Preparation method of active peptide with antioxidant function

Abstract

The invention discloses a preparation method of an active peptide with an antioxidant function, and relates to the technical field of biology. The preparation method comprises the following steps: preparing polypeptide with an amino acid sequence as shown in SEQ ID NO.2 through a solid-phase synthesis method; the polypeptide is the active peptide with the antioxidant function. According to the invention, a plurality of antioxidant active peptides are obtained by using separation and purification and mass spectrum identification technologies and by means of a saury protein database; multiple rounds of screening are carried out by utilizing the DPPH free radical scavenging rate, reducing power, water solubility, biotoxicity and other properties of the active peptide; the interaction between the antioxidant active peptide and the receptor MPO is explored by combining molecular docking, and the in-vitro antioxidant activity of the polypeptide is verified through solid-phase synthesis and cell experiments. Results show that the active peptide obtained through screening has in-vitro antioxidant activity, has good water solubility and is free of biotoxicity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing an active peptide with antioxidant function. Background Technology

[0002] The human body possesses self-protective mechanisms to stabilize and eliminate excess free radicals. Excess free radicals generate reactive oxygen species (ROS), leading to aging and diseases such as cancer. Antioxidant peptides effectively maintain free radical balance in the body, offering advantages such as safety, high efficiency, and easy digestibility and absorption. Furthermore, they are widely available and have a promising market prospect. Currently, food nutrition researchers have conducted extensive studies on antioxidant peptides derived from food-based animal and plant proteins. Fish protein-derived antioxidant peptides, in particular, have attracted widespread attention due to their high safety, novel structure, and other benefits to human health.

[0003] Pacific saury is a low-value fish resource in the Northwest Pacific Ocean, known for its rapid growth, delicious taste, and rich nutritional value. Studies have shown that Pacific saury is a high-protein, high-fat fish, containing essential unsaturated fatty acids. Discovering novel antioxidant peptides from Pacific saury is significant for enriching the variety of antioxidant peptides in fish protein sources, preventing diseases, protecting human health, and promoting the deep processing of marine protein resources. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing bioactive peptides with antioxidant functions, thereby addressing the problems existing in the prior art. The bioactive peptides prepared using the method provided by this invention exhibit excellent antioxidant activity, offering new technical support for the development of functional foods.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing an active peptide with antioxidant function, comprising the step of preparing a polypeptide with an amino acid sequence as shown in SEQ ID NO.2 by solid-phase synthesis; the polypeptide is the active peptide with antioxidant function.

[0006] The present invention also provides a method for preparing a health product that helps with anti-oxidation, comprising the step of using a polypeptide with an amino acid sequence as shown in SEQ ID NO.2 as the active ingredient and adding excipients to prepare the health product.

[0007] Furthermore, the excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

[0008] The present invention discloses the following technical effects: This invention utilizes separation and purification techniques and mass spectrometry identification, along with a saury protein database, to obtain the amino acid sequences of antioxidant peptides derived from saury. Multiple rounds of screening were conducted using the DPPH free radical scavenging rate, reducing power, water solubility, and biotoxicity properties of the active peptides. Molecular docking technology was used to investigate the interaction between the antioxidant peptides and their receptor, myeloperoxidase (MPO). The in vitro antioxidant activity of the peptides was verified through solid-phase synthesis and cell experiments. Results show that the antioxidant peptides obtained in this invention possess in vitro antioxidant activity, good water solubility, and no biotoxicity, effectively preventing oxidative damage to HEK-293 cells caused by H2O2. The novel active peptide sequences obtained in this invention provide new resources for the development of functional foods. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 The interaction between NGLNRTFVGK and MPO; Figure 2 The interaction between DAPVDQMGAGK and MPO; Figure 3 This refers to the interaction between KDEPDQASSK and MPO; Figure 4 To demonstrate the protective effect of synthetic peptides against H2O2-induced oxidative damage in HEK-293 cells; Figure 5 The images are ROS flow cytometry plots, where A represents the normal group, B the damaged group, C the GSH group, D the NGLNRTFVGK group, E the DAPVDQMGAGK group, and F the KDEPDQASSK group. Figure 6 The synthetic peptide inhibits ROS production in H2O2-induced HEK-293 cells. Detailed Implementation

