Preparation method of sturgeon ovary antioxidant peptide

Abstract

The present invention belongs to the fields of food nutrition and health and high-value utilization of aquatic products, and specifically relates to a method for preparing a sturgeon ovary antioxidant peptide. Using sturgeon ovaries as raw materials, the present invention approaches the issue of obtaining antioxidant peptides by enzymatic hydrolysis of sturgeon ovaries. Using traditional separation and purification techniques combined with bioinformatics, two sturgeon ovary protein peptides with antioxidant activity were screened out, whose amino acid sequences are: Phe-Asp-Trp-Asp-Arg-Leu; Phe-Glu-Gly-Pro-Pro-Phe-Lys-Phe. The preparation process of the present invention is simple, low-cost, and employs mild conditions, effectively maintaining the antioxidant activity of the sturgeon peptides. This method can address the issue of high-value utilization of sturgeons and can also be used as a functional factor in functional products. It has the potential to develop new health foods and contribute to the high-value utilization of sturgeons.

Description

Technical Field

[0001] The invention belongs to the fields of food nutrition and health and high-value utilization of aquatic products, and particularly relates to a method for preparing sturgeon ovary antioxidant peptides. Background Art

[0002] Free radicals (ROS) (such as hydroxyl radicals (·OH) and superoxide anion radicals (O 2- )), and the free radicals produced play an important regulatory role in the body's physiological activities, such as regulating cell proliferation, apoptosis and signal transduction. Under normal circumstances, excessive free radicals will be cleared by the endogenous antioxidant defense system (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), etc.) and the body's non-enzymatic antioxidant system (ascorbic acid, tocopherol, carotene, etc.), so the production and elimination of free radicals are in a state of balance. However, when free radicals are produced excessively or the body's antioxidant system is damaged, this balance will be broken. In addition, a large number of studies have found that excessive free radicals can directly or indirectly damage important biological molecules in cells (proteins, lipids, DNA and carbohydrates, etc.) and cause oxidative stress in the body, thereby promoting tissue inflammation and the occurrence of chronic diseases such as cancer, atherosclerosis, Alzheimer's disease and osteoporosis.

[0003] When endogenous antioxidants and repair systems cannot effectively reduce the amount of free radicals in the body, exogenous antioxidants can be used to alleviate or limit the body's oxidative stress by providing electrons or hydrogen atoms to free radicals to form complexes, thereby reducing the incidence of disease. Common synthetic antioxidants include butylated hydroxyanisole, propyl gallate, butylated hydroxytoluene and tert-butylhydroquinone, which have the advantages of high efficiency and low cost, but potential toxic side effects limit their application in the food industry. Natural antioxidants (peptides, polysaccharides, polyphenols, vitamins, etc.) not only have the characteristics of high efficiency and low cost, but also have the characteristics of wide source, easy absorption, long-term use and no obvious toxic side effects, and have received widespread attention in recent years.

[0004] Fish resources are abundant, and their high protein content makes them a valuable raw material for bioactive peptides. Besides the edible parts, other byproducts also contain considerable amounts of protein, oil, and other substances. The extraction of high-value active substances such as fish oil and peptides using sophisticated and cost-effective methods has also become a focus of current research. Sturgeons (Acipenser), belonging to the class Acipenser and Acipenser, are biologically and economically important fish. While renowned for their caviar and skin, sturgeons are known for their firm and woody flesh. Consequently, the flesh, along with other byproducts, is often discarded and not fully utilized, resulting in significant waste. Therefore, finding a way to maximize the value of sturgeon byproducts is a pressing technical challenge.

[0005] At present, there are no literature reports on the research of functional active substances in sturgeon ovaries at home and abroad. Therefore, developing the optimal enzymatic purification process and optimizing the evaluation method of antioxidant peptide activity are crucial links in the preparation of sturgeon ovary antioxidant peptides. Summary of the Invention

[0006] At present, there are no literature reports on the research of functional active substances in sturgeon ovaries at home and abroad. Regarding how to prepare sturgeon ovary antioxidant peptides, its enzymatic hydrolysis, purification process, and optimization of antioxidant peptide activity are crucial links in the preparation of sturgeon ovary antioxidant peptides.

