Antioxidant peptides derived from xuanwei ham and methods for preparation and activity measurement thereof

Abstract

To provide antioxidant peptides derived from Xuanwei ham, and to provide methods for preparation and activity measurement thereof.SOLUTION: The present invention provides an antioxidant peptide with a specific amino acid sequence derived from Xuanwei ham. The present invention uses a rigid linking peptide and a flexible linking peptide to link a hexapeptide derived from Xuanwei ham, growing the peptide chain to make it suitable for genetic recombinant expression, while increasing the proportion of a target polypeptide in a recombinant protein and significantly increasing the expression level. The rigid linking peptide has an α-helical structure, which makes it easier for the antioxidant peptide to form a stable secondary structure after serial linking, and can effectively separate the active functional domain.The flexible linking peptide is mainly composed of glycine Gly, and since it has a low molecular weight, the flexibility of a polypeptide backbone structure is ensured.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the technical field of antioxidant peptides, in particular to antioxidant peptides derived from Xuanwei ham, their preparation methods and activity assay methods. [Background technology]

[0002] In today's society, people are increasingly interested in health and longevity, and antioxidants have attracted widespread interest in this field. Oxidative stress is a biochemical imbalance caused by the excessive accumulation of radicals and oxidants, which can lead to lipid oxidation, protein oxidation, DNA damage, etc. These oxidation processes are closely related to the development of many diseases, such as cardiovascular disease, diabetes, atherosclerosis, Alzheimer's disease, and cancer. In recent years, researchers have continued to pay attention to bioactive peptides, and various bioactive peptides have been isolated and identified from various raw hams, which have rich amino acid sequences and various biological functions such as antioxidant, blood pressure lowering, lipid lowering, and blood glucose lowering effects, and are believed to have potential health benefits. For example, Non-Patent Document 1 finds that peptide extracts with molecular weights of 1700 Da or less extracted from Spanish raw ham exhibit antihypertensive and antioxidant effects, and Non-Patent Document 2 finds that natural peptides extracted and isolated from Jinhua ham have high radical scavenging activity. These active peptides have a molecular weight of 400-2000 Da and a sequence length of 5-20 amino acids, and can be used as functional foods and medicines for diseases. Patent Document 1 discloses an antioxidant peptide complex derived from raw ham containing 42 peptide segments with a molecular weight of <3.0 kDa, and the three peptide segments with the highest proportions are bioactive peptide 1, which accounts for 23.56%, bioactive peptide 2, which accounts for 13.64%, and bioactive peptide 3, which accounts for 12.98%, respectively, and 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, the polypeptides isolated and extracted from ham have low yields, high costs, and a time-consuming isolation process, making them difficult to prepare on a large scale. At present, most antioxidant peptides are still in the laboratory-scale research stage, and it has been shown that obtaining antioxidant peptides with this separation and extraction method cannot meet the requirements for industrial production. In addition, the sequences of antioxidant peptides isolated from ham usually contain 5 to 20 amino acids, which are relatively short and easily degraded by proteases and peptidases in the E. coli expression system. Therefore, it is difficult to directly express these peptides. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Elizabeth Escudero(Escudero,E.,Aristoy,M.-C.,Nishimura,H.,Arihara,K.,&Toldra(a with acute accent),F.(2012).Antihypertensive effect and antioxidant activity of peptide fractions extracted from Spanish dry-cured ham.Meat Science,91(3),306-311.) [Non-Patent Document 2] 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) [Patent documents]

[0005] [Patent Document 1] Chinese Patent Application Publication No. 115260288A Summary of the Invention [Problem to be solved by the invention]

[0006] In order to solve the problems that oligopeptides are difficult to transcribe and translate normally during direct recombinant expression and are easily degraded by proteases and peptidases in the E. coli expression system, and to improve the activity of polypeptides, the first objective of the present invention is to provide an antioxidant peptide derived from Xuanwei ham.

[0007] The second object of the present invention is to provide a method for preparing antioxidant peptides from Xuanwei ham.

[0008] The third object of the present invention is to provide a method for measuring the activity of antioxidant peptides derived from Xuanwei ham.

