Tilapia skin collagen active peptide with antioxidant effect and application thereof

Abstract

The present invention relates to the technical field of bioactive peptides, and in particular to an active peptide of tilapia skin collagen with antioxidant effect and its application. The active peptide comprises amino acid fragment 1: TMQTEADEE, amino acid fragment 2: EADIDGDGQ, and amino acid fragment 3: SVDDAINMA. The three active peptides have good antioxidant activity and can be used to prepare medicines with antioxidant efficacy. In the preparation process, trypsin and pepsin are used to enzymatically cut tilapia collagen to obtain peptide segments of specific length. The active peptide of tilapia skin collagen provided by the present invention is safe and non-toxic, easy to digest and absorb, and provides a new choice for solving the problems of potential toxicity and carcinogenicity of existing antioxidants. The implementation of the present invention not only helps to improve the added value of tilapia skin, but also provides a new source of raw materials for the development of antioxidant functional products, and has broad market prospects and application value.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive peptides, and in particular to a tilapia skin collagen active peptide with antioxidant effect and application thereof. Background Art

[0002] Tilapia, also known as African crucian carp, is an important freshwater fish breeding variety in my country. It has the characteristics of omnivorous diet, fast growth rate, strong stress resistance and short breeding cycle. In 2022, China's tilapia breeding output was 1.7389 million tons, making it the largest breeding, consumption and export trade country in the world. Among them, Hainan's tilapia breeding output was about 310,000 tons, accounting for 19% of the domestic tilapia production. Tilapia skin is rich in collagen, accounting for 63.30% of the total protein, and is a good raw material for the production of bioactive peptides. Collagen peptides are a class of small molecule amino acid sequences with multiple biological functions, with absorption mechanisms and functional properties that amino acids cannot match. Therefore, collagen peptides are widely used as biomaterials in pharmaceuticals, cosmetics, and biomedicine. As a medicine and cosmetic with special effects, collagen peptides play an important role in the field of beauty. It is widely used to improve the appearance and texture of the skin, reduce wrinkles and fine lines, enhance skin elasticity and firmness, and provide moisturizing and nourishing effects. Among them, there is a close connection between beauty and anti-oxidation, and anti-oxidation plays an important role in maintaining skin health and improving appearance. The main goal of anti-oxidation is to neutralize the activity of free radicals to reduce the damage of oxidative stress to the skin. In the process of free radical formation in the skin, it can cause oxidative damage to cells and tissues through oxidation reactions, which in turn leads to the degradation of collagen and elastic fibers, causing the appearance of signs of aging such as skin sagging, wrinkles and fine lines. The application of antioxidants can neutralize free radicals and reduce the damage of oxidative stress to the skin, thereby delaying the process of skin aging. In addition, antioxidants can also enhance the skin barrier function. The oxidative stress process will damage the skin barrier, making it easier for external irritants to enter the skin and induce inflammatory reactions. The application of antioxidants can enhance the resistance of the skin barrier and reduce the adverse effects of external factors on the skin, thereby maintaining the health of the skin.

[0003] The most widely used antioxidants at present are chemically synthesized butyl hydroxyanisole, propyl gallate, butyl hydroxytoluene and tert-butyl hydroquinone, etc. Although they have the advantages of high efficiency and low price, their potential toxicity and carcinogenicity limit their application in medicines, so people focus more on natural antioxidants. Antioxidant peptides are a class of natural bioactive peptides that have been widely studied in recent years. Antioxidant peptides can not only delay lipid oxidation in food, reduce oxidative stress response in the body, but also provide additional essential amino acids for the human body. As a natural antioxidant, antioxidant peptides have a relatively simple structure, are easy to absorb, have good stability and no immunoreactivity, which makes antioxidant peptides more and more popular in the field of medical drugs. Therefore, the present invention provides three active peptides with antioxidant effects derived from tilapia skin collagen, and is used to prepare medicines with antioxidant effects. Summary of the invention

[0004] In view of this, the present invention proposes a tilapia skin collagen active peptide with antioxidant effect and its application to solve the above problems.

[0005] The technical solution of the present invention is achieved as follows: a tilapia skin collagen active peptide with antioxidant effect comprises an amino acid fragment 1: TMQTEADEE, whose amino acid sequence is shown in SEQ ID No.1, an amino acid fragment 2: EADIDGDGQ, whose amino acid sequence is shown in SEQ ID No.2, and an amino acid fragment 3: SVDDAINMA, whose amino acid sequence is shown in SEQ ID No.3.

[0006] Preferably, the sequence listing of SEQ ID No. 1 is Thr-Met-Gln-Thr-Glu-Ala-Asp-Glu-Glu;

[0007] Preferably, the sequence listing of SEQ ID No. 2 is Glu-Ala-Asp-Ile-Asp-Gly-Asp-Gly-Gln;

[0008] Preferably, the sequence listing of SEQ ID No. 3 is Ser-Val-Asp-Asp-Ala-Ile-Asn-Met-Ala.

[0009] Furthermore, the tilapia skin collagen active peptide has antioxidant activity.

