Preparation method of anti-oxidation polypeptide

Abstract

A method of preparing an anti-oxidation polypeptide having an amino acid sequence of SEQ ID NO:1 includes enzymatic hydrolysis of black shark skins, which serve as the raw material, with alkali protease, separation, purification, freezing, and drying to obtain the anti-oxidation polypeptide. Enzymatic hydrolysis conditions include 7.0-9.0 pH value, 40-50° C. temperature, 4.0-6.0 h enzymatic hydrolysis time, 2.0-4.0% primer concentration, and 9.0-10.0 wt % of enzymes.

Claims

1-3. (canceled)

4. A method of preparing an anti-oxidation polypeptide having an amino acid sequence of SEQ ID NO:1, comprising the steps of: (a) enzymatically hydrolysing black shark skins having alkali protease; (b) separating the enzymatically hydrolysed black shark skins to obtain a plurality of enzymatic hydrolysis products; (c) purifying the enzymatic hydrolysis products; (d) freezing the enzymatic hydrolysis products; and (e) drying the enzymatic hydrolysis products to obtain an anti-oxidation polypeptide; wherein step (a) is taken under a plurality of conditions including 7.0-9.0 pH value, 40-50° C. temperature, 4.0-6.0 h enzymatic hydrolysis time, 2.0-4.0% primer concentration, and 9.0-10.0 wt % of enzymes.

5. The method of claim 4, step (b) further comprising the sub-steps of: (b-1) separating a plurality of ingredients of the enzymatic hydrolysis products; (b-2) monitoring an anti-oxidation activity of the ingredients; and (b-3) collecting the ingredients having a maximum anti-oxidation activity by means of RP-HPLC (reversed-phase high-performance liquid chromatography); wherein in sub-step (b-3) an acetonitrile solution having a concentration gradient of 0%-40% containing 0.05% trifluoroacetic acid (TFA) by volume is used as eluent for linear eluting, and a chromatographic column Gemini 5μ C18 is used to determine an eluent antioxidant activity corresponding to each of a plurality of absorption peaks, and the ingredients having the maximum anti-oxidation activity under a plurality of conditions including 100 μL feeding volume, 1 mL/min flow rate, and 214 nm detection wavelength are collected.

6. The method of claim 4, wherein the amino acid sequence of SEQ ID NO:1 is identified by means of Q-TOF LC-MS.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 is a curve diagram of the ABTS radical elimination rate of the FIG. 1 is a curve diagram of the ABTS radical elimination rate;

[0041] FIG. 2 is a curve diagram of the DPPH radical elimination rate of the protease hydrolysate of black shark skins;

[0042] FIG. 3 is a diagram of the lipid peroxidation inhibition activity of the protease hydrolysate of black shark skins;

[0043] FIG. 4 is a curve diagram of the hydroxyl radical elimination rate of the protease hydrolysate of black shark skins;

[0044] FIG. 5 is a RP-HPLC elution diagram of the protease hydrolysate of black shark skins;

[0045] FIG. 6 is the curve diagram of the DPPH radical elimination rate of the RP-HP LC eluting ingredient of the protease hydrolysate of black shark skins;

[0046] FIG. 7 is a total ions chromatogram of F19;

[0047] FIG. 8 is a grade-1 mass chromatogram of the anti-oxidation polypeptide;

[0048] FIG. 9 is a grade-2 mass chromatogram of the anti-oxidation polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

[0049] The anti-oxidation polypeptide of the invention has an amino acid sequence of

TABLE-US-00001 (SEQ ID NO: 1) Gly-Ala-Ala-Val-Ala-Leu.

[0050] The preparation method is as follows:

[0051] Black shark skins which serve as the raw material are enzymatically hydrolyzed using alkali protease; and then the enzymatic hydrolysis products are separated, purified, frozen and dried to obtain the anti-oxidation polypeptide. The enzymatic hydrolysis conditions include: 8.0 pH value, 45 C. temperature, 4.9 h enzymatic hydrolysis time, 3% primer concentration and 9.6 wt. % enzymes. RP-HPLC (reversed-phase high-performance liquid chromatography) is used to separate the enzymatic hydrolysis products, monitor the anti-oxidation activity of separated ingredients, and collect ingredients with the maximum anti-oxidation activity. In the process of separation using the RP-HPLC, an acetonitrile solution in a concentration gradient of 0%-40% that contains 0.05% trifluoroacetic acid (TFA) by volume is used as eluent for linear eluting, and a chromatographic column Gemini 5μ C18 is used to determine the eluent anti-oxidation activity corresponding to each absorption peak, collect and obtain the ingredients with the maximum anti-oxidation activity under the conditions of 100 μL feeding volume, 1 mL/min flow rate and 214 nm detection wavelength.

