Xuanwei ham-derived antioxidant peptide, preparation method and activity determination method thereof

Abstract

The present application relates to the technical field of antioxidant peptides, and particularly relates to a Xuanwei ham-derived antioxidant peptide, a preparation method and an activity determination method of the Xuanwei ham-derived antioxidant peptide, and an amino acid sequence of the Xuanwei ham-derived antioxidant peptide is shown as SEQ ID NO:1 or SEQ ID NO:2. The Xuanwei ham-derived hexapeptide is connected by using rigid connection peptides and flexible connection peptides, so that the peptide chain is lengthened, and the Xuanwei ham-derived hexapeptide is suitable for gene recombination expression, and meanwhile, the proportion of the target polypeptide in the recombinant protein is increased, and the expression is greatly increased. Secondly, the rigid connection peptides have alpha helix structures, so that the antioxidant peptides after connection can easily form stable secondary structures, and meanwhile, the active functional domains can be effectively separated. The flexible connection peptides are mainly glycines Gly, and the molecular mass is small, so that the freedom of the polypeptide skeleton conformation is ensured, the protein has sufficient space folding to obtain original biological activity, so that the activity of the fusion protein is higher than that of the parent protein.

Description

Technical Field

[0001] This invention relates to the field of antioxidant peptide technology, specifically to an antioxidant peptide derived from Xuanwei ham, its preparation method, and its activity assay method. Background Technology

[0002] In today's society, people are paying increasing attention to health and longevity, and antioxidants have attracted widespread interest in this field. Oxidative stress is a biochemical imbalance caused by the excessive accumulation of free radicals and oxidizing substances, which can lead to lipid oxidation, protein oxidation, and DNA damage. These oxidative processes are closely related to the development of various diseases, including cardiovascular disease, diabetes, atherosclerosis, Alzheimer's disease, and cancer. In recent years, with researchers' continued focus on bioactive peptides, a variety of bioactive peptides have been isolated and identified from different types of cured hams. These peptides possess rich amino acid sequences and multiple biological functions, such as antioxidation, lowering blood pressure, lowering lipids, and lowering blood sugar, and are considered to have potential health benefits. For example, Elizabeth Escudero (Escudero, E., Aristoy, M.-C., Nishimura, H., Arihara, K., & Toldrá, F. (2012). Antihypertensive effect and antioxidant activity of peptide fractions extracted from Spanish dry-cured ham. Meat Science, 91(3), 306-311.) found that a peptide extract with a molecular weight not exceeding 1700 Da extracted from Spanish dry-cured ham had hypotensive and antioxidant activities; Chao-Zhi (Zhu, CZ, Zhang, WG, Kang, ZL, Zhou, GH, & Xu, XL (2014). Stability of an antioxidant peptide extracted from Jinhua ham. Meat Science, 96(2), 783-789) found that the natural peptide extracted and isolated from Jinhua ham had high free radical scavenging activity. These bioactive peptides have molecular weights ranging from 400 to 2000 Da and sequence lengths between 5 and 20 amino acids. They can be used as functional foods or drugs for treating diseases. CN115260288A discloses an antioxidant peptide complex derived from dried cured ham, containing 42 peptides with molecular weights <3.0 kDa. The three peptides with the highest proportions are bioactive peptide 1 (23.56%), bioactive peptide 2 (13.64%), and bioactive peptide 3 (12.98%). The amino acid sequence of bioactive peptide 1 is: LGEHNIDVLEGNEQFINAAK, the amino acid sequence of bioactive peptide 2 is: GHYTEGAELVDSVLDVVR, and the amino acid sequence of bioactive peptide 3 is: DLVILLYETALLSSGFSLEDPQTHANR.

[0003] However, like most antioxidant peptides, large-scale preparation is difficult due to the low yield, high cost, and time-consuming separation process of peptides isolated from ham. Currently, most antioxidant peptides are still in the laboratory-scale research stage, indicating that obtaining antioxidant peptides through this separation and extraction method cannot meet the requirements of industrial production. Furthermore, the sequences of antioxidant peptides isolated from ham typically consist of 5 to 20 amino acids, which are relatively short and easily degraded by proteases or peptidases in E. coli expression systems. Therefore, direct expression of these peptides is challenging. Summary of the Invention

[0004] To address the challenges of normal transcription and translation during direct recombinant expression of oligopeptides, their susceptibility to degradation by proteases or peptidases in E. coli expression systems, and to improve peptide activity, the primary objective of this invention is to provide an antioxidant peptide derived from Xuanwei ham.

