Antioxidant peptides and preparation methods and applications thereof

Abstract

The present invention relates to the field of biotechnology, and discloses an antioxidant peptide, a preparation method and application thereof. The amino acid sequence of the antioxidant peptide of the present invention is shown in SEQ ID NO: 1. The preparation method comprises the following steps: firstly enzymatically hydrolyzing white mullet to obtain an enzymatic hydrolysate, using ultrafiltration to separate an ultrafiltration component with a molecular weight cutoff of less than 3000Da, and further separating and purifying the ultrafiltration component by RP-HPLC method after gel chromatography to obtain the antioxidant peptide. The antioxidant peptide prepared by the present invention has high antioxidant activity, can be used in the preparation of antioxidants, and improve the overall antioxidant performance of the product.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an antioxidant peptide and a preparation method and application thereof. Background Art

[0002] Superoxide (O 2- ) free radicals, hydroxyl radicals (OH - The generation of reactive oxygen free radicals (ROS) such as free radicals and hydrogen peroxide is one of the main causes of cellular oxidative stress damage. Modern non-infectious diseases such as cancer, diabetes, inflammation, and liver disease are all related to it. Among the antioxidant products that can remove ROS, compared with synthetic drugs, antioxidants made from natural antioxidant ingredients have attracted much attention due to their safety and high efficiency. For example, natural antioxidant ingredients such as vitamin C, vitamin E, and melatonin have been successfully discovered and extracted from edible plants such as tomatoes, kiwis, and wheat. Peptides are small molecule compounds formed by α-amino acids connected together by peptide bonds, and are also intermediate products of protein hydrolysis. Among them, antioxidant peptides are a group of small functional bioactive peptides that can effectively remove the above ROS. Therefore, the discovery of antioxidant peptides is of great significance to human health and the food industry.

[0003] White mullet ( Opniocepnalus argus var Kimnra ) is a very rare and high-quality fish among my country's freshwater fish. It is a special aquatic product independently developed by Neijiang City, Sichuan Province. It has tender meat, few bones, delicious taste, and has the effects of nourishing the spleen and stomach, promoting lactation, etc. White mullet contains a large amount of protein and amino acids, calcium, iron and other elements required by the human body. It can supplement the human body with a variety of nutrients, especially phosphorus, iron and other elements. After consumption, it is beneficial to the normal functioning of the spleen and stomach and has the effect of nourishing the spleen and stomach. However, the current research on white mullet is mainly focused on product development, and the research on peptides in white mullet has not yet been reported. Summary of the invention

[0004] The purpose of the present invention is to provide an antioxidant peptide and a preparation method and application thereof. The antioxidant peptide can be used in the preparation of antioxidants to improve their antioxidant capacity.

[0005] To achieve the above object, the first technical solution adopted by the present invention is:

[0006] Antioxidant peptide, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] Preferably, the antioxidant peptides are extracted from white mullet.

[0008] The second technical solution adopted by the present invention is:

[0009] The preparation of antioxidant peptides comprises the following steps:

[0010] homogenizing the white mullet and subjecting it to enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0011] The enzymatic hydrolysate is subjected to ultrafiltration to separate the ultrafiltration component with a molecular weight cut-off less than 3000Da;

[0012] performing gel chromatography on the ultrafiltration fraction; and

[0013] The components obtained after chromatography are separated and purified.

[0014] The third technical solution adopted by the present invention is:

[0015] Application of antioxidant peptides in antioxidant products.

[0016] The fourth technical solution of the present invention is:

[0017] Antioxidant, containing antioxidant peptide of the first technical solution.

[0018] The above antioxidant peptides can also be synthesized using amino acids as raw materials by chemical methods, which is well known in the art.

