A polypeptide prepared from zanthoxylum bungeanum seed protein

Abstract

The application provides a polypeptide with antioxidant efficacy prepared from Zanthoxylum bungeanum Maxim seed protein, and the polypeptide is prepared by subjecting Zanthoxylum bungeanum Maxim seed protein to enzymolysis, centrifuging the enzymolysis solution, subjecting the centrifuged solution to ultrafiltration, and drying the filtrate. The polypeptide prepared by the application has good antioxidant efficacy after purification. The prepared polypeptide has high activity and can be used in the fields of medicines and functional foods.

Description

Technical Field

[0001] This invention belongs to the field of functional polypeptide preparation technology, specifically relating to a polypeptide with antioxidant effects prepared from Sichuan pepper seed protein. Background Technology

[0002] Sichuan peppercorn( Zanthoxylum bungeanum Maxim Zanthoxylum L. is a deciduous shrub or small tree belonging to the Rutaceae family and the Zanthoxylum genus. The fruit is spherical, 4-6 mm in diameter, with a brown or purplish-red pericarp when ripe, containing seeds (Zanthoxylum seeds). Mature seeds consist of a hard pericarp and a kernel, accounting for 30% and 70% of the total weight, respectively. It is conservatively estimated that my country produces over one million tons of Zanthoxylum seeds annually. However, the residue from oil extraction is often used as fuel, returned to fields as fertilizer, or even discarded as waste, wasting resources and polluting the environment. Therefore, deep processing of Zanthoxylum seeds or their processing byproducts to ensure their rational and full utilization has become a challenge for my country's Zanthoxylum industry. Summary of the Invention

[0003] The purpose of this invention is to provide a polypeptide with antioxidant effects prepared from Sichuan pepper seed protein, thereby improving the economic value of Sichuan pepper seed deep-processed products.

[0004] The present invention provides a polypeptide with antioxidant effects prepared from Sichuan pepper seeds. The polypeptide is prepared by enzymatic hydrolysis of Sichuan pepper seed protein, centrifugation and ultrafiltration of the hydrolysate, and drying of the filtrate.

[0005] Furthermore, the enzymatic hydrolysis involves using papain to hydrolyze the protein in the Sichuan pepper seeds.

[0006] The enzymatic hydrolysis was carried out in a water bath shaker at a constant temperature of 48°C for 4 hours.

[0007] Furthermore, the centrifugation involves heating the enzymatic hydrolysate to 98°C for 12 minutes to inactivate the protease, followed by centrifugation at 3000g for 20 minutes.

[0008] Furthermore, the ultrafiltration involves passing the enzymatic hydrolysate sequentially through ultrafiltration membranes with molecular weight cutoffs of 3kDa, 2kDa, and 1kDa.

[0009] Furthermore, the polypeptide has an amino acid sequence of either Ser-Glu-Cys-Val-His-Leu or Pro-Gly-Arg-His-Trp.

[0010] The present invention also provides the application of the aforementioned polypeptide in the preparation of functional foods or pharmaceuticals.

[0011] The peptides prepared by this invention exhibit good antioxidant properties after purification. The prepared peptides have high activity and can be used in functional foods and healthcare fields. Attached Figure Description

[0012] Figure 1 Flowchart of the extraction process of Sichuan pepper seed protein

[0013] Figure 2 This is a schematic diagram illustrating the free radical scavenging ability of the polypeptide prepared in Example 2 of the present invention at different concentrations;

[0014] Figure 3 This is a schematic diagram of the antioxidant enzyme activity of HepG2 cells in Example 2 of the present invention;

[0015] Figure 4 : This is the chromatogram of ab-p-1 ion exchange in Example 3 of the present invention;

[0016] Figure 5 : This is the RP-HPLC chromatogram of ab-p-1-B2 in Example 3 of the present invention. Detailed Implementation

[0017] The terminology used in this invention is described below:

[0018] 1. Antioxidant: Any substance that can effectively inhibit the oxidation reaction of free radicals when present in low concentration. Its mechanism of action can be to act directly on free radicals or to indirectly consume substances that easily generate free radicals, preventing further reactions. While the human body inevitably produces free radicals, it also naturally produces antioxidants to resist free radicals and counteract the oxidative attack of free radicals on human cells.

[0019] 2. Free radicals: Free radicals are atoms or groups with unpaired electrons formed when covalent bonds of a compound molecule undergo homolytic cleavage under external conditions such as light and heat. In the human body, free radicals (ROS) are substances lacking electrons (unsaturated electron substances). After entering the human body, they compete for electrons everywhere. If they take electrons from cellular protein molecules, they cause the proteins to attach branches and undergo alkylation, forming distorted molecules that can cause cancer. Because these distorted molecules lack electrons themselves, they will also take electrons from neighboring molecules, causing neighboring molecules to also become distorted and carcinogenic.

