Hydrolyzed polypeptide as well as preparation method and application thereof

Abstract

The invention provides a hydrolyzed polypeptide as well as a preparation method and application thereof, and relates to the technical field of biology. According to the invention, the oligopeptide with antioxidant activity is separated and purified from pomegranate seeds by using a composite enzymolysis technology for the first time. Antioxidant activity identification shows that the active oligopeptide has good anti-free radical activity, can effectively remove residual DPPH free radicals and ABTS free radicals in an organism, and has a great application prospect.

Description

Technical Field

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

[0002] Antioxidants are any substances that can effectively inhibit the oxidation reaction of free radicals at low concentrations. Their mechanism of action can be to act directly on free radicals, or to indirectly consume substances that easily generate free radicals to prevent further reactions. While the human body inevitably produces free radicals, it also naturally produces antioxidant substances that resist free radicals to offset 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, which are natural antioxidants. They can help prevent the formation of free radicals and damage to cells, thereby slowing down the aging process, improving immunity and reducing the risk of disease. Many studies have shown that pomegranates also have anti-inflammatory, anti-cardiovascular disease, lower blood pressure and improve memory, so they are considered a very healthy fruit.

[0004] However, the identification of antioxidant substances in pomegranates has yet 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] In view of the technical problems existing in the prior art, the present invention provides a hydrolyzed polypeptide and a preparation method and application thereof. The present invention separates and purifies short peptides with antioxidant activity from pomegranate seeds for the first time. After verification and analysis, the short peptides have good anti-free radical activity and can effectively remove residual DPPH free radicals and ABTS free radicals in the body, and have 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 is a combination of neutral protease and papain.

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

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

[0010] Further preferably, the amino acid sequence of the hydrolyzed polypeptide is 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: Preparation: Wash the pomegranate, peel it and leave the pomegranate seeds, air-dry them and set aside; 2) Physical crushing: crush the pomegranate seeds air-dried in step 1) into fine powder by breaking the wall for later use; 3) Ultrasonic-assisted extraction: add 20-40% ethanol solution to the fine powder of step 2), perform ultrasonic extraction at a temperature of 45-60° C., an ultrasonic power of 100-120 W, and an extraction time of 3-5 h; 4) Composite enzymatic hydrolysis: When the temperature of step 3) is cooled to room temperature, 120-150U composite protease is added, and the enzymatic hydrolysis is carried out at 37°C with continuous stirring for 5-10h, and the temperature is raised to 100°C and kept constant for 10-20min to inactivate the enzyme, followed by centrifugation at 3500rpm for 15-30min, and the supernatant is separated to obtain pomegranate seed peptide enzymatic hydrolysate; wherein the composite protease is a combination of neutral protease and papain, and the mass ratio of the two is (3-5):(2-4); 5) Filtration and concentration: The pomegranate seed peptide hydrolysate of step 4) is filtered using an ultrafiltration membrane to retain polypeptide molecules with a molecular weight of 1500-3500Da; the filtered pomegranate seed peptide is vacuum concentrated to a concentration of 15-18° Bé to obtain a pomegranate seed peptide concentrate; 6) Identification of pomegranate seed peptides: The pomegranate seed peptide concentrate from step 5) was sequenced by Obitrap Fusion Lumos mass spectrometer; 7) Purity identification of pomegranate seed peptide: The pomegranate seed peptide concentrate is transferred 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 moisture content of the pomegranate seed peptide dry powder is 5-10%, and the dry powder is collected to obtain pomegranate seed peptide.

