A hydrolyzed polypeptide, its preparation method and application

Abstract

The present invention provides a hydrolyzed polypeptide, a preparation method and an application thereof, relating to the field of biotechnology. For the first time, the present invention separates and purifies a short peptide with antioxidant activity from pomegranate seeds by using a composite enzymolysis technology. Through antioxidant activity identification, the active short peptide has good free radical scavenging activity, can effectively scavenge residual DPPH free radicals and ABTS free radicals in the body, and has great application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a hydrolyzed polypeptide and its preparation method and application. Background Art

[0002] Antioxidation refers to any substance that can effectively inhibit the oxidation reaction of free radicals at a low concentration. Its mechanism of action can be either directly acting on free radicals or indirectly consuming substances that are prone to generating free radicals to prevent further reactions. While the human body inevitably generates free radicals, it also naturally produces antioxidant substances that resist free radicals to counteract the oxidative attack of free radicals on human cells.

[0003] Pomegranates have powerful antioxidant effects. Pomegranates are rich in polyphenolic compounds such as anthocyanins, flavonoids, and tannins, and these substances are all natural antioxidants. They can help prevent the formation of free radicals and damage to cells, thereby slowing down the aging process, enhancing immunity, and reducing the risk of disease. Multiple studies have shown that pomegranates also have anti-inflammatory, anti-cardiovascular disease, blood pressure-lowering, and memory-improving effects, so they are regarded as a very healthy fruit.

[0004] However, at present, the identification of antioxidant substances in pomegranates still needs to be further clarified, and there is an urgent need to isolate a natural antioxidant active substance for skin anti-aging. Summary of the Invention

[0005] Aiming at the technical problems existing in the prior art, the present invention provides a hydrolyzed polypeptide and its preparation method and application. The present invention first isolates and purifies a short peptide with antioxidant activity from pomegranate seeds. Through verification and analysis, the short peptide has good free radical scavenging activity, can effectively scavenge residual DPPH free radicals and ABTS free radicals in the body, and has great application prospects.

[0006] Specifically, the present invention first provides a hydrolyzed polypeptide, which is a composite enzymatic hydrolysis product from pomegranate seeds.

[0007] Preferably, the composite enzymatic hydrolysis uses a compound of neutral protease and papain.

[0008] Preferably, the mass ratio of neutral protease to papain in the composite enzyme is (3 - 5):(2 - 4).

[0009] Preferably, the mass ratio of neutral protease to papain in the composite enzyme is 3:2.

[0010] More preferably, the amino acid sequence of the hydrolyzed polypeptide is as shown in any one of SEQ ID NO.1 - 2.

[0011] Further preferably, the amino acid sequence of the hydrolyzed polypeptide is as shown in SEQ ID NO.1.

[0012] On the other hand, the present invention also provides a method for preparing the hydrolyzed polypeptide, comprising the following steps:

[0013] Material preparation: After cleaning the pomegranates, peel them to leave the pomegranate seeds, and air-dry them for later use;

[0014] 2) Physical pulverization: Pulverize the air-dried pomegranate seeds in step 1) by wall-breaking to a fine powder for later use;

[0015] 3) Ultrasonic-assisted extraction: Add an ethanol solution with a concentration of 20-40% to the fine powder in step 2), and perform ultrasonic extraction at a temperature of 45-60 °C, with an ultrasonic power of 100-120 W and an extraction time of 3-5 h;

[0016] 4) Complex enzymatic hydrolysis: When the temperature in step 3) cools down to room temperature, add 120-150 U of complex protease, continuously stir and enzymatically hydrolyze at 37 °C for 5-10 h, then raise the temperature to 100 °C and keep it constant for 10-20 min to inactivate the enzyme, and then centrifuge at 3500 rpm for 15-30 min to separate the supernatant to obtain the pomegranate seed peptide enzymatic hydrolysate; wherein, the complex protease is a compound of neutral protease and papain, and the mass ratio of the two is (3-5):(2-4);

[0017] 5) Filtration and concentration: Filter the pomegranate seed peptide enzymatic hydrolysate in step 4) using an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 1500-3500 Da; Vacuum-concentrate the filtered pomegranate seed peptide to a concentration of 15-18 °Bé to obtain the pomegranate seed peptide concentrate;

[0018] 6) Identification of pomegranate seed peptide: Take the pomegranate seed peptide concentrate in step 5) for sequencing with an Obitrap Fusion Lumos mass spectrometer;

[0019] 7) Purity identification of pomegranate seed peptide: Transfer the pomegranate seed peptide concentrate to a spray dryer, with an inlet air temperature of 80-90 °C and an outlet air temperature of 60-70 °C for concentration until the water content of the pomegranate seed peptide dry powder is 5-10%, collect the dry powder, and thus obtain the pomegranate seed peptide.