[0007] In response to the existing technical problems, the present invention uses sturgeon ovary as raw material, approaches the issue of obtaining antioxidant peptides by enzymatic hydrolysis of sturgeon ovary, and adopts traditional separation and purification combined with bioinformatics technology to provide a simple and efficient method for preparing sturgeon antioxidant peptides. It also screens out two sturgeon ovary protein peptides with antioxidant activity, providing a feasible solution for the utilization of other by-products and a feasible path for their application as functional products in the food and pharmaceutical industries.

[0008] The present invention first provides a sturgeon ovary antioxidant peptide, comprising two polypeptides, the amino acid sequences of which are:

[0009] Phe-Asp-Trp-Asp-Arg-Leu;

[0010] Phe-Glu-Gly-Pro-Pro-Phe-Lys-Phe.

[0011] The present invention also provides a method for preparing sturgeon ovary antioxidant peptide, which comprises the following steps:

[0012] (1) The ovaries of sturgeons were de-roed, washed and drained, and minced to obtain a minced product; and the crude protein content of the sturgeon ovaries was determined by the Kjeldahl method;

[0013] (2) adding distilled water to the minced material in step (1), ultrasonically treating the mixture to obtain a mixed solution, adjusting the pH of the mixed solution, and then adding alkaline protease to perform enzymatic hydrolysis in a constant temperature water bath, performing enzyme inactivation treatment after enzymatic hydrolysis, cooling to room temperature after treatment, and collecting the supernatant by centrifugation and vacuum freeze-drying to obtain a powdered sturgeon ovary protein hydrolysate;

[0014] (3) ultrafiltration: using an ultrafiltration membrane to fractionate the powdered sturgeon ovary protein hydrolysate obtained in (2) to obtain components, and freeze-drying the components to obtain freeze-dried components;

[0015] (4) Sephadex G-25 gel column purification: purify the freeze-dried component in step (3), elute with distilled water, collect the eluate, and freeze-dry to obtain the freeze-dried component;

[0016] (5) Preparative reversed-phase liquid chromatography column purification: The freeze-dried components in (4) are further purified. After purification, the components are eluted using a linear gradient of an eluent, collected, and freeze-dried to finally obtain the sturgeon ovary antioxidant peptide.

[0017] Preferably, in step (2), the ratio of the minced material to distilled water is 1 g:3 mL; and the pH of the mixed solution is adjusted to 10.0 using 1 mol / L NaOH.

[0018] Preferably, in step (2), the ultrasonic treatment is performed by placing the sample in a 37°C water bath, adjusting the triple-frequency ultrasonic wave to 25 / 50 / 75 Hz, the ultrasonic power to 240 W, the ultrasonic mode to 10 s on and 10 s off, and the ultrasonic time to 30 min.

[0019] Preferably, in step (2), alkaline protease is added at 4000 U / g for enzymatic hydrolysis according to the crude protein content in the minced material measured by Kjeldahl nitrogen determination and the amount of protein used.

[0020] Preferably, in step (2), the conditions for enzymatic hydrolysis in a constant temperature water bath are: temperature 50° C., and enzymatic hydrolysis time 240 min.

[0021] Preferably, in step (2), the enzyme inactivation treatment condition is: inactivation of the enzyme at 95° C. for 15 minutes.

[0022] Preferably, in step (2), the centrifugal condition is 10000 r / min, 10-15 min.