[0009] The present invention uses Xuanwei ham peptide as raw material, repeatedly expresses it through tandem ligation, and uses different ligation methods to repeatedly ligate the same peptide sequence to form a polypeptide molecule, thereby increasing its stability, biological activity and yield. The present invention achieves high-level expression of Xuanwei ham peptide by constructing an expression vector of Xuanwei ham peptide gene in E. coli. [Means for solving the problem]

[0010] To achieve the above objectives, the present invention provides the following technical solutions:

[0011] 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. The corresponding gene sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0012] A method for preparing antioxidant peptides from Xuanwei ham, comprising the steps of: Step (1): According to the amino acid sequence of Xuanwei ham peptide NPPKFD, use rigid linking peptide (EAAAK)2 or flexible linking peptide (GGGGS)2 to link Xuanwei ham peptide NPPKFD to a hexaploid form to obtain a target gene, the sequences of the Xuanwei ham peptide, rigid linking peptide and flexible linking 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; (2) double-enzymatically cleaving the target gene and the prokaryotic expression plasmid pET-28a(+) with restriction endonucleases Nde I and Xho I and connecting them via DNA ligase to obtain the prokaryotic recombinant expression vectors pET-28a(+)-G6 and pET-28a(+)-R6; (3) transforming the prokaryotic recombinant expression vector into E. coli BL21(DE3) competent cells by heat shock method to construct recombinant engineering strains BL21(DE3)-pET-28a(+)-G6, BL21(DE3)-pET-28a(+)-R6; (4) verifying successful transformation of the recombinant expression vector by colony PCR; (5) inducing expression of the His-tagged tandemly linked recombinant polypeptides in the expression engineered strains BL21(DE3)-pET-28a(+)-G6 and BL21(DE3)-pET-28a(+)-R6; The expressed His-tagged fusion protein was purified using a nickel ion affinity chromatography column. When the protein was loaded, the His-tagged fusion protein specifically bound to the nickel column, while the impurity proteins flowed out due to non-specific binding to the nickel column. An imidazole gradient elution was used, and the His-tagged fusion protein was purified using a nickel ion affinity chromatography column. 2+ competitively binding to the nickel column, thereby releasing the fusion protein, and collecting the eluate (6); (7) obtaining high purity tandemly linked recombinant antioxidant peptides using RP-HPLC for purity testing; Protein concentration was measured using the Bradford method, detecting absorbance at 595 nm, and plotting the protein concentration using a standard curve (y = 0.6361x + 0.477, R 2 and (8) measuring the protein concentration, calculating the total protein concentration of the sample according to (A = 0.9923).

[0013] The rigid linking peptide (EAAAK)2 used in the present invention has an α-helical structure, which makes it easier for the antioxidant peptides to form a stable secondary structure after tandem linking, and at the same time, the active functional domains can be effectively separated, so that the fusion protein has higher activity than the parent protein.

[0014] The flexible linking peptide (GGGGS)2 used in the present invention is composed mainly of glycine Gly and has a small molecular weight, which ensures maximum flexibility of the polypeptide backbone structure, thereby allowing the protein to obtain sufficient spatial folding to obtain its inherent biological activity.

[0015] The present invention uses the pET-28a(+) plasmid, which contains two His tags, which are polypeptide sequences with six histidine residues that facilitate subsequent purification and localization of the protein.

[0016] In the method for preparing antioxidant peptides derived from Xuanwei ham of the present invention, the primers used in colony PCR are: XHP-G6 upstream primer: 5'-TGAATCCGCCGAAATTCGACGAAG-3', downstream primer: 5'-TCTTTCGCAGCCGCTTCATCG-3', XHP-R6: upstream primer: 5'-CATATGAATCCGCCGAAATTCG-3', downstream primer: 5'-CTCGAGATCGAATTTTGGCGG-3', and the sequences are shown in SEQ ID NOs:5 to 8.

[0017] In the preparation method of antioxidant peptide derived from Xuanwei ham of the present invention, the fermentation conditions for inducing the expression of the polypeptide are to add isopropyl thiogalactopyranoside to a final concentration of 0.5 mmol / L and culture at 37°C and 220 r / min for 8 hours.

