White mullet polypeptide, preparation method and application

Abstract

The present invention relates to the field of biotechnology, and discloses a white mullet polypeptide, a preparation method and an application. The white mullet polypeptide of the present invention is extracted from white mullet, and its amino acid sequence is shown in SEQ ID NO: 1. The preparation method of the white mullet polypeptide comprises: simulating human in vitro digestion to obtain an enzymatic hydrolyzate from the white mullet, using ultrafiltration to separate ultrafiltration components with a molecular weight cutoff of less than 3000Da, and further separating and purifying the chromatographic components with antioxidant properties by RP-HPLC method after gel chromatography to obtain the white mullet polypeptide. The polypeptide prepared by the present invention has a strong antioxidant effect and can be used to prepare antioxidant products.

Description

Technical Field

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

[0002] Peptides are small molecule compounds formed by α-amino acids linked together by peptide bonds. They are intermediate products of protein hydrolysis and have the most common functionality of bioactive peptides, namely antioxidant properties. 2- Free radicals, OH-free radicals and other active oxygen free radicals have a certain scavenging function. Food-derived plants and animals are important sources of peptides. At present, natural antioxidant peptides have been successfully discovered and extracted from edible plants such as millet, wheat, hericium erinaceus, red beans, black beans, red amaranth, lentils, and oysters, Arctic sweet shrimp, loach, crayfish, silkworm pupae, sheep bones, crucian carp, krill, clams and other edible animals. White mullet ( Opniocepnalus argus var Kimnra ), also known as white-armored black snakehead, commonly known as white black stick, is a very rare and high-quality fish among freshwater fish in my country. It is a special aquatic product independently developed by Neijiang City, Sichuan Province. White mullet not only has tender meat, few bones, and delicious taste, but also has the effects of nourishing the spleen and stomach, promoting lactation, etc. It also contains a large amount of protein and amino acids, phosphorus, calcium, iron and other elements required by the human body. It can supplement the human body with a variety of nutrients needed. After consumption, it is beneficial to the normal functioning of the spleen and stomach and has the effect of nourishing the spleen and stomach. At present, the 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

[0003] The object of the present invention is to provide a white mullet polypeptide and a preparation method thereof, wherein the white mullet polypeptide is extracted from white mullet. The present invention also provides an application of the white mullet polypeptide, which can be applied to the preparation of antioxidant products.

[0004] To achieve the above object, the first technical solution adopted by the present invention is:

[0005] The amino acid sequence of the white mullet polypeptide is shown in SEQ ID NO: 1.

[0006] Preferably, the white mullet polypeptide is extracted from white mullet.

[0007] The second technical solution adopted by the present invention is:

[0008] The preparation of white mullet polypeptide comprises the following steps:

[0009] The white mullet is homogenized and subjected to enzymatic hydrolysis treatment simulating human in vitro digestion to obtain an enzymatic hydrolysate;

[0010] The enzymatic hydrolysate is subjected to ultrafiltration to separate the ultrafiltration component with a molecular weight cut-off less than 3000Da;

[0011] performing gel chromatography on the ultrafiltration fraction; and

[0012] The components obtained after chromatography are separated and purified.

[0013] The third technical solution adopted by the present invention is:

[0014] Application of white mullet peptide in antioxidant products.

[0015] The fourth technical solution of the present invention is:

[0016] Antioxidant, containing white mullet peptide of the first technical solution.

[0017] The above-mentioned white mullet polypeptide can also be synthesized according to the disclosed amino acid sequence using amino acids as raw materials by chemical solid phase synthesis, which is well known in the art.

[0018] The white mullet polypeptide provided by the present invention can effectively remove active oxygen free radicals such as DPPH free radicals, superoxide free radicals, hydroxyl free radicals, etc., and has a strong removal effect on hydroxyl free radicals. The white mullet polypeptide has good antioxidant activity and can be used in the preparation of various antioxidant products such as antioxidants, antioxidant foods, and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] 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;

[0021] Figure 2-5 The results of different antioxidant activities of components B1, B2, and B3;

[0022] Figure 6 is the ultra-high performance liquid chromatography separation spectrum of the chromatography component B3;

[0023] Figure 7 is the secondary mass spectrum of sequence FSYGRAL;

[0024] Figure 8 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION CHARACTERISTICS FOR PURITY CONFIRMATION OF THE SEQUENCE FSYGRAL;

[0025] Figures 9 to 12The results show the different antioxidant activities of FSYGRAL and glutathione. DETAILED DESCRIPTION

[0026] 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.

[0027] The first embodiment of the present invention provides a white mullet polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0028] Specifically, the white mullet polypeptide is extracted from white mullet.