[0005] The second objective of this invention is to provide a method for preparing antioxidant peptides derived from Xuanwei ham.

[0006] The third objective of this invention is to provide a method for determining the activity of antioxidant peptides derived from Xuanwei ham.

[0007] This invention uses Xuanwei ham peptide as a raw material and employs tandem repeat expression, utilizing different linkage methods to repeatedly link the same peptide sequence to form polypeptide molecules, thereby increasing its stability, bioactivity, and yield. This invention achieves high-level expression of Xuanwei ham peptide by constructing an expression vector for the Xuanwei ham peptide gene in *E. coli*.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] An antioxidant peptide derived from Xuanwei ham, the amino acid sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2. Its corresponding gene sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0010] A method for preparing antioxidant peptides derived from Xuanwei ham includes the following steps:

[0011] (1) Based on the amino acid sequence of Xuanwei ham peptide NPPKFD, the Xuanwei ham peptide NPPKFD is hexaploidized by using rigid linker peptide (EAAAK)2 or flexible linker peptide (GGGGS)2 to obtain the target gene; the sequences of the Xuanwei ham peptide, rigid linker peptide and flexible linker peptide are shown in SEQ ID NO: 9-11, and the sequence of the target gene is shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0012] (2) The target gene was double-digested with the prokaryotic expression plasmid pET-28a(+) using restriction endonucleases Nde I and Xho I, and then ligated with DNA ligase to obtain the prokaryotic recombinant expression vectors pET-28a(+)-G6 and pET-28a(+)-R6.

[0013] (3) The prokaryotic recombinant expression vector was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method to construct recombinant engineered strains BL21(DE3)-pET-28a(+)-G6 and BL21(DE3)-pET-28a(+)-R6;

[0014] (4) Colony PCR confirmed that the recombinant expression vector was successfully transformed;

[0015] (5) The engineered bacteria BL21(DE3)-pET-28a(+)-G6 and BL21(DE3)-pET-28a(+)-R6 were induced to express tandem recombinant peptides with His tags;

[0016] (6) The expressed His-tagged fusion protein was purified using a nickel affinity chromatography column: After protein loading, the His-tagged fusion protein specifically binds to the nickel column, while other proteins elute due to non-specific binding; elution is performed using an imidazole gradient, as imidazole reacts with Ni... 2+ The fusion protein is released by competitively binding to a nickel column, and the eluent is collected.

[0017] (7) The purity was determined by RP-HPLC to obtain high-purity tandemly recombinant antioxidant peptides.

[0018] (8) Protein concentration determination: Protein concentration was determined using the Bradford method, measuring the absorbance at 595 nm, and calculated according to the standard curve (y = 0.6361x + 0.477, R0). 2 =0.9923) Calculate the total protein concentration of the sample.

[0019] The rigid linker peptide (EAAAK)2 used in this invention has an α-helix structure, which makes it easy for the tandem antioxidant peptides to form a stable secondary structure. At the same time, it can effectively separate the active functional domains, thereby enabling the fusion protein to have higher activity than the parent protein.

[0020] The flexible linker peptide (GGGGS)2 used in this invention is mainly composed of glycine (Gly), which has a small molecular weight and can maximize the freedom of peptide skeletonization, allowing the protein to have sufficient space to fold and obtain its original biological activity.

[0021] This invention uses pET-28a(+) plasmid containing two His tags. The His tag is a polypeptide sequence with 6 histidine residues, which facilitates subsequent protein purification and localization.

[0022] In the preparation method of the antioxidant peptide derived from Xuanwei ham in this invention, the primers used for colony PCR are:

[0023] XHP-G6 upstream primer: 5'-TGAATCCGCCGAAATTCGACGAAG-3', downstream primer: 5'-TCTTTCGCAGCCGCTTCATCG-3';

[0024] XHP-R6 upstream primer: 5'-CATATGAATCCGCCGAAATTCG-3', downstream primer: 5'-CTCGAGATCGAATTTTGGCGG-3'; sequences are shown in SEQ ID NO: 5-8.

[0025] In the preparation method of the antioxidant peptide derived from Xuanwei ham in this invention, the fermentation conditions for inducing expression of the peptide are as follows: isopropyl thiogalactoside is cultured at 37°C at 220 r / min for 8 h until the final concentration is 0.5 mmol / L.

