ACE inhibitory peptide from Pacific squid viscera and its preparation method and application

Abstract

The present invention provides an ACE inhibitory peptide from Pacific pleated squid viscera, a preparation method and an application thereof, and belongs to the technical field of marine bioactive peptides. The present invention uses Pacific pleated squid viscera to prepare a highly active ACE inhibitory peptide, and the obtained polypeptide has excellent ACE inhibitory activity. Through mass spectrometry identification, a total of 8 small molecule polypeptides were screened, and these 8 polypeptides have different degrees of ACE inhibitory activity. The ACE inhibitory peptide from Pacific pleated squid viscera prepared by the present invention has an ACE inhibitory ability that is several times or even dozens of times that of the currently confirmed ACE inhibitory ability of Pacific pleated squid visceral protein peptides, and has an excellent ACE inhibitory effect. The present invention makes high-value and precise use of the processing byproduct resource of Pacific pleated squid viscera, which can effectively improve resource utilization and industrial value, and also provides new ways and methods for the prevention and treatment of hypertension.

Description

Technical Field

[0001] The invention relates to the technical field of marine bioactive peptides, and in particular to an ACE inhibitory peptide of Pacific squid viscera mass and a preparation method and application thereof. Background Art

[0002] Hypertension is one of the common chronic diseases that have affected the health of Chinese residents in recent years. Its main clinical feature is the continuous increase in arterial blood pressure, which is an important risk factor for cardiovascular and cerebrovascular diseases such as stroke, coronary heart disease, and heart failure. In the absence of antihypertensive drugs, if the blood pressure values ​​measured on three different days are higher than normal, that is, systolic blood pressure ≥140mmHg or diastolic blood pressure ≥90mmHg, it is considered hypertension.

[0003] Angiotensin-I converting enzyme (ACE, EC 3.2.1.41) plays an important role in regulating blood pressure balance. It causes blood pressure to rise by converting angiotensin I into angiotensin II (vasoconstrictor), inactivating bradykinin with vasodilating effect, and promoting aldosterone secretion. The effect of treating hypertension can be achieved by inhibiting ACE activity. Currently, clinically used antihypertensive drugs such as chemically synthesized ACE inhibitors such as captopril and enalapril have obvious therapeutic effects, but long-term use can easily cause adverse reactions such as kidney damage, angioedema, and hyperkalemia. It is particularly urgent to find natural, safe and reliable ACE inhibitors.

[0004] Bioactive peptides have multiple physiological functions and can be divided into antioxidant peptides, immune active peptides, antihypertensive peptides, uric acid-lowering peptides, antimicrobial peptides, etc. Compared with chemically synthesized ACE inhibitors, food-derived polypeptide ACE inhibitors have the advantages of higher safety, stronger physiological activity, and better absorption, and can provide a new way to prevent and treat hypertension.

[0005] my country is one of the most important squid production, processing and consumption countries in the world. According to the "2023 National Fisheries Economic Statistical Bulletin", my country's cephalopod catch output in 2023 was 593,600 tons, accounting for 25.56% of the total catch of distant-water fisheries. In recent years, my country's annual squid processing volume has remained at 400,000 to 500,000 tons, and the industry output value has exceeded 10 billion yuan. The development quality of the squid industry affects the quality upgrade of my country's distant-water fishing industry and aquatic product processing industry. However, the squid currently caught is mainly used to process and produce primary processed products such as squid shreds, dried squid, and squid crisps, and the degree of deep processing and utilization is low. In addition, a large number of by-products generated in the production process of primary processed squid products, including visceral balls, skin, eyes, etc., account for about 25% of the total weight of the squid, and this part of the resources is usually simply processed into squid paste, squid oil and other products or directly used in the feed industry. It has not yet been processed and utilized most effectively, resulting in problems such as waste of resources and environmental pollution, which to a certain extent affects the high-quality development of the industry. Pacific Todarodes pacificus, also known as Pacific Swinhoe's squid and Japanese flying squid, is mainly distributed in the northwest Pacific Ocean. It is rich in resources and is the main economic squid species in my country. The visceral mass produced during the processing of Pacific Todarodes pacificus has a protein content of about 50% of the dry weight, and the amino acid composition meets the high-quality protein standards stipulated by the Food and Agriculture Organization of the United Nations / World Health Organization. It is a good raw material for the preparation of bioactive peptides. Scientifically utilizing the high-quality protein in the visceral mass of Pacific Todarodes pacificus to develop polypeptide ACE inhibitors can provide a new way to promote the comprehensive high-value utilization of squid resources, and can expand new ideas for the development of food-derived polypeptide antihypertensive products, which has important theoretical significance and application value. Summary of the invention

[0006] One of the purposes of the present invention is to provide Pacific squid visceral mass ACE inhibitory peptide, and another purpose is to provide a preparation method and specific application thereof to make up for the deficiencies of the prior art.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0008] A Pacific squid visceral mass ACE inhibitory peptide, the ACE inhibitory peptide comprising: GFAGDDAPR, or GTALP, or WCPSPD, or SVPTDV, or GVAVP, or LGGLP, or SVAVP, or SVIENP one of the above.

