Antioxidant peptides derived from Xuanwei ham and their preparation and activity assay methods

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, particularly to antioxidant peptides derived from Xuanwei ham, their preparation methods and activity assay methods. [Background technology]

[0002] In today's society, people are increasingly concerned about their 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, and other conditions. These oxidative processes are closely related to the development of many diseases, including cardiovascular disease, diabetes, atherosclerosis, Alzheimer's disease, and cancer. In recent years, researchers have continued to focus on bioactive peptides. A variety of bioactive peptides have been isolated and identified from various cured hams. These peptides possess rich amino acid sequences and various biological functions, including antioxidant, antihypertensive, hypolipidemic, and hypoglycemic effects, and are believed to have potential health benefits. For example, Non-Patent Document 1 discovered that peptide extracts with molecular weights of less than 1700 Da extracted from Spanish cured ham exhibited antihypertensive and antioxidant effects, while Non-Patent Document 2 discovered that natural peptides extracted and isolated from Jinhua ham possessed high radical scavenging activity. These active peptides have molecular weights of 400-2000 Da and sequence lengths of 5-20 amino acids, and can be used as functional foods or therapeutic drugs. Patent document 1 discloses an antioxidant peptide complex derived from prosciutto containing 42 peptide segments with molecular weights of less than 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. The amino acid sequence of bioactive peptide 1 is LGEHNIDVLEGNEQFINAAK (SEQ ID NO: 12) The amino acid sequence of the bioactive peptide 2 is GHYTEGAELVDSVLDVVR (SEQ ID NO: 13)The amino acid sequence of the bioactive peptide 3 is DLVILLYETALLSSGFSLEDPQTHANR (SEQ ID NO:14) is.

[0003] However, like most antioxidant peptides, the yield of polypeptides isolated and extracted from ham is low, the cost is high, and the isolation process is time-consuming, making large-scale preparation difficult. Currently, most antioxidant peptides are still in the laboratory research stage, and it has been shown that even if antioxidant peptides are obtained using this isolation and extraction method, they cannot meet the requirements for industrial production. Furthermore, the sequences of antioxidant peptides isolated from ham typically contain 5–20 amino acids, which are relatively short and easily degraded by proteases and peptidases in E. coli expression systems. Therefore, direct expression of these peptides is difficult. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Elizabeth Escudero (Escudero, E., Aristoy, M.-C., Nishimura, H., Arihara, K., & Toldra, 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] The first objective of the present invention is to provide an antioxidant peptide derived from Xuanwei ham 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 the polypeptide.

[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 a raw material, and expresses it repeatedly through tandem linkage, and different linkage methods can be used to repeatedly link 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 for the Xuanwei ham peptide gene in E. coli. [Means for solving the problem]

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

[0011] The antioxidant peptide derived from Xuanwei ham has an amino acid sequence 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: According to the amino acid sequence of Xunweiham peptide NPPKFD, rigid linking peptide (EAAAK)2 (SEQ ID NO: 15) or flexible linking peptide (GGGGS)2 (SEQ ID NO:16) Step (1): Linking the Xuanwei ham peptide NPPKFD to a hexaploid using the Xuanwei ham peptide NPPKFD 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 to 11, and the sequence of the target gene is shown in SEQ ID NO: 3 or SEQ ID NO: 4; (2) double-cleaving the target gene and the prokaryotic expression plasmid pET-28a(+) with restriction endonucleases NdeI and XhoI and ligating them via DNA ligase to obtain 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 and 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. Upon loading, the His-tagged fusion protein specifically bound to the nickel column, while impurity proteins flowed out due to nonspecific binding to the nickel column. An imidazole gradient elution was used, and the His-tagged fusion protein was purified using a mixture of imidazole and Ni. 2+ competitively binding the combination to the nickel column, thereby releasing the fusion protein, and collecting the eluate (6); (7) using RP-HPLC for purity testing to obtain highly pure tandemly linked recombinant antioxidant peptides; Protein concentration was measured using the Bradford method, detecting absorbance at 595 nm, and a standard curve (y = 0.6361x + 0.477, R 2 and a protein concentration measurement step (8) for calculating the total protein concentration of the sample according to ΔΨ = 0.9923.

