Tilapia skin kidney protection peptide as well as preparation method and application thereof

Abstract

The invention discloses a tilapia skin kidney protection peptide as well as a preparation method and application thereof, and belongs to the technical field of polypeptides. The active oligopeptide is extracted from the tilapia skin, the active oligopeptide prepared by the separation method has high purity, activity analysis shows that the active oligopeptide has a good antioxidant effect and a good kidney protection function, and the economic value of the tilapia skin is expanded.

Description

Technical Field

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

[0002] Tilapia is a general term for fish of the genus Tilapia, which is a widely saline tropical fish. Tilapia is naturally distributed in fresh and brackish water areas of Africa, and in the brackish water sea areas along the coast of the Middle East Atlantic Ocean. It can reach as far north as Israel and Jordan. Tilapia has a strong adaptability to the environment, a relatively high suitable water temperature, and a stronger tolerance to low oxygen than general cyprinid fish. The optimal pH range for survival is 7-8.

[0003] Renal protection peptides are a class of functional peptide segments with renal protection effects and have received extensive attention in recent years. In the pathogenesis of kidney diseases, processes such as inflammation, oxidative stress, and fibrosis play key roles. Renal protection peptides can exert their protective effects through multiple mechanisms, providing new ideas for the prevention and treatment of kidney diseases. Summary of the Invention

[0004] Aiming at the technical problems existing in the prior art, the present invention provides a tilapia fish skin kidney protection peptide, a preparation method thereof, and an application thereof. The present invention extracts 1 kind of active short peptide from tilapia fish skin, and the active short peptide prepared by the separation method has a high purity.

[0005] Specifically, the present invention first provides a preparation method of a tilapia fish skin kidney protection peptide, comprising the following steps:

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

[0007] (2) Add 2-4 times the volume of ultrapure water to the tilapia fish skin obtained in step (1), carry out high-temperature steaming for 30-60 min, and perform pulping treatment after steaming;

[0008] (3) Add a composite protease with a volume ratio of 50-65% to step (2), continuously stir and enzymatically hydrolyze at 60-70 °C, and centrifuge to separate the supernatant to obtain a tilapia fish skin protein solution; wherein, the composite protease is composed of neutral protease and pepsin as a combined enzyme, and the enzyme activity dosage ratio of the two is (2-5) :(3-7); after enzymatic hydrolysis, perform enzyme inactivation treatment at 100 °C and cool it to room temperature;

[0009] (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 5-15 min, take the supernatant; and filter the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 3000-5000 Da;

[0010] (5) Sequence the retained polypeptide molecules using an Obitrap Fusion Lumos mass spectrometer.

[0011] Preferably, the method for preparing the tilapia fish skin kidney protection peptide comprises the following steps:

[0012] (1) Take 10 g of tilapia fish skin and remove the residual adipose tissue.

[0013] (2) Add 2-fold volume of ultrapure water to the tilapia fish skin obtained in step (1), perform high-temperature cooking for 30 min, and perform pulping treatment after cooking.

[0014] (3) Add compound protease with a volume ratio of 50% to step (2), continuously stir and enzymatically hydrolyze at 60 °C, and centrifuge to separate the supernatant to obtain tilapia fish skin protein solution; wherein, the compound protease is composed of neutral protease and pepsin as a combined enzyme, and the enzyme activity dosage ratio of the two is 2:3; after enzymatic hydrolysis, perform enzyme inactivation treatment at 100 °C and cool it to room temperature.

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

[0016] (5) Sequence the retained polypeptide molecules using an Obitrap Fusion Lumos mass spectrometer.

[0017] Preferably, the method for preparing the tilapia fish skin kidney protection peptide comprises the following steps:

[0018] (1) Take 15 g of tilapia fish skin and remove the residual adipose tissue.

[0019] (2) Add 3-fold volume of ultrapure water to the tilapia fish skin obtained in step (1), perform high-temperature cooking for 45 min, and perform pulping treatment after cooking.

[0020] (3) Add compound protease with a volume ratio of 60% to step (2), continuously stir and enzymatically hydrolyze at 65 °C, and centrifuge to separate the supernatant to obtain tilapia fish skin protein solution; wherein, the compound protease is composed of neutral protease and pepsin as a combined enzyme, and the enzyme activity dosage ratio of the two is 3:5; after enzymatic hydrolysis, perform enzyme inactivation treatment at 100 °C and cool it to room temperature.