[0009] Among them, the specific sequence of GFAGDDAPR is SEQ No.1:

[0010] Gly-Phe-Ala-Gly-Asp-Asp-Ala-Pro-Arg;

[0011] The specific sequence of GTALP is SEQ No. 2: Gly-Thr-Ala-Leu-Pro;

[0012] The specific sequence of WCPSPD is SEQ No. 3: Trp-Cys-Pro-Ser-Pro-Asp;

[0013] The specific sequence of SVPTDV is SEQ No. 4: Ser-Val-Pro-Thr-Asp-Val;

[0014] The specific sequence of GVAVP is SEQ No. 5: Gly-Val-Ala-Val-Pro;

[0015] The specific sequence of LGGLP is SEQ No.6: Leu-Gly-Gly-Leu-Pro;

[0016] The specific sequence of SVAVP is SEQ No.7: Ser-Val-Ala-Val-Pro;

[0017] The specific sequence of SVIENP is SEQ No. 8: Ser-Val-Ile-Glu-Asn-Pro.

[0018] Preferably, a Pacific squid visceral mass ACE inhibitory peptide comprises: GFAGDDAPR, or GTALP, or WCPSPD, or SVPTDV.

[0019] A method for preparing an ACE inhibitory peptide from Pacific squid viscera, comprising the following steps:

[0020] (1) Raw material pretreatment;

[0021] (2) Enzymatic hydrolysis;

[0022] (3) High temperature inactivation of enzymes;

[0023] (4) solid-liquid separation to obtain the enzymatic hydrolysis supernatant of Pacific squid viscera;

[0024] (5) desalting the supernatant of the enzymatic hydrolysis of the Pacific squid visceral mass obtained in step (4), spray drying or freeze drying the treated solution to obtain a dry powder of the enzymatic hydrolysis of the Pacific squid visceral mass, i.e., a Pacific squid visceral mass ACE inhibitory peptide mixture 1;

[0025] (6) The Pacific squid visceral mass ACE inhibitory peptide mixture 1 obtained in step (5) is reconstituted with deionized water and then purified, and the retentate is collected by ultrafiltration, and spray-dried or freeze-dried to obtain the preliminarily purified Pacific squid visceral mass ACE inhibitory peptide mixture 2;

[0026] (7) The Pacific squid visceral mass ACE inhibitory peptide mixture 2 obtained in step (6) is re-dissolved in deionized water and purified again, gel filtration chromatography is used to collect the chromatographic separation components, and spray-drying or freeze-drying is performed to obtain the Pacific squid visceral mass ACE inhibitory peptide mixture 3;

[0027] (8) The Pacific squid visceral mass ACE inhibitory peptide mixture 3 obtained in the step (7) was identified by liquid chromatography-mass spectrometry, and finally 8 Pacific squid visceral mass ACE inhibitory peptides were obtained: GFAGDDAPR, GTALP, WCPSPD, SVPTDV, GVAVP, LGGLP, SVAVP, and SVIENP.

[0028] Preferably, in the step (1), the Pacific squid viscera is added to a buffer solution at a material-liquid ratio of 1:4 to 1:8 g / mL and mixed, and the Pacific squid viscera is fully homogenized by a high-speed shear homogenization method for 1 to 15 minutes; preferably, the material-liquid ratio is 1:6 and the processing time is 5 minutes.

[0029] Preferably, the step (2) is performed by enzymolysis, the pH of the buffer solution is adjusted to 2.0-12.0, 200-8000U / g of protease is added according to the mass of the substrate to start the enzymolysis reaction, the enzymolysis time is 1-6h, and the enzymolysis temperature is 25-75°C; the protease is selected from a mixture of one or more of alkaline protease, trypsin, pepsin, flavor protease, and neutral protease; alkaline protease is preferred, the addition amount is 4000U / g, the enzymolysis pH is 7.5, the enzymolysis time is 3.5h, and the enzymolysis temperature is 55°C.

[0030] Preferably, the enzyme inactivation temperature in step (3) is 90-100°C, and the enzyme inactivation time is 10-20 min; preferably, the enzyme inactivation temperature is 100°C, and the enzyme inactivation time is 20 min.

[0031] Preferably, the centrifugal speed in step (4) is 3000-8000 r / min, and the centrifugal time is 10-30 min; preferably, the centrifugal speed is 7500 r / min, and the centrifugal time is 20 min.

[0032] Preferably, the desalination method in step (5) is: desalting the supernatant of the enzymatic hydrolysis of the Pacific squid viscera mass using a nanofiltration membrane with a molecular weight cutoff of 200Da, with a treatment pressure of 0.6 to 1.4MPa and a cycle treatment of 1 to 5 times; preferably, the treatment pressure is 1.2MPa and the cycle treatment is 3 times.