[0029] The second embodiment of the present invention provides a method for preparing a white mullet polypeptide, comprising the following steps:

[0030] 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;

[0031] Step 2: adjust the pH of the homogenized material to 2.0 with HCl, add pepsin according to (3%-4%) of the mass of the homogenized material, and simulate gastric digestion enzymolysis at 37°C in a water bath for 2.5 h; then adjust the pH of the above solution to 5.33 with 0.9 M NaHCO3, and then adjust the pH to 7.5 with 1 M NaOH, add pancreatic enzyme according to (3%-4%) of the mass of the homogenized material, simulate intestinal digestion at 37°C in a water bath for 2.5 h, place in boiling water for 10 min to terminate digestion, and cool to room temperature to obtain enzymolysis solution; centrifuge the above enzymolysis solution, take the supernatant and vacuum freeze-dry to obtain crude white mullet peptide powder;

[0032] Step 3: Dissolve the crude peptide powder in distilled water, use a 3000Da ultrafiltration tube for ultrafiltration separation, retain the ultrafiltration component B with a molecular weight cutoff of less than 3000Da, freeze-dry and store at -18°C;

[0033] Step 4: The ultrafiltration fraction B is prepared into a solution with a concentration of (20-30) mg / mL, and chromatographed using Sephdex G-15 dextran gel chromatography. The obtained fraction is freeze-dried and stored at -18°C;

[0034] 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 polypeptide with the following amino acid sequence:

[0035] Phe-Ser-Tyr-Gly-Arg-Ala-Leu (FSYGRAL).

[0036] In a specific embodiment, the centrifugation conditions in step 2 are 4°C, 8000xg, and time for 20 min.

[0037] In a specific 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.

[0038] Furthermore, the sample loading volume is 4 mL.

[0039] 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.

[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 solution 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] 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;

[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] 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.

[0053] Example 1

[0054] A method for extracting, separating and purifying white mullet polypeptide, 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 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 vacuum freeze-drying it to obtain a crude white mullet peptide powder;

[0057] Step (3): dissolving the crude white mullet peptide powder in distilled water, performing ultrafiltration separation using a 3000Da ultrafiltration tube, retaining an ultrafiltration component B with a molecular weight below 3000Da, freeze-drying the obtained component B, and storing it 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 dextran 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, components B1, B2, and B3 have an effect on DPPH free radicals, ABTS free radicals, and O 2- The free radicals and OH-free radicals had good scavenging effects, indicating that components B1, B2, and B3 all had good antioxidant activity. Component B3 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 B3 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 RP-HPLC separation spectrum of the chromatographic component B3 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 6 The component B4 at 45-55 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 white mullet polypeptide is:

[0065] Phe-Ser-Tyr-Gly-Arg-Ala-Leu (FSYGRAL).

[0066] like Figure 7 , is the mass spectrum of this component. The following table is the information of this white mullet polypeptide:

[0067] Table 1 White mullet polypeptide information

[0068] .

[0069] Example 2

[0070] In order to further verify the antioxidant activity of the above-mentioned white mullet polypeptide, the sequence was synthesized using the solid phase synthesis method to obtain the sequence FSYGRAL synthetic product.

[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 FSYGRAL synthesis is ≥98%.

[0072] Using glutathione as a positive control, the antioxidant activity of the above sequence FSYGRAL synthetic 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 Figure 9-12 As shown, GSH is the glutathione sample and FSYGRAL is the sequence FSYGRAL synthetic sample.

[0073] according to Figure 9-12 The results show that the free radical scavenging rate of the two samples, that is, the antioxidant activity, increases with the increase of sample concentration. 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 significantly higher than those in the control group. 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 FSYGRAL sequence synthetic compound were 53.13%, 53.08%, 33.53% and 39.79%, respectively. Its OH-free radical scavenging rate was even higher than that of the positive control sample, indicating that the FSYGRAL sequence 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. White mullet polypeptide, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

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

3. The method for preparing the white mullet polypeptide according to claim 1 or 2, characterized in that: The following steps are involved: Homogenize the white mullet to obtain a homogenous material, adjust the pH to 2.0 with HCl, add pepsin at 3%-4% of the mass of the homogenous material, simulate gastric digestion and enzymolysis at 37°C in a water bath for 2.5 h, then adjust the pH to 5.33 with 0.9 M NaHCO3, and then adjust the pH to 7.5 with 1 M NaOH, add pancreatin at 3%-4% of the mass of the homogenous material, simulate intestinal digestion at 37°C in a water bath for 2.5 h, place in boiling water for 10 min to terminate digestion, and cool to room temperature to obtain an enzymolysis solution; 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 white mullet polypeptide as claimed in claim 1 or 2 in the preparation of antioxidant products.

5. Use of the white mullet polypeptide as claimed in claim 1 or 2 in the preparation of an antioxidant.

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

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