Donkey-hide gelatin protein source hexapeptide with antioxidant activity and application thereof

Abstract

The invention relates to the technical field of bioactive peptides, and provides a donkey-hide gelatin protein source hexapeptide with antioxidant activity and application thereof, the amino acid sequence of the donkey-hide gelatin protein source hexapeptide is Gly-Pro-Ser-Gly-Pro-Gln, the donkey-hide gelatin protein source hexapeptide has the beneficial effects that the donkey-hide gelatin protein source hexapeptide is synthesized for the first time, and the donkey-hide gelatin protein source hexapeptide can effectively remove DPPH and ABTS free radicals, so that the antioxidant activity of the donkey-hide gelatin protein source hexapeptide is improved, and the antioxidant activity of the donkey-hide gelatin protein source hexapeptide is improved. The product has an anti-oxidation effect and can be applied to the fields of anti-oxidation supplements and functional foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and particularly relates to an Asini Corii Colla protein-derived hexapeptide with antioxidant activity and its application. Background Art

[0002] Asini Corii Colla is a solid glue made from the skin of Equus asinus L. (donkey) of the Equidae family after decocting and concentrating, containing various active ingredients such as gelatin, protein, and trace elements, and having various effects such as anti-fatigue, anti-tumor, enhancing immunity, and promoting hematopoietic function. Currently, Asini Corii Colla preparations are usually made by directly adding Asini Corii Colla powder, such as oral liquids, effervescent tablets, soft capsules, etc. However, Asini Corii Colla protein needs to be degraded into polypeptides by proteases before it can be absorbed by the body. For people with weak spleen and stomach and low digestive ability, taking Asini Corii Colla or its preparations directly not only brings a greater burden to the gastrointestinal tract, but also the medicinal effect of Asini Corii Colla cannot be fully exerted. Therefore, hydrolyzing Asini Corii Colla into protein peptides by controlled enzymatic hydrolysis technology has become the key to solving this problem.

[0003] Currently, domestic and foreign scholars have isolated and identified bioactive peptides from various animal and plant raw materials such as corn, potatoes, and egg whites, and there are also research reports that Asini Corii Colla enzymolysis products have functions such as blood-enriching and white blood cell-raising, and immune regulation. However, the research on the isolation and identification of Asini Corii Colla protein peptides and their active action mechanisms is relatively less, especially in terms of antioxidant function, and there are few relevant experimental studies reported. Therefore, exploring the potential active functions of Asini Corii Colla has important theoretical and practical significance for the industrialization of high-quality Asini Corii Colla series products, and at the same time provides new ideas and scientific bases for the modernization of traditional Chinese foods. Summary of the Invention

[0004] In view of this, the present invention provides an Asini Corii Colla protein-derived hexapeptide with antioxidant activity and its application, synthesizes the Asini Corii Colla protein-derived hexapeptide for the first time, and the Asini Corii Colla protein-derived hexapeptide can effectively scavenge DPPH and ABTS free radicals, has an antioxidant effect, and can be applied in the fields of antioxidant supplements and functional foods.

[0005] The technical solution is as follows: An Asini Corii Colla protein-derived hexapeptide with antioxidant activity, the amino acid sequence of the Asini Corii Colla protein-derived hexapeptide is Gly-Pro-Ser-Gly-Pro-Gln, and the molecular weight is 541.25 Da; wherein,

[0006] Gly represents the corresponding residue of the amino acid with the English name Glycine and the Chinese name glycine;

[0007] Pro represents the corresponding residue of the amino acid with the English name Proline and the Chinese name proline;

[0008] Ser represents the corresponding residue of the amino acid with the English name Serine and the Chinese name serine;

[0009] Gln represents the corresponding residue of the amino acid with the English name Glutarnine and the Chinese name glutamine.

[0010] During the use of the above technical solution: An ejiao protein-derived hexapeptide was synthesized for the first time.

[0011] Preferably, the ejiao protein-derived hexapeptide is synthesized by solid-phase synthesis using a peptide synthesizer, purified by high-performance liquid chromatography, and finally obtained by freeze-drying.