[0023] Preferably, in step (3), the molecular weight cut-off (MW) of the ultrafiltration membrane is 100 kDa, 10 kDa and 3 kDa; four polypeptide components with different MW are separated: MW>100 kDa, 10 kDa and 3 kDa. <MW<100kDa,3kDa<MW<10kDa,MW<3kDa。

[0024] Preferably, in step (4), the filler used for gel column chromatography is Sephadex G-25, and the chromatography conditions are: column: Sephadex G-25 gel column, column size 1.6 cm × 60 cm, column temperature: room temperature, flow rate: 1 mL / min, sample concentration: 25 mg / mL, injection volume: 2.0-5.0 mL, detection wavelength: 280 nm, eluent: distilled water.

[0025] Preferably, in step (5), the preparative reversed-phase liquid chromatography column has a column size of 21.2 cm×250 mm; methanol and deionized water are selected as the mobile phase, and the elution parameters are: 0.01-20 min, 10% methanol; 20-20.01 min, 50% methanol; 20.01-40 min: 50% methanol; 40-40.01 min: 10% methanol; 40.01-45 min: 10% methanol; column temperature: room temperature, flow rate: 5 mL / min, sample concentration: 200 mg / mL, injection volume: 1.5 mL, and detection wavelength: 280 nm.

[0026] The method for detecting antioxidant activity was to establish a H2O2-damaged MC3T3-E1 cell injury model and measure cell viability using the CCK-8 method.

[0027] Screening method for antioxidant peptides:

[0028] S1. LC-MS / MS identification of peptide sequences: The components obtained in step (5) above were separated using a Nano-HPLC liquid phase system Ulti Mate 3000RSLCnano (Thermo Fisher Scientific) and then subjected to mass spectrometry analysis using a Q-Exactive plus mass spectrometer (Thermo Fisher Scientific); the obtained mass spectrometry data were analyzed using the MAXQUANT software and the obtained peptide sequences were compared with the Uniprot-Acipenser database to determine the protein origin of the peptide sequences;

[0029] S2. Antioxidant peptide screening: The peptides obtained in step S1 were scored for activity prediction using PertideRanker software, and high-scoring peptides were docked with receptor proteins using Zdock software to ultimately determine the target peptide sequence.

[0030] S3. Peptide synthesis and activity verification: The peptide sequences screened in step S2 were synthesized, and their antioxidant potential was verified through cytotoxicity and cell viability experiments, and finally two peptides with antioxidant potential were obtained.

[0031] Preferably, the bioinformatics analysis technology includes using MAXQUANT software and the Uniprot-Acipenser database to combine mass spectrometry results analysis, using PeptideRanker software to predict the activity of the peptide; using Zdock software to dock the peptide with the receptor protein to ultimately determine the target peptide sequence; using BIOPEP-UWM (http: / / www.uwm.edu.pl / biochemia / index.php / pl / biopep) software to evaluate the novelty of the target peptide.

[0032] Preferably, in step S2, the amino acid sequences of the two polypeptides with antioxidant potential screened out are: Phe-Asp-Trp-Asp-Arg-Leu; Phe-Glu-Gly-Pro-Pro-Phe-Lys-Phe.

[0033] Preferably, in step S3, the specific operation of the cytotoxicity is: when the cell viability of the peptide-treated group is equal to or higher than that of the blank group (p>0.05), the peptide is considered to have no obvious cytotoxicity; the specific operation of the cell activity is: establishing a H2O2-induced MC3T3-E1 cell oxidative damage model, when the cell viability of the peptide-treated group is equal to or lower than that of the model group (H2O2 group) (p>0.05), the peptide is considered to have no antioxidant activity.

[0034] The antioxidant activity assay in the above steps includes: establishing a H2O2-induced MC3T3-E1 cell oxidative damage model, and determining cell viability using the CCK-8 method.

[0035] Cell viability was determined by CCK-8 assay: MC3T3-E1 cells were cultured in α-MEM medium containing 10% fetal bovine serum and 1% double-antibody. When the cell confluence reached 80%, they were digested and passaged. When the cell confluence reached 80%, 5×10 4 The cells were plated at a density of 100 μg / mL in a 96-well plate and cultured for 24 h. Then, different concentrations of polypeptide components were added and cultured for another 24 h. That is, 48 ​​h after plating, 400 μmol / L H2O2 was added to induce the cell damage model for 4 h. Subsequently, the cell viability was determined using the CCK-8 kit.

