A preparation method of tussah pupa ACE inhibitory protein peptide

Abstract

A method for preparing an ACE inhibitory protein peptide from tussah pupa is to prepare a protein solution by adding a buffer solution with a pH of 10.5 to tussah pupa protein powder as a raw material, firstly using alkaline protease for the first step of hydrolysis, and then using pepsin for the second step of hydrolysis. The ACE inhibitory protein peptide prepared by the invention using tussah pupa as a raw material has a smaller molecular weight distribution range after separation and purification, and the ACE inhibition rate can reach up to 79.42%, and also has relatively excellent antioxidant reduction ability, the ABTS free radical scavenging rate is 95.17%, and the DPPH free radical scavenging rate is 95.64%.

Description

Technical Field

[0001] The invention relates to the technical field of protein peptide preparation, and in particular to a method for preparing tussah pupa ACE inhibitory protein peptide. Background Art

[0002] my country is a major tussah industry country. Tussah resources are distributed throughout the country, mostly in the northeast. The annual output of tussah cocoons is about 90,000 tons, with an output value of about 4 billion yuan. For a long time, the processing and utilization rate of silkworm pupae in my country has been low. Except for most of them being directly used as livestock and poultry feed and nutritional additives after drying and crushing, some are even used as fertilizers or directly discarded, resulting in a huge waste of resources. With the development of the silk industry and the continuous increase in the output of silkworm cocoons, the comprehensive utilization and deep development of silkworm pupae have become an urgent problem to be solved.

[0003] The protein of tussah silkworm pupa is a complete protein with a protein content of more than 50%. It contains 18 kinds of amino acids. The 17 kinds of amino acids commonly found in tussah silkworm pupae account for 34.57% of the total amino acid mass fraction. Among them, the essential amino acids that cannot be synthesized by the human body and must be supplied from the outside, such as methionine, lysine, leucine, valine, isoleucine, threonine, phenylalanine, etc., are all in high content in silkworm pupae, with a total amount of 14.59%, accounting for 42.20% of the total amino acids. The nutritional value is very optimistic and it is one of the high-quality raw materials for the preparation of polypeptides. The protein of tussah silkworm pupa has high nutritional and functional properties, but due to its complex molecular structure and large molecular weight (MW), the human body cannot effectively absorb it, resulting in allergic effects. Therefore, the protein must be hydrolyzed to produce low molecular weight fragments to improve biological functions, solubility and bioavailability.

[0004] Hypertension is one of the major cardiovascular public diseases in recent years, which seriously affects human health. Hypertension is mainly caused by the increased concentration of angiotensin I converting enzyme (ACE, EC 3.4.15.1). Angiotensin I converts the enzyme (ACE, EC 3.4.15.1) carboxypeptidase with a zinc-dependent dipeptidyl structure, which not only promotes the production of vasoconstrictive octapeptides, but also inactive bradykinin. At present, many artificial chemicals have good ACE inhibitor capabilities, such as captopril, enalapril and lisinopril. However, these drugs have obvious negative effects, usually causing dry cough, angioedema, taste disorders and rash. Therefore, finding non-toxic natural sources of ACE inhibitors has become an effective way to treat hypertension. Silkworm pupa protein hydrolysate (SPP hydrolysate) has an inhibitory effect on ACE. Meng et al. (2016) discovered a new ACE inhibitory peptide from silkworm pupa protein. The silkworm pupa protein was hydrolyzed with neutral protease, and the silkworm pupa peptide obtained had a molecular weight of 603.7Da.

[0005] The preparation of peptides is mainly obtained from natural animal and plant proteins through food biotechnology methods such as enzymatic methods and fermentation methods. It has the advantages of stable antioxidant activity, no toxic side effects and convenient sources. The most important factors in enzymatic hydrolysis are the selection of enzyme types, enzyme concentration, temperature, time, pH and substrate concentration. In order to obtain ideal hydrolyzed products, it is also necessary to optimize the enzymatic hydrolysis process parameters. Acid hydrolysis and alkaline hydrolysis are violent reactions accompanied by the formation of carcinogens, while enzymatic hydrolysis is a mild reaction and has always been the main process for producing active peptides from proteins. The development of bioactive peptides with health-care effects has become an active research field in the biochemical industry in the 21st century.

[0006] Bioactive peptides can be obtained by enzymatic hydrolysis, but they are all blind enzymatic hydrolysis of proteins, and the effect of the hydrolysis products is unstable. In view of this, we have developed a tussah pupa protein peptide with better antioxidant and antihypertensive activities. Summary of the invention

[0007] The present invention aims to provide a method for preparing ACE inhibitory protein peptide from tussah pupa.