[0012] Preferably, the amino acid sequence of the ejiao protein-derived hexapeptide is obtained by Fmoc solid-phase synthesis technology. The C-terminal carboxyl group of the target polypeptide is covalently linked to a high-molecular resin, and then the amino group of this amino acid is condensed with the carboxyl group of another amino acid. Amino acids are sequentially added in the solid-phase synthesis order from the C-terminus to the N-terminus. After the target polypeptide sequence is formed, the peptide chain is separated from the resin, and finally the target polypeptide product is obtained. After the synthesis is completed, it is purified by high-performance liquid chromatography, and after quick-freezing in liquid nitrogen and vacuum freeze-drying, the target polypeptide is obtained.

[0013] Use of a kind of ejiao protein-derived hexapeptide with antioxidant activity in an antioxidant supplement, and the antioxidant supplement is a functional health product or a nutrient-fortified food.

[0014] An antioxidant supplement. When the concentration of the ejiao protein-derived hexapeptide is 10 mg / mL, the scavenging rate of DPPH free radicals can reach 81.29 ± 2.18%, and the scavenging rate of ABTS free radicals can reach 93.95 ± 0.86%.

[0015] During the use of the above technical solution: The ejiao protein-derived hexapeptide can effectively scavenge DPPH and ABTS free radicals, has an antioxidant effect, and can be used in the fields of antioxidant supplements and functional foods.

[0016] After adopting the above technical solution, the beneficial effects of the present invention are: An ejiao protein-derived hexapeptide was synthesized for the first time, and the ejiao protein-derived hexapeptide can effectively scavenge DPPH and ABTS free radicals, has an antioxidant effect, and can be used in the fields of antioxidant supplements and functional foods. Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0018] Figure 1 This is the LC-MS-MS spectrum of the six-peptide Gly-Pro-Ser-Gly-Pro-Gln from donkey-hide gelatin protein source of the present invention;

[0019] Figure 2 This is the HPLC spectrum of the six-peptide Gly-Pro-Ser-Gly-Pro-Gln from donkey-hide gelatin protein source of the present invention;

[0020] Figure 3 This is the MS spectrum of the six-peptide Gly-Pro-Ser-Gly-Pro-Gln from donkey-hide gelatin protein source of the present invention;

[0021] Figure 4 This is the DPPH free radical scavenging graph of the six-peptide Gly-Pro-Ser-Gly-Pro-Gln from donkey-hide gelatin protein source of the present invention;

[0022] Figure 5 This is the ABTS free radical scavenging graph of the six-peptide Gly-Pro-Ser-Gly-Pro-Gln from donkey-hide gelatin protein source of the present invention; Detailed implementation manners

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0024] Embodiment 1

[0025] As Figures 1 to 5 shown, a six-peptide from donkey-hide gelatin protein source with antioxidant activity, the sequence of the six-peptide from donkey-hide gelatin protein source is: Gly-Pro-Ser-Gly-Pro-Gln, and the molecular weight is 541.25 Da. Among them,

[0026] Gly represents the corresponding residue of the amino acid with the English name Glycine and the Chinese name Glycine;

[0027] Pro represents the corresponding residue of the amino acid with the English name Proline and the Chinese name Proline;

[0028] Ser represents the corresponding residue of the amino acid with the English name Serine and the Chinese name serine;

[0029] Gln represents the corresponding residue of the amino acid with the English name Glutarnine and the Chinese name glutamine.

[0030] The amino acid sequence of the colla-corii-asini protein-derived hexapeptide was identified from colla-corii-asini protein peptides with a molecular weight < 1 kDa by LC-MS / MS technology. Then it was obtained by Fmoc solid-phase synthesis technology. The C-terminal carboxyl group of the target polypeptide was covalently linked to the high-molecular resin, and then the amino group of this amino acid was condensed with the carboxyl group of another amino acid. Amino acids were sequentially added in the solid-phase synthesis order from the C-terminal to the N-terminal to form the target polypeptide sequence. After that, the peptide chain and the resin were separated, and finally the target polypeptide product was obtained. After the synthesis was completed, it was purified by high-performance liquid chromatography, quick-frozen in liquid nitrogen and freeze-dried in vacuum to obtain a polypeptide finished product with a purity of 96.49%.