[0020] The method for detecting free radical scavenging ability is as follows:

[0021] 1) DPPH· free radical scavenging capacity determination: Add 2mL x 10 -4 After mixing the mol / L DPPH solution and reacting it in the dark at room temperature for 20 min, centrifuge at 4000 r / min for 10 min, zero the instrument with 95% ethanol, and measure its absorbance A at 517 nm. iSimultaneously, the absorbance A of a mixture of 2 mL of 95% ethanol and 2 mL of sample solution was measured. j ; and the absorbance A0 of 2 mL DPPH solution mixed with 2 mL deionized water; its scavenging rate (%) = ;

[0022] 2) 02 - • Free radical scavenging capacity determination: Take 4.5 mL of 50 mmol / L pH 8.2 Tris-HCl buffer solution, add 0.1 mL of sample solutions of different concentrations, incubate at 25℃ for 10 min, then add 0.3 mL of 5 mmol / L pyrogallol pre-warmed at 25℃, mix well, and quickly add to a dry cuvette. Measure the absorbance at 320 nm every 20 mm, starting from when the absorbance value reaches 0.2. Zero the instrument with an equal volume of 10 mmol / L HCl instead of pyrogallol. For the blank group, use an equal volume of deionized water instead of the sample solution, and follow the same steps. Plot the absorbance values ​​of the sample solution and pyrogallol, and the mixture of deionized water and pyrogallol, at 320 nm over time. The slope of the regression equation is taken as the auto-oxidation rate V of pyrogallol. 样品 , Its clearance rate (%) = .

[0023] 3. Papain: Papain is a protease with a molecular weight of 23,406. It is composed of a single polypeptide chain and can break down proteins in acidic, neutral, and alkaline environments.

[0024] 4. Ultrafiltration: Ultrafiltration refers to the process of allowing small molecule solutes and solvents to pass through a specially designed membrane with a certain pore size under a certain pressure, while large molecule solutes cannot pass through and remain on one side of the membrane, thereby partially purifying the large molecules.

[0025] This invention produces a polypeptide from pepper seed protein through enzymatic hydrolysis, centrifugation, and ultrafiltration. After purification, the polypeptide exhibits good antioxidant properties.

[0026] The invention will be further described in detail below with reference to implementation examples.

[0027] Example 1: Preparation of polypeptides from Sichuan pepper seed protease hydrolysis

[0028] 1) After adjusting the pH of the 3-5% Sichuan pepper seed protein solution to 7.0, use papain to enzymatically hydrolyze it; after digesting in a water bath shaker at a constant temperature of 48℃ for 4 hours, heat the hydrolysate to 98℃ for 12 minutes to inactivate the protease, then centrifuge at 3000g for 20 minutes and collect the supernatant.

[0029] The Sichuan pepper seed protein was prepared using a conventional alkali extraction method, and the process of one preparation method is as follows: Figure 1 As shown, other methods for extracting Sichuan pepper seed protein can also be used.

[0030] 2) The supernatant enzymatic hydrolysate obtained by centrifugation was passed sequentially through nanofiltration membranes with molecular weight cutoffs of 3kDa, 2kDa, and 1kDa to obtain four different molecular weight hydrolysate components with molecular weights less than 3 kDa (ab-p-4), 3kDa-2kDa (ab-p-3), 2kDa-1kDa (ab-p-2), and less than 1kDa (ab-p-1), respectively; then the four enzymatic hydrolysates were freeze-dried to obtain peptide powder.

[0031] Example 2: Detection of the free radical scavenging ability of Sichuan pepper seed peptides

[0032] 1) Ability to scavenge free radicals

[0033] The peptide powder was dissolved in distilled water at concentrations of 5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL, respectively. The free radical scavenging abilities of the pre-ultrafiltration ab-p sample, ab-p-1, ab-p-2, ab-p-3, and ab-p-4 at different concentrations are shown in the figure. Figure 2 .

