Waste milk source ACE (angiotensin converting enzyme) inhibitory peptide as well as enzymolysis method and application thereof

Abstract

According to the waste milk source ACE inhibitory peptide and the enzymolysis method and application thereof, neutral protease is added into cow milk protein liquid for enzymolysis reaction, the pH value of the cow milk protein liquid in the enzymolysis process is adjusted through three stages to obtain enzymatic hydrolysate, the enzymatic hydrolysate is subjected to ultrafiltration through a filter membrane, and then the ACE inhibitory peptide is obtained. The inhibition rate of the obtained ACE inhibitory peptide reaches 90% or above, and the yield of the ACE inhibitory peptide (1kDa to 5kDa) in each gram of protein reaches 41.5%. Compared with a polypeptide preparation process in the prior art, the method provided by the invention has the advantages that by adjusting the pH in the cow milk proteolysis process, the inhibition rate of the ACE inhibitory peptide can be ensured to be 90% or above, and the yield of the ACE inhibitory peptide (a polypeptide component with the molecular weight of 1kDa to 5kDa) in each gram of protein is increased to 41.5%; the cow milk protein liquid is subjected to enzymolysis in three stages creatively provided by the invention, so that the yield of the ACE inhibitory peptide is effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of ACE inhibitory peptides, and in particular to a waste milk-derived ACE inhibitory peptide, and an enzymatic hydrolysis method and application thereof. Background Art

[0002] Hypertension is the most common chronic disease in my country and even in the world, and is also the main predisposing factor for various cardiovascular and cerebrovascular diseases. Usually, patients with hypertension need to take antihypertensive drugs for a long time to control their blood pressure. At the same time, there will be certain toxic side effects, such as being detrimental to vascular health, increasing uric acid and increasing the burden on the liver and kidneys.

[0003] With the increase of fat and sterol components in daily diet, the accelerated pace of life and increased pressure, as well as the pollution and destruction of the ecological environment, the number of hypertension patients in my country has continued to increase in recent years, which has brought heavy economic and nursing burdens to the patients' families and society. ACE is a protein that can convert angiotensin I (Ang I) into angiotensin II (Ang II) and passivate the antihypertensive substance bradykinin. For a long time, the mechanism of action of clinical treatment drugs for hypertension mainly includes beta-blockers, diuretics, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blockers, calcium antagonists and alpha-blockers. Although the antihypertensive mechanism is diverse, long-term use will produce drug resistance and some side effects.

[0004] Milk is a daily drink with rich nutritional value and easy to obtain. It is rich in casein. Since Brantle et al. obtained a short peptide composed of 7 amino acids from the enzymatic hydrolysis of bovine casein in 1979, milk crude protein, especially casein, has become one of the main raw materials for the preparation of bioactive peptides. ACEI, as an active peptide with significant effect on the treatment of hypertension, has been the focus of researchers for many years.

[0005] In the prior art, a Chinese invention with publication number CN118580311A discloses an ACE inhibitory peptide and its preparation method and application. The peptide segment provided in the invention binds to ACE through hydrogen bond interaction, van der Waals force and electrostatic interaction, and through specific compounding and synergistic enhancement, jointly improves its affinity with ACE, thereby obtaining excellent ACE inhibition ability, and can increase blood vessel diameter, reduce blood flow velocity, and have excellent rate ability. The larger the molecular weight of the polypeptide, the weaker its ACE inhibition rate. When the molecular weight of the polypeptide is <1kDa, the ACE rate of the polypeptide is optimal. However, those skilled in the art also know that The hydrophobic structure of natural protein is often hidden inside its molecule. As the hydrolysis proceeds, when the molecular weight gradually decreases, the hydrophobic group is gradually exposed, and the obtained polypeptide product will show bitterness. Based on this, in order to obtain a polypeptide with a good ACE rate, the taste of the polypeptide will not show severe bitterness and affect the palatability. Therefore, polypeptides with a molecular weight of 1kDa to 5kDa are generally selected. However, in the prior art, in the process of preparing polypeptides by enzymatic hydrolysis of proteins with proteases, the yield of polypeptides with a molecular weight of 1kDa to 5kDa is only about 20%, resulting in a low yield of polypeptides with a molecular weight of 1kDa to 5kDa. Summary of the invention