[0020] Preferably, the method for preparing the hydrolyzed polypeptide comprises the following steps:

[0021] Material preparation: After cleaning the pomegranates, peel them to leave the pomegranate seeds, and air-dry them for later use;

[0022] 2) Physical pulverization: Pulverize the air-dried pomegranate seeds in step 1) by wall-breaking to a fine powder for later use;

[0023] 3) Ultrasonic-assisted extraction: Add ethanol solution with a concentration of 20% to the fine powder in step 2), and perform ultrasonic extraction at a temperature of 45°C, with an ultrasonic power of 100 W and an extraction time of 3 h;

[0024] 4) Complex enzymatic hydrolysis: When the temperature in step 3) cools down to room temperature, add 120 U of complex protease, continuously stir and hydrolyze at 37°C for 5 h, then raise the temperature to 100°C and keep it constant for 10 min to inactivate the enzyme, and then centrifuge at 3500 rpm for 15 min to separate the supernatant to obtain the pomegranate seed peptide hydrolysate; among them, the complex protease is a compound of neutral protease and papain, and the mass ratio of the two is 3:2;

[0025] 5) Filtration and concentration: Filter the pomegranate seed peptide hydrolysate in step 4) with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 1500 - 3500 Da; Vacuum-concentrate the filtered pomegranate seed peptide to a concentration of 15° Bé to obtain the pomegranate seed peptide concentrate;

[0026] 6) Identification of pomegranate seed peptide: Take the pomegranate seed peptide concentrate in step 5) for sequencing with an Obitrap Fusion Lumos mass spectrometer;

[0027] 7) Purity identification of pomegranate seed peptide: Transfer the pomegranate seed peptide concentrate to a spray dryer, with an inlet air temperature of 80°C and an outlet air temperature of 60°C for concentration until the water content of the pomegranate seed peptide dry powder is 5%, collect the dry powder, and thus obtain the pomegranate seed peptide.

[0028] Another object of the present invention is to provide a kit for scavenging human free radicals, and the kit uses the hydrolyzed polypeptide as the main active ingredient, and the amino acid sequence of the hydrolyzed polypeptide is as shown in any one of SEQ ID NO.1 - 2.

[0029] Further preferably, the amino acid sequence of the hydrolyzed polypeptide is as shown in SEQ ID NO.1.

[0030] Furthermore, the present invention also provides the use of the hydrolyzed polypeptide in the preparation of a kit for scavenging human free radicals, and the amino acid sequence of the pomegranate seed peptide is as shown in any one of SEQ ID NO.1 - 2.

[0031] Further preferably, the amino acid sequence of the hydrolyzed polypeptide is as shown in SEQ ID NO.1.

[0032] The advantages of the present invention are as follows: The present invention first isolates and purifies a short peptide with antioxidant activity from pomegranate seeds. Through verification and analysis, the short peptide has good free radical scavenging activity, can effectively scavenge residual DPPH free radicals and ABTS free radicals in the body, and has great application prospects. Detailed implementation mode

[0033] 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.

[0034] 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 belong to the protection scope of the present invention.

[0035] 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 all conventional means well-known to those skilled in the art.

[0036] Example 1 Preparation, Separation and Identification of Hydrolyzed Polypeptides

[0037] The preparation method of the hydrolyzed polypeptide includes the following steps:

[0038] Material preparation: After washing the pomegranates clean, remove the peels and leave the pomegranate seeds, and dry them for later use;

[0039] 2) Physical pulverization: Pulverize the dried pomegranate seeds in step 1) by wall-breaking to a fine powder for later use;

[0040] 3) Ultrasonic-assisted extraction: Add a 20% ethanol solution to the fine powder in step 2), and perform ultrasonic extraction at a temperature of 45 °C, with an ultrasonic power of 100 W and an extraction time of 3 h;

[0041] 4) Composite enzymatic hydrolysis: When the temperature in step 3) cools to room temperature, add 120 U of composite protease, continuously stir and enzymatically hydrolyze at 37 °C for 5 h, then raise the temperature to 100 °C and keep it constant for 10 min to inactivate the enzyme, and then centrifuge at 3500 rpm for 15 min to separate the supernatant to obtain the pomegranate seed peptide enzymatic hydrolysate; among them, the composite protease is a compound of neutral protease and papain, and the mass ratio of the two is 3:2;

[0042] 5) Filtration and concentration: Filter the pomegranate seed peptide enzymatic hydrolysate in step 4) with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 1500 - 3500 Da; Vacuum-concentrate the filtered pomegranate seed peptide to a concentration of 15 °Bé to obtain the pomegranate seed peptide concentrate;

[0043] 6) Identification of pomegranate seed peptide: Take the pomegranate seed peptide concentrate in step 5) for sequencing with an Obitrap Fusion Lumos mass spectrometer;

[0044] 7) Purity identification of pomegranate seed peptide: Transfer the pomegranate seed peptide concentrate to a spray dryer, with an inlet air temperature of 80 °C and an outlet air temperature of 60 °C for concentration until the water content of the pomegranate seed peptide dry powder is 5%, collect the dry powder, and thus obtain the pomegranate seed peptide.

