, the complex enzyme is replaced by flavor protease.

[0077] Example 4

[0078] S1. Weigh whey protein (commercially available from Milk Specialties Global, batch number FL2003A1) and pure water in proportion to make a 3% whey protein solution, put it into a hydrolysis tank for mixing, and heat it to 55° C. in a water bath;

[0079] S2, weigh nattokinase powder (made by the company) at a ratio of 6000 IU / g, add it to the S1 enzymolysis tank, stir thoroughly to dissolve, and enzymolysis time is 5 hours;

[0080] S3. After the enzymatic hydrolysis is completed, the temperature of the hydrolyzate is raised to 90°C for 10 minutes to inactivate the enzyme, and after the enzyme is inactivated, the hydrolyzate is centrifuged and filtered;

[0081] The supernatant after centrifugation in steps S4 and S3 is spray-dried, the spray-drying air inlet temperature is 155° C., and the air outlet temperature is 75° C., to obtain whey protein polypeptides.

[0082] Example 5

[0083] S1. Weigh whey protein (commercially available from Milk Specialties Global, batch number FL2003A1) and pure water in proportion to make a 3% whey protein solution, put it into a hydrolysis tank for mixing, and heat it to 55° C. in a water bath;

[0084] S2, weigh nattokinase powder (self-made by the company, purity ≥ 95%) at a ratio of 6000 IU / g, add it to the S1 enzymolysis tank, stir thoroughly to dissolve, and enzymolysis time 5h;

[0085] S3. After the enzymatic hydrolysis is completed, the temperature of the hydrolyzate is raised to 90°C for 10 minutes to inactivate the enzyme, and then the temperature is lowered to 45°C;

[0086] S4, adding a composite protease of carboxypeptidase (purchased from Jiangsu Enming Bioengineering Technology Co., Ltd.) and flavor protease (purchased from Novozymes Biotechnology Co., Ltd.) in a ratio of 1:1 at 6000 U / g, and performing two-step enzymolysis for 2 h;

[0087] S5. The hydrolyzate after enzymatic hydrolysis of S4 was heated to 90°C, and the enzyme was inactivated for 10 minutes. After the enzyme was inactivated, the hydrolyzate was centrifuged and filtered;

[0088] S6. The liquid after centrifugation is filtered through a 5KD filter membrane, and the supernatant and the permeate are collected. The supernatant is spray-dried to obtain whey protein peptide powder with a molecular weight of >5KD, referred to as >5KD type.

[0089] S7, the permeate continues to pass through the 2KD filter membrane, and the supernatant and the permeate are collected. The supernatant is spray-dried to obtain 2KD-5KD whey protein peptide, referred to as 2KD-5KD type.

[0090] S8. The permeate is spray-dried to obtain <2KD whey protein peptides, referred to as <2KD type.

[0091] Comparative Example 1

[0092] The implementation steps are the same as those in Example 1, except that nattokinase is replaced by neutral protease (derived from Bacillus subtilis, purchased from Novozymes Biotech Co., Ltd.).

[0093] Comparative Example 2

[0094] S1. Weigh whey protein (commercially available from Milk Specialties Global, batch number FL2003A1) and pure water in proportion to make a 3% whey protein solution, put it into a hydrolysis tank for mixing, and heat it to 55° C. in a water bath;

[0095] S2, adding compound protease, carboxypeptidase (purchased from Jiangsu Enming Bioengineering Technology Co., Ltd.) and flavor protease (purchased from Novozymes Biotechnology Co., Ltd.) at a ratio of 6000 U / g, with a compound enzyme ratio of 1:1, and performing enzymolysis for 2 h;

[0096] S3, heating the hydrolyzate after enzymatic hydrolysis of S2 to 90°C, inactivating the enzyme for 10 minutes, and centrifugally filtering after inactivation of the enzyme;

[0097] After centrifugation in steps S4 and S3, spray drying is performed, and the spray drying air inlet temperature is 155° C. and the air outlet temperature is 75° C. to obtain whey protein polypeptides.

[0098] Experimental Example 1

[0099] Sensory evaluation of whey protein peptides

[0100] 5 g of the whey protein peptides prepared in the example and the control example were respectively taken and added with 95 g of purified water to obtain a 5 wt % whey protein peptide solution.