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

[0009] A method for preparing tussah pupa ACE inhibitory protein peptides is characterized in that: tussah pupa protein powder is used as a raw material, a buffer solution with a pH of 10.5 is added to prepare a protein solution, alkaline protease is firstly used for the first step of hydrolysis, and then pepsin is used for the second step of hydrolysis.

[0010] Furthermore, in the first step of enzymatic hydrolysis, the protein solution concentration was 3.5 wt.%, alkaline protease with an enzyme activity of 250 U / mg was added at a concentration of 6.5 wt.%, the hydrolysis temperature was 50° C., and constant temperature hydrolysis was performed for 4 h after adding ultrapure water.

[0011] Furthermore, the second step of hydrolysis is to adjust the pH of the enzymatic hydrolyzate after the first step of hydrolysis to 1.5-3.5, add pepsin with an enzyme activity of 30 U / mg and a concentration of 1-3.5 wt.%, and hydrolyze at 35-42° C. for 10-20 min.

[0012] Preferably, the added concentration of pepsin is 3 wt.%, the hydrolysis temperature is 37° C., and the hydrolysis time is 15 min.

[0013] Preferably, before the second step of hydrolysis, sodium sulfate is added to the enzymatic hydrolyzate obtained in the first step of hydrolysis at a concentration of 0.4-0.8 wt.%, and the solution is heated to 40-50° C. and kept warm for 30-60 min.

[0014] Different proteases have different specific hydrolysis sites, and different peptide products can be obtained by acting on the same protein substrate, so the selection of proteases is particularly important. First, a high concentration of alkaline protease is used for hydrolysis for a long time to make the tussah pupa protein hydrolysis more complete. After the first step of hydrolysis, a small amount of sodium sulfate is added to adjust the solubility of the hydrolyzate. When it is hydrolyzed by pepsin, it is found that the amino acids at the C-terminus and N-terminus of the hydrolyzed peptide are arranged to form a higher biological activity. Pepsin is used for secondary hydrolysis in a very short time, which effectively preserves the overall structure of the peptide chain that has been completely hydrolyzed by the alkaline protease in a very short hydrolysis time, and effectively reduces the molecular weight of the peptide and the distribution range of the molecular weight.

[0015] Specifically, a method for preparing ACE inhibitory protein peptide from tussah pupa is characterized by following the steps:

[0016] (1) Using tussah pupa protein powder as a raw material, adding a buffer solution with a pH of 10.5 to prepare a protein solution with a concentration of 3.5 wt.%, adding alkaline protease with an enzyme activity of 250 U / mg to make its concentration reach 6.5 wt.%, the hydrolysis temperature is 50° C., and the first step of hydrolysis is performed for 4 h;

[0017] (2) adjusting the pH of the enzymatic hydrolyzate after the hydrolysis in step (1) to 1.5-3.5, adding pepsin with an enzyme activity of 30 U / mg at a concentration of 1-3.5 wt.%, and performing the second step of hydrolysis at 35-42° C. for 10-20 min;

[0018] (3) The hydrolyzed enzymatic solution was centrifuged at 1000 rpm for 15 min, the supernatant was taken, and ultrafiltration was performed using a 3 kDa filter membrane to obtain a tussah pupa protein peptide solution with a molecular weight of less than 3 kDa, which was then lyophilized to obtain silkworm pupa ACE inhibitory peptide powder.

[0019] Preferably, a method for preparing ACE inhibitory protein peptide from tussah pupa is characterized by following the steps:

[0020] (1) Using tussah pupa protein powder as a raw material, adding a buffer solution with a pH of 10.5 to prepare a protein solution with a concentration of 3.5 wt.%, adding alkaline protease with an enzyme activity of 250 U / mg to make its concentration reach 6.5 wt.%, the hydrolysis temperature is 50° C., and the first step of hydrolysis is performed for 4 h;

[0021] (2) adding sodium sulfate to the enzymatic hydrolyzate obtained in the first step of hydrolysis at a concentration of 0.4-0.8 wt.%, heating to 40-50° C., and keeping warm for 30-60 min;

[0022] (3) adjusting the pH of the enzymatic hydrolyzate after treatment in step (2) to 1.5-3.5, adding pepsin with an enzyme activity of 30 U / mg at a concentration of 1-3.5 wt.%, and performing a second step of hydrolysis at 35-42° C. for 10-20 min;

[0023] (4) The hydrolyzed enzymatic solution was centrifuged at 1000 rpm for 15 min, the supernatant was taken, and ultrafiltration was performed using a 3 kDa filter membrane to obtain a tussah pupa protein peptide solution with a molecular weight of less than 3 kDa, which was then lyophilized to obtain silkworm pupa ACE inhibitory peptide powder.