[0045] The results showed that a total of two pomegranate seed peptides 1 and 2 were obtained through sequencing and identification analysis, and the sequences were as shown in SEQ ID NO.1-2. After purity determination, the purities of the obtained pomegranate seed peptides 1 and 2 could reach 95.8% and 92.5% in sequence.

[0046] Example 2 Analysis of antioxidant activity of hydrolyzed polypeptides

[0047] Determination of DPPH free radical scavenging activity

[0048] The pomegranate seed peptides 1 and 2 separated in Example 1 were respectively prepared into solutions with a concentration of 100 μg / mL using sterile distilled water, and stirred and mixed evenly to make them fully dissolved.

[0049] Treatment group 1: Respectively take 1 mL of the aqueous solution of pomegranate seed peptide 1, add 1 mL of 0.5 mol / L DPPH solution, mix evenly, react in the dark at room temperature for 60 min, centrifuge at 3500 revolutions per minute for 10 minutes, and measure the absorbance value A1 at a wavelength of 517 nm.

[0050] Treatment group 2: Respectively take 1 mL of the aqueous solution of pomegranate seed peptide 2, add 1 mL of 0.5 mol / L DPPH solution, mix evenly, react in the dark at room temperature for 60 min, centrifuge at 3500 revolutions per minute for 10 minutes, and measure the absorbance value A1 at a wavelength of 517 nm.

[0051] Blank group: 1 mL of deionized water was added to 1 mL of 0.5 mol / L DPPH solution. After mixing evenly, react in the dark at room temperature for 60 min, centrifuge at 3500 revolutions per minute for 10 minutes, and measure the absorbance value A0 at a wavelength of 517 nm.

[0052] The calculation formula is as shown in Formula I:

[0053] DPPH free radical scavenging rate (%) = (1 - (A1÷A0)) × 100% (Formula I).

[0054] Determination of ABTS free radical scavenging activity

[0055] The pomegranate seed peptides 1 and 2 separated in Example 1 were prepared into solutions with a concentration of 100 μg / mL using sterile distilled water, and stirred and mixed evenly to make them fully dissolved.

[0056] Prepare 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution with distilled water. Mix the ABTS solution and potassium persulfate solution in a volume ratio of 1:1, place it in the dark at 25 °C for 12 h to form an ABTS cation radical stock solution, and then store it at 4 °C. Before use, appropriately dilute the ABTS cation radical solution with distilled water so that the absorbance of the diluted solution at a wavelength of 734 nm is 0.68 ± 0.05 to obtain the ABTS cation radical working solution.

[0057] Treatment group 1: Take 150 μL of pomegranate seed peptide 1 aqueous solution and add it to 150 μL of ABTS cation radical working solution and mix well. React at 35 °C for 15 min, and immediately measure the absorbance B1 at 734 nm.

[0058] Treatment group 2: Take 150 μL of pomegranate seed peptide 2 aqueous solution and add it to 150 μL of ABTS cation radical working solution and mix well. React at 35 °C for 15 min, and immediately measure the absorbance B1 at 734 nm.

[0059] Blank group: Take 150 μL of deionized water and add it to 150 μL of ABTS cation radical working solution and mix well. React at 35 °C for 15 min, and immediately measure the absorbance B0 at 734 nm.

[0060] The calculation formula is as shown in Formula II:

[0061] ABTS radical scavenging rate (%) = (1 - (B1 ÷ B0)) × 100% (Formula II).

[0062] The results are shown in Table 1. Pomegranate seed peptide 1 has superior antioxidant activity compared to pomegranate seed peptide 2, can effectively scavenge residual DPPH radicals and ABTS radicals in the body, and has great application prospects.

[0063] Table 1. Analysis of antioxidant activities of pomegranate seed peptides 1 and 2

[0064] IC50 value of DPPH radical scavenging activity (mmol / L) IC50 value of ABTS radical scavenging activity (mmol / L) Pomegranate seed peptide 1 2.65±0.21 3.25±0.28 Pomegranate seed peptide 2 3.89±0.45 5.72±0.18

[0065] It is necessary to point out here that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not further limitations on the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme and is not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, 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 hydrolyzed polypeptide, characterized in that, The hydrolyzed polypeptide is a composite enzymolysis product derived from pomegranate seeds, and the amino acid sequence of the hydrolyzed polypeptide is as shown in SEQ ID NO.