[0006] Based on this, the present invention provides a waste milk-derived ACE inhibitory peptide, an enzymatic hydrolysis method and application thereof, to solve the technical problem of low yield of polypeptides with a molecular weight of 1 kDa to 5 kDa existing in the prior art.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] A method for enzymatic hydrolysis of waste milk-derived ACE inhibitory peptides, comprising the following steps:

[0009] S10 degreases and concentrates the waste milk to obtain milk protein liquid;

[0010] S20: adjusting the pH of the milk protein solution to 8.2 to 9, adding a neutral protease to perform an enzymolysis reaction, reacting for 2 h to 3 h to obtain a first enzymolysis product, adjusting the pH of the first enzymolysis product to 5 to 5.8, continuing the enzymolysis reaction, reacting for 2 h to 3 h to obtain a second enzymolysis product, adjusting the pH of the second enzymolysis product to 6.5 to 7.5, continuing the enzymolysis reaction, reacting for 2 h to 3 h to obtain an enzymolysis solution;

[0011] S30: ultrafiltration of the enzymatic hydrolysate through a filter membrane to collect components with a molecular weight of 1 kDa to 5 kDa to obtain an ACE inhibitory peptide.

[0012] Preferably, in step S20, the amount of neutral protease added is 5200 U / g to 6800 U / g.

[0013] Preferably, in step S20, the pH of the bovine milk protein solution is adjusted to 8.5 to 8.7, and then a neutral protease is added to carry out an enzymatic hydrolysis reaction, and the reaction is carried out for 2 hours to obtain a first enzymatic hydrolysis product, and the pH of the first enzymatic hydrolysis product is adjusted to 5.5 to 5.7, and then the enzymatic hydrolysis reaction is continued, and the reaction is carried out for 2 hours to obtain a second enzymatic hydrolysis product, and the pH of the second enzymatic hydrolysis product is adjusted to 7, and then the enzymatic hydrolysis reaction is continued, and the enzymatic hydrolysis solution is obtained.

[0014] Preferably, in step S20, the step of “adjusting the pH of the milk protein solution to 8.2 to 9 and then adding a neutral protease to perform an enzymatic hydrolysis reaction” specifically includes the following steps:

[0015] The pH of the milk protein solution is adjusted to 8.2 to 9, and then a mixed enzyme of neutral protease and papain is added to carry out enzymolysis reaction.

[0016] Preferably, the mass ratio of the neutral protease to the papain is (0.15 to 0.25):(0.05 to 0.08).

[0017] Preferably, in step S20, the amount of the mixed enzyme added is 6000 U / g to 7500 U / g.

[0018] Preferably, the S30 further comprises the following steps: taking the component with a molecular weight of 1 kDa to 5 kDa obtained by ultrafiltration and performing gel chromatography column separation to collect the polypeptide component with a retention time of 25 min to 30 min;

[0019] The polypeptide component with a retention time of 25 min to 30 min is separated by a high performance liquid chromatography column, and the polypeptide component with a retention time of 4 min to 11 min is collected to obtain the ACE inhibitory peptide.

[0020] Preferably, in S20, the reagent used to adjust the pH value is 1.5M sodium hydroxide solution or 1.5M hydrochloric acid solution.

[0021] A waste milk ACE inhibitory peptide is prepared by the enzymatic hydrolysis method of the waste milk ACE inhibitory peptide described in any one of the above.

[0022] A use of the above-mentioned waste milk-derived ACE inhibitory peptide in the preparation of a drug for treating hypertension.

[0023] Compared with the prior art, the present invention has at least the following advantages:

[0024] The present invention provides a kind of waste milk source ACE inhibitory peptide, its enzymatic hydrolysis method and application, neutral protease is added to bovine milk protein solution for enzymatic hydrolysis reaction, and the pH of the enzymatic hydrolysis process of the bovine milk protein solution is adjusted in three stages to obtain an enzymatic hydrolyzate, and the enzymatic hydrolyzate is ultrafiltered through a filter membrane to obtain an ACE inhibitory peptide, and the inhibition rate of the obtained ACE inhibitory peptide reaches more than 90%, and the yield of ACE inhibitory peptide (1kDa to 5kDa) per gram of protein reaches 41.5%. The present invention proposes that by adjusting the pH during the enzymatic hydrolysis of bovine milk, compared with the process for preparing polypeptides in the prior art, the inhibition rate of ACE inhibitory peptides can be guaranteed to be more than 90%, and the yield of ACE inhibitory peptides (molecular weight 1kDa to 5kDa polypeptide components) per gram of protein is increased to 41.5%, and the bovine milk protein solution is enzymatically hydrolyzed in three stages proposed by the present invention, effectively improving the yield of ACE inhibitory peptides. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The technical solution of the present invention will be further described below in conjunction with the embodiments of the present invention, and the present invention is not limited to the following specific implementation methods.