[0036] The beneficial technical effects of the present invention are:

[0037] (1) Compared with the existing methods for preparing active peptides, the preparation process of the present invention is simple, low-cost, and mild, effectively maintaining the antioxidant activity of sturgeon peptides. In addition, the preparation method is mature and the technology is stable and reliable.

[0038] (2) The present invention uses computer simulation software to perform virtual screening of directed binding peptides, which not only reduces the actual screening intensity but also increases the probability of successful screening. Through online peptide database retrieval, the screened peptide sequences have not been reported in any papers, which improves the innovation of sturgeon ovary antioxidant peptides.

[0039] (3) The present invention is the first to use sturgeon ovaries to obtain two new polypeptide sequences; the obtained antioxidant peptides expand the application range of sturgeon, which can not only solve the problem of high-value utilization of sturgeon, but also can be used as a functional factor in functional products, and has the potential to develop new health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The ABTS free radical scavenging abilities of the sturgeon ovary protein hydrolysate at different concentrations and its four ultrafiltration fractions in Example 1.

[0041] Figure 2 The hydroxyl radical scavenging abilities of the sturgeon ovary protein hydrolysate at different concentrations and its four ultrafiltration fractions in Example 1.

[0042] Figure 3 The three peptide fractions F1, F2, and F3 were obtained after the <3 kDa fraction was separated by Sephadex G-25.

[0043] Figure 4 The protective effects of three peptide fractions of different concentrations obtained after separation of <3 kDa fractions by Sephadex G-25 on MC3T3-E1 cells damaged by H2O2; among them, ### represents p<0.008 compared with the control group; * represents p<0.05 compared with the model group; ** represents p<0.01 compared with the model group; *** represents p<0.008 compared with the model group; **** represents p<0.001 compared with the model group.

[0044] Figure 5 These are 16 peptide components: F1-1 to F1-16, obtained after the F3 component was separated by a preparative reversed-phase liquid chromatography column.

[0045] Figure 6 The protective effects of 16 peptide fractions obtained after separation of the F3 component by a preparative reversed-phase liquid chromatography column on MC3T3-E1 cells damaged by H2O2 at 50 μg / mL; among them, ### represents p<0.008 compared with the control group; * represents p<0.05 compared with the model group; ** represents p<0.01 compared with the model group; *** represents p<0.008 compared with the model group; **** represents p<0.001 compared with the model group.

[0046] Figure 7Figure A shows the docking postures of five peptides with myeloperoxidase molecules; Figure B shows the docking model and two-dimensional structure diagram of FL6 with myeloperoxidase molecules; Figure C shows the docking model and two-dimensional structure diagram of FF8 with myeloperoxidase molecules.

[0047] Figure 8 The cytotoxicity results of synthetic peptides are shown; # represents p < 0.05 compared with the control group.

[0048] Figure 9 The improvement results of the synthetic peptides on H2O2-damaged MC3T3-E1 cells at concentrations of 12.5-200 μM; among them, ### represents p<0.008 compared with the control group; * represents p<0.05 compared with the model group; ** represents p<0.01 compared with the model group; *** represents p<0.008 compared with the model group; **** represents p<0.001 compared with the model group. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] Example 1:

[0055] (1) Preparation of sturgeon ovary protein peptide:

[0056] Sturgeon ovaries were roe-free, washed, and drained. The ovaries were minced and dissolved in water at a material-liquid ratio of 1:3 (g / mL). The pH was adjusted to 10.0 with 1 mol / L NaOH. The mixture was ultrasonicated for 30 min in a 37°C waterbath (ultrasonic frequencies of 25 / 50 / 75 Hz, ultrasonic power of 240 W, and ultrasonic mode set to 10 s on, 10 s off). Alkaline protease was then added at a concentration of 4000 U / g protein based on the protein content of the substrate (9.36%). The mixture was then subjected to constant temperature extraction in a 50°C waterbath for 240 min. Following enzymatic hydrolysis, the hydrolyzate was heated in a 95°C waterbath for 15 min, cooled to room temperature, and centrifuged at 10,000 rpm for 15 min. The supernatant was separated and freeze-dried to a dry powder, resulting in sturgeon ovary enzymatic hydrolysate, which was stored at -20°C.