[0018] The method for measuring the activity of the antioxidant peptide derived from Xuanwei Ham of the present invention includes a hydroxyl radical scavenging method and a DPPH radical scavenging method. The hydroxyl radical (·OH) is a highly reactive oxygen radical that can cause oxidative stress in the body and lead to cell damage and disease. Therefore, measuring the hydroxyl radical scavenging ability of an antioxidant can indirectly evaluate its antioxidant activity. The DPPH radical scavenging method evaluates the antioxidant ability of a sample by measuring the scavenging ability of the sample's 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The DPPH solution is purple in color and has a maximum absorption peak, which fades when it reacts with an antioxidant, and the scavenging rate is calculated by measuring the absorption change of the solution using a spectrophotometer.

[0019] Among them, the hydroxyl radical elimination method is specifically as follows: 1 mL of recombinant polypeptide sample is mixed with 1 mL of ferrous sulfate and 1 mL of hydrogen peroxide, and the mixture is kept at 37°C for 10 minutes, and then the solution is mixed with 1 mL of salicylic acid; in the control group, distilled water is used instead of the sample solution; and after incubation for 30 minutes, the absorbance is measured at 510 nm; Hydroxyl radical scavenging activity was calculated according to the following formula:

number

[0020] Here, the DPPH radical scavenging method is specifically as follows: weigh a certain amount of DPPH, use absolute ethanol to prepare a 0.04mg / mL DPPH solution, take 2mL of recombinant polypeptide solution of different concentrations of 1mg / mL, add 2mL of DPPH solution, mix evenly, leave at room temperature for 30 minutes, then centrifuge at 5000r / min for 10 minutes, take the supernatant, measure the absorbance at 517nm, and use Vc as the positive control. The scavenging rate of DPPH radical by the sample is calculated using the following formula:

number

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses a rigid linking peptide (EAAAK)2 and a flexible linking peptide (GGGGS)2 to link the hexapeptide derived from Xuanwei ham, so that the peptide chain can be grown and made suitable for recombinant expression. At the same time, the proportion of the target polypeptide in the recombinant protein is increased, and the expression level is greatly increased. Secondly, the rigid linking peptide has an α-helical structure, which makes it easier for the antioxidant peptide to form a stable secondary structure after tandem linking, and can effectively separate the active functional domains. The flexible linking peptide is mainly composed of glycine Gly, and has a small molecular weight, which ensures the maximum degree of freedom of the polypeptide backbone structure, which allows the protein to obtain sufficient spatial folding to obtain its original biological activity, and thus the fusion protein can obtain a higher activity than the parent protein.

[0022] The present invention utilizes the smaller His tag on pET-28a(+) for affinity chromatography purification, resulting in isolated and purified proteins with high purity and strong specificity, improving purification efficiency and reducing the difficulty of polypeptide isolation.

[0023] The E. coli expression system used in the present invention has a clear expression background, a high expression level, simple operation, a short culture period, and a strong anti-pollution ability. Secondly, the raw materials for the medium for growing E. coli are inexpensive, and it is easy to produce antioxidant peptide substances in large quantities and on a large scale, which can reduce the economical cost compared with the conventional enzymatic degradation method.

[0024] The preparation method of the present invention is simple to operate, and an engineered strain for prokaryotic expression of recombinant tandemly linked antioxidant peptide fusion gene can be rapidly constructed. In the examples, it has been demonstrated that the hydroxyl radical scavenging ability of the antioxidant peptide after tandem linkage is about three times higher than that before modification, which lays the foundation for the subsequent application of small molecule active peptides in the health food field.