[0024] The present invention has the following technical effects:

[0025] The ACE inhibitory protein peptide prepared by using tussah pupae as raw materials has a smaller molecular weight distribution range after separation and purification, and the ACE inhibition rate can reach up to 79.42%. It also has excellent antioxidant reduction ability, the ABTS free radical scavenging rate is 95.17%, and the DPPH free radical scavenging rate is 95.64%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : The tussah pupa protein peptide powder with a molecular weight greater than 10kDa prepared by the present invention.

[0027] Figure 2 : The tussah pupa protein peptide powder with a molecular weight greater than 10kDa-3kDa is prepared by the present invention.

[0028] Figure 3 : The tussah pupa protein peptide powder with a molecular weight less than 3kDa prepared by the present invention. DETAILED DESCRIPTION

[0029] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0030] The tussah pupa protein powder used in the present invention comes from Jilin Province Sericultural Science Research Institute.

[0031] Example 1

[0032] A method for preparing ACE inhibitory protein peptide from tussah pupa is carried out according to the following steps:

[0033] (1) Using tussah pupa protein powder as a raw material, adding a buffer solution with a pH of 10.5 to prepare a protein solution with a concentration of 3.5 wt.%, adding alkaline protease with an enzyme activity of 250 U / mg to make its concentration reach 6.5 wt.%, the hydrolysis temperature is 50° C., and the first step of hydrolysis is performed for 4 h;

[0034] (2) adjusting the pH of the enzymatic hydrolyzate obtained in step (1) to 3, adding pepsin with an enzyme activity of 30 U / mg at a concentration of 3 wt.%, and performing a second step of hydrolysis at 37° C. for 15 min;

[0035] (3) The hydrolyzed enzymatic solution was centrifuged at 1000 rpm for 15 min, the supernatant was taken, and ultrafiltration was performed using 10 kDa and 3 kDa filter membranes to obtain tussah pupa protein peptide solutions with molecular weights greater than 10 kDa, 10 kDa-3 kDa, and less than 3 kDa, which were then lyophilized to obtain tussah pupa ACE inhibitory peptide powders with different molecular weights.

[0036] Example 2

[0037] A method for preparing ACE inhibitory protein peptide from tussah pupa is carried out according to the following steps:

[0038] (1) Using tussah pupa protein powder as a raw material, adding a buffer solution with a pH of 10.5 to prepare a protein solution with a concentration of 3.5 wt.%, adding alkaline protease with an enzyme activity of 250 U / mg to make its concentration reach 6.5 wt.%, the hydrolysis temperature is 50° C., and the first step of hydrolysis is performed for 4 h;

[0039] (2) adding sodium sulfate to the enzymatic hydrolyzate obtained in the first step of hydrolysis at a concentration of 0.5 wt.%, heating to 45° C., and keeping the temperature for 40 min;

[0040] (3) adjusting the pH of the enzymatic hydrolyzate after treatment in step (2) to 3, adding pepsin with an enzyme activity of 30 U / mg at a concentration of 3 wt.%, and performing the second step of hydrolysis at 37° C. for 15 min;

[0041] (4) The hydrolyzed enzymatic solution was centrifuged at 1000 rpm for 15 min, the supernatant was taken, and ultrafiltration was performed using 10 kDa and 3 kDa filter membranes to obtain tussah pupa protein peptide solutions with molecular weights greater than 10 kDa, 10 kDa-3 kDa, and less than 3 kDa, which were then lyophilized to obtain tussah pupa ACE inhibitory peptide powders with different molecular weights.

[0042] Determination of hydrolysis degree: Take 1mL of the enzymatic hydrolyzate and centrifuge at 12000r / min for 5min, take the supernatant and dilute it 20 times, determine the hydrolysis degree by OPA method, measure the OD value at 340nm by microplate reader, and calculate the hydrolysis degree. Hydrolysis degree measurement formula:

[0043]

[0044] In the formula,

[0045] L1: the amount of free amino acids released after hydrolysis;

[0046] L0: the amount of free amino acids in the original hydrolysate;

[0047] L max : The total amount of free amino groups in the original hydrolyzate obtained after acid hydrolysis.

