CN118240018A - White mullet antioxidant peptide and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, and discloses an antioxidant peptide of white cuttlefish, and a preparation method and application thereof. The antioxidant peptide of the white cuttlefish is extracted from the white cuttlefish, and the amino acid sequence is shown in SEQ ID NO: 1. The preparation method comprises the steps of firstly simulating in-vitro digestion of a human body to obtain enzymolysis liquid from the white cuttlefish, separating an ultrafiltration component with the interception molecular weight smaller than 3000Da by ultrafiltration, and further separating and purifying a chromatographic component with antioxidant activity by adopting an RP-HPLC method after gel chromatography to obtain the white cuttlefish antioxidant peptide. The antioxidant peptide of the croaker prepared by the invention has a strong antioxidant effect, and can be applied to the preparation of antioxidant products.

Description

White mullet antioxidant peptide and preparation method and application thereof

Technical Field

The invention belongs to the field of biotechnology, and in particular to a white mullet antioxidant peptide and a preparation method and application thereof.

Background technique

Peptides are small molecule compounds formed by α-amino acids linked together by peptide bonds. They are also intermediate products of protein hydrolysis. Among them, antioxidant peptides are a group of small functional bioactive peptides that can achieve antioxidant effects by scavenging free radicals, inhibiting lipid peroxidation, accelerating peroxide decomposition, and enhancing the activity of intracellular antioxidant enzymes. In recent years, the extraction of antioxidant peptides from food has been widely studied. The extracted antioxidant peptides are composed of 2-20 amino acids and can be used as natural antioxidants, but their antioxidant activity is affected by factors such as relative molecular mass, amino acid type, arrangement and spatial structure. Obtaining antioxidant peptides from aquatic products and plants is the most common way. At present, antioxidant peptides have been extracted from a variety of aquatic products and functional peptide beverages have been prepared, such as tuna, sea urchin, mackerel, krill, bighead carp, etc.

White mullet ( Opniocepnalus argus var Kimnra ) is a new freshwater fish species recognized by the China Fisheries Association in 2022. It is a very rare and high-quality fish among China'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 tonifying the spleen and stomach, and promoting lactation. 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 tonifying the spleen and stomach. However, the current research on white mullet is mainly focused on product development, and research on peptides in white mullet has not yet been reported.

Summary of the invention

The present invention aims to provide a white mullet antioxidant peptide and a preparation method thereof, wherein the white mullet antioxidant peptide is extracted from the white mullet. The present invention also provides an application of the white mullet antioxidant peptide, which can be applied to the preparation of antioxidants.

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

The amino acid sequence of the white mullet antioxidant peptide is shown in SEQ ID NO: 1.

Preferably, the white mullet antioxidant peptide is extracted from white mullet.

The second technical solution adopted by the present invention is:

The preparation of white mullet antioxidant peptide comprises the following steps:

The white mullet is homogenized and subjected to enzymatic hydrolysis treatment simulating human in vitro digestion to obtain an enzymatic hydrolysate;

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.

The third technical solution adopted by the present invention is:

Application of antioxidant peptides from white mullet in antioxidant products.

The fourth technical solution of the present invention is:

Antioxidant, containing the first technology solution of white mullet antioxidant peptide.

The above-mentioned white mullet antioxidant peptide can also be synthesized by chemical methods using amino acids as raw materials, which is well known in the art.

The white mullet antioxidant peptide provided by the present invention can effectively remove active oxygen free radicals such as superoxide free radicals and hydroxyl free radicals, has high antioxidant activity, and can be applied to various antioxidant products such as antioxidants, antioxidant foods, and medicines.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG1 is a chromatogram of ultrafiltration fractions with a molecular weight below 3000 Da after separation by Sephadex G-15 gel chromatography;

Figures 2-5 show the different antioxidant activity results of components B1, B2, and B3;

FIG6 is an ultra-high performance liquid chromatography separation spectrum of chromatography component B2;

FIG7 is a secondary mass spectrum of the sequence YTYSGLL;

FIG8 is an ultra-high performance liquid chromatography separation spectrum of the purity verification of the YTYSGLL synthetic compound;

Figures 9 to 12 show the different antioxidant activity results of YTYSGLL and glutathione.

Detailed ways

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.

The first embodiment of the present invention provides a white mullet antioxidant peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

The second embodiment of the present invention provides a method for preparing white mullet antioxidant peptides, comprising the following steps:

Step 1: remove the byproducts of the white mullet, add ultrapure water (2-6) times the weight of the fish meat, and homogenize to obtain a homogenous material;

Step 2: Use HCl to adjust the pH of the homogenous material to 2.0, add pepsin according to (1%-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 NaOH to adjust the pH to 7.5, add pancreatic enzyme according to (1%-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 and vacuum freeze-dry to obtain white mullet crude peptide powder;

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;

Step 4: The ultrafiltration fraction B is prepared into a solution with a concentration of (10-50) mg/mL, and chromatographed using Sephdex G-15 dextran gel chromatography. The obtained fraction is freeze-dried and stored at -18°C;

Step 5: Select the components with antioxidant activity after chromatography (antioxidant activity determination), separate and purify the components by RP-HPLC method to obtain the white mullet antioxidant peptide with the following amino acid sequence:

Tyr-Thr-Tyr-Ser-Gly-Leu-Leu (YTYSGLL).

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

In a further embodiment, the Sephadex G-15 dextran 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.

Furthermore, the sample loading volume is 4 mL.