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

[0022] (5) Sequence the retained polypeptide molecules using an Obitrap Fusion Lumos mass spectrometer.

[0023] Preferably, the method for preparing the tilapia fish skin kidney protection peptide comprises the following steps:

[0024] (1) Take 20 g of tilapia fish skin and remove the residual adipose tissue;

[0025] (2) Add 4 times the volume of ultrapure water to the tilapia fish skin obtained in step (1), perform high-temperature cooking for 60 min, and perform pulping after cooking;

[0026] (3) Add a 70% composite protease by volume to step (2), continuously stir and enzymatically hydrolyze at 60 °C, and centrifuge to separate the supernatant to obtain a tilapia fish skin protein solution; wherein, the composite protease is composed of neutral protease and pepsin as a combined enzyme, and the enzyme activity dosage ratio of the two is 5:7; after enzymatic hydrolysis, perform enzyme inactivation treatment at 100 °C and cool it to room temperature;

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

[0028] (5) Sequence the retained polypeptide molecules using an Obitrap Fusion Lumos mass spectrometer.

[0029] Preferably, after identification, 1 active peptide is prepared by the method, and the sequence is as shown in SEQ ID NO.1 in sequence, and the specific sequence is: STERTDPERQDDNRDKKRRKKSHETESDESHKKC; and it is reserved for use after spray drying or vacuum freeze-drying at low temperature.

[0030] Another aspect of the present invention provides the use of the tilapia fish skin kidney protection peptide in the preparation of an antioxidant active drug, and the tilapia fish skin kidney protection peptide is the main active short peptide prepared by the above method.

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

[0032] Another aspect of the present invention also provides the use of the tilapia fish skin kidney protection peptide in the preparation of a kidney protection agent drug, and the tilapia fish skin kidney protection peptide is the main active short peptide prepared by the above method.

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

[0034] Advantages of the invention: One active short peptide is extracted from tilapia fish skin. The active short peptide prepared by the separation method has a high purity. Through activity analysis, it is known that the active short peptide has good antioxidant effects and good kidney protection functions, expanding the economic value of tilapia fish skin. Description of the drawings

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

[0036] Figure 2 It is a graph of the results of ABTS free radical scavenging activity.

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

[0038] Figure 4 It is a graph of the results of the analysis I of the proliferation activity of the kidney protection peptide from tilapia fish skin on mouse glomerular mesangial cells. Detailed implementation manners

[0039] The present invention will be further described in detail below in conjunction with 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 efforts fall within the protection scope of the present invention.

[0041] In the embodiments 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 embodiments of the present invention, unless specifically specified, the technical means used are conventional means well-known to those skilled in the art.

[0042] Example 1

[0043] A preparation method of a kidney protection peptide from tilapia fish skin includes the following steps:

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

[0045] (2) Add 2 volumes of ultrapure water to the tilapia fish skin obtained in step (1), perform high-temperature steaming for 30 min, and perform pulping treatment after steaming;

[0046] (3) Add compound protease with a volume ratio of 50% to step (2), continuously stir and enzymatically hydrolyze at 60°C, and centrifuge to separate the supernatant to obtain tilapia fish skin protein solution; wherein, the compound protease is composed of neutral protease and pepsin as a combined enzyme, and the enzyme activity dosage ratio of the two is 2:3; after enzymatic hydrolysis, perform enzyme inactivation treatment at 100°C and cool it to room temperature;

[0047] (4) Centrifuge the cooled enzymatic hydrolysate at 2000g for 5 min, and take the supernatant; and filter the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 3000 - 5000 Da;

[0048] (5) Sequence the retained polypeptide molecules with an Obitrap Fusion Lumos mass spectrometer. After identification and analysis, a total of 1 main active peptide is obtained, and the sequence is shown in SEQ ID NO.1 in sequence, and it is reserved for use after spray drying or vacuum freeze-drying at low temperature. After identification by high-performance liquid chromatography analysis, the purity of the main active peptide prepared by the method of this example reaches 90.6%.