[0010] Furthermore, the active peptides were virtually digested by trypsin (EC3.4.21.4) and pepsin (EC3.4.23.1) on the obtained sequences.

[0011] Furthermore, the tilapia skin collagen active peptide is used in the preparation of medicines with antioxidant effects.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The identification of the antioxidant peptides from tilapia skin collagen of the present invention mainly includes three processes, namely: primary structure identification and computer virtual screening of tilapia skin collagen peptides; determination of antioxidant capacity of tilapia skin collagen peptides; determination of three antioxidant peptides; screening of antioxidant active peptides TMQTEADEE, EADIDGDGQ and SVDDAINMA from tilapia skin collagen, and clarifying the molecular inhibition mechanism of the active peptides and Keap1 active sites. The active peptide has a strong antioxidant activity, can effectively remove free radicals, inhibit the occurrence of oxidation reactions, and protect cells and tissues from oxidative damage. This characteristic makes it have broad application prospects in the preparation of drugs with antioxidant effects.

[0014] The present invention successfully obtains active peptide fragments with specific amino acid sequences by virtual enzymatic digestion of tilapia skin collagen with trypsin and pepsin. This enzymatic hydrolysis method can maintain the biological activity of the peptide fragments, while improving the purity and stability of the peptide fragments, providing reliable raw materials for subsequent applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure 2 is a diagram of the peptide TMQTEADEE-Keap1 interaction, where a is a 2D diagram and b is a 3D diagram;

[0016] Figure 2 Figure 2 is a diagram of the peptide EADIDGDGQ-Keap1 interaction, where a is a 2D diagram and b is a 3D diagram;

[0017] Figure 3 Figure 2 is a diagram of the peptide SVDDAINMA-Keap1 interaction, where a is a 2D diagram and b is a 3D diagram. DETAILED DESCRIPTION

[0018] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0019] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0020] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0021] Example 1 - Primary structure identification and computer virtual screening of tilapia skin collagen peptides

[0022] The present invention uses liquid chromatography-mass spectrometry tandem mass spectrometry technology to analyze tilapia skin collagen peptides, and clarifies the primary structure of tilapia skin collagen peptides (molecular weight 700-6000) according to database retrieval comparison. The online enzyme cutting tool ExPASyPeptideCutter is used to perform virtual enzyme cutting. The water solubility and toxicity of the polypeptide are predicted by the online programs Peptidepropertycalculator and ToxinPred. The non-toxic and water-soluble polypeptides screened are defined as ligands, and the X-ray crystal structure of the Kelch region (PDBID: 2FLU) of the receptor target Keap1 protein is obtained from the ProteinDataBank (PDB) database. Molecular docking is performed by DiscoveryStudio (DS) software to screen peptides that can tightly bind to the Keap1 active site, and potential active peptides with antioxidant effects are preliminarily determined. The following is a specific analysis:

[0023] The present invention uses liquid chromatography-mass spectrometry tandem mass spectrometry technology to analyze tilapia skin collagen peptides, and a total of 299 tilapia skin collagen peptides are identified through software library search qualitative analysis. The number of amino acids in the tilapia skin collagen peptides ranges from 9 to 43, and the molecular weight ranges from 799.4 to 4832.2 Da. The online enzyme cutting tool ExPASyPeptideCutter (http: / / web.expasy.org / peptide_cutter / ) is used to virtually cut the obtained sequence using trypsin (EC3.4.21.4) and pepsin (EC3.4.23.1), and a total of 1127 tilapia skin collagen peptides are obtained. The online programs Peptidepropertycalculator and ToxinPred are used to predict the toxicity and solubility of tilapia skin collagen peptides. The results show that 711 tilapia skin collagen peptides have good water solubility, non-mutagenicity, non-toxicity and non-sensitization. The X-ray crystal structure of the Kelch region of the receptor target Keap1 protein (PDBID: 2FLU) was obtained through the RCSBPDB database. After removing water molecules and adding hydrogen atoms from the Keap1 protein using DiscoveryStudio (DS) 2017R2 (Dassault Systemes Biovia, San Diego, CA, USA) software, its active center was defined in the "Receptor-Ligand Interactions" module: x = -2.330167, y = 6.213780, z = 1.648807, r = 22.351954 with a radius of The structure of the peptide was constructed by the "Macromolecules" module of the DS software, and the "MinimizeLigands" and "PrepareLigands" tools were used to optimize the energy of the peptide structure under the CHARMm force field. The receptor and peptide were molecularly docked by the "CDOCKER" tool of the "Receptor-LigandInteractions" module, and the active peptides with good potential antioxidant effects were screened according to the "–CDOCKER_Energy" value. The known antioxidant peptide YSNQNGRF (122.698 kcal / mol) was used as a positive control. As shown in Table 1, the top 10 tilapia skin collagen peptides with higher "–CDOCKER_Energy" values ​​binding to Keap1 are: TMQTEADEE, EVGDDQEE, SVDDAINMA, QVTEISMGE, GEPGEQGQK, SEENQSTWK, EADIDGDGQ, VVMISDTEK, EEIDSEGR and NAEVQEK

[0024] Table 1 Docking energy values ​​of tilapia skin collagen peptides and receptor Keap1

[0025]

[0026] Example 2-Determination of antioxidant capacity of tilapia skin collagen peptides

[0027] 1. DPPH free radical scavenging activity assay

[0028] Take a 96-well plate, add 0.5mL of DPPH solution (dissolved in 95% ethanol solution, with a concentration of 0.4mmol / L, prepared and used immediately, stored in the dark at 0-4°C), add 50μL of 0.1mg / mL sample and 150μLDPH reaction solution to each well, mix well and react at room temperature in the dark for 30min, measure the absorbance at 517nm, where the absorbance of deionized water mixed with DPPH reaction solution is the blank control, and each sample is paralleled to 3 groups, and the average value is taken. The calculation of DPPH free radical scavenging rate is shown in Formula 1.