In the following specific embodiments of the present invention, the ultrafiltration fraction is chromatographed by Sephadex G-15 dextran gel chromatography, and the fraction having antioxidant activity after chromatography (antioxidant activity assay) is selected and further separated by RP-HPLC.

Among them, the antioxidant activity is expressed by DPPH free radical scavenging rate, ABTS free radical scavenging rate, ^O2- free radical scavenging rate and OH- free radical scavenging rate. The specific determination method of each free radical scavenging rate is as follows:

(1) DPPH free radical scavenging rate and ABTS free radical scavenging rate

The determination was carried out according to GB/T 39100-2020 "Determination of the antioxidant activity of polypeptides - DPPH and ABTS method";

(2) O ^2- free radical scavenging rate

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 solution to the reaction mixture, incubate at 25°C for 5 min, measure the absorbance at 560 nm, and calculate the ^O2- free radical scavenging rate (%) according to the following formula:

O ^2- free radical scavenging rate (%) = (A0-A1)/A0×100%

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

Then, 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 was established, and the half-clearance concentration was calculated;

(3) OH- free radical scavenging rate

Take 0.15 mL of 5.0 mM 1,10-phenanthroline, 0.15 mL of 5.0 mM FeSO ₄ , 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% H ₂ O ₂ . After incubating 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:

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

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} ;

Then, 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 was established, and the half-clearance concentration was calculated.

Example 1

A method for extracting, separating and purifying white mullet antioxidant peptides, the specific steps are as follows:

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;

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 NaOH, 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 enzymatic solution; centrifuging the enzymatic solution at 8000 x g and 4°C for 20 min, taking the supernatant and freeze-drying it in vacuum to obtain crude white mullet peptide powder;

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

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, and chromatographed using Sephadex G-15 dextran gel chromatography, with double distilled water as the eluent, the chromatography method is an elution flow rate of 1 mL/min, a detection wavelength of 220 nm, and a sample volume of 4 mL. The chromatographic results are shown in Figure 1, where the abscissa represents the elution time (min) and the ordinate represents the absorbance A.

As shown in FIG1 , three separated components were obtained in the chromatography collection and elution process, which were labeled B1, B2, and B3 in sequence, and freeze-dried and stored at -18°C.

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

As shown in Figure 2-5, components B1, B2, and B3 have good scavenging effects on DPPH free radicals, ABTS free radicals, O ^2- free radicals, and OH- free radicals, indicating that components B1, B2, and B3 all have good antioxidant activity. Component B2 was selected for the next step of RP-HPLC separation and purification.

Step (6): RP-HPLC was used to further separate and purify the B2 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.

The RP-HPLC separation spectrum of the chromatographic component B2 is shown in Figure 6. In the figure, the abscissa represents the elution time (min), and the ordinate represents the response value (AU).

Step (7): The component purified in step (6), i.e., component B4 at 15-20 min in Figure 6, was identified by liquid chromatography-mass spectrometry. 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, 56-66 min 95% B; analytical column was packed with Acclaim PepMapRPLC C18 (3 μm, 150 μm i.d. × 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 white mullet antioxidant peptide is:

Tyr-Thr-Tyr-Ser-Gly-Leu-Leu (YTYSGLL).

Figure 7 shows the mass spectrum of this component. The following table shows the information of the antioxidant peptide of white mullet:

Table 1 Antioxidant peptide information of white mullet

.

Example 2

In order to further verify the antioxidant activity of the above-mentioned white mullet antioxidant peptide, the sequence was synthesized using the solid phase synthesis method to obtain the sequence YTYSGLL synthetic product.

The method of step (6) in Example 1 was used for detection. The detection result is shown in FIG8 . The purity of the compound of sequence YTYSGLL is ≥ 98%.

Using glutathione as a positive control, the antioxidant activity of the above sequence YTYSGLL compound and glutathione at different concentrations (0.5 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL, 8 mg/mL) was measured. The results are shown in Figures 9-12, where GSH is the glutathione sample and YTYSGLL is the sequence YTYSGLL compound sample.

According to the results in Figures 9-12, the higher the concentration of the two samples, the higher their free radical scavenging rates. When the concentration was 8 mg/mL, the DPPH free radical scavenging rate, ABTS free radical scavenging rate, O ^2- free radical scavenging rate and OH- free radical scavenging rate of glutathione in the control group were 94.83%, 91.08%, 89.01% and 36.72%, respectively, and the free radical scavenging rates corresponding to the YTYSGLL synthetic compound were 60.34%, 74.04%, 45.11% and 21.79%, respectively, indicating that the sequence YTYSGLL has strong antioxidant activity.

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 (6)

1. Bai Wuyu antioxidant peptide, which is characterized in that the amino acid sequence is shown in SEQ ID NO: 1.

2. The antioxidant peptide of croaker of claim 1, wherein the antioxidant peptide is extracted from croaker.

3. The method for preparing the antioxidant peptide of croaker as claimed in claim 1 or 2, comprising the steps of:

homogenizing the white cuttlefish, and simulating in-vitro digestion and enzymolysis treatment of a human body to obtain an enzymolysis liquid;

ultrafiltering the enzymolysis liquid to separate ultrafiltered component with molecular weight cut-off smaller than 3000 Da;

carrying out chromatography on the ultrafiltration component by adopting a gel chromatography method; and

And separating and purifying the components obtained after chromatography.

4. Use of an antioxidant peptide of croaker according to claim 1 or 2 in an antioxidant product.

5. Use of an antioxidant peptide of croaker according to claim 1 or 2 in an antioxidant.

6. An antioxidant comprising the antioxidant peptide of croaker according to claim 1 or 2.