[0026] In one embodiment of the present invention, a method for enzymatic hydrolysis of waste milk-derived ACE inhibitory peptides comprises the following steps:

[0027] S10 degreases and concentrates the waste milk to obtain milk protein liquid.

[0028] Specifically, the preparation of milk protein liquid: the waste milk is defatted and concentrated, and centrifuged at (5±1)°C to remove the upper fat and precipitate to obtain milk protein liquid; in this step, the centrifugation conditions are: (6000±600) r / min centrifugation for 25 minutes.

[0029] S20 adjusts the pH of the milk protein solution to 8.2 to 9, then adds a neutral protease to carry out enzymolysis reaction, reacts for 2h to 3h to obtain a first enzymolysis product, adjusts the pH of the first enzymolysis product to 5 to 5.8, continues the enzymolysis reaction, reacts for 2h to 3h to obtain a second enzymolysis product, adjusts the pH of the second enzymolysis product to 6.5 to 7.5, then continues the enzymolysis reaction, reacts for 2h to 3h to obtain an enzymolysis solution.

[0030] Specifically, the amount of neutral protease added is 5200U / g to 6800U / g. Preferably, the pH of the milk protein solution is adjusted to 8.5 to 8.7, and then the neutral protease is added for enzymolysis reaction. The first enzymolysis product is obtained after the reaction is carried out for 2 hours. The pH of the first enzymolysis product is adjusted to 5.5 to 5.7, and then the enzymolysis reaction is continued. The second enzymolysis product is obtained after the reaction is carried out for 2 hours. The pH of the second enzymolysis product is adjusted to 7, and then the enzymolysis reaction is continued. The enzymolysis solution is obtained after the reaction is carried out for 2 hours.

[0031] In the step of "adjusting the pH of the milk protein solution to 8.2 to 9 and then adding a neutral protease to carry out an enzymatic hydrolysis reaction", the pH of the milk protein solution is adjusted to 8.2 to 9, and then a mixed enzyme of neutral protease and papain is added to carry out an enzymatic hydrolysis reaction, the mass ratio of the neutral protease to the papain is (0.15 to 0.25):(0.05 to 0.08), and the amount of the mixed enzyme added is 6000U / g to 7500U / g.

[0032] The reagent used to adjust the pH value is 1.5M sodium hydroxide solution or 1.5M hydrochloric acid solution.

[0033] S30: ultrafiltration of the enzymatic hydrolysate through a filter membrane to collect components with a molecular weight of 1 kDa to 5 kDa to obtain an ACE inhibitory peptide.

[0034] Specifically, the pH of the bovine milk protein solution is adjusted to 8.2 to 9, and then a neutral protease is added for enzymolysis reaction. The reaction is carried out for 2h to 3h to obtain a first enzymolysis product. The pH of the first enzymolysis product is adjusted to 5 to 5.8, and then the enzymolysis reaction is continued. The reaction is carried out for 2h to 3h to obtain a second enzymolysis product. The pH of the second enzymolysis product is adjusted to 6.5 to 7.5, and then the enzymolysis reaction is continued. The reaction is carried out for 2h to 3h to obtain an enzymolysis solution, which is then separated by ultrafiltration, column chromatography, and high performance liquid chromatography to obtain the ACE inhibitory peptide.

[0035] In a preferred embodiment, the component with a molecular weight of 1 kDa to 5 kDa obtained by ultrafiltration is subjected to gel chromatography column separation, and the polypeptide component with a retention time of 25 min to 30 min is collected;

[0036] The polypeptide component with a retention time of 25 min to 30 min is separated by a high performance liquid chromatography column, and the polypeptide component with a retention time of 4 min to 11 min is collected to obtain the ACE inhibitory peptide;

[0037] Specifically, the gel chromatography column includes one of Sephadex G25 Flash chromatography column, Sephadex G15 Flash chromatography column or Sephadex G50 Flash chromatography column, and is more preferably Sephadex G25 Flash chromatography column.