[0013] Preferably, the method for preparing the hydrolyzed polypeptide comprises the following steps: Preparation: Wash the pomegranate, peel it and leave the pomegranate seeds, air-dry them and set aside; 2) Physical crushing: crush the pomegranate seeds air-dried in step 1) into fine powder by breaking the wall for later use; 3) Ultrasonic-assisted extraction: add 20% ethanol solution to the fine powder in step 2), perform ultrasonic extraction at 45°C, ultrasonic power 100W, and extraction time 3h; 4) Composite enzymatic hydrolysis: When the temperature of step 3) is cooled to room temperature, 120U composite protease is added, and the mixture is stirred and hydrolyzed at 37°C for 5 hours, and the temperature is raised to 100°C and kept constant for 10 minutes to inactivate the enzyme, followed by centrifugation at 3500 rpm for 15 minutes, and the supernatant is separated to obtain pomegranate seed peptide hydrolysate; wherein the composite protease is a combination of neutral protease and papain, and the mass ratio of the two is 3:2; 5) Filtration and concentration: The pomegranate seed peptide hydrolysate of step 4) is filtered by an ultrafiltration membrane, with a molecular weight cutoff of 1500-3500Da polypeptide molecules; the filtered pomegranate seed peptide is vacuum concentrated to a concentration of 15° Bé to obtain a pomegranate seed peptide concentrate; 6) Identification of pomegranate seed peptides: The pomegranate seed peptide concentrate from step 5) was sequenced by Obitrap Fusion Lumos mass spectrometer; 7) Purity identification of pomegranate seed peptide: The pomegranate seed peptide concentrate was transferred to a spray dryer with an inlet air temperature of 80°C and an outlet air temperature of 60°C for concentration until the moisture content of the pomegranate seed peptide dry powder was 5%. The dry powder was collected to obtain pomegranate seed peptide.

[0014] Another object of the present invention is to provide a kit for removing free radicals from the human body, wherein the kit contains the hydrolyzed polypeptide as a main active ingredient, and the amino acid sequence of the hydrolyzed polypeptide is shown in any one of SEQ ID NO.1-2.

[0015] Further preferably, the amino acid sequence of the hydrolyzed polypeptide is as shown in SEQ ID NO.1.

[0016] Furthermore, the present invention also provides the use of the hydrolyzed polypeptide in preparing a kit for removing free radicals from the human body, and the amino acid sequence of the pomegranate seed peptide is shown in any one of SEQ ID NO.1-2.

[0017] Further preferably, the amino acid sequence of the hydrolyzed polypeptide is as shown in SEQ ID NO.1.

[0018] The advantages of the present invention are as follows: the present invention separates and purifies short peptides with antioxidant activity from pomegranate seeds for the first time. After verification and analysis, the short peptides have good anti-free radical activity, can effectively remove residual DPPH free radicals and ABTS free radicals in the body, and have great application prospects. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0020] 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 ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0021] 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.

[0022] Example 1 Preparation, separation and identification of hydrolyzed polypeptides The preparation method of hydrolyzed polypeptide comprises the following steps: Preparation: Wash the pomegranate, peel it and leave the pomegranate seeds, air-dry them and set aside; 2) Physical crushing: crush the pomegranate seeds air-dried in step 1) into fine powder by breaking the wall for later use; 3) Ultrasonic-assisted extraction: add 20% ethanol solution to the fine powder in step 2), perform ultrasonic extraction at 45°C, ultrasonic power 100W, and extraction time 3h; 4) Composite enzymatic hydrolysis: When the temperature of step 3) is cooled to room temperature, 120U composite protease is added, and the mixture is stirred and hydrolyzed at 37°C for 5 hours, and the mixture is heated to 100°C and kept at a constant temperature for 10 minutes to inactivate the enzyme, and then centrifuged at 3500rpm for 15 minutes to separate the supernatant to obtain pomegranate seed peptide hydrolysate; wherein the composite protease is a combination of neutral protease and papain, and the mass ratio of the two is 3:2; 5) Filtration and concentration: The pomegranate seed peptide hydrolysate of step 4) is filtered by an ultrafiltration membrane, with a molecular weight cutoff of 1500-3500Da polypeptide molecules; the filtered pomegranate seed peptide is vacuum concentrated to a concentration of 15° Bé to obtain a pomegranate seed peptide concentrate; 6) Identification of pomegranate seed peptides: The pomegranate seed peptide concentrate from step 5) was sequenced by Obitrap Fusion Lumos mass spectrometer; 7) Purity identification of pomegranate seed peptide: The pomegranate seed peptide concentrate was transferred to a spray dryer with an inlet air temperature of 80°C and an outlet air temperature of 60°C for concentration until the moisture content of the pomegranate seed peptide dry powder was 5%. The dry powder was collected to obtain pomegranate seed peptide.