1.

2. The hydrolyzed polypeptide according to claim 1, wherein, The composite enzymolysis is carried out using a composite enzyme composed of neutral protease and papain for enzymolysis.

3. The hydrolyzed polypeptide according to claim 2, wherein The mass ratio of neutral protease to papain in the composite enzyme is (3 - 5):(2 - 4).

4. The hydrolyzed polypeptide according to claim 3, wherein, The mass ratio of neutral protease to papain in the composite enzyme is 3:

2.

5. The preparation method of the hydrolyzed polypeptide according to any one of claims 1-4, characterized in that, The method includes the following steps: 1) Preparation of materials: After cleaning the pomegranates, remove the peels and leave the pomegranate seeds, and air-dry them for later use; 2) Physical pulverization: Pulverize the air-dried pomegranate seeds in step 1) by wall-breaking to a fine powder for later use; 3) Ultrasonic-assisted extraction: Add an ethanol solution with a concentration of 20 - 40% to the fine powder in step 2), and carry out ultrasonic extraction at a temperature of 45 - 60 °C, with an ultrasonic power of 100 - 120 W and an extraction time of 3 - 5 h; 4) Composite enzymolysis: When the temperature in step 3) cools to room temperature, add 120 - 150 U of composite protease, continuously stir and enzymolyze at 37 °C for 5 - 10 h, then raise the temperature to 100 °C and keep it constant for 10 - 20 min to inactivate the enzyme, and then centrifuge at 3500 rpm for 15 - 30 min to separate the supernatant to obtain the pomegranate seed peptide enzymolysis solution; among them, the composite protease is obtained by mixing neutral protease and papain according to a mass ratio of (3 - 5):(2 - 4); 5) Filtration and concentration: Filter the pomegranate seed peptide enzymolysis solution in step 4) using an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 1500 - 3500 Da; Vacuum-concentrate the filtered pomegranate seed peptide to a concentration of 15 - 18 °Bé to obtain the pomegranate seed peptide concentrated solution; 6) Identification of pomegranate seed peptide: Take the pomegranate seed peptide concentrated solution in step 5) for sequencing with an Obitrap Fusion Lumos mass spectrometer; 7) Purity identification of pomegranate seed peptide: Transfer the pomegranate seed peptide concentrated solution to a spray dryer, with an inlet air temperature of 80 - 90 °C and an outlet air temperature of 60 - 70 °C for concentration until the water content of the pomegranate seed peptide dry powder is 5 - 10%, collect the dry powder, and thus obtain the pomegranate seed peptide.

6. The preparation method of the hydrolyzed polypeptide according to claim 5, characterized in that, The preparation method of the hydrolyzed polypeptide includes the following steps: S1) Preparation of materials: After cleaning the pomegranates, remove the peels and leave the pomegranate seeds, and air-dry them for later use; S2) Physical pulverization: Pulverize the air-dried pomegranate seeds in step S1) by wall-breaking to a fine powder for later use; S3) Ultrasonic-assisted extraction: Add an ethanol solution with a concentration of 20% to the fine powder in step S2), and carry out ultrasonic extraction at a temperature of 45 °C, with an ultrasonic power of 100 W and an extraction time of 3 h; S4) Composite enzymolysis: When the temperature in step S3) cools to room temperature, add 120 U of composite protease, continuously stir and enzymolyze at 37 °C for 5 h, then raise the temperature to 100 °C and keep it constant for 10 min to inactivate the enzyme, and then centrifuge at 3500 rpm for 15 min to separate the supernatant to obtain the pomegranate seed peptide enzymolysis solution; among them, the composite protease is obtained by mixing neutral protease and papain according to a mass ratio of 3:2; S5) Filtration and concentration: The pomegranate seed peptide hydrolysate from step S4) is filtered using an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 1500 - 3500 Da; the filtered pomegranate seed peptide is vacuum concentrated to a concentration of 15 °Bé to obtain a concentrated pomegranate seed peptide solution; S6) Identification of pomegranate seed peptide: Take the concentrated pomegranate seed peptide solution from step S5) for sequencing with an Obitrap Fusion Lumos mass spectrometer; S7) Purity identification of pomegranate seed peptide: Transfer the concentrated pomegranate seed peptide solution to a spray dryer, with an inlet air temperature of 80 °C and an outlet air temperature of 60 °C for concentration until the water content of the pomegranate seed peptide dry powder is 5%, collect the dry powder, and thus obtain the pomegranate seed peptide.

7. An antioxidant kit, characterized in that, The said kit uses the hydrolyzed polypeptide described in claim 1 as the main active ingredient.

8. Use of the hydrolyzed polypeptide according to any one of claims 1 - 4 in the preparation of an antioxidant kit.