[0025] The study of the present invention can further 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. [Brief description of the drawings]

[0026] [Figure 1] Restriction enzyme electrophoresis of XHP-G6 (left) and XHP-R6 (right). M: DNA standard molecular weight, lane 1: original plasmid electrophoresis, lane 2: molecular weight of the fragment after digestion with NdeI / XhoI enzymes. [Diagram 2] Agarose electrophoresis. Lane M: DNA standard molecular weight, lanes 1 and 2 are the molecular weights of the target genes of XHP-G6 and XHP-R6, respectively. [Diagram 3]SDS-PAGE electrophoresis. Lane M: protein marker. Lanes 1 and 2 are recombinant proteins of XHP-G6 and XHP-R6, respectively. [Figure 4] SDS-PAGE electrophoresis. Lane M: protein marker. Lanes 1 and 2 are purified proteins of XHP-G6 and XHP-R6, respectively. [Diagram 5] Figure 5(a) and Figure 5(b) are RP-HPLC profiles of XHP-G6 and XHP-R6. [Figure 6] FIG. 6(a) and FIG. 6(b) are antioxidant activity analysis graphs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The technical solutions in the embodiments of the present invention will be described below clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, obviously, the described embodiments are only a part of the embodiments of the present invention, not all the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are included in the scope of protection of the present invention.

[0028] Example 1 Gene design and prokaryotic expression method of antioxidant peptides derived from recombinant Xuanwei ham (1) Design of tandemly linked antioxidant peptide sequences derived from Xuanwei ham The method expands the NPPKFD peptide sequence and constructs a design containing the NPPKFD sequence suitable for recombinant expression, and the specific operation is to use a rigid linking peptide (EAAAK)2 and a flexible linking peptide (GGGGS)2 to link the hexaploid NPPKFD respectively, and thus the new amino acid sequence of the designed peptide segment is finally shown in SEQ ID NO:1 or SEQ ID NO:2. (2) Construction of tandemly linked antioxidant peptide target genes According to the amino acid sequence of the tandemly linked antioxidant peptides, the mRNA sequence corresponding to the amino acid sequence of the tandemly linked antioxidant peptides was designed according to the codon preference of E. coli using computer software Primer Premier 5.0. The gene sequence designed for tandem linkage using the rigid linking peptide is shown in SEQ ID NO: 3. The gene sequence designed for tandem linkage using the flexible linking peptide is shown in SEQ ID NO: 4. The 86 amino acid protein sequence was optimized to better express the protein in E. coli. The codon usage preference was adjusted to fit the highest expression profile of the target host, and the CAI (codon adaptation index) was upgraded to 0.87 and 0.9, respectively. (A CAI between 0.8 and 1.0 is considered to be a suitable value for high expression). The average GC content was adjusted to 55.07% and 64.86%, respectively, to remove unfavorable peaks. The repeated regions of the original sequence were deleted to avoid stem-loop structures in the mRNA and facilitate the synthesis process. Unnecessary motifs, such as restriction enzyme cleavage sites for subcloning and negative cis-acting sites, were modified. The entire sequence was fine-tuned to improve translation efficiency and extend the half-life of the mRNA. The optimized gene sequence was synthesized by Shanghai Biotechnology Co., Ltd. (3) Construction of gene expression vector The enzyme cleavage sites added to both ends of the active peptide are the restriction endonucleases Nde I and Xho I, respectively. As shown in the electrophoretic diagram in Figure 1, after double enzyme cleavage of pET-28a(+) with Nde I and Xho I, the target gene was cloned into the pET-28a(+) plasmid using DNA ligase to construct the recombinant expression plasmid. Take a small amount of recombinant plasmid pET-28a(+)-G6 and E. coli BL21(D E3) competent cells, mix lightly and homogeneously, and keep in an ice bath for 30 minutes. After the ice bath, give a heat shock at 42 °C for 90 seconds, and immediately put it into ice to cool for 1 to 2 minutes. Add 900 microliters of LB liquid medium, mix gently and evenly, and culture at 37°C with shaking for 1 hour to fully revive the bacteria. Take an appropriate amount of the bacterial liquid and spread it on LB solid medium (containing 100 mg / L kanamycin) to screen for positive plasmids, then culture upside down at 37°C overnight. Confirm the culture colonies, and DNA sequencing confirms that the recombinant plasmid was correctly constructed. LB medium: 10.0 g tryptone, 5.0 g yeast powder, 10.0 g NaCl. If solid medium was used, 20.0 g powdered agar was added. (4) Colony PCR Verification Using a sterile 10 µL pipette tip, select a monoclonal colony from the overnight grown screening plate, place it in a PCR tube containing 10 µL of sterile ddHO, and validate the lineage by colony PCR.

