Donkey-hide gelatin peptide beneficial to removing free radicals of human body and preparation method thereof

Abstract

The invention provides colla corii asini peptide beneficial for removing free radicals of a human body and a preparation method thereof. According to the invention, three active oligopeptides are separated and purified from donkey-hide gelatin for the first time, and the donkey-hide gelatin peptide has good anti-free radical activity, can effectively remove residual DPPH free radicals and ABTS free radicals in a body, and has a great application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to donkey-hide gelatin peptide which is beneficial to clearing free radicals from human body and a preparation method thereof. Background Art

[0002] The aging process of an organism is the result of the accumulation of free radicals continuously produced by the body's tissue cells. Free radicals can cause DNA damage, leading to mutations and inducing tumor formation. Free radicals are intermediate products of normal metabolism and have strong reactivity. They can oxidize a variety of substances in cells and damage biological membranes. They can also cross-link macromolecules such as proteins and nucleic acids, affecting their normal functions.

[0003] Donkey hide gelatin is a solid gelatin made by boiling and concentrating the dried or fresh skin of the donkey, an equine animal. It is known as the "national treasure of nourishment and holy medicine for blood tonic". Donkey hide gelatin is rich in various amino acids and is the basic material for building cells and repairing tissues. It is used to make antibody proteins to fight against bacterial and viral infections, to make hemoglobin to transport oxygen, and to make enzymes and hormones to maintain and regulate metabolism. Long-term use of donkey hide gelatin can keep the free radicals in our body balanced. Long-term use of donkey hide gelatin can dilate blood vessels, improve microcirculation, delay aging, resist fatigue, supplement calcium, prevent osteoporosis, and fight cancer, among other benefits.

[0004] At present, enzymatic hydrolysis and fermentation are usually used to decompose the protein in donkey-hide gelatin into small molecular peptides to improve bioavailability, but the donkey-hide gelatin peptides prepared by existing methods have problems such as long time consumption, low purity, and low activity. The above factors restrict the application of donkey-hide gelatin in anti-aging. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a donkey-hide gelatin peptide that is beneficial to the function of removing free radicals in the human body and a preparation method thereof. The present invention separates and purifies three active short peptides from donkey-hide gelatin for the first time, and the donkey-hide gelatin peptide has good anti-free radical activity, can effectively remove residual DPPH free radicals and ABTS free radicals in the body, and has great application prospects.

[0006] Specifically, the present invention first provides a donkey-hide gelatin peptide that is beneficial to the function of removing free radicals from the human body, characterized in that the amino acid sequence of the donkey-hide gelatin peptide is selected from one or more combinations shown in SEQ ID NO.1-3.

[0007] Preferably, the amino acid sequence of the donkey-hide gelatin peptide is shown in SEQ ID NO.1.

[0008] Preferably, the amino acid sequence of the donkey-hide gelatin peptide is shown in SEQ ID NO.2.

[0009] Preferably, the amino acid sequence of the donkey-hide gelatin peptide is shown in SEQ ID NO.3.

[0010] On the other hand, the present invention also provides a method for preparing donkey-hide gelatin peptide, the method comprising the following steps:

[0011] 1) Preparation: Wash the donkey-hide gelatin, cut it into pieces, and air-dry it for later use;

[0012] 2) Physical crushing: Put the air-dried donkey-hide gelatin pieces from step 1) into a wall-breaking machine and crush them into granules;

[0013] 3) Acid water treatment: 15-35 mg of the fine donkey-hide gelatin particles obtained in step 2) were added to 50-1001 mL of a citric acid aqueous solution with a pH of 3-5, and the mixture was heated to boiling and maintained for 60-120 min, with continuous stirring during the period;

[0014] 4) Composite enzymatic hydrolysis: when the temperature of step 3) is cooled to room temperature, 100-210 U of composite protease is added for enzymatic hydrolysis; after enzymatic hydrolysis, the enzyme is inactivated, and the supernatant is separated by centrifugation to obtain donkey-hide gelatin enzymatic hydrolyzate;