[0101] The sensory evaluation was carried out according to the following method:

[0102] According to the sensory evaluation scores in Table 1, 20 evaluators were selected as the evaluation team to conduct sensory evaluation on the samples. The evaluation indicators included the bitterness and astringency of whey protein peptides. Before the evaluation, the evaluators were trained on the evaluation standards so that they could evaluate objectively. Before the evaluation, the evaluators should avoid contact with strong-flavored foods, avoid discussion during the evaluation, and rinse their mouths with water between sample evaluations. The whey protein peptide solution was provided to the evaluators in an unknown order for objective evaluation and filling in the sensory evaluation form.

[0103] Table 1 Sensory evaluation standard table of whey protein peptides

[0104] Sensory characteristics Scoring criteria bitterness The bitterness brought by whey protein peptides: no bitterness 5 points; slightly bitter 3 points; very strong bitterness 1 point. Astringency The feeling of astringency on the tongue: no astringency 5 points; slight astringency 3 points; strong astringency 1 point.

[0105] Table 2 Sensory evaluation of whey protein peptides

[0106] sample bitterness Astringency Example 1 4.5 4.5 Example 2 3.8 3.5 Example 3 3.0 3.0 Example 4 2.5 2.6 Comparative Example 1 4.5 4.5 Comparative Example 2 4.6 4.7

[0107] Sensory evaluation is the most intuitive evaluation of the taste of whey protein peptides, and it is evaluated from two indicators: bitterness and astringency. From the results of the sensory evaluation, Example 1, Comparative Example 1, and Comparative Example 2 use composite proteases, and the comprehensive scores are high, and the prepared whey protein peptides have the best taste. The taste of the whey protein peptide prepared by using carboxypeptidase (Example 2) is inferior to that of the composite protease, and the taste of the whey protein peptide prepared by using flavor protease (Example 3) is inferior to that of carboxypeptidase. The whey protein peptide prepared by enzymolysis without using carboxypeptidase or flavor protease (Example 4) has the strongest bitterness and astringency.

[0108] The combination of the composite protease carboxypeptidase and flavor protease has the most obvious improvement on the taste of whey protein peptides. Carboxypeptidase can remove the hydrophobic amino acid at the C-terminus of the bitter peptide. The bitter removal effect of carboxypeptidase combined with flavor protease is better than that of the single enzyme preparation flavor protease which is commonly used to improve the taste.

[0109] Experimental Example 2

[0110] The relative molecular mass distribution of whey protein peptides is determined according to GB / T 22492-2008.

[0111] Insulin, bacitracin, glycine-glycine-tyrosine-arginine, glycine-glycine-glycine four standard substances were respectively prepared into solutions of certain concentrations, mixed in a certain proportion and tested to obtain the chromatogram of the standard. The molecular mass standard curve and its equation were obtained by linear regression of the logarithm of the relative molecular mass against the retention time, and then the chromatographic retention time of the test sample was brought into the calibration curve to calculate the relative molecular weight of the test sample and the distribution range of the molecular weight.

[0112] Experimental instruments and parameters:

[0113] High performance liquid chromatograph: Fuli LC5090;

[0114] Chromatographic column: TSKgelG2000swxl 300mm×7.8mm;

[0115] Column temperature: 30°C; flow rate: 0.5 mL / min; detection wavelength: 220 nm;

[0116] Mobile phase and elution conditions: acetonitrile: water: trifluoroacetic acid = 10:90:0.1; detection time: 30 min.

[0117] Table 3 Molecular weight distribution of whey protein peptide samples prepared in Examples and Comparative Examples

[0118] sample >5000Da 2000-5000Da <2000Da Example 1 5.87％ 7.70％ 86.43％ Example 2 6.27％ 9.63％ 84.10％ Example 3 7.12％ 12.57％ 80.31％ Example 4 7.58％ 21.68％ 70.74％ Example 5 (>5KD type) 100％ —— —— Example 5 (2KD-5KD type) —— 100％ —— Example 5 (<2KD type) —— —— 100％ Comparative Example 1 21.12％ 41.91％ 36.97％ Comparative Example 2 29.31％ 41.33％ 29.36％

[0119] Example 1 uses two-step enzymolysis (nattokinase, composite protease), Example 2 uses two-step enzymolysis (nattokinase, carboxypeptidase), Example 3 uses two-step enzymolysis (nattokinase, flavor protease), and Example 4 uses only one-step enzymolysis (nattokinase). From the experimental results, it can be seen that on the basis of selecting one-step enzymolysis of nattokinase, adding two-step enzymolysis can increase the proportion of small molecule peptides (<2KD), and the smaller the molecular weight, the higher the activity of the peptide.