[0029] The PCR amplification program was an initial denaturation at 94 ° C for 4 minutes, a total of 35 amplification cycles at 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 40 seconds, and an extension at 72 ° C for 10 minutes. After the PCR reaction was completed, nucleic acid electrophoresis was performed. The upstream primer of XHP-G6 was 5'-TGAATCCGCCGAAATTCGACGAAG-3', the downstream primer was 5'-TCTTTCGCAGCCGCTTCATCG-3', the upstream primer of XHP-R6 was 5'-CATATGAATCCGCCGAAATTCG-3', and the downstream primer was 5'-CTCGAG ATCGAATTTTGGCGG-3'. The sequences are shown in SEQ ID NOs: 5 to 8. The electrophoretic diagram is shown in FIG. 2.

[0030] [Table 1]

[0031] (5) Inducible expression of recombinant E. coli strains The selected recombinant strain verified by PCR was inoculated into 3 ml of LB medium containing resistance, and the bacterial seed solution was cultured at 37 ° C and 220 rpm for 12 hours. The bacterial seed solution was inoculated into 100 ml of LB liquid medium at a ratio of 1% to expand the culture, and kanamycin was added to a working concentration of 100 μg / ml at the same time. When the OD600nm of the bacterial solution was 0.6, the final concentration of isopropyl-β-D-thiogalactoside (IPTG) was 0.5 mmol / L, and it was cultured at 37 ° C and 220 r / min for 8 hours. The bacterial solution after the induction was centrifuged at 5000 r / min for 10 minutes, the supernatant was discarded, the bacterial precipitate was collected, the bacterial cells were resuspended in phosphate buffer, and mixed uniformly, and then ultrasonicated in an ice water bath. The ultrasonic disruption conditions were 250 W, ultrasonic for 3 seconds, interval of 5 seconds, and 15 minutes. At this time, the total bacterial protein and the bacterial solution were clear and transparent. The ultrasonically disrupted bacterial solution was centrifuged at 12,000 r / min for 15 minutes, the supernatant was the supernatant protein, and the supernatant and precipitate were separated. The obtained total bacterial solution, supernatant, and precipitate samples were each analyzed by SDS-PAGE to measure the expression of the fusion protein.

[0032] SDS-PAGE analysis: The flat / concave glass plate was taken out, washed, and dried, and the glass plate was fixed to the glue maker with a holder. The separation gel was prepared, and after each component was added, it was blown using a pipette to mix evenly, and pasted on the wall along the gap of the glass plate, and the rubber surface was sealed using distilled water, flattened, and left at room temperature until a clear dividing line appeared on the liquid surface, and the distilled water was discarded, and the preparation of the separation gel was completed. The upper layer of the concentrated gel was prepared, and after each component was added and mixed evenly, the upper layer gel was added along the gap, a comb was inserted, and the upper layer gel was left at room temperature until it solidified. After polymerization, two short gel plates were attached to the grooves facing inward, and the inner groove fixing bracket was assembled. Place the whole fixation apparatus into the electrophoresis tank, add electrophoresis buffer (electrophoresis buffer formulation: Tris buffer (25 mM, pH 8.3), glycine (190 mM), SDS (0.1%)), pull out the comb, add 5 μl of standard protein and sample to the gel lane, add loading buffer if there is an extra sampling hole, turn on the power, the starting voltage is 80 V, about 30 min, after entering the concentrating gel, change the voltage to 120 V, when the front of bromophenol blue reaches the bottom of the electrophoresis groove, turn off the power (about 2-3 h).