[0038] The high performance liquid chromatography column comprises a Flash C18 chromatography column or a CAPCELL PAK C18AQ reverse phase chromatography column, and is more preferably a Flash C18 chromatography column.

[0039] The separation includes the following steps:

[0040] (1) adjusting the pH of the milk protein solution to 8.2 to 9, adding a neutral protease to perform an enzymolysis reaction, stirring, and enzymolysis for 2 h to 3 h to obtain a first enzymolysis product, and then adjusting the pH of the first enzymolysis product to 5.5 to 5.7 and continuing the enzymolysis reaction, stirring, and enzymolysis for 2 h to 3 h to obtain a second enzymolysis product, adjusting the pH of the second enzymolysis product to 6.5 to 7.5 and continuing the enzymolysis reaction, stirring, and enzymolysis for 2 h to 3 h, then inactivating the enzyme, filtering, centrifuging, filtering with a ceramic membrane, desalting with a nanofiltration membrane, and concentrating and drying to obtain an enzymolysis solution;

[0041] The neutral protease is added to the milk protein solution in an amount of 5200U / g to 6800U / g;

[0042] (2) preparing the enzymatic hydrolysate obtained in step (1) into a 60 mg / mL to 155 mg / mL solution, and ultrafiltration to obtain a polypeptide component with a molecular weight of 1 kDa to 5 kDa;

[0043] (3) preparing a 5 mg / mL to 13 mg / mL solution of the polypeptide component with a molecular weight of 1 kDa to 5 kDa in step (2), separating the polypeptide component with a retention time of 25 min to 30 min by using a Sephadex G25 Flash column;

[0044] The eluent for separation in the gel chromatography column is water, and the elution flow rate is 4.5 mL / min to 6.5 mL / min;

[0045] (4) The polypeptide component with a retention time of 25 min to 30 min (for example, 26 min, 26.5 min, 28 min, 28.5 min, 29 min, etc.) in step (3) is prepared into a 18 mg / mL to 28 mg / mL solution, separated by a Flash C18 column, and the polypeptide component with a retention time of 4 min to 11 min (for example, 5 min, 5.5 min, 6 min, 7.5 min, 8 min, etc.) is collected to obtain the ACE inhibitory peptide.

[0046] A waste milk ACE inhibitory peptide is prepared by the enzymatic hydrolysis method of the waste milk ACE inhibitory peptide described in any one of the above.

[0047] A use of the above-mentioned waste milk-derived ACE inhibitory peptide in the preparation of a drug for treating hypertension.

[0048] The present invention adds a neutral protease to a bovine milk protein solution for enzymolysis reaction, and adjusts the pH of the enzymolysis process of the bovine milk protein solution in three stages to obtain an enzymolysis solution, and the enzymolysis solution is ultrafiltered through a filter membrane to obtain an ACE inhibitory peptide, and the inhibition rate of the obtained ACE inhibitory peptide reaches more than 90%, and the yield of ACE inhibitory peptide (1kDa to 5kDa) per gram of protein reaches 41.5%. The present invention proposes that by adjusting the pH during the enzymolysis of bovine milk protein, compared with the process of preparing polypeptides by enzymolysis of proteins by protease in the prior art, the inhibition rate of ACE inhibitory peptides reaches more than 90%, and the yield of ACE inhibitory peptides (molecular weight 1kDa to 5kDa polypeptide components) per gram of protein is increased to 41.5%. The three-stage enzymolysis of the bovine milk protein solution creatively proposed by the present invention effectively improves the yield of ACE inhibitory peptides.

[0049] The present invention will be further described below in conjunction with specific embodiments.

[0050] Test equipment: Gel chromatography column (Sephadex G25 Flash chromatography column);

[0051] High performance liquid chromatography column (Flash C18 column).

[0052] Experimental materials: waste milk, 1.5M sodium hydroxide solution, 1.5M hydrochloric acid solution, all of which are domestically produced commercially available reagents.

[0053] Detection method:

[0054] Inhibition rate:

[0055] The ACE inhibition ability test indirectly measures ACE activity by measuring the content of hippuric acid.