[0033] Preferably, the ultrafiltration conditions in the step (6) are: the Pacific squid visceral mass ACE inhibitory peptide mixture 1 is re-dissolved with deionized water to a solution with a mass concentration of 0.5-10 g / L, and an ultrafiltration membrane with a molecular weight cutoff of 1 KDa, 3 KDa or 5 KDa is used for ultrafiltration treatment, with a treatment pressure of 0.5-1.5 MPa and a treatment volume of 0.5-10 L / h; preferably, the solution mass concentration is 5 g / L, the ultrafiltration membrane molecular weight cutoff is 1 KDa, the treatment pressure is 1.0 MPa, and the treatment volume is 3 L / h.

[0034] Preferably, the gel filtration chromatography conditions in the step (7) are as follows: the Pacific squid visceral mass ACE inhibitory peptide mixture 2 is reconstituted with deionized water to a solution with a mass concentration of 10 to 100 mg / mL, and purified using the AKTA protein separation and purification system through the dextran gel Sephadex G-15, with a sample volume of 1 to 10 mL, an eluent of deionized water, and an elution flow rate of 0.2 to 1.0 mL / min; preferably, the solution mass concentration is 30 mg / mL, the sample volume is 5 mL, and the elution flow rate is 0.5 mL / min.

[0035] Preferably, the LC-MS method in step (8) specifically comprises the following steps:

[0036] 1) Sample pretreatment: The ACE inhibitory peptide 3 from Pacific squid viscera was desalted, vacuum freeze-dried, and then re-dissolved and filtered through a 0.45 μm microporous filter membrane for detection;

[0037] 2) Instrument detection conditions: After injection into a C18 capillary capture column (100 μm×20 mm, 5 μm, Dr. Maisch GmbH), the sample was passed through a C18 separation column (75 μm×150 mm, 3 μm, Dr. Maisch GmbH) for gradient separation; mobile phase A was 0.1% (v / v) formic acid solution, and mobile phase B was 80% (v / v) acetonitrile solution (containing 0.1% formic acid); the chromatographic column was fully equilibrated with 92% mobile phase A and gradient elution was performed, with a volume ratio of A to B of 92:8 at 0 min, a volume ratio of A to B of 76:28 at 98 min, a volume ratio of A to B of 63:37 at 113 min, a volume ratio of A to B of 0:100 at 117 min, a volume ratio of A to B of 0:100 at 120 min, and a volume ratio of A to B of 0:100 at 125 min. The volume ratio of A to B was 92:8, and the elution program ended after 130 min; the flow rate was 300 nL / min; the injection volume was 1 μL; and the detection mode was positive ion mode.

[0038] 3) The mass-to-charge ratios of the peptide and its fragments were collected according to the following method: 20 fragment spectra (MS2scan) were collected after each full scan; scanning range: 400-1800 m / z; primary resolution: 60000@m / z200, secondary resolution: 150000@m / z200; collision energy CE28 eV.

[0039] The application of the Pacific squid visceral mass ACE inhibitory peptide in the preparation of products inhibiting angiotensin converting enzyme (ACE); the products include ordinary food, health food, medicine and the like.

[0040] The application of the Pacific squid visceral mass ACE inhibitory peptide in the preparation of products with blood pressure lowering efficacy; the products include ordinary foods, health foods, medicines, etc.

[0041] Advantages and beneficial effects of the present invention:

[0042] The present invention uses Pacific squid viscera to prepare ACE inhibitory peptides, and the obtained polypeptides have excellent ACE inhibitory activity. Through mass spectrometry identification, a total of 8 small molecule polypeptides were screened: GFAGDDAPR, GTALP, WCPSPD, SVPTDV, GVAVP, LGGLP, SVAVP and SVIENP, and these 8 polypeptides have different degrees of ACE inhibitory activity.

[0043] The ACE inhibitory peptide of Pacific pleated squid visceral mass prepared by the present invention has an ACE inhibitory ability that is several times or even dozens of times that of the currently confirmed ACE inhibitory ability of Pacific pleated squid visceral mass protein peptide, and has an excellent ACE inhibitory effect. The ACE inhibitory peptide of Pacific pleated squid visceral mass can be applied to the fields of ordinary food, health food, medicine, etc. The present invention makes high-value and precise use of the processing by-product resource of Pacific pleated squid visceral mass, which can effectively improve resource utilization and industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Example 11 Gel filtration chromatography separation profile.

[0045] Figure 2 LC-MS total ion chromatogram of the ACE inhibitory peptide from the visceral mass of Pacific squid in Example 12.

[0046] Figure 3 Secondary mass spectrum of the ACE inhibitory peptide with the amino acid sequence of GFAGDDAPR.

[0047] Figure 4 Secondary mass spectrum of the ACE inhibitory peptide with the amino acid sequence of GTALP.

[0048] Figure 5Secondary mass spectrum of the ACE inhibitory peptide with the amino acid sequence of WCPSPD.