[0013] The rigid-linked peptide (EAAAK)2 used in the present invention has an α-helical structure, which facilitates the formation of a stable secondary structure for the antioxidant peptides after tandem linkage and simultaneously effectively separates the active functional domains, resulting in a fusion protein with 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 in the polypeptide backbone structure, allowing the protein to achieve sufficient spatial folding to achieve 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 protein purification and localization.

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

[0018] The methods for measuring the activity of the antioxidant peptides derived from Xuanwei ham of the present invention include the hydroxyl radical scavenging method and the DPPH radical scavenging method. The hydroxyl radical (·OH) is a highly reactive oxygen radical that can cause oxidative stress in the body, potentially resulting in cellular 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 capacity of a sample by measuring its ability to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The DPPH solution is purple in color with a maximum absorption peak, which fades upon reaction with an antioxidant. The scavenging rate is calculated by measuring the change in absorption of the solution using a spectrophotometer.

[0019] Specifically, the hydroxyl radical scavenging method involves mixing 1 mL of a recombinant polypeptide sample with 1 mL of ferrous sulfate and 1 mL of hydrogen peroxide, and then incubating the mixture at 37°C for 10 minutes. The mixture is then mixed with 1 mL of salicylic acid. In the control group, distilled water is used instead of the sample solution. After incubation for 30 minutes, the absorbance is measured at 510 nm. The hydroxyl radical scavenging activity is 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 0.04 mg / mL DPPH solution, take 2 mL of 1 mg / mL recombinant polypeptide solution of different concentrations, add 2 mL of DPPH solution, 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 the positive control. The DPPH radical scavenging rate of 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: This invention uses a rigid linking peptide (EAAAK)2 and a flexible linking peptide (GGGGS)2 to link a hexapeptide derived from Xuanwei ham, growing the peptide chain and making it suitable for recombinant expression. At the same time, the proportion of the target polypeptide in the recombinant protein is increased, significantly increasing the expression level. Second, the rigid linking peptide has an alpha-helical structure, which makes it easier for the antioxidant peptide to form a stable secondary structure after tandem linking, effectively isolating the active functional domains. The flexible linking peptide is mainly composed of glycine Gly, and its small molecular weight ensures maximum flexibility of the polypeptide backbone structure, allowing the protein to achieve sufficient spatial folding to achieve its original biological activity. As a result, the fusion protein has higher activity than the parent protein.

[0022] The present invention utilizes the smaller His tag on pET-28a(+) for affinity chromatography purification, resulting in highly 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, high expression level, simple operation, short culture period, and strong anti-pollution ability. Secondly, the raw materials for the medium used to grow E. coli are inexpensive, making it easy to mass-produce antioxidant peptide substances on a large scale, and enabling economical cost reduction compared to conventional enzymatic degradation methods.

[0024] The preparation method of the present invention is simple to operate and allows for the rapid construction of engineered strains that prokaryotically express recombinant tandemly linked antioxidant peptide fusion genes. In the examples, it has been demonstrated that the hydroxyl radical scavenging ability of the tandemly linked antioxidant peptides is approximately 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 explanation 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 fragment after digestion with NdeI / XhoI enzymes. [Figure 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. [Figure 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. [Figure 5] Figure 5(a) and Figure 5(b) are RP-HPLC profiles of XHP-G6 and XHP-R6. [Figure 6] Figure 6(a) and Figure 6(b) are antioxidant activity analysis charts. DETAILED DESCRIPTION OF THE INVENTION