[0034] Table 1: O2 - Free radical scavenging ability record table

[0035] concentration ab-p sample before ultrafiltration (%) ab-p-1 (%) ab-p-2 (%) ab-p-3 (%) ab-p-4 (%) 5 mg / mL 38.5 36.3 34.7 31.8 22.6 10 mg / mL 51.2 54.3 51.6 43.4 35.8 15 mg / mL 55.1 63.2 57.3 53.4 42.6 20 mg / mL 62.1 74.4 58.8 55.9 44.3

[0036] Table 2: Record of DPPH·Free Radical Scavenging Capacity

[0037] concentration ab-p sample before ultrafiltration (%) ab-p-1 (%) ab-p-2 (%) ab-p-3 (%) ab-p-4 (%) 5 mg / mL 38.2 35.1 32.1 31.3 25.4 10 mg / mL 49.8 51.6 50.8 44.3 39.2 15 mg / mL 55.3 63.8 57.2 51.8 44.2 20 mg / mL 62.6 73.7 63.4 58.2 47.6

[0038] As shown in Tables 1-2 and 2, all four prepared enzymatic hydrolysates exhibited antioxidant activity, capable of scavenging the different types of free radicals tested. Furthermore, the antioxidant capacity of the hydrolysates increased with increasing dosage. Among the four hydrolysates, ab-p-1 exhibited the highest free radical scavenging ability.

[0039] 2) The effect of ab-p-1 on the antioxidant enzyme activity of HepG2 cells

[0040] Human liver cancer cells were used as a model to study the mechanism of antioxidant stress, and cell viability was measured to check whether ab-p-1 caused any cell damage.

[0041] Cell culture: Human liver cancer cells (HepG2 cells, Chinese Academy of Sciences Type Culture Collection, catalog number: SCSP-510) were cultured in DMEM medium supplemented with 2.5% (v / v) fetal bovine serum, 50 mg / L gentamicin, and 50 mg / L penicillin G at 37°C and 5% CO2.

[0042] Cell viability assay: HepG2 cell viability was determined using a CCK-8 assay kit. CCK-8 reagent was added to the wells of HepG2 cells that had been incubated with different concentrations of short peptides (KGP and VPD). After the reaction was complete, the absorbance was measured at 450 nm using a microplate reader. Cells not treated with the sample and AAPH served as the control group. Cell viability was expressed as the percentage of surviving cells in the sample group relative to the surviving cells in the control group.

[0043] HepG2 cells (1×10⁵ / mL) were cultured in 96-well plates. They were cultured at 37°C and 5% CO₂ for 24 h. Then, 100 μL of DMEM serum-free medium containing different types (KGP or VPD) and concentrations (100 mg / L, 200 mg / L, 400 mg / L, 500 mg / L) of peptides and 10 μmol / L DCFH-DA was added, and the cells were incubated at 37°C for 90 min. After discarding the medium, the cells were washed three times with serum-free medium. 100 μL of AAPH (200 μM) was added, and the cells were incubated for 0, 25, 50, 90, and 180 min. The cells were then measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0044] like Figure 3 As shown, ab-p-1 has a slight effect on the growth of HepG2 cells; however, ab-p-1 did not show cytotoxicity in HepG2 cells at concentrations as high as 400 µg / mL (cell viability > 90%).

[0045] Example 3: Purification of Sichuan pepper seed polypeptides

[0046] 1) The peptide with the highest antioxidant activity after ultrafiltration (ab-p-1) was dissolved at a concentration of 40 mg / mL in sodium acetate buffer (20 mM, pH 3.6) and loaded onto a cation exchange column (25 mm × 500 mm, Shanghai Huxi Analytical Instruments Co., Ltd., Shanghai, China). The column was equilibrated with sodium acetate buffer and then eluted with Tris-HCl (pH 7.2) at a constant flow rate of 0.8 mL / min. The eluent (4 mL) was collected and monitored at 214 nm.

[0047] ab-p-1 produced two distinct peaks (labeled ab-p-1-A and ab-p-1-B). Each collected fraction was combined, lyophilized, and subjected to DPPH and free radical scavenging assays. Figure 4 As shown, ab-p-1-B was found ( Figure 4 a) compared to ab-p-1-A ( Figure 4 b) Better activity.

[0048] 2) The ab-p-1-B fraction was further purified using a Sephadex G-15 packed gel filtration column (25 mm × 500 mm). Elution was performed with distilled water at a constant flow rate of 0.8 mL / min. Each fraction (4 mL) was collected and monitored at 214 nm. The ab-p-1-B fraction was aliquoted into two parts (labeled ab-p-1-B1 and ab-p-1-B2, respectively). Ab-p-1-B2 was found to be more active than ab-p-1-B1, and the ab-p-1-B2 fraction was lyophilized for further purification.