[0015] 5) Filtration and concentration: The donkey-hide gelatin peptide hydrolysate in step 4) is filtered using an ultrafiltration membrane with a molecular weight cutoff of 1000-3500 Da polypeptide molecules; the filtered donkey-hide gelatin peptide is vacuum concentrated to obtain a donkey-hide gelatin peptide concentrate;

[0016] 6) Identification of donkey-hide gelatin peptides: The donkey-hide gelatin peptide concentrate from step 5) was sequenced by Obitrap Fusion Lumos mass spectrometer;

[0017] 7) Purity identification of donkey-hide gelatin peptide: The donkey-hide gelatin peptide concentrate is transferred to a spray dryer with an inlet air temperature of 95-100°C and an outlet air temperature of 75-85°C for concentration until the moisture content of the donkey-hide gelatin peptide dry powder is 4-6%. The dry powder is collected to obtain donkey-hide gelatin peptide.

[0018] Preferably, in step 1), the size of the fragments is 0.5-1.0 cm in length, width and height.

[0019] Preferably, the cell breaking conditions in step 2) are 60000-100000 r / min for 15-30 min.

[0020] Preferably, in step 4), the composite protease is a combination of neutral protease, papain and collagenase, and the mass ratio of the three is (1-2):(2-5):(1-2);.

[0021] Preferably, the enzymatic hydrolysis conditions in step 4) are 37-45° C. with continuous stirring for 10-12 h.

[0022] Preferably, the method of inactivating the enzyme in step 4) comprises heating the enzymatic hydrolysate to 90-100° C. and maintaining the temperature for 15-20 minutes to inactivate the enzyme.

[0023] Preferably, the centrifugal separation in step 4) is performed at 5000-12000 r / min for 10-60 min.

[0024] Preferably, the vacuum concentration in step 5) is performed to a concentration of 10-18° Bé.

[0025] Preferably, the amino acid sequences of the donkey-hide gelatin peptides identified in step 6) are shown in SEQ ID NOs. 1-3.

[0026] Another object of the present invention is to provide a product or kit for removing free radicals from the human body, wherein the product or kit uses donkey-hide gelatin peptide as the main active ingredient, and the amino acid sequence of the donkey-hide gelatin peptide is any one or a combination of SEQ ID NO.1-3.

[0027] Furthermore, the present invention also provides the use of the donkey-hide gelatin peptide in the preparation of a product or a kit for removing free radicals from the human body, wherein the amino acid sequence of the donkey-hide gelatin peptide is any one or a combination of SEQ ID NO. 1-3.

[0028] The advantages of the present invention are as follows: the present invention separates and purifies three active short peptides from donkey-hide gelatin for the first time, and the donkey-hide gelatin 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

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Example 1 Preparation, separation and identification of donkey-hide gelatin peptide

[0033] The preparation method of donkey-hide gelatin peptide comprises the following steps:

[0034] 1) Preparation: Wash the donkey-hide gelatin, cut it into pieces with a length, width and height of 0.5 cm, and air-dry it for later use;

[0035] 2) Physical crushing: put the air-dried donkey-hide gelatin pieces obtained in step 1) into a wall-breaking machine and crush them into particles at a speed of 60,000 r / min for 15 min;

[0036] 3) Acid water treatment: 15 mg of the fine donkey-hide gelatin particles obtained in step 2) were added to 50 mL of a pH=3 citric acid aqueous solution, and the mixture was heated to boiling and kept for 75 min, with continuous stirring during the period;