[0027] The following clearly and completely describes the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention, and obviously, the described embodiments are only a part of the embodiments of the present invention, and are not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art 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 involves extending the NPPKFD peptide sequence and constructing a design containing the NPPKFD sequence suitable for recombinant expression. The specific procedure involves using a rigid linking peptide (EAAAK)2 and a flexible linking peptide (GGGGS)2 to link the hexaploid NPPKFD, respectively. Therefore, 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 Based on the amino acid sequences of the tandemly linked antioxidant peptides, mRNA sequences corresponding to the amino acid sequences of the tandemly linked antioxidant peptides were designed according to the codon preference of E. coli using the computer software Primer Premier 5.0. The gene sequence designed for tandem linkage using rigid linking peptides is shown in SEQ ID NO: 3. The gene sequence designed for tandem linkage using flexible linking peptides is shown in SEQ ID NO: 4. The 86-amino acid protein sequence was optimized for better protein expression in E. coli. The codon usage preference was adjusted to match 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 appropriate for high expression.) The average GC content was adjusted to 55.07% and 64.86%, respectively, and undesirable peaks were removed. Repeated regions in 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 vectors The enzyme cleavage sites added to both ends of the active peptide were NdeI and XhoI restriction endonucleases, respectively. As shown in the electrophoresis diagram in Figure 1, pET-28a(+) was double-cleaved with NdeI and XhoI, and then the target gene was cloned into the pET-28a(+) plasmid using DNA ligase to construct the recombinant expression plasmid. A small amount of the recombinant plasmid pET-28a(+)-G6 and E. coli BL21(D E3) competent cells were mixed gently and homogenously. The mixture was then placed in an ice bath for 30 minutes, heat-shocked at 42°C for 90 seconds, and then immediately placed on ice to cool for 1-2 minutes. Add 900 microliters of LB liquid medium, mix gently and evenly, and incubate 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. Incubate upside down at 37°C overnight. Confirm the culture colonies, and DNA sequencing confirms that the recombinant plasmid was correctly constructed. LB medium: tryptone 10.0 g, yeast powder 5.0 g, NaCl 10.0 g. When using solid medium, add 20.0 g of powdered agar. (4) Colony PCR verification Using a sterile 10 µL pipette tip, select a monoclonal colony from the overnight-cultured screening plate, place it in a PCR tube containing 10 µL of sterile ddHO, and verify the lineage by colony PCR.

[0029] The PCR amplification program consisted of an initial denaturation at 94°C for 4 minutes, a total of 35 amplification cycles (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 for XHP-G6 was 5'-TGAATCCGCCGAAATTCGACGAAG-3', the downstream primer was 5'-TCTTTCGCAGCCGCTTCATCG-3', and the upstream primer for XHP-R6 was 5'-CATATGAATCCGCCGAAATTCG-3', the downstream primer was 5'-CTCGAG ATCGAATTTTGGCGG-3'. These primers are shown in SEQ ID NOs: 5 to 8. The electropherograms are shown in Figure 2.

[0030] [Table 1]

[0031] (5) Inducible expression of recombinant E. coli strains The selected recombinant strains, verified by PCR, were inoculated into 3 ml of LB medium containing the resistance gene and grown for 12 hours at 37°C and 220 rpm. The resulting seed solution was then inoculated into 100 ml of LB liquid medium at a 1% ratio and expanded. At the same time, kanamycin was added to a working concentration of 100 μg / ml. When the OD600nm of the culture reached 0.6, a final concentration of isopropyl-β-D-thiogalactoside (IPTG) of 0.5 mmol / L was added and cultured for 8 hours at 37°C and 220 rpm. After induction, the 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 uniformly, and sonicated in an ice-water bath for 15 minutes at 250 W for 3 seconds with a 5-second interval. The culture was clear and transparent, with total cell protein. The sonicated bacterial solution was centrifuged at 12,000 r / min for 15 minutes, and the supernatant was the supernatant protein. The supernatant and precipitate were separated, and the resulting whole 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 and concave glass plates were removed, washed, and dried. The plates were then attached to a glue maker with a holder. The separation gel was prepared by adding each component and spraying it evenly onto the glass plate along the gaps. The rubber surface was sealed with distilled water, flattened, and left at room temperature until a clear dividing line appeared on the surface. The distilled water was then discarded, completing the preparation of the separation gel. The upper layer of the concentration gel was prepared by adding each component and mixing it evenly. The upper layer gel was then added along the gaps, inserted with a comb, and left at room temperature until the upper layer gel solidified. After polymerization, two short gel plates were attached to the grooves facing inward, and the inner groove fixing brackets were assembled. Place the entire immobilization 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 are extra sampling holes, turn on the power, start voltage is 80 V, about 30 minutes, after entering the stacking gel, change the voltage to 120 V, and when the bromophenol blue tip reaches the bottom of the electrophoresis groove, turn off the power (about 2-3 hours).

