Cottonseed antioxidant peptide K13P and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a cottonseed antioxidant peptide K13P and application thereof. According to the invention, the cottonseed antioxidant peptide with higher antioxidant activity and an amino acid sequence of KVVPWRRGHEGSCNP is separated from cottonseed protein, and the cottonseed antioxidant peptide can relieve cell oxidative stress induced by tert-butyl hydroperoxide. The compound can be used as a natural antioxidant to be applied to food, cosmetics and feed additives.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to cottonseed antioxidant peptide K13P and its applications. Background Technology

[0002] Naturally derived antioxidants are gaining increasing attention due to their wide availability, easy absorption, and safety. Antioxidant peptides, as an important class of natural antioxidants, have been successfully isolated from various proteins; however, their extraction often faces challenges such as complex methods, low efficiency, and high costs. Therefore, finding efficient, safe, and economical production processes for natural antioxidant peptides is a key need in the health industry.

[0003] Cottonseed protein is obtained from cottonseed meal through low-temperature oil extraction and defatting. It has a crude protein content of 40-70% and is an important plant protein resource containing abundant potential antioxidant peptides.

[0004] While studies have reported the isolation of antioxidant peptides from cottonseed protein, truly bioactive antioxidant peptides cannot be obtained simply by optimizing enzymatic hydrolysis conditions and purification parameters. Their formation is influenced by multiple factors, including the structure of the raw protein, the specificity of the enzyme cleavage sites, the stability of small peptides, and the extraction method. In other words, the antioxidant activity of cottonseed peptides stems from the precise release and enrichment of specific functional peptides. Furthermore, not all antioxidant peptides exhibiting free radical scavenging capabilities in vitro can successfully enter cells to exert their antioxidant effects. The molecular weight, polarity, stability, and cellular uptake mechanisms of peptides all affect their bioavailability at the cellular level. Therefore, developing antioxidant peptides with actual intracellular activity requires a systematic strategy, far exceeding simple optimization of process parameters. Summary of the Invention

[0005] According to the present invention, the cottonseed antioxidant peptide has the amino acid sequence shown (SEQ ID NO: 1).

[0006] SEQ ID NO:1:

[0007] KVVPWRRGHEGSCNP.

[0008] The present invention also provides a method for preparing the above-mentioned cottonseed antioxidant peptides, comprising the following steps: enzymatic hydrolysis using serine protease to obtain hydrolysate, wherein the amount of proteinase K used is 4000 U / g cottonseed protein, and the amount of enzyme added is calculated based on the weight of cottonseed protein; the enzymatic hydrolysis temperature of proteinase K is 60℃, pH is 7, rotation speed is 200 rpm, material-to-liquid ratio is 1:5, and reaction time is 4 hours; after hydrolysis, the reaction solution is boiled for 10 min to inactivate the protease activity and the supernatant is collected.

[0009] Furthermore, the supernatant is subjected to molecular weight cutoff ultrafiltration, anion exchange chromatography, gel chromatography column separation, reversed-phase high-performance liquid chromatography, and mass spectrometry sequencing to obtain the cottonseed antioxidant peptide of this application.

[0010] The present invention also provides applications of the cottonseed antioxidant peptides, such as as active ingredients in antioxidant products, preferably including food antioxidants, feed additives, or cosmetics.

[0011] The beneficial effects of this invention are:

[0012] This invention employs proteolytic hydrolysis to selectively hydrolyze cottonseed protein, yielding a cottonseed antioxidant peptide with the amino acid sequence KVVPWRRGHEGSCNP exhibiting high antioxidant activity. This peptide can alleviate cellular oxidative stress induced by tert-butyl hydroperoxide and can be used as a natural antioxidant in food, cosmetics, and feed additives. Attached Figure Description

[0013] Figure 1 This demonstrates the free radical scavenging ability of cottonseed peptides with different molecular weights;

[0014] Figure 2 Display an anion exchange chromatogram;

[0015] Figure 3 This demonstrates the free radical scavenging ability of the ion-exchange components;

[0016] Figure 4 Display size exclusion chromatogram;

[0017] Figure 5 Display size exclusion separation component free radical scavenging ability;

[0018] Figure 6 Display reverse high performance liquid chromatogram;

[0019] Figure 7 It demonstrates the ability to scavenge free radicals in reverse high-performance liquid phase separation components;

[0020] Figure 8 The mass spectrometry detection pattern of peptide KVVPWRRGHEGSCNP is shown.

[0021] Figure 9 The effect of the peptide on the viability of HepG2 cells stimulated with tert-butyl hydrogen peroxide was shown.