[0056] HHL (Hip-His-Leu) is rapidly decomposed by ACE to produce hippuric acid (Hip) and dipeptide (His-Leu, HL). Hippuric acid has a maximum absorption at 228nm. When ACEI samples are added, the activity of ACE enzyme is inhibited and the amount of hippuric acid generated is reduced. Therefore, the inhibition rate of ACE activity by the sample can be evaluated by measuring the amount of hippuric acid generated by high performance liquid chromatography.

[0057] ACE activity was indirectly determined by measuring the content of hippuric acid; 45 μL of sample solutions of different concentrations were mixed with 30 μL of 0.1.5 U / mL ACE (0.1 mol / L boric acid buffer solution), and then pre-incubated at 38°C for 12 min, and then 55 μL of 7 mM HHL (Hip-His-Leu) solution was added, and after incubation at 38°C for 35 min, 88 μL of 1.5 M hydrochloric acid was added to terminate the reaction, centrifuged (10,000 × g, 22 min), filtered through a 0.50 μm filter membrane, and analyzed by liquid chromatography. Control tube: 42 μL of boric acid buffer (pH 8.3 0.3 mol / L NaCl) was used to replace the sample solution of the test group. Blank tube: 25 μL of boric acid buffer (pH 8.3 0.3 mol / L NaCl) was used to replace the ACE solution of the control group. Waters-C18, 5 μm, 4.5 mm×250 mm; mobile phase: A acetonitrile: B water = 1:4.2 (containing 0.05% acetic acid).

[0058] Comparative Example 1

[0059] (1) defatting and concentrating the waste milk, and centrifuging at 6000 r / min for 25 min at 5° C. to remove the upper fat layer and the precipitate to obtain a 150 mg / ml milk protein solution;

[0060] (2) taking 30 ml of milk protein solution, adjusting the pH of the milk protein solution to 8.5, adding 6200 U / g neutral protease to carry out enzymatic hydrolysis reaction, the enzymatic hydrolysis reaction time is 6 hours, and obtaining enzymatic hydrolyzate. After the enzymatic hydrolysis time is up, boiling water bath is used to inactivate the enzyme for 10 minutes, thereby completing the enzymatic hydrolysis, centrifuging the enzymatic hydrolyzate at 8000 r / min for 15 minutes, and taking the supernatant;

[0061] (3) The supernatant obtained after centrifugation was subjected to ultrafiltration to obtain a polypeptide component with a molecular weight of 1 kDa to 5 kDa. The polypeptide component with a molecular weight of 1 kDa to 5 kDa was prepared into an 8 mg / mL solution using a Biotage Selekt rapid preparative chromatograph, and separated by a dextran gel G25 Flash column (3.5 cm*10 cm). The eluent for separation by the gel column was water, the amount was 18 CV (column volume), the elution flow rate was 5 mL / min, and the polypeptide component with a retention time of 30 min was collected. The polypeptide component with a retention time of 30 min was prepared into a 20 mg / mL solution, separated by a Flash C18 column, and the polypeptide component with a retention time of 7 min was collected to obtain an ACE inhibitory peptide.

[0062] The ACE inhibitory peptides obtained were tested using the above inhibition rate calculation method. The test results are shown in Table 1.

[0063] Comparative Example 2

[0064] The same as comparative example 1, except that in step (2), the pH of the milk protein solution is adjusted to 5.5.

[0065] Comparative Example 3

[0066] The same as comparative example 1, except that in step (2), the pH of the milk protein solution is adjusted to 7.

[0067] Table 1

[0068]

[0069] Note: The yield of ACE inhibitory peptide per gram of protein is the ACE inhibitory peptide obtained by per gram of milk protein, that is, the yield per gram of protein = weight of ACE inhibitory peptide / weight of milk protein.

[0070] It can be seen from Comparative Examples 1, 2 and 3 that when the enzymatic hydrolysis component is neutral protease and the pH of the milk protein solution is 8.5, 5.5 and 7 respectively, there is basically no effect on the inhibition rate of ACE inhibitory peptides and the yield of ACE inhibitory peptides per gram of protein.