[0049] Figure 6 Secondary mass spectrum of the ACE inhibitory peptide with the amino acid sequence of SVPTDV.

[0050] Figure 7 Secondary mass spectrum of the ACE inhibitory peptide with the amino acid sequence of GVAVP.

[0051] Figure 8 Secondary mass spectrum of the ACE inhibitory peptide with the amino acid sequence of LGGLP.

[0052] Fig. 9 Secondary mass spectrum of the ACE inhibitory peptide with the amino acid sequence of SVAVP.

[0053] Fig.10 Secondary mass spectrum of the ACE inhibitory peptide with the amino acid sequence of SVIENP.

[0054] Fig.11 Process flow chart for the preparation of ACE inhibitory peptides from Pacific squid viscera. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with embodiments. The specific examples described herein are only for explaining the present invention and are not limited thereto.

[0056] In the following examples, the experimental method for determining the ACE inhibition rate of each sample is as follows:

[0057] (1) Solution preparation

[0058] 0.1mol / L borate buffer (pH 8.3, containing 0.3mol / L sodium chloride): weigh 1.730g of boric acid, heat to dissolve, and dilute to 100mL for use; weigh 1.907g of borax (Na2B4O7·10H2O), heat to dissolve, and dilute to 100mL for use; measure 32.5mL of the above boric acid solution and 17.5mL of the above borax solution, mix well, adjust the pH to 8.3, and then add 1.753g of sodium chloride and dilute to 100mL.

[0059] 0.125U / mL ACE enzyme solution: Take 0.5U ACE enzyme and dilute to 4mL with deionized water.

[0060] 0.25mmol / L N-[3-(2-furyl)acryloyl]-L-phenylalanamide-glycine-glycine solution (FAPGG): weigh 5.0mg FAPGG powder, dissolve it with the above 0.1mol / L borate buffer (pH 8.3, containing 0.3mol / L sodium chloride) and make up to 50mL.

[0061] (2) Experimental methods

[0062] Pipette 550 μL of sample solution, add 275 μL of the above FAPGG solution, mix, then add 275 μL of the above ACE enzyme solution, measure the absorbance at 340 nm immediately after mixing, then incubate at 37°C for 30 min, and measure the absorbance at 340 nm again after the reaction is completed; use deionized water instead of the sample solution as a control.

[0063] (3) Calculation formula

[0064] The ACE inhibitory activity was calculated as follows:

[0065]

[0066] In the formula: A1 represents the initial absorbance value of the sample group; A2 represents the absorbance value of the sample group after 30 minutes of reaction; A3 represents the initial absorbance value of the control group; A4 represents the absorbance value of the control group after 30 minutes of reaction.

[0067] Embodiment 1:

[0068] A method for preparing an ACE inhibitory peptide from Pacific squid viscera, the method comprising the following steps:

[0069] (1) Raw material pretreatment: add an appropriate amount of Pacific squid viscera to phosphate buffer at a material-liquid ratio of 1:6, and homogenize at high speed for 5 minutes;

[0070] (2) Enzymatic hydrolysis: adjust the pH to 7.5, add alkaline protease to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000U / g, the enzymatic hydrolysis temperature is 55°C, and the enzymatic hydrolysis reaction is carried out for 3.5h;

[0071] (3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

[0072] (4) solid-liquid separation: centrifugation at 7500 r / min for 20 min to obtain the enzymatic hydrolysis supernatant of Pacific squid viscera;

[0073] (5) Desalination: The supernatant of the enzymatic hydrolysis of the Pacific squid viscera was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle treatment of 3 times. The treated liquid was spray-dried or freeze-dried to obtain a Pacific squid viscera ACE inhibitory peptide mixture 1.

[0074] Embodiment 2:

[0075] A method for preparing an ACE inhibitory peptide from Pacific squid viscera, the method comprising the following steps:

[0076] (1) Raw material pretreatment: add an appropriate amount of Pacific squid viscera to phosphate buffer at a material-liquid ratio of 1:6, and homogenize at high speed for 5 minutes;

[0077] (2) Enzymatic hydrolysis: adjust the pH to 8.0, add trypsin to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000U / g, the enzymatic hydrolysis temperature is 37°C, and the enzymatic hydrolysis is carried out for 3.5h;

[0078] (3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

[0079] (4) solid-liquid separation: centrifugation at 7500 r / min for 20 min to obtain the enzymatic hydrolysis supernatant of Pacific squid viscera;

[0080] (5) Desalination: The supernatant of the enzymatic hydrolysis of the Pacific squid viscera was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle treatment of 3 times. The treated liquid was spray-dried or freeze-dried to obtain a Pacific squid viscera ACE inhibitory peptide mixture 1.