[0048] Calculations show that the degree of hydrolysis in the first step of hydrolysis using alkaline protease is 30.032%, and pepsin is used for secondary hydrolysis. According to the relationship between the C / N termini and functions of different polypeptides, in order to ensure that the terminal amino acids are amino acids with better biological activity, the hydrolysis time is controlled to be short, and the degree of hydrolysis is 34.274%.

[0049] The antihypertensive and antioxidant activities of ACE inhibitory peptide powders from tussah pupa of different molecular weights were determined.

[0050] 1. Determination of blood pressure lowering activity (ACE inhibitory activity)

[0051] The reaction was carried out in 0.1 M sodium borate buffer solution (pH 8.3, containing 0.3 M sodium chloride). The sample solution (20 μL) and ACE (20 μL of 1 mU) for initialization of the reaction were pre-incubated at 37°C for 10 minutes. Then 20 μL of HHL (5 mM) was added, and the mixture was further incubated at 37°C for 30 minutes. HCl (100 μL, 1 M) was added, ethyl acetate (200 μL) was added, and after shaking, the solution was centrifuged at 800 g and 4°C for 15 minutes. The upper layer solution (80 μL) was transferred to a 1.5 mL centrifuge tube and incubated at 90°C for 10 minutes to remove the ethyl acetate. Hippuric acid was dissolved in ultrapure water (100 μL) and the absorbance was measured at 228 nm using a UV spectrophotometer.

[0052] Inhibitory activity calculation formula:

[0053] ACE inhibitory peptide activity (%) = [(A-B) / (A-C)] × 100%

[0054] In the formula,

[0055] A is the negative control (ACE without inhibitor);

[0056] B is the sample and positive control (ACE and inhibitor);

[0057] C is the blank control (without ACE and inhibitor).

[0058] The final measurement results are shown in Table 1.

[0059] Table 1: ACE inhibitory activity of protein peptides with different molecular weights in Example 1 and Example at different concentrations

[0060]

[0061] 2. Antioxidant activity detection

[0062] (1) Determination of ABTS free radical scavenging rate

[0063] Take 0.02g ABTS and 0.0034g potassium persulfate and dissolve them in 5ml pure water. Let it stand for 24 hours to prepare the mother solution. Dilute the mother solution with anhydrous ethanol to OD 734 The value of the test solution is 0.7±0.02. Take 100uL of the test solution and add it to a 1.5mL EP tube, add 900uL of distilled water, mix thoroughly, and measure the OD value at 734nm (Ac). Take 100uL of samples of different concentrations and molecular weights and mix them evenly with 900uL of the test solution, react at room temperature and avoid light for 5 minutes, and measure the OD value at 734nm (A1). 100uL of the sample solution to be tested (the same dilution factor as the sample of A1) and 900uL of anhydrous ethanol solution are measured at 734nm OD value (A2).

[0064] ABTS free radical scavenging rate calculation formula:

[0065] [1-(A1-A2) / Ac]×100%

[0066] The measurement results are shown in Table 2.

[0067] Table 2: ABTS free radical scavenging rate of protein peptides with different molecular weights and concentrations

[0068]

[0069] (2) Determination of DPPH free radical scavenging rate

[0070] Take an appropriate amount of DPPH powder and place it in a 100ml volumetric flask, add anhydrous ethanol and ultrasonicate for 5 minutes to fully dissolve it, then use anhydrous ethanol to make it 100ml. The solution is purple and must be fully shaken to keep the color of the upper and lower parts consistent. Keep it away from light and prepare it now (OD value is between 0.221 and 0.689). Take 400uL of DPPH anhydrous ethanol solution and add it to a 1.5mL EP tube, add 400uL of distilled water, mix it thoroughly, and measure the OD value (Ac) at 517nm. Take 400uL of samples of different concentrations and 400uLDPPPH anhydrous ethanol solution, mix them evenly, react at room temperature and avoid light for 30min, and measure the OD value (Ai) at 517nm. 400uL of the sample solution to be tested (the dilution multiple is the same as that of the Ai sample) and 400uL of anhydrous ethanol solution are measured at 517nm OD value (Aj).

[0071] DPPH free radical scavenging rate calculation formula:

[0072] [1-(A i -A j ) / A c ]×100%

[0073] The measurement results are shown in Table 3.