[0019] The antioxidant peptide provided by the present invention can effectively remove active oxygen free radicals such as superoxide free radicals and hydroxyl free radicals, and has high antioxidant activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is the chromatogram of the ultrafiltration fraction with a molecular weight below 3000Da after being separated by Sephadex G-15 gel chromatography;

[0022] Figure 2-5 The results of different antioxidant activities of chromatographic fractions B1, B2, and B3;

[0023] Figure 6 is the ultra-high performance liquid chromatography separation spectrum of the chromatography component B1;

[0024] Figure 7 is the secondary mass spectrum of the sequence LKKNSYEESLEHL;

[0025] Figure 8 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION CHARACTERISTICS FOR PURITY CONFIRMATION OF THE SEQUENCE LKKNSYEESLEHL;

[0026] Figures 9 to 12 The results of different antioxidant activities of the compound with sequence LKKNSYEESLEHL and glutathione. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and accompanying drawings. It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. In the embodiment, those who do not indicate specific conditions are carried out according to the conditions recommended by normal conditions or manufacturers. Those who do not indicate manufacturers for reagents or instruments used are conventional products that can be obtained by commercial purchase.

[0028] The first embodiment of the present invention provides an antioxidant peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0029] The antioxidant peptide is extracted from white mullet.

[0030] The second embodiment of the present invention provides a method for preparing an antioxidant peptide, comprising the following steps:

[0031] Step 1: Remove the byproducts of the white mullet, add ultrapure water (3-5 times the weight of the fish meat) to homogenize and obtain a homogenous material;

[0032] Step 2: adjust the pH of the homogenous material to 2.0 with HCl, add pepsin according to (3%-4%) of the mass of the homogenous material, and simulate gastric digestion enzymolysis at 37°C water bath for 2.5 hours; then use 0.9 M NaHCO3 to adjust the pH of the above solution to 5.33, and then use 1 M sodium hydroxide to adjust the pH to 7.5, add pancreatic enzyme according to (3%-4%) of the mass of the homogenous material, simulate intestinal digestion at 37°C water bath for 2.5 hours, place in boiling water for 10 minutes to terminate digestion, and cool to room temperature to obtain enzymolysis solution; centrifuge the above enzymolysis solution to obtain the supernatant, vacuum freeze-dry to obtain white mullet crude peptide powder, and store at -18°C;

[0033] Step 3: The crude peptide powder is subjected to ultrafiltration separation, and the ultrafiltration fraction B with a molecular weight cutoff of less than 3000Da is retained, freeze-dried, and stored at -18°C;

[0034] Step 4: The ultrafiltration fraction B is prepared into a solution with a concentration of (20-30) mg / mL, and chromatographed using Sephadex G-15 gel chromatography. The obtained fraction is freeze-dried and stored at -18°C;

[0035] Step 5: Select the component with the highest antioxidant activity obtained after chromatography (antioxidant activity determination), separate and purify the component by RP-HPLC method to obtain the antioxidant peptide isolated from white mullet with the following amino acid sequence:

[0036] Leu-Lys-Lys-Asn-Ser-Tyr-Glu-Glu-Ser-Leu-Glu-His-Leu (LKKNSYEESLEHL).

[0037] In a further embodiment, the centrifugation conditions in step 2 are 4° C., 8000×g, and time for 20 min.

[0038] In a further embodiment, the Sephadex G-15 gel chromatography method in step 4 is as follows: the loading volume is (2-4) mL, the elution flow rate is (1-2) mL / min, and the detection wavelength is 220 nm.

[0039] In the following specific embodiments of the present invention, the ultrafiltration fractions were chromatographed using Sephadex G-15 gel chromatography, and the fractions with the highest antioxidant activity (antioxidant activity assay) were selected, which were further separated using RP-HPLC.

[0040] Among them, antioxidant activity was measured using DPPH free radical scavenging rate, ABTS free radical scavenging rate, O 2- The free radical scavenging rate and OH-free radical scavenging rate are used to express it. The specific determination methods of each free radical scavenging rate are as follows:

[0041] (1) DPPH free radical scavenging rate and ABTS free radical scavenging rate

[0042] The determination was carried out according to GB / T 39100-2020 "DPPH and ABTS method for determination of antioxidant activity of polypeptides";

[0043] (2) O 2- Free radical scavenging rate

[0044] Take 0.1 mL of the above sample, add 0.1 mL of 2.52 mM nitro blue tetrazolium chloride and 0.1 mL of 624 mM NADH to obtain a reaction mixture, add 0.1 ml of 120 µM phenazine N-methylsulfate to the reaction mixture, incubate at 25°C for 5 min, measure the absorbance at 560 nm, and calculate O according to the following formula: 2- Free radical scavenging rate (%) was calculated:

[0045] O 2- Free radical scavenging rate (%) = (A0-A1) / A0×100%

[0046] Where: A0 is the absorbance without sample, A1 is the absorbance with sample;

[0047] Half-clearance concentration: With the concentration of the sample solution as the horizontal axis and the clearance rate as the vertical axis, a linear equation between the sample solution and the clearance rate is established, and then the calculation is performed;

[0048] (3) OH- free radical scavenging rate

[0049] Take 0.15 mL of 5.0 mM 1,10-phenanthroline, 0.15 mL of 5.0 mM FeSO4, 0.15 mL of 15 mM ethylenediaminetetraacetic acid and 0.1 mL of pH 7.4 phosphate buffer and mix them. Add 0.15 mL of sample and 0.2 ml of 1% H2O2. After incubation at 37°C for 1 h, measure the absorbance at 536 nm and calculate the OH- free radical scavenging rate (%) according to the following formula:

[0050] OH- free radical scavenging rate (%) = (AS-A0) × 100 / (AC-A0)

[0051] Where: AS is the absorbance of the sample; A0 is the absorbance of the blank solution using distilled water instead of the sample; AC is the absorbance of the control solution in the absence of H2O2;

[0052] Half-clearance concentration: With the concentration of the sample solution as the horizontal axis and the clearance rate as the vertical axis, a linear equation between the sample solution and the clearance rate is established, and then calculations are performed.

[0053] Example 1

[0054] A method for extracting, separating and purifying antioxidant peptides, the specific steps are as follows:

[0055] Step (1): removing the byproducts of the white mullet, adding ultrapure water 4 times the weight of the fish meat for homogenization to obtain a homogenous material;

[0056] Step (2): adjusting the pH of the homogenized material to 2.0 with HCl, adding pepsin at 4% of the mass of the homogenized material, and simulating gastric digestion enzymolysis at 37°C in a water bath for 2.5 h; then adjusting the pH of the solution to 5.33 with 0.9 M NaHCO3, and then adjusting the pH to 7.5 with 1 M sodium hydroxide, adding pancreatic enzyme at 4% of the mass of the homogenized material, simulating intestinal digestion at 37°C in a water bath for 2.5 h, placing in boiling water for 10 min to terminate digestion, and cooling to room temperature to obtain an enzymolysis solution; centrifuging the enzymolysis solution, taking the supernatant, and vacuum freeze-drying to obtain a crude white mullet peptide powder, which is stored at -18°C;

[0057] Step (3): ultrafiltration separation is performed on the crude white mullet peptide powder to retain the ultrafiltration component B with a molecular weight cutoff of less than 3000 Da, and the obtained component B is freeze-dried and stored at -18°C;

[0058] Step (4): The ultrafiltration component B is prepared into a solution with a concentration of 25 mg / mL and filtered through a 0.22 μm aqueous microporous filter membrane. The solution is chromatographed using Sephadex G-15 gel chromatography, with double distilled water as the eluent. The chromatography method is as follows: the elution flow rate is 1 mL / min, the detection wavelength is 220 nm, and the sample volume is 4 ml. The chromatographic spectrum results are as follows: Figure 1 As described above, the abscissa represents the elution time (min), and the ordinate represents the absorbance A.

[0059] like Figure 1 As shown, the three separated components obtained in the chromatography collection and elution process are labeled B1, B2, and B3 in sequence, and are freeze-dried and stored at -18°C.

[0060] Step (5): The antioxidant activity of the B1, B2, and B3 fractions obtained after chromatography was determined. The results are shown in Figure 2-5 , the horizontal axis represents each component, and the vertical axis represents the half-scavenging concentration of each component for each free radical.

[0061] like Figure 2-5 As shown, component B1 has the lowest half-scavenging concentration for each free radical, indicating that it has the highest antioxidant activity. Therefore, component B1 was selected for the next step of RP-HPLC separation and purification.

[0062] Step (6): RP-HPLC was used to further separate and purify the B1 component. The separation conditions of RP-HPLC were as follows: mobile phase A was water containing 0.1% TFA; mobile phase B was acetonitrile containing 0.1% TFA; gradient elution, i.e. 2% B at 0 min, 2%-10% B at 0-18 min, 10%-20% B at 18-36 min, 20%-30% B at 36-55 min, 30%-40% B at 55-70 min; analytical column was C18 (3 μm, 100A); injection volume was 10 uL; flow rate was 1 mL / min.