[0057] (2) Ultrafiltration purification of sturgeon ovary protein hydrolysate;

[0058] The sturgeon ovary protein hydrolysate dry powder obtained in step (1) was prepared into an aqueous solution and ultrafiltered through 3kDa, 10kDa and 100kDa ultrafiltration membranes to collect the retentate; the retentate was freeze-dried for 48 hours to obtain <3kDa, 3-10kDa, 10-100kDa and >100kDa component polypeptide powders, which were used to detect the antioxidant effects of polypeptides with different molecular weights.

[0059] Figure 1 and 2 The ABTS free radical scavenging ability and hydroxyl free radical scavenging ability of sturgeon ovary protein hydrolysate and its four ultrafiltration fractions at different concentrations, where hydrolysates refers to sturgeon ovary protein hydrolysate, and Vc is used as a positive control.

[0060] The results are as follows Figure 1 and Figure 2 As shown in the results of ABTS and hydroxyl radical scavenging experiments, the sturgeon ovary protein peptide stock solution and its ultrafiltration fractions with different molecular weights all have the ability to scavenge free radicals, but the <3kDa fraction has a more outstanding ability to scavenge free radicals than the stock solution and other fractions; therefore, the <3kDa fraction was selected for the next step of separation and purification.

[0061] The component with the best antioxidant effect was selected and further separated and purified by gel column chromatography. The gel column chromatography filler was Sephadex G-25, and the chromatography conditions were as follows: column: Sephadex G-25 gel column (1.6×60 cm), column temperature: room temperature (25°C), flow rate: 1 mL / min, sample concentration: 25 mg / mL, injection volume: 2 mL, detection wavelength: 280 nm, eluent: distilled water.

[0062] like Figure 3 As shown in Figure 2, after purification of the <3kDa fraction by gel column chromatography, three fractions were collected, which were named as: F1, F2, and F3. The three fractions were prepared into different concentrations (20, 50, 100, 200, and 400 μg / mL) and applied to the H2O2-induced MC3T3-E1 cell injury model. The cell viability was shown in Figure 2. Figure 4 As shown in the results, compared with the model group, both F2 and F3 fractions alleviated cellular oxidative damage (P<0.05), and the antioxidant capacity of these two fractions was positively correlated with concentration at concentrations of 50-200 μg / mL. However, compared with F2, low concentrations of F3 fraction showed a better ability to alleviate cellular oxidative damage; therefore, F3 fraction was selected for further purification.

[0063] The fraction with the best antioxidant activity after gel column chromatography was further purified using a preparative reversed-phase liquid chromatography column. The column parameters were: methanol and deionized water as the mobile phase, with elution parameters of: 10% methanol from 0.01 to 20 minutes; 50% methanol from 20 to 20.01 minutes; 50% methanol from 20.01 to 40 minutes; 10% methanol from 40 to 40.01 minutes; and 10% methanol from 40.01 to 45 minutes. Column temperature: room temperature (approximately 25°C), flow rate: 5 mL / min, sample concentration: 200 mg / mL, injection volume: 1.5 mL, and detection wavelength: 280 nm.

[0064] like Figure 5 As shown in Figure 2, after the F3 fraction was purified by preparative reverse phase liquid chromatography column, a total of 16 fractions were collected, named F3-1, F3-2, ..., F3-16. The 16 fractions were prepared into 50 μg / mL concentrations and acted on the H2O2-induced MC3T3-E1 cell injury model. The cell viability was shown in Figure 2. Figure 6 As shown in the results, compared with the H2O2-induced model group, the F3-15 fraction showed the strongest ability to alleviate oxidative damage (cell viability after treatment with the F15 fraction was 76.1%). Therefore, the F3-15 fraction was determined to be the fraction with the strongest antioxidant activity among the sturgeon ovary proteolytic hydrolysates.