[0074] Table 3: DPPH free radical scavenging rate of protein peptides with different molecular weights and concentrations

[0075]

[0076] (3) Determination of total reducing power of antioxidant activity

[0077] Prepare 0.2M pH 6.6 PBS, 1% potassium ferricyanide, 10% trichloroacetic acid, and 0.1% ferric chloride as solutions 1-4 respectively. After preparation, place them in a 50°C water bath and wait for use. Add 100uL of solution 1, 100uL of sample, and 100uL of solution 2 to a 1.5ml EP tube (A 样 ), add 100uL solution 1, 100uL ultrapure water, and 100uL sample (A0), react in a water bath at 50℃ for 20min, then add 100ul solution 3, centrifuge at 10000rpm for 5min, take 200ul supernatant, 200ul ultrapure water, and 40uL solution 4 and add them to a 1.5mL EP tube, react at room temperature for 10min, and measure the OD value at 700nm.

[0078] The total reducing power calculation formula is: A 样 -A0

[0079] The measurement results are shown in Table 4.

[0080] Table 4: Total reducing power of protein peptides of different molecular weights and concentrations

[0081]

[0082] It can be seen from the above table that the ACE inhibitory peptides with a molecular weight distribution of less than 3 kDa have the best ACE inhibition rate and antioxidant activity.

[0083] Comparative Example 1:

[0084] Compared with Example 1, after one-step hydrolysis with alkaline protease, ultrafiltration was directly performed through a 3 kDa filter membrane to prepare a protein inhibitory peptide. The antioxidant activity was determined, and its ABTS free radical scavenging rate, DPPH free radical scavenging rate and total reducing power are shown in Table 5.

[0085] Table 5: ACE inhibitory activity and antioxidant activity of protein peptides prepared in Comparative Example 1 at different concentrations

[0086]

[0087] Regarding the ACE inhibition rate, the changes in pH during the alkaline protease hydrolysis in Comparative Example 1, Example 1 and Example 2 have an effect on the ACE inhibition rate (5 mg / mL) of the finally prepared peptide as shown in Table 6.

[0088] Table 6: Effect of alkaline protease hydrolysis pH on ACE inhibition rate of protein peptides

[0089]

[0090] As can be seen from the above table, the ACE inhibition of the hydrolysis product of alkaline protease alone increases first and then decreases with the increase of pH, and the inhibition rate is highest when the pH is 9.5. On this basis, the ACE inhibition rate of the hydrolysis product after the first step of hydrolysis by alkaline protease at pH 10.5 and the secondary hydrolysis by pepsin is the highest, which is more than 1 times higher than that of the hydrolysis product of alkaline protease alone.

[0091] Comparative Example 2

[0092] On the basis of Example 1, pepsin was used for the second step hydrolysis, and the hydrolysis time was extended to 30 minutes, and other parameters were consistent with Example 1. The ACE inhibition rate of the prepared protein peptide was 41.26%, which was seriously reduced compared with Example 1, and as the pepsin hydrolysis time was extended to more than 1 hour, the ACE inhibition rate of the prepared protein peptide was lower than that of the protein peptide prepared by single alkaline protease hydrolysis, and the ACE inhibition rate was 35.14%.

[0093] In addition, after the first step of hydrolysis with alkaline protease (pH 10.5), the second step of hydrolysis was directly carried out with common trypsin, neutral protease and flavor protease, respectively. The hydrolysis results were not satisfactory. The ACE inhibition rates of the prepared protein peptides were 40.26%, 33.68% and 39.71%, respectively. The ACE inhibition rate did not significantly improve the ACE inhibition effect compared with the single alkaline protease hydrolysis product. After the neutral protease hydrolysis, the ACE inhibition rate decreased.

[0094] Example 3

[0095] A method for preparing ACE inhibitory peptide from tussah pupa is carried out according to the following steps:

[0096] (1) Using tussah pupa protein powder as a raw material, adding a buffer solution with a pH of 10.5 to prepare a protein solution with a concentration of 3.5 wt.%, adding alkaline protease with an enzyme activity of 250 U / mg to make its concentration reach 6.5 wt.%, the hydrolysis temperature is 50° C., and the first step of hydrolysis is performed for 4 h;

[0097] (2) adding sodium sulfate to the enzymatic hydrolyzate obtained in the first step of hydrolysis at a concentration of 0.4 wt.%, heating to 40° C., and keeping the temperature for 30 min;

[0098] (3) adjusting the pH of the enzymatic hydrolyzate after treatment in step (2) to 1.5, adding pepsin with an enzyme activity of 30 U / mg at a concentration of 3.5 wt.%, and performing a second step of hydrolysis at 42° C. for 20 min;

[0099] (4) The hydrolyzed enzymatic solution was centrifuged at 1000 rpm for 15 min, the supernatant was taken, and ultrafiltration was performed using 10 kDa and 3 kDa filter membranes to obtain tussah pupa protein peptide solutions with molecular weights greater than 10 kDa, 10 kDa-3 kDa, and less than 3 kDa, which were then lyophilized to obtain tussah pupa ACE inhibitory peptide powders with different molecular weights.