[0033] Sample processing: Take the E. coli lysate and the supernatant after centrifugation, mix with loading buffer, add 40 μl of PBS buffer and 10 μl of 5xSDS loading buffer to the precipitate, mix 40 μl of the supernatant after centrifugation with 10 μl of 5xSDS loading buffer, boil in a boiling water bath for 5 minutes, centrifuge at 12,000 rpm for 5 minutes, take the supernatant, and take 5 μl of the sample for protein electrophoresis. After the electrophoresis is completed, take out the glass plate, carefully pry open the glass plate, cut off the stacking gel, peel off the gel from the gel making board, put it in deionized water, wash the gel, put the protein gel into a staining box, and stain with Coomassie Brilliant Blue R-250 for 2 hours. After staining, put the gel into the destaining solution, diffuse destain until the blue color of the background plate disappears, and after watching the bands, take a picture and record it using gel imaging. The SDS-PAGE electrophoresis diagram is shown in Figure 3.

[0034] Example 2 Purification of Recombinant Tandem Antioxidant Peptides A 5 mL Ni-IDA prepacked column was taken out and the storage buffer was allowed to drain by gravity. The column was equilibrated with 2x column volume of Wash Buffer (20 mM Tris-HCl, 8 M urea, 500 mM NaCl, 5 mM imidazole, pH 8.0) and the resin was slowly drained from the buffer using a flow rate of 0.5-1 mL / min. The supernatant was passed through a 0.45 μm microporous filter membrane and added to the column, the flow-through was collected in a centrifuge tube, the column was then washed with 2x column volume of Wash Buffer and the flow-through was collected, and finally, the histidine-tagged proteins on the column were eluted using 2x column volume of Elution Buffer (20 mM Tris-HCl, 8 M urea, 500 mM NaCl, 500 mM imidazole, pH 8.0), and each eluate was stored separately until the absorbance of the eluate at 280 nm was close to the baseline. The collected eluates were subjected to SDS-PAGE and RP-HPLC to analyze the purity. The SDS-PAGE electrophoretic pattern is shown in Figure 4, and the RP-HPLC pattern is shown in Figure 5(a) and Figure 5(b).

[0035] RP-HPLC conditions: Acquity (Watts Corporation) was used. The HPLC system was equipped with a reversed-phase BEH C18 analytical column (1.7 μm, 2.1 × 100 mm, Watts Corporation). 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°C, UV detection wavelength: 220 nm. Measurement of protein concentration: Measure protein concentration using the Bradford method, detect absorbance at 595 nm, and follow the standard curve (y=0.6361x+0.477, R 2 = 0.9923), the total protein concentration of the sample was calculated to be 11.95 mg / L after conversion.

[0036] Example 3: Activity verification of tandemly linked antioxidant peptides derived from Xuanwei ham (1) Measurement of DPPH radical scavenging activity A certain amount of DPPH was weighed and 0.04 mg / mL DPPH solution was prepared using absolute ethanol. 2 mL of recombinant polypeptide solution with different concentrations of 1 mg / mL was taken, and 2 mL of DPPH solution was added and mixed uniformly. The solution was left at room temperature for 30 minutes, and then centrifuged at 5000 r / min for 10 minutes. The supernatant was taken and the absorbance at 517 nm was measured. Vc was used as a positive control. The scavenging rate of DPPH radical by the sample was calculated using the following equation:

number

number

[0037] The results show that the hydroxyl radical and DPPH radical scavenging capacities of the antioxidant peptides prepared in tandem using the rigid linking peptides of the present invention are 79.46% and 57.25%, respectively, and the hydroxyl radical and DPPH radical scavenging capacities of the antioxidant peptides prepared in tandem using the flexible linking peptides of the present invention are 87.42% and 68.05%, respectively, indicating that the antioxidant capacity of the polypeptides prepared by the present invention is approximately three times higher than that of the polypeptides isolated and extracted from Xuanwei ham.