[0031] The steps for obtaining the sequence of the colla-corii-asini protein-derived hexapeptide Gly-Pro-Ser-Gly-Pro-Gln are as follows:

[0032] S1. Preparation of colla-corii-asini protease hydrolysate: The colla-corii-asini block was crushed, passed through an 80-mesh sieve and then formulated into a 4% colla-corii-asini solution, and slowly stewed in a water bath at 80 °C for 30 min. After cooling to the temperature required for enzymatic hydrolysis, 65 °C, the pH of the colla-corii-asini solution was adjusted to 10. Then Alcalase 2.4L was added to the solution, and the enzyme-to-substrate ratio was 9% for enzymatic hydrolysis. During the enzymatic hydrolysis process, the pH range was maintained at 10 ± 0.05. After enzymatic hydrolysis for 2.5 h, the enzyme activity was inactivated in a water bath at 90 °C for 10 min, and then centrifuged at 4 °C (104 r / min) for 15 min, and the supernatant was collected and the pH was adjusted to 7.0;

[0033] S2. Preparation of colla-corii-asini protein peptides with a molecular weight < 1 kDa: The above-mentioned colla-corii-asini enzymatic hydrolysate was decolorized and defatted by an activated carbon filter, and then filtered through an ultrafiltration membrane with a cut-off molecular weight of 1 kDa, and colla-corii-asini protein peptides with a molecular weight < 1 kDa were obtained after freeze-drying;

[0034] S3. LC-MS / MS identification of colla-corii-asini protein peptides: The peptide sequence of the antioxidant active peptide described in step S2 was identified using a Nano LC-MS / MS mass spectrometer, and the peptide sequence was retrieved from the established database through MaxQuant 1.5.5.1 software, and a colla-corii-asini protein-derived hexapeptide was identified.

[0035] Experimental results: The LC-MS / MS spectrum is as Figure 1As shown, an ejiao protein-derived hexapeptide containing two Gly, two Pro, one Ser, and one Gln residues with a molecular weight of 541.25 Da was identified. Its amino acid sequence is Gly-Pro-Ser-Gly-Pro-Gln, which contains potential antioxidant activity.

[0036] The solid-phase synthesis steps of the ejiao protein-derived hexapeptide Gly-Pro-Ser-Gly-Pro-Gln are as follows:

[0037] S1. Solid-phase synthesis was carried out using a high-molecular resin according to the amino acid sequence Gly-Pro-Ser-Gly-Pro-Gln. First, the carboxyl terminus of Gln was covalently linked to the resin, then the amino terminus of Gln was condensed with the carboxyl terminus of Pro, and then the amino acids Gly → Ser → Pro → Gly were added in sequence from right to left. After the last amino acid was linked, the resin was removed, and the target polypeptide was finally obtained.

[0038] S2. Purification was carried out using high-performance liquid chromatography. The column model was Kromasil100-5C18, with a size of 4.6 * 250 mm. Mobile phase A was acetonitrile containing 0.1% (v / v) trifluoroacetic acid; mobile phase B was water containing 0.1% (v / v) trifluoroacetic acid. The elution conditions were as follows: 0 - 20.0 min: mobile phase A increased from 3% to 30%; 20.0 - 20.1 min: mobile phase A increased from 30% to 100%; the flow rate was 1.0 mL / min, and the detection wavelength was 220 nm. It was quickly frozen in liquid nitrogen and freeze-dried to obtain the ejiao protein-derived hexapeptide, with a required purity of over 95%. The actual purity was 96.49%, and the structure was identified by ESI-MS.

[0039] Example 2

[0040] Based on Example 1, as Figure 4 shown, the application of an ejiao protein-derived hexapeptide with antioxidant activity in an antioxidant supplement, where the antioxidant supplement is a functional health product or a nutrient-fortified food.

[0041] For an antioxidant supplement, when the concentration of the ejiao protein-derived hexapeptide is 10 mg / mL, the scavenging rate of DPPH free radicals can reach 81.29 ± 2.18%, and the scavenging rate of ABTS free radicals can reach 93.95 ± 0.86%.