[0023] The results showed that two pomegranate seed peptides 1 and 2 were obtained through sequencing and identification analysis, and the sequences were shown in SEQ ID NO.1-2. After purity determination, the purities of the obtained pomegranate seed peptides 1 and 2 were 95.8% and 92.5%, respectively.

[0024] Example 2 Analysis of Antioxidant Activity of Hydrolyzed Peptides DPPH free radical scavenging activity assay The pomegranate seed peptides 1 and 2 separated in Example 1 were respectively prepared with sterile distilled water to a concentration of 100 μg / mL, and stirred to be fully dissolved.

[0025] Treatment group 1: Take 1 mL of pomegranate seed peptide 1 aqueous solution, add 1 mL of 0.5 mol / L DPPH solution, mix well, react at room temperature in the dark for 60 min, centrifuge at 3500 rpm for 10 min, and measure the absorbance A1 at a wavelength of 517 nm.

[0026] Treatment group 2: Take 1 mL of pomegranate seed peptide 2 aqueous solution, add 1 mL of 0.5 mol / L DPPH solution, mix well, react at room temperature in the dark for 60 min, centrifuge at 3500 rpm for 10 min, and measure the absorbance A1 at a wavelength of 517 nm.

[0027] Blank group: 1 mL of 0.5 mol / L DPPH solution was added to 1 mL of deionized water. After mixing evenly, the mixture was reacted at room temperature in the dark for 60 min, centrifuged at 3500 rpm for 10 min, and the absorbance A0 was measured at a wavelength of 517 nm.

[0028] The calculation formula is shown in Formula I: DPPH free radical scavenging rate (%) = (1-(A1÷A0)) × 100% (Formula I).

[0029] ABTS free radical scavenging activity assay The pomegranate seed peptides 1 and 2 separated in Example 1 were prepared with sterile distilled water to a concentration of 100 μg / mL, and stirred to fully dissolve.

[0030] Prepare 7mmol / L ABTS solution and 2.45mmol / L potassium persulfate solution with distilled water. Mix ABTS solution and potassium persulfate solution at a volume ratio of 1:1, place at 25℃ for 12 hours in a dark place to form ABTS cation radical stock solution, and then store at 4℃. Dilute the ABTS cation radical solution appropriately with distilled water before use, so that the absorbance of the diluted solution at 734nm wavelength is 0.68±0.05, and obtain ABTS cation radical working solution.

[0031] Treatment group 1: 150 μL of pomegranate seed peptide 1 aqueous solution was added to 150 μL ABTS cation free radical working solution, mixed, reacted at 35°C for 15 min, and the absorbance B1 was immediately measured at 734 nm.

[0032] Treatment group 2: 150 μL of pomegranate seed peptide 2 aqueous solution was added to 150 μL ABTS cation free radical working solution, mixed, reacted at 35°C for 15 min, and the absorbance B1 was immediately measured at 734 nm.

[0033] Blank group: 150 μL of deionized water was added to 150 μL of ABTS cation radical working solution and mixed evenly. The mixture was reacted at 35°C for 15 min and the absorbance B0 was immediately measured at 734 nm.

[0034] The calculation formula is shown in Formula II: ABTS free radical scavenging rate (%) = (1-(B1÷B0)) × 100% (Formula II).

[0035] The results are shown in Table 1. Pomegranate seed peptide 1 has superior antioxidant activity compared to pomegranate seed peptide 2, can effectively remove residual DPPH free radicals and ABTS free radicals in the body, and has great application prospects.

[0036] Table 1. Antioxidant activity analysis of pomegranate seed peptides 1 and 2 DPPH free radical scavenging activity IC50 value (mmol / L) ABTS free radical scavenging activity IC50 value (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 It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of 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 modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hydrolyzed polypeptide, characterized in that: The hydrolyzed polypeptide is a composite enzymatic hydrolysis product from pomegranate seeds, and the amino acid sequence of the hydrolyzed polypeptide is shown in SEQ ID NO.

1.

