Tilapia skin kidney-protective peptide, preparation method and application thereof

Abstract

The present invention discloses a tilapia skin kidney-protective peptide, its preparation method, and application, belonging to the technical field of polypeptides. The present invention extracts an active short peptide from tilapia skin. The active short peptide prepared by the separation method has high purity. Activity analysis shows that the active short peptide has good antioxidant effects and good kidney-protective function, expanding the economic value of tilapia skin.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and more particularly to a tilapia skin kidney protective peptide and a preparation method and application thereof. Background Art

[0002] Tilapia, a general term for fish of the genus Tilapia, is a euryhaline tropical fish. Tilapia is naturally distributed in freshwater and brackish waters of Africa and along the Atlantic coast of the Middle East, as far north as Israel and Jordan. Tilapia is highly adaptable to its environment, preferring higher water temperatures and a greater tolerance to hypoxia than other carp species. Its optimal pH range for survival is 7-8.

[0003] Nephroprotective peptides, a class of functional peptides with renal protective effects, have garnered widespread attention in recent years. Inflammation, oxidative stress, and fibrosis play key roles in the pathogenesis of kidney disease. Nephroprotective peptides can exert their protective effects through multiple mechanisms, providing new insights into the prevention and treatment of kidney disease. Summary of the Invention

[0004] In response to the technical problems existing in the prior art, the present invention provides a tilapia skin renal protective peptide and its preparation method and application. The present invention extracts an active short peptide from tilapia skin, and the active short peptide prepared by the separation method has high purity.

[0005] Specifically, the present invention first provides a method for preparing tilapia fish skin kidney protective peptide, comprising the following steps:

[0006] (1) Take 10-20g of tilapia skin and remove the residual fat tissue;

[0007] (2) adding 2-4 times the volume of ultrapure water to the tilapia skin obtained in step (1), performing high-temperature cooking for 30-60 min, and performing beating treatment after cooking;

[0008] (3) adding a composite protease with a volume ratio of 50-65% to step (2), stirring continuously for enzymolysis at 60-70° C., and centrifuging the supernatant to obtain a tilapia skin protein solution; wherein the composite protease is composed of a neutral protease and a pepsin as a combined enzyme, and the ratio of the enzyme activity of the two is (2-5): (3-7); after enzymolysis, performing an enzyme inactivation treatment at 100° C. and cooling the mixture to room temperature;

[0009] (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 5-15 min, and collect the supernatant; filter the resulting supernatant using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Da polypeptide molecules;

[0010] (5) The retained polypeptide molecules are sequenced using an Obitrap Fusion Lumos mass spectrometer.

[0011] Preferably, the preparation method of the tilapia fish skin kidney protective peptide comprises the following steps:

[0012] (1) Take 10 g of tilapia skin and remove the remaining fat tissue;

[0013] (2) adding 2 times the volume of ultrapure water to the tilapia skin obtained in step (1), performing high-temperature cooking for 30 min, and performing beating treatment after cooking;

[0014] (3) adding a 50% by volume composite protease to step (2), stirring continuously at 60° C. for enzymolysis, and centrifuging the supernatant to obtain a tilapia skin protein solution; wherein the composite protease is composed of a neutral protease and a pepsin as a combined enzyme, and the ratio of the active enzymes of the two is 2:3; after enzymolysis, performing an enzyme inactivation treatment at 100° C. and cooling the solution to room temperature;

[0015] (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 5 min, and collect the supernatant; and filter the resulting supernatant using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Da polypeptide molecules;

[0016] (5) The retained polypeptide molecules are sequenced using an Obitrap Fusion Lumos mass spectrometer.

[0017] Preferably, the preparation method of the tilapia fish skin kidney protective peptide comprises the following steps:

[0018] (1) Take 15g of tilapia skin and remove the residual fat tissue;

[0019] (2) adding 3 times the volume of ultrapure water to the tilapia skin obtained in step (1), performing high-temperature cooking for 45 minutes, and performing beating treatment after cooking;

[0020] (3) adding a 60% by volume composite protease to step (2), stirring continuously at 65° C. for enzymolysis, and centrifuging the supernatant to obtain a tilapia skin protein solution; wherein the composite protease is composed of a neutral protease and a pepsin as a combined enzyme, and the ratio of the enzyme activity of the two is 3:5; after enzymolysis, performing an enzyme inactivation treatment at 100° C. and cooling the solution to room temperature;

[0021] (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 8 min, and collect the supernatant; and filter the resulting supernatant using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Da polypeptide molecules;

[0022] (5) The retained polypeptide molecules are sequenced using an Obitrap Fusion Lumos mass spectrometer.