[0042] The steps for scavenging DPPH free radicals of the ejiao protein-derived hexapeptide Gly-Pro-Ser-Gly-Pro-Gln are as follows:

[0043] S1. Prepare sample solutions of the donkey-hide gelatin protein-derived hexapeptide Gly-Pro-Ser-Gly-Pro-Gln with concentration gradients of 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, and 12 mg / mL;

[0044] S2. Prepare a 0.6 mM DPPH free radical solution;

[0045] S3. Take 100 μL of the sample solution, 100 μL of the DPPH free radical solution prepared in S2, and 100 μL of methanol into a 96-well plate, shake well, and incubate in the dark at 25 °C for 30 min. Then, measure the absorbance at 515 nm (using methanol as the blank). Finally, the scavenging ability of the sample against DPPH free radicals is calculated by the following formula:

[0046] DPPH free radical scavenging rate (%) = (1 - A_sample / A_blank) × 100

[0047] The sample described in S3 is the donkey-hide gelatin protein-derived hexapeptide Gly-Pro-Ser-Gly-Pro-Gln with different concentration gradients prepared in step S1.

[0048] Experimental results: As Figure 4 shown, with the increase in concentration, the DPPH free radical scavenging rate of the donkey-hide gelatin protein-derived hexapeptide Gly-Pro-Ser-Gly-Pro-Gln shows an upward trend. When the concentration is 10 mg / mL, the DPPH free radical scavenging rate can reach 81.29 ± 2.18%.

[0049] When the enzymatic hydrolysis pH is 11, the degree of hydrolysis reaches the maximum value of 13.57 ± 0.99%.

[0050] The steps for the ABTS free radical scavenging properties of the donkey-hide gelatin protein-derived hexapeptide Gly-Pro-Ser-Gly-Pro-Gln are as follows:

[0051] S1. Prepare solution A as a 7 mM ABTS solution and solution B as a 2.45 mM potassium persulfate solution. Then, take 10 mL of each of solution A and solution B, mix them evenly, and generate ABTS radical cations in the dark for 16 h. Dilute to an absorbance of 0.70 ± 0.02 at 734 nm before use;

[0052] S2. Prepare sample solutions of the donkey-hide gelatin protein-derived hexapeptide Gly-Pro-Ser-Gly-Pro-Gln with concentration gradients of 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, and 12 mg / mL;

[0053] S3. Take 50 μL of the sample solution and 150 μL of the diluted ABTS solution and mix them well on a 96-well plate (deionized water is used as the blank). After standing in the dark for 6 min, measure the absorbance at 734 nm. The scavenging activity of different samples against ABTS radicals is calculated as follows:

[0054] Scavenging rate of ABTS radicals (%) = (1 - A_sample / A_blank) × 100

[0055] The sample described in S3 is the hexapeptide Gly-Pro-Ser-Gly-Pro-Gln from donkey-hide gelatin protein source with different concentration gradients prepared in step S1.

[0056] Experimental results: As Figure 5 shown, with the increase of the concentration, the scavenging rate of ABTS radicals by the hexapeptide Gly-Pro-Ser-Gly-Pro-Gln from donkey-hide gelatin protein source shows an upward trend. When the concentration is 10 mg / mL, the scavenging rate of ABTS radicals can reach 93.95 ± 0.86%.

[0057] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A gelatin-derived hexapeptide with antioxidant activity, characterized in that: The amino acid sequence of the donkey-hide gelatin protein-derived hexapeptide is Gly-Pro-Ser-Gly-Pro-Gln.

2. The donkey-hide gelatin protein-derived hexapeptide with antioxidant activity according to claim 1, characterized in that: The donkey-hide gelatin protein-derived hexapeptide is synthesized by using a peptide synthesizer and a solid phase synthesis method, purified by high-performance liquid chromatography, and finally freeze-dried.

3. The use of the donkey-hide gelatin protein-derived hexapeptide with antioxidant activity in an antioxidant supplement according to any one of claims 1 to 2, characterized in that: The antioxidant supplement is a functional health product and a nutritionally fortified food.

4. An antioxidant supplement, comprising the use of the donkey-hide gelatin-derived hexapeptide with antioxidant activity according to claim 3 in an antioxidant supplement, characterized in that: When the concentration of the donkey-hide gelatin protein-derived hexapeptide is 10 mg / mL, the scavenging rate of DPPH free radicals can reach 81.29±2.18%, and the scavenging rate of ABTS free radicals can reach 93.95±0.86%.

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