[0120] When neutral protease (derived from Bacillus subtilis) was used to replace nattokinase (Comparative Example 1), short peptides <2KD accounted for 36.97%, which was much lower than that in Example 1, and the enzymolysis efficiency was lower than that of nattokinase.

[0121] Experimental Example 3 In vitro detection of angiotensin converting enzyme (ACE) inhibition rate

[0122] The angiotensin converting enzyme inhibitor activity detection kit was purchased from Solebow, catalog number: BC5570. The detection was performed using the ultraviolet spectrophotometric method according to the operating steps in the kit manual.

[0123] The test samples were Example 5 (>5KD type), Example 5 (2KD-5KD type), and Example 5 (<2KD type), which were respectively prepared into 15 mg / mL peptide solutions.

[0124] Table 4 ACE inhibition rate test results

[0125]

[0126] The main function of angiotensin converting enzyme (ACE) is to inactivate bradykinin and catalyze the conversion of angiotensin I into angiotensin II, which can induce strong vasoconstriction and promote the synthesis and release of adrenal cortical hormone aldosterone. Inhibiting angiotensin converting enzyme can reduce the generation of angiotensin II and increase the activity of bradykinin, making it an ideal choice for treating diseases such as hypertension.

[0127] From the experimental results (see Table 4), it can be seen that under the same peptide concentration conditions, whey protein peptides <2KD have the highest ACE inhibition rate activity, reaching 88.58%, and the highest antihypertensive activity. As the molecular weight of the peptide increases, the ACE inhibition rate activity decreases, and the antihypertensive activity decreases.

[0128] Experimental Example 4 Experiment on the blood pressure lowering effect of whey protein peptide in vivo

[0129] Spontaneously hypertensive model rats (SHR), 48 males, 10-12 weeks old, weighing 210-230g; normotensive rats (WKY), 6 males, 10-12 weeks old, weighing 210-230g. The temperature of the breeding environment was maintained at (25±1)℃, the humidity was maintained at 55%±5%, the light / dark cycle was 12 hours per day, and the lighting time was from 8:00 am to 20:00 pm. Rats could eat standard feed and tap water at will, and the breeding environment was disinfected regularly. Before the experiment, SHR and WKY had a one-week environmental adaptation period. After the adaptation period, the weight and blood pressure of the rats were measured, and they were divided into different groups according to the measurement results to ensure the uniformity of the baseline data of the rats in each group.

[0130] SHR rats were divided into 8 groups, 6 rats in each group, namely the positive control group intervened by captopril, the intervention group of Example 1, the intervention group of Example 2, the intervention group of Example 3, the intervention group of Example 4, the intervention group of control group 1, the intervention group of control group 2, and the hypertension model control group. WKY rats served as the blank control group with 6 rats.

[0131] Table 5. Table of oral administration dosage for each group

[0132] Group <![CDATA[Gavage administration / mg·kg -1 > Blank control group An equal volume of saline Hypertension model control group An equal volume of saline Example 1 Whey protein peptide prepared in Example 1 500 mg / kg Example 2 Example 2 Whey protein peptide 500mg / kg Example 3 Example 3 Whey protein peptide prepared 500mg / kg Example 4 Example 4 Whey protein peptide 500mg / kg Comparative Example 1 Comparative Example 1 Whey protein peptide 500 mg / kg Comparative Example 2 Comparative Example 2 Whey protein peptide 500 mg / kg Positive control group 10 mg / kg captopril