[0022] Figure 10 This study demonstrates the effect of the peptide on intracellular ROS in HepG2 cells stimulated with tert-butyl hydrogen peroxide. Detailed Implementation

[0023] Implementation Case 1: Isolation of Antioxidant Peptides from Cottonseed

[0024] The cottonseed protein is preferably 65% ​​cottonseed protein, and the specific source of the cottonseed protein is not particularly limited in this invention. Preferably, before enzymatic hydrolysis, the process also includes mixing the cottonseed protein with water to prepare a suspension, wherein the material-to-liquid ratio of the suspension is 1:5, and the mixture is homogenized at 60°C for 1 hour. The amount of proteinase K is preferably 4000 U / g cottonseed protein, the enzymatic hydrolysis temperature is 60°C, the hydrolysis time is preferably 4 hours, and the pH is preferably 7. Enzyme inactivation is then performed, preferably by heating at 100°C for 20 minutes. After enzyme inactivation, the supernatant is collected as the cottonseed protein hydrolysate. More preferably, the supernatant is subjected to vacuum freeze-drying to obtain the cottonseed protein hydrolysate. The method of vacuum freeze-drying is not particularly limited in this invention; conventional vacuum freeze-drying methods in the art are acceptable.

[0025] Preliminary separation of cottonseed peptides was based on molecular weight using ultrafiltration. CPH was separated into 10 kDa, 5 kDa, and 3 kDa fractions using ultrafiltration tubes. At the same concentration, the fractions with molecular weights below 3 kDa showed the highest free radical scavenging capacity in the antioxidant activity assay. Figure 1 Therefore, the <3kDa component was selected for subsequent experiments.

[0026] Cottonseed protein hydrolysate was ultrafiltered to collect peptides with a molecular weight <3 kDa, which were then lyophilized. The cottonseed antioxidant peptides were then reconstituted in Tris-HCl buffer at pH 9.0, and negatively charged peptides were collected by anion exchange chromatography. The anion exchange chromatography column used in this invention is a Hitrap column. TM After the Q HP, CPH-3k sample flowed through the ion exchange column, it was separated into three components, named H1, H2, and H3. Figure 2 At the same concentration, all three antioxidant assays showed that component H1 had the highest free radical scavenging capacity. Figure 3 Therefore, the H1 fraction was collected for further purification.

[0027] Size exclusion chromatography was used to further separate the peptides in fraction H1. Fraction H1 was separated into fraction one and fraction two in descending order of molecular weight. Figure 4 The results showed that component one exhibited significantly higher scavenging abilities against ABTS and DPPH radicals than the other two components. Therefore, component one was collected for further separation. The gel chromatography column used in this invention is... 200 Increase 10 / 300GL

[0028] The peptides of component one were further separated using RP-HPLC, and eight components were collected at time points. Figure 6The collected small peptides were mainly concentrated in component 1, which was enriched with ABTS free radical scavenging activity. Figure 7 Ultimately, the component with the highest antioxidant activity was obtained and prepared for subsequent analysis. The reversed-phase high-performance liquid chromatography column used in this invention is an Agilent TC-C18(2) (5μm 4.6×150mm) column.

[0029] Example 2: Determination of antioxidant peptides in cottonseed

[0030] More than 64 small peptides were identified from component 1; however, cottonseed peptides do not generally possess antioxidant properties. For example, as shown in Table 1, 10 small peptides from component 1 were chemically synthesized, and their ABTS and DPPH free radical scavenging abilities were determined.

[0031] The specific method is as follows:

[0032] The hydroxyl radical scavenging activities of 1,1-diphenyl-2-picrylhydrazine (DPPH), 2,2-azobis(3-ethylbenzothiazole)-6-sulfonic acid (ABTS), and cottonseed peptide were determined using the following method: 0.2 mL of polypeptide aqueous solutions of different concentrations were added to 0.2 mL of 1 mM DPPH anhydrous ethanol solution in a 2 mL centrifuge tube. The mixture was thoroughly mixed and reacted in the dark for 30 min. The absorbance was then measured at 517 nm (A). s Take 0.2 ml of the polypeptide aqueous solution in a test tube, add 0.2 ml of anhydrous ethanol, and measure its absorbance at 517 nm (A). b ); Take 0.2 mL of 1 mM DPPH anhydrous ethanol solution and 0.2 mL of water as a reference, and measure its absorbance at 517 nm (A). c ).

[0033] Accurately prepare 7.0 mM ABTS solution and 2.45 mM potassium persulfate solution, mix well, and let stand for 12 h at room temperature in the dark to obtain the ABTS stock solution. Dilute the ABTS stock solution with deionized water to obtain an absorbance of 0.70 ± 0.02 at 734 nm, and equilibrate at 30 °C for 30 min to obtain the ABTS working solution. Add 0.1 mL of peptide solution of different concentrations and 0.9 mL of ABTS working solution to test tubes, mix well, and let stand at room temperature in the dark for 5 min. Measure the absorbance at 734 nm. Use an equal volume of deionized water instead of peptide I solution as a control. Set up 3 replicates and calculate the average value.

[0034] Absorbance was measured using a microplate reader. The formula for calculating the cleaning ability is as follows:

[0035] Scavenging capacity (%) = ((A) s -A b ) / A c)×100%

[0036] Where A s A represents the absorbance of the sample to be tested. c A represents the absorbance of the blank control. b Absorbance for the negative control. Half-maximal effective concentration (EC50) 50 The concentration of a sample that removes 50% of free radicals can be calculated using a nonlinear regression analysis of the relationship between the removal rate and concentration using GraphPad Prism 10 software.