[0011] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0012] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0013] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0014] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0015] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0016] The instruments and chemicals used in the following examples are as follows: AKTA Purifier 100 protein purification system, Agilent 1260 Infinity high performance liquid chromatograph, MALDI-TOF / TOF mass spectrometer; HEK-293 cells were purchased from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; acetonitrile (chromatographic grade) and trifluoroacetic acid (chromatographic grade) were purchased from Merck Biotechnology (China) Co., Ltd.; DPPH (analytical grade) was purchased from Fuzhou Feijing Biotechnology Co., Ltd.; ferric chloride (analytical grade) and trichloroacetic acid (analytical grade) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0017] To obtain components with higher purity and stronger activity, the enzymatic hydrolysate needs to be separated and purified. Ultrafiltration, gel chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC) are currently the most commonly used separation and purification methods. Mass spectrometry identification technology has the advantages of simple operation, high detection sensitivity, and accurate results. By comparing mass spectrometry data with protein databases, the amino acid sequence of active peptides can be obtained. Molecular docking technology is a theoretical simulation method that uses bioinformatics analysis to study the interaction between ligands and receptors and predict their binding modes and affinities. This method is low-cost, short-cycle, and can achieve efficient ligand screening. Myeloperoxidase (MPO) is a peroxidase that is expressed and secreted in large quantities by activated polymorphonuclear leukocytes (neutrophils). Since extracellular myeloperoxidase can catalyze the peroxidation of lipoproteins and other molecules in the blood, MPO is considered one of the main driving factors of lipid peroxidation in vivo.

[0018] Example 1: Isolation and purification of active peptides This invention uses saury enzymatic hydrolysate as raw material and employs ultrafiltration, gel chromatography, and RP-HPLC to separate and purify it. By measuring the DPPH free radical scavenging rate and reducing power of each component, components with strong antioxidant activity are screened.

[0019] Enzymatic hydrolysis process: material-to-liquid ratio 1:2.67 (m / v, g / mL), enzyme dosage 0.6%, hydrolysis time 4 h, temperature 50 ℃, pH 7.0. After hydrolysis, the enzyme was inactivated by heating in a 100 ℃ water bath for 20 min, centrifuged for 10 min, and the supernatant was collected and filtered to remove suspended solids and residues, yielding saury enzymatic hydrolysate.

[0020] Separation and purification methods: (1) Ultrafiltration: A 50 mg / mL saury enzymatic hydrolysate was prepared using ultrapure water and passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 10,000 Da and 3,000 Da. Solution fractions with molecular weights greater than 10,000 Da, 3,000 ~ 10,000 Da, and less than 3,000 Da were collected, and the fraction with the strongest antioxidant activity was screened out. (2) Gel chromatography: The column packing material was Sephadex G-25 dextran gel resin; the sample concentration was 20 mg / mL; the injection volume was 2 mL; the flow rate was 1 mL / min; and the UV detection wavelength was 214 nm. The collected solutions of the same peak were mixed, concentrated, and freeze-dried. The fraction with the strongest antioxidant activity was screened and separated by RP-HPLC.

[0021] (3) RP-HPLC: The chromatographic column was a YMC-Pack ODS-AQ C18 column (250×10 mm, 5 μm); the mobile phase was (A: acetonitrile, B: 0.1% v / v TFA); the flow rate was 1 mL / min; the UV detection wavelength was 215 nm; the sample loading volume was 100 μL; isocratic elution was performed (10% A, 90% B); the elution time was 50 min. Each peak component was collected, concentrated, freeze-dried, and stored at -80 ℃ for later use.

[0022] Calculate the DPPH radical scavenging rate using the following formula: Experimental group: The sample solution was mixed thoroughly with an equal volume of DPPH. Control group 1: The sample solution was mixed thoroughly with an equal volume of 95% ethanol solution. Control group 2: DPPH was mixed thoroughly with an equal volume of distilled water. Blank group: Distilled water was mixed thoroughly with an equal volume of 95% ethanol solution.

[0023] Example 2 Identification of the amino acid sequence of the active peptide The five antioxidant components obtained by RP-HPLC separation and purification were analyzed by MADIL-TOF / TOF MS. By comparison with the saury protein database, the amino acid sequences of 20 novel antioxidant active peptides were obtained.

[0024] Example 3 Screening of bioactive peptides The water solubility and toxicity of the 20 novel antioxidant peptides obtained in Example 2 were predicted and screened using the online tools Prot Param (https: / / web.expasy.org / protparam / ) and Toxin Pred (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php). Sixteen peptides with good water solubility and no biotoxicity were obtained, and the results are shown in Table 1.