[0133] The rats were fed with ordinary feed and given drugs by gavage once a day according to the dosage gavage table for 8 consecutive weeks. The body weight, food intake and blood pressure of the rats were collected every week during the experiment. When measuring the blood pressure and heart rate of the rats, in order to ensure the accuracy of the measured data, the rats to be tested need to be placed in a warm box (37°C) in advance and left to stand for about 15 minutes. After the rats were quiet and the blood pressure was stable, the systolic blood pressure, diastolic blood pressure and heart rate of SHR and WKY rats were monitored using the BP-2006A small animal non-invasive blood pressure meter. The rats were fixed in the heating hood, and a blackout cloth was put on the outer layer of the heating hood to avoid the stress response of the rats affecting the accuracy of the test data. The rats were placed in the heating hood and covered with a blackout cloth. The temperature of the heating hood was set to 37-39°C to ensure that the tail artery of each rat was fully dilated during the measurement. The sphygmomanometer air bag was put on 3 cm from the tail root of the fixed rat, and the "pulse signal sensor" in the air bag was kept directly above the tail artery. When the sensor shows that the pulse wave is in a stable wave shape, blood pressure measurement begins. The system was connected to a computer, and the test results were recorded using BP98AWU software, recording three to five readings with heart rate changes <10 beats / min and blood pressure changes <20 mmHg, and the measurements were repeated three times for each rat to obtain the average value.

[0134] Each group was observed by gavage for 8 weeks. During the experiment, the animals in each group showed normal growth, development, and activity, and were in good mental state, with no signs of poisoning or death.

[0135] Table 6 Changes in body weight of rats in each group

[0136]

[0137] Table 6 shows the changes in body weight of rats in each group after 1 week, 4 weeks, and 8 weeks of administration. In the 8th week of intervention, compared with the blank control group, the body weight of the hypertension model control group, comparative example 1, and comparative example 2 was significantly reduced (P < 0.05). Although the body weight of Examples 1, 2, 3, and 4 decreased slightly, there was no significant difference compared with the blank control group. This shows that the rats are affected by hypertension, their food intake is reduced, and their spirits are depressed, which will lead to weight loss.

[0138] Table 7 Systolic blood pressure (SBP) of rats in each group after 8 weeks of intervention

[0139]

[0140]

[0141] Table 7 shows the weekly systolic blood pressure test values ​​of rats in each group during the experiment. In the 0th week of intervention, the systolic blood pressure of the hypertension model group, embodiment, control example and positive control group was significantly higher than that of the blank control group (P < 0.05), and the hypertension model was established.

[0142] After 8 weeks of intervention, the systolic blood pressure of rats in each example and the comparative example was lower than that in the hypertension model control group, but the systolic blood pressure of the comparative examples 1 and 2 was not significantly reduced. Examples 1, 2, 3, and 4 significantly reduced the systolic blood pressure of the hypertension model group (P < 0.05), and the systolic blood pressure of Example 1 was the largest.

[0143] Table 8 Diastolic blood pressure (DBP) of rats in each group after 8 weeks of intervention

[0144]

[0145]

[0146] Table 8 shows the weekly diastolic blood pressure test values ​​of rats in each group. In the 0th week of intervention, the diastolic blood pressure of the hypertension model group, the embodiment, the control example and the positive control group was significantly higher than that of the blank control group (P < 0.05), and the hypertension model was established.

[0147] At the 8th week of intervention, the diastolic pressure of rats in each example and comparison example decreased compared with the hypertension model control group, but the diastolic pressure of comparison examples 1 and 2 did not decrease significantly. The diastolic pressure of examples 1, 2, 3, and 4 was significantly lower than that of the hypertension model group (P < 0.05), and the diastolic pressure of example 1 decreased the most.

[0148] Table 9 Heart rate (HR) of rats in each group after 8 weeks of intervention

[0149]

[0150] Table 9 shows the heart rate values ​​of rats in each group after 8 weeks of intervention. The hypertension model control group significantly increased compared with the blank control group (P < 0.05), while Examples 1, 2, 3, 4 and the positive control group significantly decreased compared with the hypertension model control group (P < 0.05). Comparative Examples 1 and 2 compared with the hypertension model group, the heart rate did not decrease significantly.