[0081] Embodiment 3:

[0082] A method for preparing an ACE inhibitory peptide from Pacific squid viscera, the method comprising the following steps:

[0083] (1) Raw material pretreatment: add an appropriate amount of Pacific squid viscera to phosphate buffer at a material-liquid ratio of 1:6, and homogenize at high speed for 5 minutes;

[0084] (2) Enzymatic hydrolysis: adjust the pH to 2.5, add pepsin to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000 U / g, the enzymatic hydrolysis temperature is 40°C, and the enzymatic hydrolysis reaction is carried out for 3.5 h;

[0085] (3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

[0086] (4) solid-liquid separation: centrifugation at 7500 r / min for 20 min to obtain the enzymatic hydrolysis supernatant of Pacific squid viscera;

[0087] (5) Desalination: The supernatant of the enzymatic hydrolysis of the Pacific squid viscera was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle treatment of 3 times. The treated liquid was spray-dried or freeze-dried to obtain a Pacific squid viscera ACE inhibitory peptide mixture 1.

[0088] Embodiment 4:

[0089] A method for preparing an ACE inhibitory peptide from Pacific squid viscera, the method comprising the following steps:

[0090] (1) Raw material pretreatment: add an appropriate amount of Pacific squid viscera to phosphate buffer at a material-liquid ratio of 1:6, and homogenize at high speed for 5 minutes;

[0091] (2) Enzymatic hydrolysis: adjust the pH to 7.0, add neutral protease to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000U / g, the enzymatic hydrolysis temperature is 45°C, and the enzymatic hydrolysis reaction is carried out for 3.5h;

[0092] (3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

[0093] (4) solid-liquid separation: centrifugation at 7500 r / min for 20 min to obtain the enzymatic hydrolysis supernatant of Pacific squid viscera;

[0094] (5) Desalination: The supernatant of the enzymatic hydrolysis of the Pacific squid viscera was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle treatment of 3 times. The treated liquid was spray-dried or freeze-dried to obtain a Pacific squid viscera ACE inhibitory peptide mixture 1.

[0095] Embodiment 5:

[0096] A method for preparing an ACE inhibitory peptide from Pacific squid viscera, the method comprising the following steps:

[0097] (1) Raw material pretreatment: add an appropriate amount of Pacific squid viscera to phosphate buffer at a material-liquid ratio of 1:6, and homogenize at high speed for 5 minutes;

[0098] (2) Enzymatic hydrolysis: adjust the pH to 7.0, add flavor protease to start the enzymatic hydrolysis reaction, the enzyme addition amount is 4000U / g, the enzymatic hydrolysis temperature is 50°C, and the enzymatic hydrolysis reaction is carried out for 3.5h;

[0099] (3) High temperature inactivation of enzymes: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is heated in a boiling water bath at 100°C for 20 min to inactivate the enzymes;

[0100] (4) solid-liquid separation: centrifugation at 7500 r / min for 20 min to obtain the enzymatic hydrolysis supernatant of Pacific squid viscera;

[0101] (5) Desalination: The supernatant of the enzymatic hydrolysis of the Pacific squid viscera was desalted using a nanofiltration membrane with a molecular weight cutoff of 200 Da, with a treatment pressure of 1.2 MPa and a cycle treatment of 3 times. The treated liquid was spray-dried or freeze-dried to obtain a Pacific squid viscera ACE inhibitory peptide mixture 1.

[0102] Embodiment 6:

[0103] In this example, the activity of the mixture of ACE inhibitory peptides of Pacific pleated squid viscera obtained in Examples 1 to 5 was determined by spectrophotometry, and the results are shown in Table 1. The results show that the product obtained by alkaline protease hydrolysis in Example 1 has significantly higher ACE inhibitory activity than the hydrolysis products obtained by other conditions in Examples 2 to 5; using alkaline protease to hydrolyze Pacific pleated squid viscera is more conducive to the preparation of highly active Pacific pleated squid viscera ACE inhibitory peptides.

[0104] Table 1 ACE inhibition rate of the mixture of ACE inhibitory peptides of Pacific squid viscera obtained in Examples 1 to 5

[0105]

[0106] Embodiment 7:

[0107] A method for preparing an ACE inhibitory peptide from the visceral mass of Pacific squid, the preparation method comprises further purifying the ACE inhibitory peptide mixture 1 from the visceral mass of Pacific squid in Example 1, comprising the following steps:

[0108] (1) The Pacific squid visceral mass ACE inhibitory peptide mixture obtained in Example 1 was redissolved in deionized water to a solution with a mass concentration of 5 g / L;

[0109] (2) The ACE inhibitory peptide mixture solution of Pacific squid viscera was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1 KDa, with a treatment pressure of 1.0 MPa and a treatment volume of 3 L / h to obtain retentates less than 1 KDa and greater than 1 KDa, respectively. The retentates less than 1 KDa were collected and spray-dried or freeze-dried to obtain the preliminarily purified Pacific squid visceral mass ACE inhibitory peptide mixture 2.