[0023] Preferably, the preparation method of the tilapia fish skin kidney protective peptide comprises the following steps:

[0024] (1) Take 20 g of tilapia skin and remove the remaining fat tissue;

[0025] (2) adding 4 times the volume of ultrapure water to the tilapia skin obtained in step (1), performing high-temperature cooking for 60 min, and performing beating treatment after cooking;

[0026] (3) adding a 70% by volume composite protease to step (2), stirring continuously at 60° C. for enzymolysis, and centrifuging the supernatant to obtain a tilapia skin protein solution; wherein the composite protease is composed of a neutral protease and a pepsin as a combined enzyme, and the ratio of the enzyme activity of the two is 5:7; after enzymolysis, performing an enzyme inactivation treatment at 100° C. and cooling the solution to room temperature;

[0027] (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 10 min, collect the supernatant, and filter the resulting supernatant using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Da polypeptide molecules;

[0028] (5) The retained polypeptide molecules are sequenced using an Obitrap Fusion Lumos mass spectrometer.

[0029] Preferably, it is identified that the method produces an active peptide, the sequence of which is shown in SEQ ID NO. 1, specifically the sequence: STERTDPERQDDNRDKKRRKKSHETESDESHKKC; the peptide is spray-dried or vacuum-freeze-dried for later use.

[0030] Another aspect of the present invention provides the use of tilapia skin kidney protective peptide in preparing antioxidant active drugs. The tilapia skin kidney protective peptide is the main active short peptide prepared by the above method.

[0031] Preferably, the sequence of the tilapia skin kidney protective peptide is shown as SEQ ID NO.1.

[0032] On the other hand, the present invention also provides the use of tilapia fish skin kidney protective peptide in the preparation of kidney protective agent medicine. The tilapia fish skin kidney protective peptide is the main active short peptide prepared by the above method.

[0033] Preferably, the sequence of the tilapia skin kidney protective peptide is shown as SEQ ID NO.1.

[0034] Beneficial effects of the invention: The present invention extracts an active short peptide from tilapia skin. The active short peptide prepared by the separation method has high purity. Activity analysis shows that the active short peptide has good antioxidant effect and good kidney protection function, thereby expanding the economic value of tilapia skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a graph showing the results of DPPH free radical scavenging activity.

[0036] Figure 2 This is the result graph of ABTS free radical scavenging activity.

[0037] Figure 3 This is a graph showing the results of hydroxyl radical scavenging activity.

[0038] Figure 4 This is a graph showing the results of an analysis of the proliferative activity of tilapia skin renal protective peptide on mouse glomerular mesangial cells. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0040] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0041] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0042] Example 1

[0043] A method for preparing tilapia skin kidney protective peptide comprises the following steps:

[0044] (1) Take 10 g of tilapia skin and remove the remaining fat tissue;

[0045] (2) adding 2 times the volume of ultrapure water to the tilapia skin obtained in step (1), performing high-temperature cooking for 30 min, and performing beating treatment after cooking;

[0046] (3) adding a 50% by volume composite protease to step (2), stirring continuously at 60° C. for enzymolysis, and centrifuging the supernatant to obtain a tilapia skin protein solution; wherein the composite protease is composed of a neutral protease and a pepsin as a combined enzyme, and the ratio of the active enzymes of the two is 2:3; after enzymolysis, performing an enzyme inactivation treatment at 100° C. and cooling the solution to room temperature;

[0047] (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 5 min, and collect the supernatant; and filter the resulting supernatant using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Da polypeptide molecules;

[0048] (5) The retained polypeptide molecules were sequenced using an Obitrap Fusion Lumos mass spectrometer. After identification and analysis, a total of one major active peptide was obtained, the sequence of which is shown in SEQ ID NO. 1. The peptide was spray-dried or freeze-dried under vacuum for later use. High-performance liquid chromatography analysis showed that the purity of the major active peptide prepared by the method described in this example reached 90.6%.