[0151] Combining the in vitro test of ACE inhibition rate and the in vivo test of animal blood pressure reduction, it can be concluded that the higher the content of small molecule peptides in whey protein peptides, the higher the ACE inhibition rate activity and the better the blood pressure reduction effect.

[0152] In this application, nattokinase is used to hydrolyze whey protein, and the hydrolysis efficiency is high. The activity of the obtained whey protein peptides is significantly better than that of whey protein peptides hydrolyzed by other proteases. The process adopts a two-step enzymolysis method, and the second step of enzymolysis uses a composite protease (carboxypeptidase, flavor protease), which can increase the flavor and enhance the taste, further increase the content of short peptides, and improve the antihypertensive effect. The whey protein peptide process provided by this application is simple and feasible, and the prepared whey protein peptides have a good taste and a good antihypertensive effect.

[0153] The whey protein peptide with antihypertensive activity provided in the examples of the present application, as well as its preparation method and application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for technicians in this field, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present application.

Claims

1. A method for preparing a whey protein peptide having blood pressure lowering activity, characterized in that: include: Using nattokinase to perform the first step of enzymatic hydrolysis on whey protein to obtain the initial enzymatic hydrolysate; The initial enzymatic hydrolysate is subjected to a second step of enzymatic hydrolysis using a protease to obtain whey protein peptides.

2. The preparation method according to claim 1, characterized in that: The protease is selected from one or more of carboxypeptidase and flavor protease.

3. The preparation method according to claim 2, characterized in that: The protease is a composite protease, which includes carboxypeptidase and flavor protease, and the mass ratio of the carboxypeptidase to the flavor protease is (0.8-1.2):

1.

4. The preparation method according to claim 1, characterized in that: The nattokinase is prepared by fermenting soybeans with Bacillus subtilis, and has a purity of ≥95%.

5. The preparation method according to claim 1, characterized in that: The first step of enzymatic hydrolysis of whey protein comprises: Weigh the whey protein and pure water in proportion, put them into a hydrolysis tank and mix them to obtain a whey protein solution; Weigh nattokinase in proportion, add it to the whey protein solution, fully dissolve it and perform the first enzymolysis.

6. The preparation method according to claim 5, characterized in that: The whey protein content in the whey protein solution is 2%-8%; and / or, The added amount of nattokinase is 4000-8000 IU / g; and / or, The enzymatic hydrolysis time of the first step is 4-6 hours, and the enzymatic hydrolysis temperature is 50-60° C.; and / or, After the first step of enzymatic hydrolysis, an enzyme inactivation treatment is further included, and the conditions of the enzyme inactivation treatment are: heating to above 95° C. and inactivating the enzyme for 10-15 minutes.

7. The preparation method according to claim 1, characterized in that: The amount of the protease added is 6000-9000 U / g; and / or, The enzymatic hydrolysis time of the second step is 1-5h, and the enzymatic hydrolysis temperature is 40-50°C; and / or, After the second step of enzymolysis, an enzyme inactivation treatment is further included, wherein the conditions of the enzyme inactivation treatment are: heating to above 95° C. and inactivating the enzyme for 10-15 minutes; and / or, After the second step of enzymatic hydrolysis, an enzyme inactivation treatment is further included. After the enzyme inactivation is completed, the solution after the enzyme inactivation is subjected to solid-liquid separation, and the clear liquid is dried to obtain the whey protein peptide.

8. A whey protein peptide having blood pressure lowering activity obtained according to the preparation method according to any one of claims 1 to 7.

9. The whey protein peptide according to claim 8, characterized in that In the whey protein peptide: The proportion of peptides with a molecular weight less than 2000 Da is greater than 30%; and / or, The peptides with molecular weight less than 2000Da account for 60%-95%; and / or, The proportion of peptides with a molecular weight of 2000-5000Da is less than 40%; and / or, The peptides with molecular weight between 2000-5000Da account for 5%-30%; and / or, The proportion of peptides with a molecular weight greater than 5000 Da is less than 20%; and / or, Peptides with a molecular weight greater than 5000Da account for 2%-10%.

10. The use of whey protein peptide according to claim 9, characterized in that: Whey protein peptides are used in foods, health foods and medicines to treat high blood pressure or maintain healthy blood pressure levels.

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