[0025] Table 1. Predicted results of water solubility and biotoxicity of antioxidant peptides derived from Pacific saury. Example 4 Molecular docking analysis of active peptides and MPO Using Discovery Studio 2017 R2 software, under the CHARMm force field, 16 antioxidant active peptides were molecularly docked with MPO (PDB ID 3F9P). By comparing CDOCKER ENERGY, three antioxidant active peptides with the strongest docking were selected, and the results are shown in Table 2. The interactions of NGLNRTFVGK (SEQ ID NO.1), DAPVDQMGAGK (SEQ ID NO.2), and KDEPDQASSK (SEQ ID NO.3) with MPO are shown in Table 2. Figure 1 , Figure 2 and Figure 3 .

[0026] Table 2. Antioxidant bioactive peptides from three Pacific saury sources. The main interactions between the active peptide and MPO include van der Waals forces, hydrogen bonds, CH bonds, electrostatic interactions, and alkyl / pialkyl interactions. The amino acid residues that bind the active peptide to the catalytic site of MPO are mainly Arg31, Arg323, Cys153, Ile160, Lys505, Phe439, Pro34, Thr159, Trp32, and Val30.

[0027] Example 5: Validation of the in vitro antioxidant activity of NGLNRTFVGK, DAPVDQMGAGK, and KDEPDQASSK Three selected antioxidant peptides were synthesized in a solid-phase manner, with a purity exceeding 98%. Using HEK-293 cells as an experimental model, H2O2 as an oxidant, and glutathione (GSH) as a positive control, the in vitro antioxidant activity of the three peptides was verified.

[0028] (1) Protective effect of synthetic peptides against H2O2-induced oxidative damage in HEK-293 cells: Experimental group: HEK-293 cells were seeded in 96-well plates and cultured for 24 h. A synthetic peptide solution with a concentration of 300 µg / mL was added, and the cells were incubated for another 24 h. Then, a H2O2 solution with a concentration of 700 µmol / L was added, and the cells were incubated for 6 h. The absorbance was then measured using a CCK-8 assay kit.

[0029] Control group: The same concentration of GSH solution was used instead of the synthetic peptide solution.

[0030] Normal group: Use an equal volume of culture medium instead of synthetic peptide solution and H2O2 solution.

[0031] Damage group: Use an equal volume of culture medium instead of synthetic peptide solution.

[0032] (2) Determination of ROS content: Experimental group: HEK-293 cells were seeded in 6-well plates and cultured for 24 h. A synthetic peptide solution with a concentration of 300 µg / mL was added, and the cells were incubated for another 24 h. The culture medium was then discarded, and a H2O2 solution with a concentration of 700 µmol / L was added. After incubation for 6 h, the cells were collected, and the intracellular ROS content was measured using the DCFH-DA fluorescent probe method.

[0033] Control group: The same concentration of GSH solution was used instead of the synthetic peptide solution.

[0034] Normal group: Use an equal volume of culture medium instead of sample solution and H2O2 solution.

[0035] Damage group: Use an equal volume of culture medium instead of synthetic peptide solution.

[0036] The results showed that NGLNRTFVGK, DAPVDQMGAGK, and KDEPDQASSK could effectively reduce the oxidative damage caused by H2O2 to HEK-293 cells. (See attached image for details.) Figure 4 , Figure 5 and Figure 6 The cell viability of the NGLNRTFVGK, DAPVDQMGAGK, and KDEPDQASSK experimental groups were 0.344, 0.369, and 0.38, respectively, and their ROS levels were 0.211, 0.354, and 0.08, respectively.

[0037] In actual production, antioxidant active peptides can be produced through solid-phase chemical synthesis and added to food, pharmaceuticals, and health products in powder or granule form. This approach enhances the antioxidant capacity of food while preserving its inherent nutritional value, providing a new avenue for the diversification of food nutrition.

[0038] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an active peptide with antioxidant function, characterized in that, The method includes the step of preparing a polypeptide with an amino acid sequence as shown in SEQ ID NO.2 by solid-phase synthesis; the polypeptide is the active peptide with antioxidant function.

2. A method for preparing a health supplement that aids in antioxidation, characterized in that, The method includes the step of preparing the health product by adding excipients to a polypeptide with an amino acid sequence as shown in SEQ ID NO.2 as the active ingredient.

3. The preparation method according to claim 2, characterized in that, The excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

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