[0052] Instruments and test methods adopted in the invention are as follows:

[0053] The invention uses the RP-HPLC (reversed-phase high-performance liquid chromatography) to realize efficient separation and purification of the anti-oxidation polypeptide with obvious activity.

[0054] Q-TOF LC-MS is used to identify the amino acid sequence of the anti-oxidation polypeptide.

[0055] In order to further clarify the content, features and effects of the invention, the following embodiment is described below as an example:

EMBODIMENT 1

[0056] 3 g of black shark skins were weighed and added into 100 ml of de-ionized water; and then 1 mol/L NaOH was added to adjust the pH value of the solution to 8.0. The solution was heated to reach a temperature of 45° C. in a water bath first, and then added with corresponding enzymes by a ratio of 9.6 wt. %. The enzymatic hydrolysis time was 4.9 hours. Next, the solution was placed in a boiling water bath to deactivate enzymes for 15 min, cooled, and centrifuged at a speed of 4000 rpm for 15 min. The supernatant was collected for later use.

[0057] The anti-oxidation activity of the prepared enzymatic hydrolysis product of black shark skins was identified, wherein the inhibitory concentration 50% of ABTS was 80 μg/mL(as shown in FIG. 1), the inhibitory concentration 50% of DPPH was 3.19 mg/mL (as shown in FIG. 2), and the product had certain lipid peroxidation inhibition activity (as shown in FIG. 3) and hydroxyl radical elimination activity (as shown in FIG. 4). Therefore, the enzymatic hydrolysis product of black shark skins has certain anti-oxidation activity.

[0058] RP-HPLC was adopted to separate enzymatic hydrolysis products (as shown in FIG. 5), monitor the anti-oxidation activity of separated ingredients, and collect ingredients with the maximum anti-oxidation activity. In the process of separation using the RP-HPLC, the acetonitrile solution in a concentration gradient of 0%-40% that contained 0.05% trifluoroacetic acid (TFA) by volume was used as eluent for linear eluting, and the chromatographic column Gemini 5μ C 18 was used to determine the eluent anti-oxidation activity corresponding to each absorption peak, collect and obtain the ingredient F19 with the maximum anti-oxidation activity under the conditions of 100 μL feeding volume, 1 mL/min flow rate and 214 nm detection wavelength (as shown in FIG. 6).

[0059] Ingredient F19 was identified using Q-TOF LC-MS (see table 1 and table 2 for the chromatographic conditions and mass spectrum conditions). The total iron flow chart of F19 can be seen in FIG. 7. Then, 12.14 min ion peak was determined using MS/MS grade-2 chromatogram to obtain the full amino acid sequence (as shown in FIGS. 8-9) of Gly-Ala-Ala-Val-Ala-Leu (SEQ ID NO:1) (molecular weight: 500 Da), namely the anti-oxidation polypeptide of the invention. The anti-oxidation activity (as shown in table 3) of anti-oxidation polypeptide was determined, wherein the elimination rate of ABTS radicals was (64.12±0.20)%, the elimination rate of DPPH radicals was (67.00±0.24)%, and the oxygen radical absorption capacity was (527.53±65.15) μmol Trolox/g peptide.

TABLE-US-00002 TABLE 1 Parameters of chromatographic conditions for F19 identification Chromatographic conditions Item F19 Mobile phase 100% acetonitrile-0.1% acetic acid Flow rate 0.4 mL/min Detection wavelength 214 nm Chromatographic column Gemini C18 (10 × 250 mm, 5 μ) Detector UV

TABLE-US-00003 TABLE 2 Parameters of mass spectrum conditions for F19 identification MS conditions Item F19 Ion mode ESI.sup.+ Capillary voltage 35 k Volts Desolvation temperature 400° C. Collision energy 6 eV Mass range 50-2000 Da Ionization temperature 100° C.

TABLE-US-00004 TABLE 3 Anti-oxidation activity of the anti-oxidation polypeptide Elimination rate of Elimination rate of Oxygen radical ABTS radicals (%) DPPH radicals (%) absorption capacity (1 mg/mL) (1 mg/mL) (μmol Trolox/g peptide) 64.12 ± 0.20 67.00 ± 0.24 527.53 ± 65.15

[0060] The above is merely preferable embodiment of the invention. All equivalent changes and modifications made on the basis of the invention shall fall within the protective scope of the invention.