[0038] Although embodiments of the present invention have been illustrated and described, it will be understood that those skilled in the art can make various changes, modifications, substitutions and variations to these embodiments without departing from the principles and spirit of the present invention, the scope of the present invention being limited 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 sequence is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

3. A method for preparing antioxidant peptides derived from Xuanwei ham according to claim 1 or 2, comprising: (1) according to the amino acid sequence of Xuanwei ham peptide, using rigid linking peptide or flexible linking peptide to hexaplex Xuanwei ham peptide to obtain target gene, the sequences of said Xuanwei ham peptide, rigid linking peptide and flexible linking peptide are shown in SEQ ID NO:9-11, and the sequence of said target gene is shown in SEQ ID NO:3 or SEQ ID NO:4; (2) double-enzymatically cleaving the target gene and the prokaryotic expression plasmid pET-28a(+) with restriction endonucleases Nde I and Xho I, and ligating them via DNA ligase to obtain the prokaryotic recombinant expression vectors pET-28a(+)-G6 and pET-28a(+)-R6; (3) transforming the prokaryotic recombinant expression vector into E. coli BL21(DE3) competent cells by heat shock method to construct recombinant engineered strains BL21(DE3)-pET-28a(+)-G6, BL21(DE3)-pET-28a(+)-R6; (4) verifying successful transformation of the recombinant expression vector by colony PCR; (5) inducing expression of the His-tagged tandem recombinant polypeptides in the engineered strains BL21(DE3)-pET-28a(+)-G6 and BL21(DE3)-pET-28a(+)-R6; The expressed His-tagged fusion protein was purified using a nickel ion affinity chromatography column. Upon loading the protein, the His-tagged fusion protein specifically bound to the nickel column, while the impurity proteins flowed out due to non-specific binding to the nickel column. An imidazole gradient elution was used, and the imidazole and Ni were separated. ２＋ competitively binding to the nickel column, thereby releasing the fusion protein, and collecting the eluate (6); (7) using RP-HPLC for purity testing to obtain high purity tandemly linked recombinant antioxidant peptides.

4. The primers used in the colony PCR in step (4) are XHP-G6 upstream primer: 5'-TGAATCCGCCGAAATTCGACGAAG-3', downstream primer: 5'-TCTTTCGCAGCCGCTTCATCG-3', XHP-R6 upstream primer: 5'-CATATGAATCCGCCGAAATTCG-3', downstream primer: 5'-CTCGAGATCGAATTTTGGCGG-3', The method for preparing antioxidant peptides derived from Xuanwei Ham according to claim 3, characterized in that the sequences are as shown in SEQ ID NO:5-8.

5. The method for preparing antioxidant peptides derived from Xuanwei ham according to claim 4, characterized in that the fermentation conditions for inducing the expression of the polypeptide are adding isopropyl thiogalactopyranoside to a final concentration of 0.5 mmol / L and culturing at 37°C and 220 r / min for 8 hours.

6. A method for measuring the activity of an antioxidant peptide derived from Xuanwei ham according to claim 1 or 2, A method comprising a hydroxyl radical scavenging method and a DPPH radical scavenging method.

7. The hydroxyl radical scavenging method is as follows: 1 mL of recombinant polypeptide sample is mixed with 1 mL of ferrous sulfate and 1 mL of hydrogen peroxide, and the mixture is kept at 37° C. for 10 minutes, and then the solution is mixed with 1 mL of salicylic acid; in the control group, distilled water is used instead of the sample solution; and after 30 minutes of incubation, the absorbance at 510 nm is measured; The hydroxyl radical scavenging activity was calculated according to the following formula: [0010] Where Ai: absorbance of the sample, The method for measuring the activity of antioxidant peptides derived from Xuanwei ham as described in claim 6, characterized in that A0: absorbance of the blank control group.

8. The DPPH radical scavenging method is as follows: weigh a certain amount of DPPH, use absolute ethanol to prepare a 0.04 mg / mL DPPH solution, take 2 mL of recombinant polypeptide solution with different concentrations of 1 mg / mL, add 2 mL of DPPH solution and mix uniformly, leave at room temperature for 30 minutes, then centrifuge at 5000 r / min for 10 minutes, take the supernatant, measure the absorbance at 517 nm, and use Vc as a positive control; the scavenging rate of DPPH radical by the sample is calculated using the following formula: [0025] A ０ is the absorbance of 2 mL absolute ethanol + 2 mL DPPH solution, A １ is the absorbance of 2 mL sample solution + 2 mL DPPH solution, A ２ The method for measuring the activity of antioxidant peptides derived from Xuanwei ham according to claim 7, characterized in that: is the absorbance of 2 mL of sample solution + 2 mL of absolute ethanol.

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