[0049] Example 2

[0050] A preparation method of tilapia fish skin kidney protection peptide, comprising the following steps:

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

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

[0053] (3) Add compound protease with a volume ratio of 60% to step (2), continuously stir and enzymatically hydrolyze at 65°C, and centrifuge to separate the supernatant to obtain tilapia fish skin protein solution; wherein, the compound protease is composed of neutral protease and pepsin as a combined enzyme, and the enzyme activity dosage ratio of the two is 3:5; after enzymatic hydrolysis, perform enzyme inactivation treatment at 100°C and cool it to room temperature;

[0054] (4) Centrifuge the cooled enzymatic hydrolysate at 2000g for 8 min, and take the supernatant; and filter the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 3000 - 5000 Da;

[0055] (5) Sequence the retained polypeptide molecules with an Obitrap Fusion Lumos mass spectrometer. After identification and analysis, a total of 1 main active peptide is obtained, and the sequence is shown in SEQ ID NO.1 in sequence.

[0056] Example 3

[0057] A preparation method of tilapia fish skin kidney protection peptide, comprising the following steps:

[0058] (1) Take 20 g of tilapia fish skin and remove the residual adipose tissue.

[0059] (2) Add 4 times the volume of ultrapure water to the tilapia fish skin obtained in step (1), perform high-temperature steaming for 30 min, and perform pulping treatment after steaming.

[0060] (3) Add compound protease with a volume ratio of 70% to step (2), continuously stir and enzymatically hydrolyze at 60 °C, and centrifuge to separate the supernatant to obtain tilapia fish skin protein solution; wherein, the compound protease is composed of neutral protease and pepsin as a combined enzyme, and the enzyme activity dosage ratio of the two is 5:7; after enzymatic hydrolysis, perform enzyme inactivation treatment at 100 °C and cool it to room temperature.

[0061] (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 10 min and take the supernatant; and filter the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 3000 - 5000 Da.

[0062] (5) Sequence the retained polypeptide molecules with an Obitrap Fusion Lumos mass spectrometer, and a total of 1 main active peptide is obtained through identification and analysis, and the sequence is shown in SEQ ID NO.1 in sequence.

[0063] Example 4

[0064] 1. Determination of DPPH free radical scavenging activity:

[0065] The tilapia fish skin kidney protection peptide separated in Example 1 was respectively formulated into concentrations of 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL with sterile distilled water, and stirred and mixed evenly to make it fully dissolve.

[0066] Treatment group: Respectively take 0.5 mL of the aqueous solution of tilapia fish skin kidney protection peptide, add 0.5 mL of 0.5 mol / L DPPH solution, mix evenly, react in the dark at room temperature for 30 min, centrifuge at 5000 revolutions per minute for 5 minutes, and measure the absorbance value A1 at a wavelength of 517 nm.

[0067] Blank group: 0.5 mL of deionized water was added to 0.5 mL of 0.5 mol / L DPPH solution. After mixing evenly, react in the dark at room temperature for 30 min, centrifuge at 5000 revolutions per minute for 5 minutes, and measure the absorbance value A0 at a wavelength of 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. Determination of ABTS free radical scavenging activity:

[0071] The tilapia fish skin kidney protection peptides isolated in Example 1 were respectively prepared into solutions with concentrations of 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL and 5 mg / mL using sterile distilled water, and stirred and mixed evenly to make them fully dissolved.

[0072] Prepare 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution with distilled water. Mix the ABTS solution and the potassium persulfate solution in a volume ratio of 1:1, place it in the dark at 23 °C for 12 h, form an ABTS cationic free radical stock solution, and store it at 4 °C. Before use, appropriately dilute the ABTS cationic free radical solution with distilled water so that the absorbance of the diluted solution at a wavelength of 734 nm is 0.70 ± 0.02 to obtain the ABTS cationic free radical working solution.

[0073] Treatment group: Respectively take 100 μL of the tilapia fish skin kidney protection peptide solution, add it to 100 μL of the ABTS cationic free radical working solution and mix evenly, react at 30 °C for 10 min, and immediately measure the absorbance B1 at 734 nm.

[0074] Blank group: Respectively take 100 μL of deionized water, add it to 100 μL of the ABTS cationic free radical working solution and mix evenly, react at 30 °C for 10 min, and immediately measure the absorbance B0 at 734 nm.

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

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

[0077] 3. Determination of hydroxyl free radical scavenging activity:

[0078] The tilapia fish skin kidney protection peptides isolated in Example 1 were respectively prepared into solutions with concentrations of 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL and 5 mg / mL using sterile distilled water, and stirred and mixed evenly to make them fully dissolved.