[0100] The ACE inhibition rate of the tussah chrysalis ACE inhibitory peptide powder prepared in this example is

[0101] Example 4

[0102] A method for preparing ACE inhibitory peptide from tussah pupa, characterized by following the steps:

[0103] (1) Using tussah pupa protein powder as a raw material, adding a buffer solution with a pH of 10.5 to prepare a protein solution with a concentration of 3.5 wt.%, adding alkaline protease with an enzyme activity of 250 U / mg to make its concentration reach 6.5 wt.%, the hydrolysis temperature is 50° C., and the first step of hydrolysis is performed for 4 h;

[0104] (2) adding sodium sulfate to the enzymatic hydrolyzate obtained in the first step of hydrolysis at a concentration of 0.8 wt.%, heating to 50° C., and keeping the temperature for 60 min;

[0105] (3) adjusting the pH of the enzymatic hydrolyzate after treatment in step (2) to 3.5, adding pepsin with an enzyme activity of 30 U / mg at a concentration of 1 wt.%, and performing a second step of hydrolysis at 35° C. for 10 min;

[0106] (4) The hydrolyzed enzymatic solution was centrifuged at 1000 rpm for 15 min, the supernatant was taken, and ultrafiltration was performed using 10 kDa and 3 kDa filter membranes to obtain tussah pupa protein peptide solutions with molecular weights greater than 10 kDa, 10 kDa-3 kDa, and less than 3 kDa, which were then lyophilized to obtain tussah pupa ACE inhibitory peptide powders with different molecular weights.

Claims

1. A method for preparing an ACE inhibitory protein peptide from tussah pupa, characterized in that: The protein solution is prepared by adding a buffer solution with a pH value of 10.5 to tussah pupa protein powder as a raw material, firstly using alkaline protease for the first step of hydrolysis, and then using pepsin for the second step of hydrolysis, wherein the concentration of the protein solution in the first step of hydrolysis is 3.5wt.%, alkaline protease with an enzyme activity of 250U / mg is added at a concentration of 6.5wt.%, the hydrolysis temperature is 50°C, and ultrapure water is added to perform constant temperature hydrolysis for 4h, and the second step of hydrolysis is to adjust the pH value of the enzymatic hydrolyzate after the first step of hydrolysis to 1.5-3.5, add pepsin with an enzyme activity of 30U / mg at a concentration of 1wt.%-3.5wt.%, and hydrolyze at 35-42°C for 10-20min.

2. The method for preparing an ACE inhibitory protein peptide from tussah pupa according to claim 1, characterized in that: Before the second step of hydrolysis, sodium sulfate is added to the enzymatic hydrolyzate obtained in the first step of hydrolysis at a concentration of 0.4 wt.% to 0.8 wt.%, and the solution is heated to 40 to 50°C and kept warm for 30 to 60 minutes.

3. A method for preparing ACE inhibitory protein peptide from tussah pupa, characterized in that: Follow these steps: (1) Using tussah pupa protein powder as raw material, adding a buffer solution with a pH of 10.5 to prepare a protein solution with a concentration of 3.5 wt.%, adding alkaline protease with an enzyme activity of 250 U / mg to make its concentration reach 6.5 wt.%, the hydrolysis temperature is 50 ° C, and the first step of hydrolysis is carried out for 4 h; (2) adjusting the pH of the enzymatic hydrolyzate after the hydrolysis in step (1) to 1.5-3.5, adding pepsin with an enzyme activity of 30 U / mg at a concentration of 1 wt.%-3.5 wt.%, and performing the second step of hydrolysis at 35-42°C for 10-20 min; (3) The hydrolyzed enzymatic solution was centrifuged at 1000 rpm for 15 min, the supernatant was taken, and ultrafiltration was performed using a 3 kDa filter membrane to obtain a tussah pupa protein peptide solution with a molecular weight of less than 3 kDa, which was then lyophilized to obtain silkworm pupa ACE inhibitory peptide powder.

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