[0033] Sample processing: The E. coli lysate and the supernatant after centrifugation were mixed with loading buffer. 40 μl of PBS buffer and 10 μl of 5x SDS loading buffer were added to the precipitate. 40 μl of the supernatant was mixed with 10 μl of 5x SDS loading buffer, boiled in a boiling water bath for 5 minutes, and centrifuged at 12,000 rpm for 5 minutes. The supernatant was removed, and 5 μl of the sample was used for protein electrophoresis. After electrophoresis was complete, the glass plates were removed, carefully pried open, and the stacking gel was discarded. The gel was peeled off the gel preparation board and placed in deionized water to rinse the gel. The protein gel was then placed in a staining box and stained with Coomassie Brilliant Blue R-250 for 2 hours. After staining, the gel was placed in destaining solution and destained until the blue background color disappeared. After viewing the bands, photographs were taken and recorded using gel imaging. The SDS-PAGE electropherogram is shown in Figure 3.

[0034] Example 2 Purification of Recombinant Tandem Antioxidant Peptides A 5 mL Ni-IDA prepacked column was removed and the storage buffer was allowed to drain by gravity. The column was equilibrated with 2 column volumes 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 applied to the column. The flow-through was collected in a centrifuge tube. Next, the column was washed with 2 column volumes of Wash Buffer and the flow-through was collected. Finally, the histidine-tagged proteins were eluted from the column using 2 column volumes of Elution Buffer (20 mM Tris-HCl, 8 M urea, 500 mM NaCl, 500 mM imidazole, pH 8.0). Each eluate was stored separately until the absorbance at 280 nm of the eluate approached baseline. The collected eluate was subjected to SDS-PAGE and RP-HPLC to analyze purity. The SDS-PAGE electropherogram is shown in Figure 4, and the RP-HPLC diagram 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, detecting absorbance at 595 nm, and plotting the concentration using a standard curve (y = 0.6361x + 0.477, R 2 = 0.9923), the total protein concentration of the sample was calculated, and the converted total protein concentration was 11.95 mg / L.

[0036] Example 3: Activity verification of tandemly linked antioxidant peptides derived from Xuanwei ham (1) Measurement of DPPH radical scavenging activity A fixed amount of DPPH was weighed and a 0.04 mg / mL DPPH solution was prepared using absolute ethanol. 2 mL of recombinant polypeptide solution (various concentrations of 1 mg / mL) was added to 2 mL of DPPH solution, mixed uniformly, and left at room temperature for 30 minutes before centrifuging at 5000 rpm for 10 minutes. The supernatant was collected and its absorbance at 517 nm was measured. Vc was used as a positive control. The scavenging rate of DPPH radicals by the samples was calculated using the following equation:

number

number

[0037] The results showed that the hydroxyl radical and DPPH radical scavenging capacities of the antioxidant peptides prepared in tandem using the rigid-linked peptides of the present invention were 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-linked peptides of the present invention were 87.42% and 68.05%, respectively. The antioxidant capacity of the polypeptides prepared in the present invention is approximately three times higher than that of the polypeptides isolated and extracted from Xuanwei ham.