[0049] 3) After separation by GFC (gel filtration chromatography), ab-p-1-B2 was further purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The lyophilized peptide was dissolved in distilled water at a concentration of 20 mg / mL and then injected into a Shimadzu LC-20A system (Shimadzu Corporation, Tokyo, Japan). The sample was eluted with an aqueous solution (A) containing 0.03% trifluoroacetic acid (TFA) and 100% acetonitrile containing 0.03% TFA (B) at a flow rate of 0.8 mL / min. The following elution gradients were used: 0–10 min A, linear gradient 0%–10% B; 10–20 min, linear gradient 10%–35% B; 20–55 min, linear gradient 35%–70% B. The fractions were detected at 280 nm, collected, lyophilized, and their antioxidant effects were determined.

[0050] like Figure 5 As shown, six fractions of ab-p-1-B2 were detected (ab-p-1-B2-1, ab-p-1-B2-2, ab-p-1-B2-3, ab-p-1-B2-4, ab-p-1-B2-5, and ab-p-1-B2-6), among which ab-p-1-B2-1 and ab-p-1-B2-3 showed higher antioxidant activity than the other fractions.

[0051] Example 4: Identification of amino acid sequences of antioxidant peptides and detection of their antioxidant effects

[0052] The fraction with the strongest antioxidant capacity after RP-HPLC purification was analyzed by LC-MS / MS using an LC / MSD Trap XCT system (Agilent Technologies). Spectra were recorded in positive ion reflectometer mode, with a mass / charge (m / z) range of 200–1000. Peptide sequencing was performed by processing the MS / MS spectra using BioTools (version 3.0, Bruker Daltonics Inc., Karlsdorf-Neuthard, Germany) and by manual calculations; the amino acid sequences of ab-p-1-B2-1 and ab-p-1-B2-3 were determined to be Ser-Glu-Cys-Val-His-Leu and Pro-Gly-Arg-His-Trp, respectively.

[0053] Synthetic peptides were obtained by solid-phase synthesis according to the amino acid sequence.

[0054] Synthetic peptides (ab-p-1-B2-1 and ab-p-1-B2-3) and ab-p-1 were prepared at concentrations of 20 mg / mL, respectively. The antioxidant activities of the synthetic peptides and ab-p-1 were detected (repeated three times, and the average value was taken). The detection methods are shown in Table 3.

[0055] DPPH free radical scavenging capacity assay: Add 2 mL x 10 -4 After mixing the mol / L DPPH solution and reacting it in the dark at room temperature for 20 min, centrifuge at 4000 r / min for 10 min, zero the instrument with 95% ethanol, and measure its absorbance A at 517 nm. i Simultaneously, the absorbance A of a mixture of 2 mL of 95% ethanol and 2 mL of sample solution was measured. j ; and the absorbance A0 of 2 mL DPPH solution mixed with 2 mL deionized water; its scavenging rate (%) = .

[0056] 02 -• Free radical scavenging capacity determination: Take 4.5 mL of 50 mmol / L pH 8.2 Tris-HCl buffer solution, add 0.1 mL of sample solutions of different concentrations, incubate at 25℃ for 10 min, then add 0.3 mL of 5 mmol / L pyrogallol pre-warmed at 25℃, mix well, and quickly add to a dry cuvette. Measure the absorbance at 320 nm every 20 mm, starting from when the absorbance value reaches 0.2. Zero the instrument with an equal volume of 10 mmol / L HCl instead of pyrogallol. For the blank group, use an equal volume of deionized water instead of the sample solution, and follow the same steps. Plot the absorbance values ​​of the sample solution and pyrogallol, and the mixture of deionized water and pyrogallol, at 320 nm over time. The slope of the regression equation is taken as the auto-oxidation rate V of pyrogallol. 样品 , Its clearance rate (%) = .

[0057] Table 3: Antioxidant Activity Record Table

[0058] polypeptide <![CDATA[O2 - Free radical scavenging rate (%) DPPH free radical scavenging rate (%) ab-p-1-B2-1 83.1 80.5 ab-p-1-B2-2 81.7 79.2 ab-p-1 74.3 73.2

[0059] Table 3 shows that the DPPH· free radical scavenging rate and O2 of the synthetic peptides - • The free radical scavenging rate is significantly better than that of ab-p-1, and the synthetic peptide has better antioxidant activity; it can be prepared into genetically engineered bacteria for application.

Claims

1. A polypeptide prepared from Sichuan pepper seeds, characterized in that, The amino acid sequence of the polypeptide is either Ser-Glu-Cys-Val-His-Leu or Pro-Gly-Arg-His-Trp.

2. The application of the polypeptide according to claim 1 in the preparation of antioxidant functional foods.

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