[0110] Embodiment 8:

[0111] A method for preparing an ACE inhibitory peptide from the visceral mass of Pacific squid, the preparation method comprises further purifying the ACE inhibitory peptide mixture 1 from the visceral mass of Pacific squid in Example 1, comprising the following steps:

[0112] (1) The Pacific squid visceral mass ACE inhibitory peptide mixture obtained in Example 1 was redissolved in deionized water to a solution with a mass concentration of 5 g / L;

[0113] (2) The ACE inhibitory peptide mixture solution of Pacific squid viscera was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 KDa, with a treatment pressure of 1.0 MPa and a treatment volume of 3 L / h to obtain retentates less than 3 KDa and greater than 3 KDa, respectively. The retentates less than 3 KDa were collected and spray-dried or freeze-dried to obtain the preliminarily purified Pacific squid visceral mass ACE inhibitory peptide mixture 2.

[0114] Embodiment 9:

[0115] A method for preparing an ACE inhibitory peptide from the visceral mass of Pacific squid, the preparation method comprises further purifying the ACE inhibitory peptide mixture 1 from the visceral mass of Pacific squid in Example 1, comprising the following steps:

[0116] (1) The Pacific squid visceral mass ACE inhibitory peptide mixture obtained in Example 1 was redissolved in deionized water to a solution with a mass concentration of 5 g / L;

[0117] (2) The ACE inhibitory peptide mixture solution of Pacific squid viscera was ultrafiltrated using an ultrafiltration membrane with a molecular weight cutoff of 5 KDa, with a treatment pressure of 1.0 MPa and a treatment volume of 3 L / h to obtain retentates less than 5 KDa and greater than 5 KDa, respectively. The retentates less than 5 KDa were collected and spray-dried or freeze-dried to obtain the preliminarily purified Pacific squid visceral mass ACE inhibitory peptide mixture 2.

[0118] Embodiment 10:

[0119] In this example, the activity of the mixture of ACE inhibitory peptides of the Pacific pleated squid viscera obtained in Example 1 and Examples 7, 8, and 9 was determined by spectrophotometry, and the results are shown in Table 2. The results show that the product obtained by first enzymatic hydrolysis with alkaline protease and further ultrafiltration purification with an ultrafiltration membrane having a molecular weight cutoff of 1 KDa in Example 7 has a significantly higher ACE inhibitory activity than the products obtained in Example 1 and Examples 8 and 9; enzymatic hydrolysis of the Pacific pleated squid viscera with alkaline protease and ultrafiltration separation and purification of the enzymatic hydrolysis product to obtain a component with a relative molecular mass of less than 1 KDa is more conducive to the preparation of highly active ACE inhibitory peptides of the Pacific pleated squid viscera.

[0120] Table 2 ACE inhibition rate of the mixture of ACE inhibitory peptides of Pacific squid viscera obtained in Examples 1, 7, 8 and 9

[0121]

[0122] The ACE inhibitory peptide of Pacific squid viscera mass prepared in the above embodiment has excellent activity effect, can be used as common food, health food or pharmaceutical raw material for preventing and treating hypertension, effectively improves the utilization rate and added value of Pacific squid viscera mass as a processing by-product resource, and uses protease enzymatic hydrolysis method to obtain active peptide powder, which is economical, environmentally friendly, highly safe and has good application prospects.

[0123] Embodiment 11:

[0124] A method for preparing an ACE inhibitory peptide from the visceral mass of Pacific squid, the preparation method comprising further purifying the ACE inhibitory peptide mixture 2 from the visceral mass of Pacific squid in Example 7, comprising the following steps:

[0125] (1) The Pacific squid visceral mass ACE inhibitory peptide mixture obtained in Example 7 was re-dissolved in deionized water to a solution with a mass concentration of 30 mg / mL;

[0126] (2) 5 mL of the ACE inhibitory peptide mixture solution of Pacific squid viscera was loaded into the AKTA protein separation and purification system, and separated and purified by Sephadex G-15 chromatography, with deionized water as the eluent and an elution flow rate of 0.5 mL / min, to obtain five components A, B, C, D, and E, respectively ( Figure 1 ); the activity of components A, B, C, D, and E was determined by spectrophotometry, and the results are shown in Table 3. The target high ACE inhibitory activity components B and C were collected and spray-dried or freeze-dried to obtain Pacific squid viscera ACE inhibitory peptide mixture 3.

[0127] Table 3 ACE inhibition rate of each component obtained by gel filtration chromatography separation in Example 7 and Example 11

[0128]

[0129] The measurement results show that in Example 11, the products B and C obtained by first using alkaline protease for hydrolysis, then using ultrafiltration to separate and recover components with a relative molecular mass of less than 1 KDa, and further using Sephadex G-15 chromatography to separate, have significantly higher ACE inhibitory activity than the product obtained in Example 7; using alkaline protease to hydrolyze the Pacific squid viscera, and then purifying the hydrolysis products by ultrafiltration and gel filtration chromatography in turn, is more conducive to the preparation of highly active Pacific squid visceral mass ACE inhibitory peptides.