[0038] While 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 which is limited by the appended claims and their equivalents. The inventions described in the claims of the original application are set forth below. [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] An antioxidant peptide derived from Xuanwei ham described in [1], characterized in that the corresponding gene sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 4. [3] [1] or [2], a method for preparing an antioxidant peptide derived from Xuanwei ham, Step (1): According to the amino acid sequence of Xuanwei ham peptide, use rigid linking peptide or flexible linking peptide to hexaploidize Xuanwei ham peptide to obtain target gene, the sequences of the Xuanwei ham peptide, rigid linking peptide and flexible linking peptide are shown in SEQ ID NO: 9 to 11, and the sequence of the target gene is shown in SEQ ID NO: 3 or SEQ ID NO: 4; (2) double-cleaving the target gene and the prokaryotic expression plasmid pET-28a(+) with restriction endonucleases NdeI and XhoI and ligating them via DNA ligase to obtain 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 and 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. Upon loading, the His-tagged fusion protein specifically bound to the nickel column, while impurity proteins flowed out due to nonspecific binding to the nickel column. An imidazole gradient elution was used, and the His-tagged fusion protein was purified using a mixture of imidazole and Ni. 2+ competitively binding the combination to the nickel column, thereby releasing the fusion protein, and collecting the eluate (6); and (7) using RP-HPLC for purity testing to obtain highly pure 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', A method for preparing antioxidant peptides derived from Xuanwei ham according to [3], characterized in that the sequences are shown in SEQ ID NOs: 5 to 8. [5] The method for preparing antioxidant peptides derived from Xuanwei ham described in [4] is characterized in that the fermentation conditions for inducing polypeptide expression are to add isopropylthiogalactopyranoside to a final concentration of 0.5 mmol / L and to culture at 37°C and 220 r / min for 8 hours. [6] [1] or [2] A method for measuring the activity of an antioxidant peptide derived from Xuanwei ham, A method characterized by comprising a hydroxyl radical scavenging method and a DPPH radical scavenging method. [7] The hydroxyl radical scavenging method involves mixing 1 mL of a recombinant polypeptide sample with 1 mL of ferrous sulfate and 1 mL of hydrogen peroxide, incubating the mixture at 37°C for 10 minutes, and then mixing the solution with 1 mL of salicylic acid. In the control group, distilled water is used instead of the sample solution. After incubation for 30 minutes, the absorbance at 510 nm is measured. The hydroxyl radical scavenging activity was calculated according to the following formula:

number

number

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: Step (1): According to the amino acid sequence of Xuanwei ham peptide, use rigid linking peptide or flexible linking peptide to hexameric linking of Xuanwei ham peptide to obtain 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-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 and 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. Upon loading, the His-tagged fusion protein specifically bound to the nickel column, while impurity proteins flowed out due to nonspecific binding to the nickel column. Using an imidazole gradient elution, the His-tagged fusion protein was purified by a gradient of imidazole and Ni. ２＋ competitively binding the combination to the nickel column, thereby releasing the fusion protein, and collecting the eluate (6); (7) using RP-HPLC for purity testing to obtain highly pure 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 polypeptide expression are adding isopropylthiogalactopyranoside 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 antioxidant peptides 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 involves mixing 1 mL of a recombinant polypeptide sample with 1 mL of ferrous sulfate and 1 mL of hydrogen peroxide, and then incubating the mixture at 37°C for 10 minutes. The mixture is then mixed with 1 mL of salicylic acid. In the control group, distilled water is used instead of the sample solution. After incubation for 30 minutes, the absorbance at 510 nm is measured. The hydroxyl radical scavenging activity was calculated according to the following formula: [Equation 1] where Ai: absorbance of the sample, The method for measuring the activity of antioxidant peptides derived from Xuanwei ham according to 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 1 mg / mL recombinant polypeptide sample solution, add 2 mL of DPPH solution, 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 DPPH radical scavenging rate of the sample is calculated using the following formula: [Equation 2] 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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