[0029]

[0030] Among them, A0 is the absorbance value of the blank control measured by mixing deionized water and DPPH solution, and A1 is the absorbance value after adding antioxidant peptide solution to DPPH solution.

[0031] 2. ABTS free radical scavenging activity assay

[0032] Prepare 7mmol / L ABTS solution and 2.45mmol / L potassium persulfate solution with distilled water. Mix ABTS solution and potassium persulfate solution in a volume ratio of 1:1, place in a dark place at 23℃ for 12-16h, form ABTS cation free radical stock solution and store at 4℃. Dilute ABTS cation free radical solution appropriately with distilled water before use (about 20-25 times), so that the absorbance of the diluted solution at 734nm wavelength is 0.70±0.02, and obtain ABTS cation free radical working solution. Add 100μL 0.1mg / mL sample and 100μL ABTS cation free radical working solution to each well of a 96-well plate, mix, react at 30℃ for 10min, and immediately measure the absorbance at 734nm. The absorbance of the mixture of deionized water and ABTS cation free radical working solution is the blank control, and each sample is paralleled for 3 groups, and the average value is taken.

[0033] The ABTS free radical scavenging rate was calculated as shown in Formula 2.

[0034]

[0035] Wherein, B0 is the absorbance value of the blank control in which the ABTS free radical determination solution is added with deionized water without adding the antioxidant peptide solution, and B1 is the absorbance value after the ABTS free radical determination solution is added with the antioxidant peptide solution.

[0036] The results showed that peptides (TMQTEADEE), (EADIDGDGQ) and (SVDDAINMA) all had antioxidant activity, and their IC50 values ​​of DPPH free radical scavenging activity were 3.68mmol / L; 6.05mmol / L and 5.85mmol / L, respectively; their IC50 values ​​of ABTS free radical scavenging activity were 7.24mmol / L; 10.32mmol / L and 7.04mmol / L, respectively.

[0037] Example 3 - Determination of three antioxidant peptides

[0038] Studies have shown that peptides with antioxidant activity are not only related to peptide length and molecular weight, but also closely related to their hydrophobicity, amino acid composition and amino acid sequence. In the peptide sequence, Pro and His play an important role in antioxidant activity. The imidazole group of the His residue has the ability to scavenge free radicals and chelate metal ions. Proteins containing this residue have serine endopeptidase inhibitor activity and can reduce the accumulation of reactive oxygen. The indole ring (NH) in Trp, the phenolic hydroxyl group (OH) in Tyr, the thiol group (-SH) in Cys and the pyrrole ring in Pro act as hydrogen donors to scavenge free radicals. In addition, aromatic amino acids can significantly improve the activity of antioxidant peptides, while Ile and Leu may enhance antioxidant activity by assisting molecules in building a suitable hydrophobic environment. The 2D and 3D visualization results of the docking of tilapia skin collagen-derived antioxidant peptides TMQTEADEE, EADIDGDGQ and SVDDAINMA with Keap1 are shown below, respectively. Figure 1 , Figure 2 and Figure 3 As shown, the results indicate that residues Arg380, Arg415, Arg483, and Asn382 in the active site of Keap1 play an important role in the binding of antioxidant active peptides to Keap1, and are the key amino acids for the binding of antioxidant active peptides to Keap1. Hydrogen bond interactions and electrostatic interactions play a key role in the binding of tilapia skin collagen peptides to Keap1.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A tilapia skin collagen active peptide with antioxidant effect, characterized in that: The amino acid fragment 1 of the active peptide: TMQTEADEE, whose amino acid sequence is shown in SEQ ID No.

1.

2. A tilapia skin collagen active peptide with antioxidant effect, characterized in that: The amino acid fragment 2 of the active peptide: EADIDGDGQ, and its amino acid sequence is shown in SEQ ID No.

2.

3. A tilapia skin collagen active peptide with antioxidant effect, characterized in that: The amino acid fragment 3 of the active peptide: SVDDAINMA, the amino acid sequence of which is shown in SEQ ID No.

3.

4. A tilapia skin collagen active peptide with antioxidant effect as described in any one of claims 1 to 3, characterized in that: The active peptide uses trypsin and pepsin to enzymatically digest tilapia collagen.

5. Use of the tilapia skin collagen active peptide with antioxidant effect as described in any one of claims 1 to 3 in the preparation of medicines with antioxidant efficacy.

Images