[0079] Take 1 mL of the tilapia fish skin kidney protection peptide solution, add 1 mL of 2 mmol / L FeSO4, 1 mL of 6 mmol / L salicylic acid, and 1 mL of 2 mmol / L H2O2 to prepare 4 mL of the test sample solution. Mix the solution thoroughly and place it at room temperature for 30 min. Take 200 µL of the test sample solution and add it to a 96-well microtiter plate, measure the absorbance at 510 nm using an automatic microplate reader and calculate the scavenging rate, with 3 parallel groups set for each group.

[0080] Calculate the hydroxyl radical scavenging rate according to formula (III). A1 is the absorbance of the test solution added with the kidney protection peptide from tilapia skin; A0 is the absorbance of the absolute ethanol sample solution without the kidney protection peptide from tilapia skin; 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 Figures 1-3 shown. Within the test concentration range, the scavenging rates of the kidney protection peptide from tilapia skin against ABTS radicals, DPPH radicals, and hydroxyl radicals all increase with the increase of peptide concentration. When the concentration is 5 mg / ml, the ABTS radical scavenging rate is 69.42%, the DPPH radical scavenging rate reaches 66.262%, and the hydroxyl radical scavenging rate reaches 40.244%.

[0083] Example 5

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

[0085] 1) Culture mouse glomerular mesangial cells CP-M057, and seed 1×10 5 cells / well in a 96-well cell culture plate, 150 μL per well, and place it in an incubator for 12 h for cell attachment treatment;

[0086] 2) Prepare the kidney protection peptide solution shown in SEQ ID NO.1 with final concentrations of 1 - 5 mg / mL respectively using cell culture medium;

[0087] 3) Sequentially add 50 μL of the kidney protection peptide solution described in step 2) to the cell culture plate shown in step 1), and incubate together for 12 h. The specific groups include: the treatment group is the kidney protection peptide solution shown in SEQ ID NO.1, the control group is not treated with the kidney protection peptide solution, and the blank group is pure culture medium.

[0088] The results are as Figure 4 shown. It can be seen that within the test concentration range, the inhibition rate of the kidney protection peptide on glomerular mesangial cells is positively correlated with the kidney protection peptide concentration at 24 h. When the kidney protection peptide concentration is 5 mg / ml, the inhibition rate at 24 h is the highest, which is 45.68%.

[0089] It is necessary to point out here that the above embodiments are only limited to further elaborating and explaining the technical solution of the present invention, and do not further limit the technical solution of the present invention. The method of the present invention is only a preferred implementation solution and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of tilapia fish skin kidney protection peptide, characterized in that It includes the following steps: (1) Take 10 - 20 g of tilapia fish skin and remove the residual adipose tissue; (2) Add 2 - 4 times the volume of ultrapure water to the tilapia fish skin obtained in step (1), perform high-temperature steaming for 30 - 60 min, and perform pulping treatment after steaming; (3) Add a compound protease with a volume ratio of 50 - 65% to step (2), continuously stir and enzymatically hydrolyze at 60 - 70 °C, and centrifuge to separate the supernatant to obtain a tilapia fish skin protein solution; wherein, the compound protease is composed of neutral protease and pepsin as a combined enzyme, and the enzyme activity dosage ratio of the two is (2 - 5) : (3 - 7); after enzymatic hydrolysis, perform enzyme inactivation treatment at 100 °C and cool it to room temperature; (4) Centrifuge the cooled enzymatic hydrolysate at 2000 g for 5 - 15 min, take the supernatant; and filter the obtained supernatant with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 3000 - 5000 Da; (5) Sequence the retained polypeptide molecules with a mass spectrometer.

2. The method according to claim 1, characterized in that, One kind of bioactive peptide is prepared by the method, and the sequence is as shown in SEQ ID NO.1, and it is reserved after spray drying or vacuum freeze-drying at low temperature.

3. A tilapia fish skin kidney protection peptide, characterized in that, The tilapia fish skin kidney protection peptide is prepared by the method described in claim 1 or 2 above.

4. The tilapia fish skin kidney protection peptide according to claim 3, characterized in that, The sequence of the tilapia fish skin kidney protection peptide is as shown in SEQ ID NO.

1.

5. Use of the tilapia fish skin kidney protection peptide described in claim 4 in the preparation of an antioxidant active drug.

6. Use of the tilapia fish skin kidney protection peptide described in claim 4 in the preparation of a kidney protection agent drug.

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