[0026] The present invention describes a method for determining the activity of antioxidant peptides derived from Xuanwei ham, comprising a hydroxyl radical scavenging method and a DPPH radical scavenging method. Hydroxyl radicals (·OH) are highly reactive oxygen free radicals that can trigger oxidative stress in the body, leading to cell damage and disease. Therefore, measuring the scavenging ability of antioxidants against hydroxyl radicals can indirectly assess their antioxidant activity. The DPPH radical scavenging method assesses the antioxidant capacity by measuring the sample's ability to scavenge 2,2-diphenyl-1-picrylhydrazine (DPPH) radicals. DPPH solution is purple with a maximum absorption peak; it fades upon reaction with antioxidants. The scavenging rate is calculated by measuring the change in absorption of the solution using a spectrophotometer.

[0027] Specifically, the hydroxyl radical scavenging method involves mixing 1 mL of recombinant polypeptide sample with 1 mL of ferrous sulfate and 1 mL of hydrogen peroxide; maintaining the mixture at 37°C for 10 min; then mixing the solution with 1 mL of salicylic acid; using distilled water instead of the sample solution as a control group; and measuring the absorbance at 510 nm after incubation for 30 min.

[0028] Hydroxyl radical scavenging activity is calculated using the following formula:

[0029] Where Ai: absorbance of the sample;

[0030] A0: Absorbance of the blank control group.

[0031] The DPPH free radical scavenging method specifically involves: weighing a certain amount of DPPH and preparing a 0.04 mg / mL DPPH solution with anhydrous ethanol; taking 2 mL of recombinant peptide solutions of different concentrations (1 mg / mL) and adding 2 mL of DPPH solution, mixing thoroughly, incubating at room temperature for 30 min, and then centrifuging at 5000 r / min for 10 min; measuring the absorbance of the supernatant at 517 nm; using vitamin C as a positive control; and calculating the DPPH free radical scavenging rate of the sample using the following formula:

[0032] DPPH removal rate = 1 - (A1 - A2) / A0 * 100%

[0033] The absorbance of A0-2mL anhydrous ethanol + 2mL DPPH solution;

[0034] A1: Absorbance of 2 mL sample solution + 2 mL DPPH solution;

[0035] A2 - Absorbance of 2 mL sample solution + 2 mL anhydrous ethanol.

[0036] Compared with the prior art, the beneficial effects of the present invention are:

[0037] This invention uses rigid linker peptide (EAAAK)2 and flexible linker peptide (GGGGS)2 to link hexapeptide derived from Xuanwei ham, which can elongate the peptide chain, making it suitable for gene recombination expression. At the same time, it increases the proportion of the target peptide in the recombinant protein, greatly increasing expression. Secondly, the rigid linker peptide has an α-helix structure, which makes it easier for the tandem antioxidant peptide to form a stable secondary structure, and can effectively separate the active functional domain. The flexible linker peptide is mainly composed of glycine (Gly), with a small molecular weight, which can maximize the freedom of peptide skeletonization, allowing the protein to have sufficient space folding to obtain the original biological activity, thereby making the fusion protein more active than the parent protein.

[0038] This invention utilizes the smaller His tag on pET-28a(+) for affinity chromatography purification, resulting in high purity and specificity of the purified protein, improving purification efficiency and reducing the difficulty of peptide separation.

[0039] This invention benefits from the fact that the *E. coli* expression system used in this invention has a clear expression background, high expression level, simple operation, short culture cycle, and strong resistance to contamination. Furthermore, the raw materials for the *E. coli* growth culture medium are inexpensive and easy to mass-produce to obtain large quantities of antioxidant peptides, resulting in cost savings compared to traditional enzymatic hydrolysis methods.

[0040] The preparation method of this invention is simple to operate and can quickly construct engineered strains that express recombinant tandem antioxidant peptide fusion genes in prokaryotes. Through examples, it has been shown that the hydroxyl radical scavenging ability of the tandem antioxidant peptides is about 3 times higher than that before modification, which lays the foundation for the application of small molecule active peptides in the field of health food.