[0071] Example 1

[0072] The same as comparative example 1, except that: in step (2), the pH of the milk protein solution is adjusted to 8.5, 6200 U / g neutral protease is added for enzymatic hydrolysis reaction, the enzymatic hydrolysis reaction is carried out for 2 hours to obtain a first enzymatic hydrolysis product, and then the pH of the first enzymatic hydrolysis product is adjusted to 7 and the enzymatic hydrolysis reaction is continued for 2 hours to obtain a second enzymatic hydrolysis product, and the pH of the second enzymatic hydrolysis product is further adjusted to 5.5, and the enzymatic hydrolysis reaction is carried out again for 2 hours to obtain an enzymatic hydrolyzate.

[0073] Example 2

[0074] Same as Example 1, except that the pH of the milk protein solution in Example 1 is adjusted to 5.5, the pH of the first enzymatic hydrolysis product is adjusted to 7, and the pH of the second enzymatic hydrolysis product is adjusted to 8.5.

[0075] Example 3

[0076] The same as Example 1, except that the pH of the milk protein solution in Example 1 is adjusted to 8.5, the pH of the first enzymatic hydrolysis product is adjusted to 5.5, and the pH of the second enzymatic hydrolysis product is adjusted to 7.

[0077] Example 4

[0078] The same as Example 1, except that the pH of the milk protein solution in Example 1 is adjusted to 5.5, the pH of the first enzymatic hydrolysis product is adjusted to 8.5, and the pH of the second enzymatic hydrolysis product is adjusted to 7.

[0079] Table 2

[0080]

[0081] It can be seen from Example 1, Example 2, Example 3, and Example 4 that the pH of the milk protein solution, the first enzymatic hydrolysis product, and the second enzymatic hydrolysis product are different, and when the enzymatic hydrolysis components are all neutral proteases, there is basically no effect on the inhibition rate of ACE inhibitory peptides;

[0082] The yield of ACE inhibitory peptides per gram of protein in Example 1 is 31.3%, and the yield of ACE inhibitory peptides per gram of protein in Example 2 is 31.9%. Compared with Example 1, the yield of ACE inhibitory peptides per gram of protein in Example 2 is improved; the yield of ACE inhibitory peptides per gram of protein in Example 3 is 34.6%, and the yield of ACE inhibitory peptides per gram of protein in Example 4 is 31.5%. Compared with Example 4, the yield of ACE inhibitory peptides per gram of protein in Example 3 is improved. It can be seen from the above examples that when the pH of the bovine milk protein solution, the first enzymatic hydrolysis product, and the second enzymatic hydrolysis product are different, the yield of ACE inhibitory peptides per gram of protein is affected to a certain extent, and the yield of ACE inhibitory peptides per gram of protein is higher when the pH of the bovine milk protein solution is 8.5, the pH of the first enzymatic hydrolysis product is 5.5, and the pH of the second enzymatic hydrolysis product is 7. Compared with the case where the pH of the bovine milk protein solution was 8.5, the pH of the first enzymatic hydrolysis product was 7, and the pH of the second enzymatic hydrolysis product was 5.5, the yield of ACE inhibitory peptides per gram of protein increased by 1.8% when the pH of the bovine milk protein solution was 5.5, the pH of the first enzymatic hydrolysis product was 7, and the pH of the second enzymatic hydrolysis product was 8.5. Compared with the case where the pH of the bovine milk protein solution was 5.5, the pH of the first enzymatic hydrolysis product was 8.5, and the pH of the second enzymatic hydrolysis product was 7, the yield of ACE inhibitory peptides per gram of protein increased by 8.9% when the pH of the bovine milk protein solution was 8.5, the pH of the first enzymatic hydrolysis product was 5.5, and the pH of the second enzymatic hydrolysis product was 7.

[0083] Example 5

[0084] Same as Example 3, except that the neutral protease in Example 3 is replaced by papain, and the added amount of papain is 6200u / g.