[0049] Example 2

[0050] A method for preparing tilapia skin kidney protective peptide comprises the following steps:

[0051] (1) Take 15g of tilapia skin and remove the residual fat tissue;

[0052] (2) adding 3 times the volume of ultrapure water to the tilapia skin obtained in step (1), performing high-temperature cooking for 30 min, and performing beating treatment after cooking;

[0053] (3) adding a 60% by volume composite protease to step (2), stirring continuously at 65° C. for enzymolysis, and centrifuging the supernatant to obtain a tilapia skin protein solution; wherein the composite protease is composed of a neutral protease and a pepsin as a combined enzyme, and the ratio of the enzyme activity of the two is 3:5; after enzymolysis, performing an enzyme inactivation treatment at 100° C. and cooling the solution to room temperature;

[0054] (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 8 min, and collect the supernatant; and filter the resulting supernatant using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Da polypeptide molecules;

[0055] (5) The retained polypeptide molecules were sequenced by Obitrap Fusion Lumos mass spectrometer, and a total of one major active peptide was obtained after identification and analysis, and the sequence was shown in SEQ ID NO.1.

[0056] Example 3

[0057] A method for preparing tilapia skin kidney protective peptide comprises the following steps:

[0058] (1) Take 20 g of tilapia skin and remove the remaining fat tissue;

[0059] (2) adding 4 times the volume of ultrapure water to the tilapia skin obtained in step (1), performing high-temperature cooking for 30 min, and performing beating treatment after cooking;

[0060] (3) adding a 70% by volume composite protease to step (2), stirring continuously at 60° C. for enzymolysis, and centrifuging the supernatant to obtain a tilapia skin protein solution; wherein the composite protease is composed of a neutral protease and a pepsin as a combined enzyme, and the ratio of the enzyme activity of the two is 5:7; after enzymolysis, performing an enzyme inactivation treatment at 100° C. and cooling the solution to room temperature;

[0061] (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 10 min, and collect the supernatant; filter the resulting supernatant using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Da polypeptide molecules;

[0062] (5) The retained polypeptide molecules were sequenced by Obitrap Fusion Lumos mass spectrometer, and a total of one major active peptide was obtained after identification and analysis, and the sequence was shown in SEQ ID NO.1.

[0063] Example 4

[0064] 1. DPPH free radical scavenging activity assay:

[0065] The tilapia skin renal protective peptide isolated in Example 1 was prepared with sterile distilled water to concentrations of 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL and 5 mg / mL, respectively, and stirred to fully dissolve.

[0066] Treatment group: Take 0.5 mL of tilapia skin kidney protective peptide aqueous solution, add 0.5 mL of 0.5 mol / L DPPH solution, mix well, react at room temperature in the dark for 30 minutes, centrifuge at 5000 rpm for 5 minutes, and measure the absorbance value A1 at a wavelength of 517 nm.

[0067] Blank group: Add 0.5 mL of 0.5 mol / L DPPH solution to 0.5 mL of deionized water. Mix thoroughly, incubate at room temperature in the dark for 30 minutes, centrifuge at 5000 rpm for 5 minutes, and measure the absorbance (A0) at 517 nm.

[0068] The calculation formula is shown in Formula I:

[0069] DPPH free radical scavenging rate (%) = (1-(A1÷A0)) × 100% (Formula I).

[0070] 2. ABTS free radical scavenging activity assay:

[0071] The tilapia skin renal protective peptide isolated in Example 1 was prepared with sterile distilled water to concentrations of 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL and 5 mg / mL, respectively, and stirred to fully dissolve.

[0072] Prepare a 7 mmol / L ABTS solution and a 2.45 mmol / L potassium persulfate solution in distilled water. Mix the ABTS and potassium persulfate solutions in a 1:1 volume ratio and store in a dark place at 23°C for 12 hours to form the ABTS cation radical stock solution. Store at 4°C. Before use, dilute the ABTS cation radical solution with distilled water to an absorbance of 0.70 ± 0.02 at 734 nm to obtain the ABTS cation radical working solution.