2. The hydrolyzed polypeptide according to claim 1, characterized in that The composite enzymolysis adopts a combination of neutral protease and papain.

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

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

2.

5. The method for preparing the hydrolyzed polypeptide according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: Preparation: Wash the pomegranate, peel it and leave the pomegranate seeds, air-dry them and set aside; 2) Physical crushing: crush the pomegranate seeds air-dried in step 1) into fine powder by breaking the wall for later use; 3) Ultrasonic-assisted extraction: add 20-40% ethanol solution to the fine powder of step 2), perform ultrasonic extraction at a temperature of 45-60° C., an ultrasonic power of 100-120 W, and an extraction time of 3-5 h; 4) Composite enzymatic hydrolysis: When the temperature of step 3) is cooled to room temperature, 120-150U composite protease is added, and the enzymatic hydrolysis is carried out at 37°C with continuous stirring for 5-10h, and the temperature is raised to 100°C and kept constant for 10-20min to inactivate the enzyme, followed by centrifugation at 3500rpm for 15-30min, and the supernatant is separated to obtain pomegranate seed peptide enzymatic hydrolysate; wherein the composite protease is obtained by mixing neutral protease and papain in a mass ratio of (3-5): (2-4); 5) Filtration and concentration: The pomegranate seed peptide hydrolysate of step 4) is filtered by an ultrafiltration membrane, with a molecular weight cutoff of 1500-3500 Da polypeptide molecules; the filtered pomegranate seed peptide is vacuum concentrated to a concentration of 15-18° Bé to obtain a pomegranate seed peptide concentrate; 6) Identification of pomegranate seed peptides: The pomegranate seed peptide concentrate from step 5) was sequenced by Obitrap Fusion Lumos mass spectrometer; 7) Purity identification of pomegranate seed peptide: The pomegranate seed peptide concentrate is transferred 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 moisture content of the pomegranate seed peptide dry powder is 5-10%, and the dry powder is collected to obtain pomegranate seed peptide.

6. The method for preparing the hydrolyzed polypeptide according to claim 5, characterized in that: The preparation method of the hydrolyzed polypeptide comprises the following steps: Preparation: Wash the pomegranate, peel it and leave the pomegranate seeds, air-dry them and set aside; 2) Physical crushing: crush the pomegranate seeds air-dried in step 1) into fine powder by breaking the wall for later use; 3) Ultrasonic-assisted extraction: add 20% ethanol solution to the fine powder in step 2), perform ultrasonic extraction at 45°C, ultrasonic power 100W, and extraction time 3h; 4) Composite enzymatic hydrolysis: when the temperature of step 3) is cooled to room temperature, 120U composite protease is added, and the enzymatic hydrolysis is carried out at 37°C with continuous stirring for 5 hours, and the temperature is raised to 100°C and kept constant for 10 minutes to inactivate the enzyme, followed by centrifugation at 3500rpm for 15 minutes, and the supernatant is separated to obtain pomegranate seed peptide enzymatic hydrolysate; wherein the composite protease is obtained by mixing neutral protease and papain in a mass ratio of 3:2; 5) Filtration and concentration: The pomegranate seed peptide hydrolysate of step 4) is filtered by an ultrafiltration membrane, with a molecular weight cutoff of 1500-3500 Da polypeptide molecules; the filtered pomegranate seed peptide is vacuum concentrated to a concentration of 15° Bé to obtain a pomegranate seed peptide concentrate; 6) Identification of pomegranate seed peptides: The pomegranate seed peptide concentrate from step 5) was sequenced by Obitrap Fusion Lumos mass spectrometer; 7) Purity identification of pomegranate seed peptide: The pomegranate seed peptide concentrate was transferred to a spray dryer with an inlet air temperature of 80°C and an outlet air temperature of 60°C for concentration until the moisture content of the pomegranate seed peptide dry powder was 5%. The dry powder was collected to obtain pomegranate seed peptide.

7. A kit for removing free radicals from human body, characterized in that: The kit contains the hydrolyzed polypeptide according to claim 1 as the main active ingredient.

8. Use of the hydrolyzed polypeptide according to any one of claims 1 to 4 in preparing a kit for removing free radicals from the human body.