[0085] Table 3

[0086]

[0087] It can be seen from Example 5 that when the enzymatic hydrolysis component is papain, it has little effect on the inhibition rate of ACE inhibitory peptides;

[0088] The yield of ACE inhibitory peptides per gram of protein in Example 5 is 31.2%, the yield of ACE inhibitory peptides per gram of protein in Comparative Example 1 is 29.5%, the yield of ACE inhibitory peptides per gram of protein in Comparative Example 2 is 29.2%, and the yield of ACE inhibitory peptides per gram of protein in Comparative Example 3 is 28.9%. The yield of ACE inhibitory peptides per gram of protein in Example 5 is slightly higher than the yields of ACE inhibitory peptides per gram of protein in Comparative Example 1, Comparative Example 2, and Comparative Example 3; the yield of ACE inhibitory peptides per gram of protein in Example 1 is 31.3%, the yield of ACE inhibitory peptides per gram of protein in Example 2 is 31.9%, the yield of ACE inhibitory peptides per gram of protein in Example 3 is 34.6%, and the yield of ACE inhibitory peptides per gram of protein in Example 4 is 31.5%. The yield of ACE inhibitory peptides per gram of protein in Example 5 is slightly lower than the yields of ACE inhibitory peptides per gram of protein in Example 1, Example 2, Example 3, and Example 4.

[0089] Example 6

[0090] Same as Example 3, except that the neutral protease in Example 3 is replaced by a mixture of neutral protease and papain, and the mass ratio of neutral protease to papain is 0.2:0.08.

[0091] Example 7

[0092] Same as Example 6, except that the mass ratio of neutral protease to papain in Example 6 is changed to 0.18:0.06.

[0093] Example 8

[0094] Same as Example 6, except that the mass ratio of neutral protease to papain in Example 6 is changed to 0.15:0.05.

[0095] Table 4

[0096]

[0097] It can be seen from Example 6 and Example 3 that when the enzymatic hydrolysis components are different, the inhibition rate of ACE inhibitory peptide is basically not affected;

[0098] The yield of ACE inhibitory peptides per gram of protein in Example 3 is 34.6%, and the yield of ACE inhibitory peptides per gram of protein in Example 6 is 41.2%. Compared with Example 3, the yield of ACE inhibitory peptides per gram of protein in Example 6 is improved. It can be seen from the above examples that when the enzymatic hydrolysis components are different, the yield of ACE inhibitory peptides per gram of protein has a certain influence, and the yield of ACE inhibitory peptides per gram of protein is higher when the enzymatic hydrolysis component is a mixed enzyme of neutral protease and papain. Compared with the case where the enzymatic hydrolysis component is a neutral protease, the yield of ACE inhibitory peptides per gram of protein is increased by 16.0% when the enzymatic hydrolysis component is a mixed enzyme of neutral protease and papain.

[0099] It can be seen from Examples 6, 7 and 8 that different enzymatic hydrolysis components have little effect on the inhibition rate of ACE inhibitory peptides;

[0100] The yield of ACE inhibitory peptides per gram of protein in Example 6 is 41.0%, the yield of ACE inhibitory peptides per gram of protein in Example 7 is 41.2%, and the yield of ACE inhibitory peptides per gram of protein in Example 8 is 41.1%. Compared with Example 6, the yield of ACE inhibitory peptides per gram of protein in Example 7 is improved; compared with Example 8, the yield of ACE inhibitory peptides per gram of protein in Example 7 is improved. It can be seen from the above examples that when the enzymatic hydrolysis components are different, the yield of ACE inhibitory peptides per gram of protein is affected to a certain extent, and the yield of ACE inhibitory peptides per gram of protein is higher when the mass ratio of neutral protease to papain is 0.18:0.06. Compared with the case where the mass ratio of neutral protease to papain was 0.2:0.08, the ACE inhibitory peptide yield per gram of protein was increased by 0.4% when the mass ratio of neutral protease to papain was 0.18:0.06; compared with the case where the mass ratio of neutral protease to papain was 0.15:0.05, the ACE inhibitory peptide yield per gram of protein was increased by 0.2% when the mass ratio of neutral protease to papain was 0.18:0.06.

[0101] Example 9

[0102] Same as Example 7, except that the added amount of the neutral protease and papain mixture in Example 7 is 6800 U / g.

[0103] Example 10

[0104] Same as Example 7, except that the added amount of the neutral protease and papain mixture in Example 7 is 7300 U / g.