[0037] 4) Composite enzymatic hydrolysis: when the temperature of step 3) is cooled to room temperature, 100U composite protease is added, and the enzymatic hydrolysis is carried out at 37°C with continuous stirring for 10h, and the temperature is raised to 90°C for 15min to inactivate the enzyme, followed by centrifugation at 5000rpm for 10min, and the supernatant is separated to obtain donkey-hide gelatin hydrolysate; wherein the composite protease is a combination of neutral protease (enzyme activity of 100U / mg), papain (enzyme activity of 100U / mg) and collagenase (enzyme activity of 25U / mg), and the mass ratio of the three is 1:2:1;

[0038] 5) Filtration and concentration: The donkey-hide gelatin peptide hydrolysate in step 4) is filtered using an ultrafiltration membrane with a molecular weight cutoff of 1000-3500Da polypeptide molecules; the filtered donkey-hide gelatin peptide is vacuum concentrated to a concentration of 14°Bé to obtain a donkey-hide gelatin peptide concentrate;

[0039] 6) Identification of donkey-hide gelatin peptides: The donkey-hide gelatin peptide concentrate from step 5) was sequenced by Obitrap Fusion Lumos mass spectrometer;

[0040] 7) Purity identification of donkey-hide gelatin peptide: The donkey-hide gelatin peptide concentrate was transferred to a spray dryer with an inlet air temperature of 95° C. and an outlet air temperature of 75° C. to concentrate the donkey-hide gelatin peptide dry powder to a water content of 4 wt %. The dry powder was collected to obtain donkey-hide gelatin peptide.

[0041] The results showed that three kinds of donkey-hide gelatin peptides were obtained through sequencing and identification analysis, and the sequences were shown in SEQ ID NO.1-3. After purity determination, the purity of the obtained donkey-hide gelatin peptides could reach 90.4%, 92.3% and 89.7% respectively.

[0042] Example 2 Analysis of Antioxidant Activity of Donkey-hide Gelatin

[0043] 1. DPPH free radical scavenging activity assay

[0044] The three kinds of donkey-hide gelatin peptides separated in Example 1 were respectively prepared with sterile distilled water to a concentration of 50 μg / mL, and stirred to be fully dissolved.

[0045] Treatment group: Take 0.5 mL of donkey-hide gelatin aqueous solution, add 0.5 mL of 0.5 mol / L DPPH solution, mix well, react at room temperature in the dark for 30 min, centrifuge at 5000 rpm for 5 min, and measure the absorbance A1 at a wavelength of 517 nm.

[0046] Blank group: 0.5 mL of 0.5 mol / L DPPH solution was added to 0.5 mL of deionized water. After mixing evenly, the mixture was reacted at room temperature in the dark for 30 min, centrifuged at 5000 rpm for 5 min, and the absorbance A0 was measured at a wavelength of 517 nm.

[0047] The calculation formula is shown in Formula I:

[0048] DPPH free radical scavenging rate (%) = (1-(A1÷A0))×100% (Formula I).

[0049] 2. ABTS free radical scavenging activity assay

[0050] The three kinds of donkey-hide gelatin peptides separated in Example 1 were respectively prepared with sterile distilled water to a concentration of 50 μg / mL, and stirred to be fully dissolved.

[0051] Prepare 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution with distilled water. Mix ABTS solution and potassium persulfate solution in a volume ratio of 1:1, place at 23°C for 12 hours in a dark place to form ABTS cation free radical stock solution, and then store at 4°C. Dilute the ABTS cation free radical solution appropriately with distilled water before use, so that the absorbance of the diluted solution at a wavelength of 734 nm is 0.70±0.02, and obtain the ABTS cation free radical working solution.

[0052] Treatment group: 100 μL of donkey-hide gelatin aqueous solution was added to 100 μL of ABTS cation free radical working solution and mixed evenly, reacted at 30°C for 10 min, and the absorbance B1 was immediately measured at 734 nm.

[0053] Blank group: 100 μL of deionized water was added to 100 μL of ABTS cation radical working solution and mixed evenly. The mixture was reacted at 30°C for 10 min and the absorbance B0 was immediately measured at 734 nm.