[0037] The results showed that only KVVPWRRGHEGSCNP exhibited scavenging ability against DPPH and ABTS radicals, with EC50% showing the highest scavenging capacity against both DPPH and ABTS radicals. 50 The values ​​were 5.5 mg / mL and 0.02 mg / mL, respectively (Table 1). Figure 8 This is a mass spectrometry detection image of KVVPWRRGHEGSCNP.

[0038] Table 1. Peptide information and free radical scavenging ability

[0039]

[0040] Note: - indicates no significant ABTS and DPPH free radical scavenging ability.

[0041] Example 4: Determination of the ability of cottonseed antioxidant peptides to alleviate cellular oxidative stress

[0042] tert-butyl hydroperoxide (tBHP) generates highly reactive tert-butyloxy radicals (tBO·) and hydroxyl radicals (·OH) within cells, initiating cellular lipid peroxidation. Simultaneously, tBHP and its free radical products aggressively consume endogenous antioxidants such as glutathione and inhibit the activity of glutathione peroxidase 4 (GPX4), leading to a surge in intracellular reactive oxygen species (ROS), collapse of the antioxidant defense system, and ultimately triggering an oxidative damage pathway dominated by ferroptosis. Compared to hydrogen peroxide, tBHP exhibits greater stability and controllability in induced cellular oxidative stress models. tBHP is not easily decomposed rapidly in the cellular environment, continuously releasing free radicals to mimic in vivo oxidative stress processes such as lipid peroxidation, and its induction mechanism more closely resembles physiological states. Furthermore, its induction process is milder and has better experimental reproducibility, thus it is widely used in cellular-level antioxidant research and mechanism exploration. The liver is a core organ for metabolism, detoxification, and biotransformation, and is also one of the main target organs for oxidative stress damage. Many exogenous substances (drugs, environmental toxins, alcohol) and endogenous metabolic processes (fatty acid oxidation, drug metabolism) produce large amounts of reactive oxygen species in the liver. HepG2 cells, derived from human hepatoblastoma, are one of the most widely used cell models in oxidative stress research.

[0043] 1. Cottonseed antioxidant peptides improve the survival rate of HepG2 cells stimulated with tert-butyl hydrogen peroxide.

[0044] After culturing HepG2 cells for 24 hours, they were injected with 1×10⁻⁶ cells. 4 Cells were seeded at a density of 100 cells / well in 96-well plates. After culturing to 70-80% confluence, different concentrations of small peptides were added for 12 hours. Except for the NC group, the other groups were treated with 100 μL of medium containing 200 μM tBHP for 12 hours. After tBHP treatment, the medium was removed, cells were washed with PBS, and medium containing CCK-8 reagent was added to each well. After incubation for 4 hours, the absorbance at 450 nm was measured using a microplate reader. The results showed that ( Figure 9 Cell viability in the tBHP-treated group was significantly lower than that in the control group (P<0.05). With the addition of 20 μg / mL of antioxidant peptides, cell viability in both peptide-added groups was significantly increased compared to the tBHP-treated group (P<0.05). Particularly with the addition of 100 μg / mL of peptides, cottonseed antioxidant peptides significantly increased cell viability compared to the tBHP-treated group (P<0.05), while showing no significant difference compared to the NC group (P>0.05).

[0045] 2. Cottonseed antioxidant peptides can reduce the intracellular ROS content stimulated by tert-butyl hydrogen peroxide.

[0046] After culturing HepG2 cells for 24 hours, they were injected with 1×10⁻⁶ cells. 4 Cells were seeded at a density of 100 cells / well in black 96-well plates. After culturing to 70-80% confluence, different concentrations of small peptides were added for 12 hours. Except for the NC group, the other groups were treated with 100 μL of medium containing 200 μM tBHP for 12 hours. After tBHP treatment, the medium was removed, cells were washed with PBS, and incubated with medium containing 10 μM DCFH-DA probe for 20 minutes. Cells were washed with PBS to thoroughly remove any unintended DCFH-DA. Fluorescence intensity was detected using an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The results showed ( Figure 10 The addition of tBHP significantly stimulated the secretion of ROS by cells (P<0.05). With the addition of 20 μg / mL of antioxidant peptide, the intracellular ROS content was significantly downregulated compared to the tBHP-treated group (P<0.05). With the addition of 100 μg / mL of peptide, the intracellular ROS content in the peptide-treated groups was significantly lower than that in the tBHP-treated groups (P<0.05).

[0047] The above results indicate that the cottonseed peptide of this invention possesses high antioxidant activity. At the cellular level, the cottonseed peptide demonstrates that it can improve cell survival under oxidative stress, reduce intracellular ROS production, and effectively alleviate cellular oxidative stress. This invention is the first to discover that the cottonseed peptide KVVPWRRGHEGSCNP exhibits antioxidant activity both in vivo and in vitro.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Cottonseed antioxidant peptides, characterized in that, The amino acid sequence of the cottonseed antioxidant peptide is shown in SEQ ID NO:

1.

2. The application of the cottonseed antioxidant peptide according to claim 1 as an antioxidant.

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