[0105] Table 4

[0106]

[0107] It can be seen from Examples 9 and 10 that when the amount of the mixture of neutral protease and papain added is different, the inhibition rate of the ACE inhibitory peptide is basically not affected;

[0108] The yield of ACE inhibitory peptides per gram of protein in Example 9 is 41.5%, and the yield of ACE inhibitory peptides per gram of protein in Example 10 is 41.4%. Compared with Example 10, the yield of ACE inhibitory peptides per gram of protein in Example 9 is improved. It can be seen from the above examples that when the enzymatic hydrolysis components are different, the yield of ACE inhibitory peptides per gram of protein has a certain influence, and the yield of ACE inhibitory peptides per gram of protein is higher when the addition amount of the mixture of neutral protease and papain is 6800U / g. Compared with the case where the addition amount of the mixture of neutral protease and papain is 7300U / g, the yield is increased by 0.2% when the addition amount of the mixture of neutral protease and papain is 6800U / g.

[0109] What is disclosed above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for enzymatic hydrolysis of waste milk-derived ACE inhibitory peptides, characterized in that: Including S10 defatting and concentrating the waste milk to obtain milk protein liquid; S20: adjusting the pH of the milk protein solution to 8.2 to 9, adding a neutral protease to perform an enzymolysis reaction, reacting for 2 h to 3 h to obtain a first enzymolysis product, adjusting the pH of the first enzymolysis product to 5 to 5.8, continuing the enzymolysis reaction, reacting for 2 h to 3 h to obtain a second enzymolysis product, adjusting the pH of the second enzymolysis product to 6.5 to 7.5, continuing the enzymolysis reaction, reacting for 2 h to 3 h to obtain an enzymolysis solution; S30: ultrafiltration of the enzymatic hydrolysate through a filter membrane to collect components with a molecular weight of 1 kDa to 5 kDa to obtain an ACE inhibitory peptide.

2. The enzymatic hydrolysis method of waste milk-derived ACE inhibitory peptide according to claim 1, characterized in that: In the step S20, the amount of the neutral protease added is 5200 U / g to 6800 U / g.

3. The enzymatic hydrolysis method of waste milk-derived ACE inhibitory peptide according to claim 1, characterized in that: In the step S20, the pH of the milk protein solution is adjusted to 8.5 to 8.7, and then a neutral protease is added to carry out an enzymolysis reaction. The reaction is carried out for 2 hours to obtain a first enzymolysis product. The pH of the first enzymolysis product is adjusted to 5.5 to 5.7, and then the enzymolysis reaction is continued. The reaction is carried out for 2 hours to obtain a second enzymolysis product. The pH of the second enzymolysis product is adjusted to 7, and then the enzymolysis reaction is continued. The enzymolysis solution is obtained.

4. The enzymatic hydrolysis method of waste milk-derived ACE inhibitory peptides as claimed in claim 1, characterized in that: In the step S20, the step of "adjusting the pH of the milk protein solution to 8.2 to 9 and then adding a neutral protease to perform an enzymatic hydrolysis reaction" specifically includes the following steps: The pH of the milk protein solution is adjusted to 8.2 to 9, and then a mixed enzyme of neutral protease and papain is added to carry out enzymolysis reaction.

5. The enzymatic hydrolysis method of waste milk-derived ACE inhibitory peptides as claimed in claim 4, characterized in that: The mass ratio of the neutral protease to the papain is (0.15 to 0.25):(0.05 to 0.08).

6. The enzymatic hydrolysis method of waste milk-derived ACE inhibitory peptides as claimed in claim 4, characterized in that: In the step S20, the added amount of the mixed enzyme is 6000 U / g to 7500 U / g.

7. The enzymatic hydrolysis method of waste milk-derived ACE inhibitory peptides as claimed in claim 1, characterized in that: The S30 further comprises the following steps: taking the component with a molecular weight of 1 kDa to 5 kDa obtained by ultrafiltration and performing gel chromatography column separation to collect the polypeptide component with a retention time of 25 min to 30 min; The polypeptide component with a retention time of 25 min to 30 min is separated by a high performance liquid chromatography column, and the polypeptide component with a retention time of 4 min to 11 min is collected to obtain the ACE inhibitory peptide.

8. The enzymatic hydrolysis method of waste milk-derived ACE inhibitory peptides as claimed in claim 1, characterized in that: In the S20, the reagent used to adjust the pH value is 1.5M sodium hydroxide solution or 1.5M hydrochloric acid solution.

9. A waste milk ACE inhibitory peptide, prepared by the enzymatic hydrolysis method of the waste milk ACE inhibitory peptide according to any one of claims 1 to 8.

10. Use of the waste milk-derived ACE inhibitory peptide as claimed in claim 9 in the preparation of a drug for treating hypertension.

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