[0054] The calculation formula is shown in Formula II:

[0055] ABTS free radical scavenging rate (%) = (1-(B1÷B0)) × 100% (Formula II).

[0056] The results are shown in Table 1. All three donkey-hide gelatin peptides have excellent antioxidant activity, especially SEQ ID NO.3, which has the most significant antioxidant activity and the smallest IC50 value.

[0057] Table 1: Antioxidant activity analysis of donkey-hide gelatin peptide

[0058]

[0059]

[0060] 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 donkey-hide gelatin peptide that is beneficial to the function of removing free radicals in the human body, characterized in that: The amino acid sequence of the donkey-hide gelatin peptide is selected from one or more combinations shown in SEQ ID NO. 1-3.

2. The method for preparing donkey-hide gelatin according to claim 1, characterized in that: The method comprises the following steps: 1) Preparation: Wash the donkey-hide gelatin, cut it into pieces, and air-dry it for later use; 2) Physical crushing: Put the air-dried donkey-hide gelatin pieces from step 1) into a wall-breaking machine and crush them into granules; 3) Acid water treatment: 15-35 mg of the fine donkey-hide gelatin particles obtained in step 2) were added to 50-1001 mL of a citric acid aqueous solution with a pH of 3-5, and the mixture was heated to boiling and maintained for 60-120 min, with continuous stirring during the period; 4) Composite enzymatic hydrolysis: when the temperature of step 3) is cooled to room temperature, 100-210 U of composite protease is added for enzymatic hydrolysis; after enzymatic hydrolysis, the enzyme is inactivated, and the supernatant is separated by centrifugation to obtain donkey-hide gelatin enzymatic hydrolyzate; 5) Filtration and concentration: The donkey-hide gelatin peptide hydrolysate in step 4) is filtered using an ultrafiltration membrane with a molecular weight cutoff of 1000-3500 Da polypeptide molecules; the filtered donkey-hide gelatin peptide is vacuum concentrated to obtain a donkey-hide gelatin peptide concentrate; 6) Identification of donkey-hide gelatin peptides: The donkey-hide gelatin peptide concentrate from step 5) was sequenced by Obitrap Fusion Lumos mass spectrometer; 7) Purity identification of donkey-hide gelatin peptide: The donkey-hide gelatin peptide concentrate is transferred to a spray dryer with an inlet air temperature of 95-100°C and an outlet air temperature of 75-85°C for concentration until the moisture content of the donkey-hide gelatin peptide dry powder is 4-6%. The dry powder is collected to obtain donkey-hide gelatin peptide.

3. The method according to claim 2, characterized in that In step 1), the size of the fragments is 0.5-1.0 cm in length, width and height.

4. The method according to claim 2, characterized in that Step 2) The cell wall breaking condition is 60000-100000 r / min for 15-30 min.

5. The method according to claim 2, characterized in that Step 4) The composite protease is a combination of neutral protease, papain and collagenase, and the mass ratio of the three is (1-2):(2-5):(1-2).

6. The method according to claim 2, characterized in that The enzymatic hydrolysis conditions in step 4) are 37-45° C. with continuous stirring for 10-12 hours.

7. The method according to claim 2, characterized in that The method for inactivating the enzyme in step 4) comprises heating the enzymatic solution to 90-100° C. and maintaining the temperature for 15-20 minutes to inactivate the enzyme.

8. The method according to claim 2, characterized in that The centrifugal separation in step 4) is performed at 5000-12000 r / min for 10-60 min.

9. The method according to claim 2, characterized in that The vacuum concentration in step 5) is carried out to a concentration of 10-18° Bé.

10. A product or kit for removing free radicals from the human body, wherein the product or kit uses donkey-hide gelatin peptide as an active ingredient, and the amino acid sequence of the donkey-hide gelatin peptide is any one or a combination of SEQ ID NO. 1-3.

Images