[0073] Treatment group: 100 μL of tilapia skin kidney protective peptide solution was added to 100 μL of ABTS cationic free radical working solution and mixed evenly. The mixture was reacted at 30°C for 10 min and the absorbance B1 was immediately measured at 734 nm.

[0074] Blank group: 100 μL of deionized water was added to 100 μL of ABTS cation radical working solution, mixed evenly, reacted at 30°C for 10 min, and the absorbance B0 was immediately measured at 734 nm.

[0075] The calculation formula is shown in Formula II:

[0076] ABTS free radical scavenging rate (%) = (1-(B1÷B0)) × 100% (Formula II).

[0077] 3. Hydroxyl radical scavenging activity assay:

[0078] The tilapia skin renal protective peptide isolated in Example 1 was prepared with sterile distilled water to concentrations of 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL and 5 mg / mL, respectively, and stirred to fully dissolve.

[0079] Prepare 4 mL of test sample solution by adding 1 mL of 2 mmol / L FeSO₄, 1 mL of 6 mmol / L salicylic acid, and 1 mL of 2 mmol / L H₂O₂ to 1 mL of tilapia skin nephroprotective peptide solution. Mix the solution thoroughly and let it stand at room temperature for 30 minutes. Add 200 µL of the test sample solution to a 96-well microtiter plate. Measure the absorbance at 510 nm using an automated microplate reader, and calculate the clearance rate. Three replicates were performed for each group.

[0080] The hydroxyl radical scavenging rate was calculated according to formula (III): A1 is the absorbance of the test solution containing tilapia skin nephroprotective peptide; A0 is the absorbance of the anhydrous ethanol sample solution without tilapia skin nephroprotective peptide; and A2 is the absorbance of distilled water without H2O2.

[0081] Hydroxyl radical scavenging rate (%) = [A0 − (A1 − A2)] / A0 × 100% (III);

[0082] The results are as follows Figure 1-3 As shown in the results, within the experimental concentration range, the ABTS free radical scavenging rate, DPPH free radical scavenging rate and hydroxyl free radical scavenging rate of tilapia skin kidney protective peptide increased with the increase of peptide concentration. When the concentration was 5 mg / ml, the ABTS free radical scavenging rate was 69.42%, the DPPH free radical scavenging rate reached 66.262%, and the hydroxyl free radical scavenging rate reached 40.244%.

[0083] Example 5

[0084] Analysis of the proliferative activity of tilapia skin nephroprotective peptide on mouse glomerular mesangial cells:

[0085] 1) Culture mouse glomerular mesangial cells CP-M057 at 1×10 5 Cells / well were seeded into a 96-well cell culture plate, 150 μL per well, and placed in an incubator for 12 h to allow for attachment;

[0086] 2) using cell culture medium to prepare a solution of the tilapia skin renal protective peptide represented by SEQ ID NO. 1 at a final concentration of 1-5 mg / mL;

[0087] 3) Add 50 μL of the tilapia skin renal protective peptide solution described in step 2) to the cell culture plate described in step 1) and incubate for 12 hours. Specifically, the treatment group is treated with the tilapia skin renal protective peptide solution described in SEQ ID NO. 1, the control group is not treated with the tilapia skin renal protective peptide solution, and the blank group is culture medium alone.

[0088] The results are as follows Figure 4 As shown in the figure, it can be seen that within the test concentration range, the inhibition rate of renal protective peptide on glomerular mesangial cells at 24 hours is positively correlated with the concentration of renal protective peptide. When the concentration of renal protective peptide is 5mg / ml, the inhibition rate at 24 hours is the highest, which is 45.68%.

[0089] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A tilapia fish skin kidney protective peptide, characterized in that The sequence of the tilapia fish skin kidney protective peptide is shown as SEQ ID NO.

1.

2. The use of tilapia fish skin kidney protective peptide in preparing products with antioxidant activity, characterized in that: The sequence of the tilapia fish skin kidney protective peptide is shown in SEQ ID NO.1.

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