[0041] The research conducted in this invention can provide new data and evidence for exploring the biological functions and nutritional value of Xuanwei ham peptides, and contribute to the development and innovation of the biotechnology industry. Attached Figure Description

[0042] Figure 1 These are restriction enzyme electrophoresis images of XHP-G6 (left) and XHP-R6 (right); M: standard DNA molecular weight; lane 1: original plasmid electrophoresis; lane 2: molecular weight of fragment after NdeI / XhoI digestion.

[0043] Figure 2 This is an agarose gel electrophoresis image; lane M: standard molecular weight of DNA; lanes 1-2 are XHP-G6, XHP-R6, and the molecular weight of the target gene, respectively.

[0044] Figure 3 This is an SDS-PAGE electrophoresis image; lane M: protein marker. Lanes 1-2 are XHP-G6 and XHP-R6 recombinant proteins, respectively.

[0045] Figure 4 This is an SDS-PAGE electrophoresis image; lane M: protein marker. Lanes 1-2 show the purified XHP-G6 and XHP-R6 proteins, respectively.

[0046] Figure 5 (a) and Figure 5 (b) shows the RP-HPLC chromatograms of XHP-G6 and XHP-R6.

[0047] Figure 6 (a) and Figure 6 (b) is a graph showing the antioxidant activity analysis. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Example 1: Recombinant Xuanwei Ham-derived Antioxidant Peptide Gene Design and Prokaryotic Expression Method

[0050] (1) Tandem design of antioxidant peptide sequences derived from Xuanwei ham

[0051] This method constructs a design containing the NPPKFD sequence, which elongates the NPPKFD peptide sequence and is suitable for gene recombination expression. Specifically, the NPPKFD is hexaploidized by using rigid linker peptide (EAAAK)2 and flexible linker peptide (GGGGS)2, respectively. Therefore, the new amino acid sequence of the designed peptide is finally shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0052] (2) Construct the target gene of tandem antioxidant peptides

[0053] Based on the amino acid sequences of the tandem antioxidant peptides, mRNA sequences corresponding to the amino acid sequences of the tandem antioxidant peptides were designed using the computer software Primer Premier 5.0, taking into account the codon bias of *E. coli*. Gene sequences designed using rigid-linked peptide tandem sequences are shown in SEQ ID NO:3. Gene sequences designed using flexible-linked peptide tandem sequences are shown in SEQ ID NO:4.

[0054] To improve protein expression in *E. coli*, the 86-amino acid protein sequence was optimized. Codon usage preferences were adjusted to suit the highest expression profile of the target host, with the CAI (codon fitness index) increased to 0.87 and 0.9, respectively (a CAI of 0.8–1.0 is considered good for high expression). The average GC content was adjusted to 55.07% and 64.86%, respectively, and unfavorable peaks were removed. Repetitive regions in the original sequence were removed to avoid stem-loop structures in the mRNA and to facilitate synthesis. Unwanted motifs, including restriction enzyme sites and negative cis-acting sites used for subcloning, were modified. The entire sequence was fine-tuned to improve translation efficiency and extend the mRNA half-life. The optimized gene sequence was then synthesized by Shanghai Sangon Biotech Co., Ltd.

[0055] (3) Constructing gene expression vectors

[0056] The restriction enzymes NdeI and XhoI were added to both ends of the active peptide. After double digestion of pET-28a(+) with NdeI and XhoI, the electrophoresis results are shown in the figure. Figure 1 As shown, the target gene was cloned into the pET-28a(+) plasmid using DNA ligase to construct a recombinant expression plasmid. A small amount of the recombinant plasmid pET-28a(+)-G6 was gently mixed with E. coli BL21(DE3) competent cells and incubated on ice for 30 min. After the ice incubation, the cells were heat-shocked at 42℃ for 90 s and immediately placed in ice to cool for 1-2 min.

[0057] Add 900 μL of LB liquid medium, mix gently, and incubate at 37°C with shaking for 1 hour to allow the bacteria to fully recover. Take an appropriate amount of bacterial culture and spread it on LB solid medium (containing 100 mg / L kanamycin) to screen for positive plasmids. Incubate at 37°C upside down overnight to confirm the culture colonies. DNA sequencing verifies that the recombinant plasmid construction is correct.

[0058] LB medium: 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl. Add 20.0g agar powder when using solid medium.

[0059] (4) Colony PCR verification

[0060] Single colonies were picked from the overnight cultured screening plate using a sterile 10 μL pipette tip and placed into a PCR tube containing 10 μL of sterile ddH2O for colony PCR verification.