[0130] Embodiment 12:

[0131] The ACE inhibitory peptide 3 from Pacific squid viscera was identified by liquid chromatography-mass spectrometry, comprising the following steps:

[0132] (1) Sample pretreatment: The ACE inhibitory peptide from the Pacific squid viscera obtained in Example 11 was desalted using a Waters SEP-PAK C18 solid phase extraction column, vacuum freeze-dried, re-dissolved in 0.1% (v / v) formic acid solution, and filtered through a 0.45 μm microporous filter membrane for detection.

[0133] (2) Instrument detection conditions: the sample was injected into a C18 capillary capture column (100 μm × 20 mm, 5 μm, Dr. Maisch GmbH) and then passed through a C18 separation column (75 μm × 150 mm, 3 μm, Dr. Maisch GmbH) for gradient separation; the mobile phase A was 0.1% (v / v) formic acid solution, and the mobile phase B was 80% (v / v) acetonitrile solution (containing 0.1% formic acid); the chromatographic column was fully equilibrated with 92% mobile phase A and gradient elution was performed, with the volume ratio of A to B being 92:8 at 0 min, 76:28 at 98 min, 63:37 at 113 min, 0:100 at 117 min, 0:100 at 120 min, and 0:100 at 125 min. The volume ratio of A to B was 92:8, and the elution program ended after 130 min; the flow rate was 300 nL / min; the injection volume was 1 μL; and the detection mode was positive ion mode.

[0134] (3) The mass-to-charge ratios of the peptide and its fragments were collected according to the following method: 20 fragment spectra (MS2scan) were collected after each full scan; scanning range: 400-1800 m / z; primary resolution: 60000@m / z200, secondary resolution: 150000@m / z200; collision energy CE28 eV.

[0135] (4) The software Proteome Discoverer 2.5 was used to search and analyze the Uniprot protein database. The parameter settings are shown in Table 4, and finally the peptide identification results were obtained.

[0136] Table 4 Proteome Discoverer analysis parameter settings

[0137]

[0138] The total ion current of ACE inhibitory peptide in Pacific squid viscera is shown in the following figure. Figure 2 After mass spectrometry data retrieval, FDR≤0.01 was used as the screening standard for credible protein sequences. A total of 10,249 peptides and 52 protein sequences were identified, of which 8 peptide sequences with high credibility were GFAGDDAPR, GTALP, WCPSPD, SVPTDV, GVAVP, LGGLP, SVAVP and SVIENP. The secondary mass spectra of the 8 peptide chains are shown in Figures 3 to 10 shown.

[0139] Embodiment 13:

[0140] The 8 polypeptides obtained in Example 12 were subjected to solid phase synthesis and activity verification, comprising the following steps:

[0141] (1) The peptides GFAGDDAPR, GTALP, WCPSPD, SVPTDV, GVAVP, LGGLP, SVAVP, and SVIENP were synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. using the Fmoc solid-phase synthesis method.

[0142] (2) The in vitro ACE inhibitory activity of the synthetic peptides was determined by spectrophotometry, and the results are shown in Table 5. The results showed that among the 8 peptides, GFAGDDAPR, GTALP, WCPSPD and SVPTDV had higher ACE inhibitory activity, among which the peptide GTALP showed the highest ACE inhibitory effect.

[0143] Table 5 Peptide information and ACE inhibition rate of 8 polypeptides in Example 12

[0144]

[0145] Furthermore, the most active polypeptide GTALP was prepared into a solution with a mass concentration of 0.01-0.25 mg / mL, and the ACE inhibition rate of polypeptide GTALP at different concentrations was determined by spectrophotometry to calculate its IC 50 The value is 0.015 mg / mL. It is worth noting that the ACE inhibitory peptide mixture of Pacific squid viscera prepared in Example 1 has an IC 50 The value is 0.332 mg / mL, that is, the ACE inhibitory activity of the polypeptide prepared by the present invention can be increased by 22.13 times, the technical improvement effect is obvious, and the product has good application prospects.

[0146] The overall preparation process of the above-mentioned embodiments 1 to 13 is as follows Fig.11 shown.

[0147] The Pacific squid viscera mass ACE inhibitory peptide mixture and the eight ACE inhibitory peptides prepared in the above embodiments all have excellent activity effects and can be used as common food, health food or pharmaceutical raw materials for preventing and treating hypertension, effectively improving the utilization rate and added value of the Pacific squid viscera mass processing by-product resource, and using the protease enzymatic hydrolysis method to obtain the active peptide powder, which is economical, environmentally friendly, highly safe and has good application prospects.

[0148] Embodiment 15:

[0149] The application of the ACE inhibitory peptide of Pacific squid viscera obtained in Examples 1 to 13 specifically includes:

[0150] The ACE inhibitory peptides of Pacific squid viscera obtained in Examples 1 to 13 can be used in the fields of ordinary food, health food, medicine, etc. for preventing and treating hypertension. The product preparations can be in the form of solid beverages, liquid beverages, capsules, compressed candies, gel candies, etc.