[0063] The liquid phase separation spectrum of the chromatographic component B1 is shown in Figure 6 In the figure, the horizontal axis represents the elution time (min), and the vertical axis represents the response value (AU).

[0064] Step (7): HPLC-MS / MS is used to purify the components purified in step (6), i.e. Figure 6The component B4 at 5-6 min in the sample was identified. The liquid phase conditions were as follows: mobile phase A was water containing 0.1% formic acid; mobile phase B was water containing 0.1% formic acid and 80% CAN; gradient elution, i.e. 0 min 4%B, 0-2 min 4%-8%B, 2-45 min 8%-28%B, 45-55 min 28%-40%B, 55-56 min 40%-95%B, 56-66min 95%B; analytical column was packed with Acclaim PepMapRPLC C18 (3 μm, 150 μm id × 150 mm); injection volume was 10 uL; flow rate was 600 nL / min. Mass spectrometry conditions: primary mass spectrometry parameters are Resolution 70000, AGCtarget 3e6, MaximumIT 100 ms, Scanrange100 to1500 m / z; secondary mass spectrometry parameters are Resolution 17500, AGCtarget 1e5, MaximumIT 50 ms, TopN20, NCE / steppedNCE28. The amino acid sequence of the identified antioxidant peptide is:

[0065] Leu-Lys-Lys-Asn-Ser-Tyr-Glu-Glu-Ser-Leu-Glu-His-Leu (LKKNSYEESLEHL).

[0066] like Figure 7 , is the secondary mass spectrum of the component, i.e. the sequence LKKNSYEESLEHL. The following table shows the information of the antioxidant peptide:

[0067] Table 1 Antioxidant peptide information

[0068] .

[0069] Example 2

[0070] In order to further verify the antioxidant activity of the above antioxidant peptide, the sequence was synthesized using solid phase synthesis to obtain the sequence LKKNSYEESLEHL synthetic compound.

[0071] The method of step (6) in Example 1 was used for detection, and the detection results were as follows: Figure 8 , the purity of the sequence LKKNSYEESLEHL synthesis is ≥98%.

[0072] Using glutathione as a positive control, the antioxidant activity of the above sequence LKKNSYEESLEHL compound and glutathione at different concentrations (0.5 mg / ml, 1 mg / ml, 2 mg / ml, 4 mg / ml, 8 mg / ml) was determined. The results are shown in Figure 9-12 As described above, GSH in the figure is a glutathione sample, and LKKNSYEESLEHL is a sample of a synthetic product of the sequence LKKNSYEESLEHL.

[0073] according to Figure 9-12 The results show that the higher the concentration of the two samples, the higher their free radical scavenging rate. When the concentration is 8 mg / mL, the DPPH free radical scavenging rate and ABTS free radical scavenging rate of glutathione in the control group are 2- The free radical scavenging rates and OH- free radical scavenging rates were 94.83%, 91.08%, 89.01% and 36.72%, respectively. The free radical scavenging rates corresponding to the synthetic sequence LKKNSYEESLEHL were 75.17%, 80.21%, 72.25% and 43.84%, respectively, indicating that the sequence LKKNSYEESLEHL has strong antioxidant activity.

[0074] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. An antioxidant peptide, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. The antioxidant peptide according to claim 1, characterized in that The antioxidant peptide is extracted from white mullet.

3. The method for preparing the antioxidant peptide according to claim 1 or 2, characterized in that: The following steps are involved: homogenizing the white mullet and subjecting it to enzymatic hydrolysis to obtain an enzymatic hydrolyzate; The enzymatic hydrolysate is subjected to ultrafiltration to separate the ultrafiltration component with a molecular weight cut-off less than 3000Da; performing gel chromatography on the ultrafiltration fraction; and The components obtained after chromatography are separated and purified.

4. Use of the antioxidant peptide according to claim 1 or 2 in the preparation of antioxidant products.

5. Use of the antioxidant peptide according to claim 1 or 2 in the preparation of antioxidants.

6. An antioxidant, characterized in that Contains the antioxidant peptide according to claim 1 or 2.

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