[0061] The PCR amplification program was as follows: 94℃ pre-denaturation for 4 min, 35 amplification cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 40 s, followed by a 10 min extension at 72℃. Nucleic acid electrophoresis was performed after the PCR reaction. The upstream primer for XHP-G6 was 5'-TGAATCCGCCGAAATTCGACGAAG-3', and the downstream primer was 5'-TCTTTCGCAGCCGCTTCATCG-3'; the upstream primer for XHP-R6 was 5'-CATATGAATCCGCCGAAATTCG-3', and the downstream primer was 5'-CTCGAGATTTTGGCGG-3'. See SEQ ID NO: 5-8. Electrophoresis images are shown below. Figure 2 As shown.

[0062] Table 1 Colony PCR Validation System

[0063]

[0064] (5) Induced expression of recombinant Escherichia coli strains

[0065] The recombinant strains validated by PCR were inoculated into 3 ml of LB medium containing the resistant strain and cultured at 37°C and 220 rpm for 12 hours to prepare a seed culture. This seed culture was then inoculated at a ratio of 1% into 100 ml of LB liquid medium for expansion culture, with kanamycin added to a working concentration of 100 μg / ml. When the OD600nm of the bacterial culture reached 0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mmol / L, and the culture was incubated at 37°C and 220 rpm for 8 hours. The induced bacterial culture was centrifuged at 5000 rpm for 10 minutes, the supernatant was discarded, and the bacterial pellet was collected. The cells were resuspended in phosphate buffer, mixed thoroughly, and then sonicated in an ice-water bath under the following conditions: 250 W, 3 seconds of sonication, 5 seconds of rest, for 15 minutes. At this point, the whole bacterial protein was obtained, and the bacterial culture was clear and transparent. The ultrasonically disrupted bacterial culture was centrifuged at 12000 r / min for 15 min, and the supernatant was the supernatant protein. The supernatant and precipitate were separated, and the obtained whole bacterial culture, supernatant and precipitate samples were analyzed by SDS-PAGE to determine the expression of the fusion protein.

[0066] SDS-PAGE Analysis: Remove the flat / concave glass plates, clean and allow them to air dry. Secure the glass plates to the gel casting apparatus using a holder. Prepare the separating gel: After adding each component, mix thoroughly with a pipette. Add the gel along the glass plate seams, pressing it flat with distilled water. Allow it to stand at room temperature until a clear dividing line appears on the liquid surface. Then, discard the distilled water to complete the separating gel preparation. Prepare the upper layer stacking gel: After adding and mixing each component, add the upper layer gel along the gaps. Insert a comb and allow it to solidify at room temperature. After polymerization, install the two gel plates with the short plates facing inwards into the upper tank and assemble the inner tank fixing frame. Place the entire fixation device into the electrophoresis tank, add electrophoresis buffer (electrophoresis buffer formula: Tris buffer (25mM, pH 8.3), glycine (190mM), SDS (0.1%)), remove the comb, add 5 μL of standard protein and sample to the gel lanes, and add sample loading buffer if there are extra wells. Turn on the power, starting at 80V, for about 30 minutes. After entering the stacking gel, change the voltage to 120V. When the bromophenol blue front reaches the bottom of the electrophoresis tank, turn off the power (about 2-3 hours).

[0067] Sample preparation: Mix the lysate of *E. coli* cells and the supernatant after centrifugation with loading buffer. Add 40 μL of PBS buffer and 10 μL of 5x SDS loading buffer to the precipitate. Mix 40 μL of the supernatant after centrifugation with 10 μL of 5x SDS loading buffer, boil in a water bath for 5 min, centrifuge at 12000 rpm for 5 min, and collect the supernatant. Take 5 μL of the sample for protein electrophoresis. After electrophoresis, remove the glass plate, carefully pry it open and discard the stacking gel. Peel the gel from the gel casting plate and place it in deionized water to wash the gel. Place the protein gel in a staining cassette and stain with Coomassie Brilliant Blue R-250 for 2 h. After staining, place the gel in destaining solution for diffusion destaining until the blue on the background plate fades. Once bands are visible, photograph and record the image using a gel imaging system. The SDS-PAGE electrophoresis image is shown below. Figure 3 As shown.