[0151] The embodiments described above are only preferred implementations of the present invention and do not constitute limitations on the scope of the claims. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are within the protection scope of the present invention.

Claims

1. An ACE inhibitory peptide from Pacific squid viscera, characterized in that: The amino acid sequence of the ACE inhibitory peptide is: GTALP, or WCPSPD, or SVPTDV.

2. The method for preparing the ACE inhibitory peptide from Pacific squid viscera according to claim 1, characterized in that: The following steps are involved: (1) Raw material pretreatment; (2) Enzymatic hydrolysis: alkaline protease was added to start the enzymatic hydrolysis reaction. The addition amount was 4000 U / g, the enzymatic hydrolysis pH was 7.5, the enzymatic hydrolysis time was 3.5 h, and the enzymatic hydrolysis temperature was 55°C. (3) High temperature inactivation of enzymes; (4) solid-liquid separation to obtain the enzymatic hydrolysis supernatant of Pacific squid viscera; (5) desalting the enzymatic hydrolysis supernatant of the Pacific squid visceral mass obtained in the step (4), spray drying or freeze drying the treated liquid to obtain a dry powder of the enzymatic hydrolysis liquid of the Pacific squid visceral mass, i.e., a Pacific squid visceral mass ACE inhibitory peptide mixture 1; the desalting treatment comprises desalting the enzymatic hydrolysis supernatant of the Pacific squid visceral mass using a nanofiltration membrane with a molecular weight cutoff of 200 Da, at a treatment pressure of 0.6-1.4 MPa, and cyclic treatment for 1-5 times; (6) The Pacific squid visceral mass ACE inhibitory peptide mixture 1 obtained in step (5) is reconstituted with deionized water and then purified, and the permeate is collected by ultrafiltration, and the filtrate is spray-dried or freeze-dried to obtain the preliminarily purified Pacific squid visceral mass ACE inhibitory peptide mixture 2; the ultrafiltration conditions are: the Pacific squid visceral mass ACE inhibitory peptide mixture 1 is reconstituted with deionized water to a solution with a mass concentration of 0.5-10 g / L, and an ultrafiltration membrane with a molecular weight cutoff of 1 KDa is used for ultrafiltration treatment, the treatment pressure is 0.5-1.5 MPa, and the treatment volume is 0.5-10 L / h; (7) The Pacific squid visceral mass ACE inhibitory peptide mixture 2 obtained in step (6) is re-dissolved in deionized water and then purified again, and the target high ACE inhibitory activity components are collected by gel filtration chromatography, and the Pacific squid visceral mass ACE inhibitory peptide mixture 3 is obtained by spray drying or freeze drying; the gel filtration chromatography conditions are: the Pacific squid visceral mass ACE inhibitory peptide mixture 2 is re-dissolved in deionized water to a solution with a mass concentration of 10-100 mg / mL, and purified by AKTA protein separation and purification system through Sephadex G-15, the sample volume is 1-10 mL, the eluent is deionized water, and the elution flow rate is 0.2-1.0 mL / min; (8) The Pacific squid visceral mass ACE inhibitory peptide mixture 3 obtained in step (7) is identified by liquid chromatography-mass spectrometry to finally obtain the Pacific squid visceral mass ACE inhibitory peptide GTALP, or WCPSPD, or SVPTDV.

3. The preparation method according to claim 2, characterized in that: The LC-MS method in step (8) specifically comprises the following steps: 1) Sample pretreatment: The ACE inhibitory peptide 3 from Pacific squid viscera was desalted, vacuum freeze-dried, and then re-dissolved and filtered through a 0.45 μm microporous filter membrane for detection; 2) Instrument detection conditions: After injection into the C18 capillary capture column, the sample was passed through the C18 separation column for gradient separation; mobile phase A was 0.1% (v / v) formic acid solution, and mobile phase B was 80% (v / v) acetonitrile solution containing 0.1% formic acid; the chromatographic column was fully equilibrated with 92% mobile phase A for gradient elution, with a volume ratio of A to B of 92:8 at 0 min, 76:28 at 98 min, 63:37 at 113 min, 0:100 at 117 min, 0:100 at 120 min, 92:8 at 125 min, and the elution program ended at 130 min; flow rate was 300 nL / min; injection volume was 1 μL; detection mode: positive ion mode; 3) The mass-to-charge ratios of peptides and peptide fragments were collected according to the following method: 20 fragmentation spectra were collected after each full scan; scanning range: 400-1800 m / z; primary resolution: 60000@m / z200, secondary resolution: 150000@m / z200; Collision energy CE28eV.

4. Use of the Pacific squid visceral mass ACE inhibitory peptide GTALP, or WCPSPD, or SVPTDV according to claim 1 in the preparation of blood pressure lowering efficacy drugs or auxiliary blood pressure lowering health foods.

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