[0068] Example 2: Purification of Recombinant Tandem Antioxidant Peptides

[0069] Take 5 mL of a pre-packed Ni-IDA column and allow the storage buffer to flow out by gravity. Equilibrate the column with two column volumes of Wash Buffer (20 mM Tris-HCl, 8 M urea, 500 mM NaCl, 5 mM imidazole, pH 8.0) at a flow rate of 0.5–1 mL / min, allowing the buffer to slowly drain from the resin. Filter the supernatant through a 0.45 μm microporous membrane and add it to the column. Collect the flow-through into a centrifuge tube. Wash the column with two column volumes of Wash Buffer and collect the flow-through. Finally, elute the histidine-tagged protein on the column with two column volumes of Elution Buffer (20 mM Tris-HCl, 8 M urea, 500 mM NaCl, 500 mM imidazole, pH 8.0). Store each eluent separately until the absorbance at 280 nm is close to the baseline. Analyze the purity of the collected eluent using SDS-PAGE and RP-HPLC. SD S-PAGE electrophoresis image as shown Figure 4 As shown, the RP-HPLC chromatogram is as follows: Figure 5 (a) and Figure 5 As shown in (b).

[0070] RP-HPLC conditions: Acquity (Watts Inc.) was used. The HPLC system was equipped with a reverse-phase BEH C18 analytical column (1.7 μm, 2.1 × 100 mm, Watts Inc.).

[0071] Mobile phase: Phase A was ultrapure water (0.1% trifluoroacetic acid), Phase B was acetonitrile (0.1% trifluoroacetic acid), flow rate was 0.8 mL / min; injection volume: 8 μL; column temperature: 30℃; UV detection wavelength: 220 nm.

[0072] Protein concentration was measured using the Bradford method, detecting the absorbance at 595 nm and applying it according to the standard curve (y = 0.6361x + 0.477, R0). 2 =0.9923) Calculate the total protein concentration of the sample. After conversion, the total protein concentration can reach 11.95 mg / L.

[0073] Example 3: Activity Verification of Xuanwei Ham-derived Tandem Antioxidant Peptides

[0074] (1) Determination of DPPH free radical scavenging activity

[0075] Weigh a certain amount of DPPH and prepare a 0.04 mg / mL DPPH solution with anhydrous ethanol. Take 2 mL of recombinant peptide solutions of different concentrations (1 mg / mL), add 2 mL of DPPH solution, mix well, incubate at room temperature for 30 min, and then centrifuge at 5000 rpm for 10 min. Take the supernatant and measure the absorbance at 517 nm. Use vitamin C as a positive control.

[0076] The scavenging rate of the sample against DPPH free radicals was calculated using the following formula:

[0077] DPPH removal rate = 1 - (A1 - A2) / A0 * 100%

[0078] Absorbance of A0-2mL anhydrous ethanol + 2mL DPPH solution:

[0079] A1 - 2mL sample solution + 2mL DPPH solution absorbance

[0080] A2 - Absorbance of 2 mL sample solution + 2 mL anhydrous ethanol.

[0081] (2) Determination of hydroxyl radical scavenging activity

[0082] 1 mL of the recombinant peptide sample was mixed with 1 mL of ferrous sulfate (9 mmol / L) and 1 mL of hydrogen peroxide (10 mmol / L). After incubation at 37°C for 10 min, the solution was mixed with 1 mL of salicylic acid (9 mmol / L). Distilled water was used instead of the sample solution for the control group. After incubation for 30 min, the absorbance was measured at 510 nm.

[0083] Hydroxyl radical scavenging activity is calculated using the following formula:

[0084]

[0085] Where Ai: absorbance of the sample;

[0086] A0: Absorbance of the blank control group.

[0087] The results showed that the antioxidant peptides prepared by rigidly linked peptides in this invention had hydroxyl radical and DPPH radical scavenging abilities of 79.46% and 57.25%, respectively; while the antioxidant peptides prepared by flexible linked peptides in this invention had hydroxyl radical and DPPH radical scavenging abilities of 87.42% and 68.05%, respectively. The antioxidant capacity of the peptides prepared in this invention was approximately three times higher than that of the peptides isolated and extracted from Xuanwei ham.

[0088] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An antioxidant peptide derived from Xuanwei ham, characterized in that: Its amino acid sequence is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. The antioxidant peptide derived from Xuanwei ham according to claim 1, characterized in that: The corresponding gene sequences are shown in SEQ ID NO:3 or SEQ ID NO:4.

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