[0065] Example 2:

[0066] Peptide analysis by liquid chromatography-mass spectrometry (LC-MS / MS)

[0067] The samples were analyzed by LC-MS / MS equipped with an online nanospray ion source. The system consisted of an EASY-nanoLC1200 coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, MA, USA). A total of 3 μL of sample was loaded onto an Acclaim PepMap C18 analytical column, 75 μm × 25 cm. The sample was separated using a 60-min gradient with a flow rate of 300 nL / min, a column temperature of 40°C, and an electrospray voltage of 2 kV. The gradient started with 4% phase B, equilibrated for 6 minutes, and then increased to 28% over 40 minutes using a nonlinear gradient, then to 90% over 5 minutes, where it was maintained for 15 minutes. The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition.

[0068] The mass spectrometry parameters were set as follows:

[0069] (1) MS: scan range (m / z): 200-1550; resolution: 60000; AGC target: 4e5, maximum injection time: 50 ms;

[0070] (2) HCD-MS / MS: resolution: 15,000; AGC target: 5e4; maximum injection time: 90ms; collision energy: 35, dynamic exclusion time: 30s. The identification results were obtained by MAXQUANT analysis and comparison with the sturgeon database (Uniprot-Acipenser).

[0071] The Peptideranker program (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict the biological activity of peptides. Bioinformatics techniques were used to analyze the properties of the high-scoring peptides. The experimental results are shown in Table 1.

[0072] Table 1 shows the molecular weight distribution of sturgeon ovary protein peptides

[0073]

[0074]

[0075] Database comparisons revealed 1,961 matching peptide sequences, primarily derived from yolk protein, actin, collagen, and ferritin. These peptides were primarily composed of 4-20 amino acids, with molecular weights ranging from 454.428 to 2118.136 Da, consistent with the finding that bioactive peptides typically contain 2-20 amino acid residues. These peptides were scored using PeptideRanker, and the 12 highest-scoring peptides were selected for further analysis.

[0076] Example 3:

[0077] Molecular docking analysis

[0078] Zdock software was used to perform molecular docking on the above 12 sturgeon ovary protein peptides with potential biological activity.

[0079] Table 2 shows the docking results of the target peptide and myeloperoxidase molecule.

[0080]

[0081]

[0082] The crystal structure of the receptor protein myeloperoxidase (hMPO) was obtained from the RCSB Protein Data Bank (https: / / www.rcsb.org / structure / ), PDB: 3f9p.

[0083] Similarly, APPTEST software (https: / / research.timmons.eu / apptest) was used to predict the tertiary structure of the target peptide sequence. Key amino acid residues such as Q91, H95, R239, F99, F336, and F407 were selected as the active pocket sites of the receptor protein. Finally, the molecular docking results were evaluated based on the Zdock score, the interaction site between the target peptide and the receptor protein, and the interaction type. The known MPO enzyme inhibitor sequence "GRRRSVQWCA" was selected as a reference. The Zdock score of GRRRSVQWCA was 1082.06; therefore, peptides with a Zdock score greater than 1082.06 were considered potential inhibitors of the MPO enzyme.

[0084] Combined with Table 2, Figure 7As shown, a total of five peptides met the requirements. Observation of the 3D structure of the peptide-receptor binding revealed that all five peptides are located in the active pocket of MPO and are able to bind to key residues with the help of non-covalent bonds. Furthermore, observation of the docking postures of the five peptides revealed that most peptides tend to dock amino acid residues containing aromatic rings near heme, and that polar amino acids in the sequence tend to form hydrogen bonds with MP. Among them, the FL6 (FDWDRL) and FF8 (FEGPPFKF) peptides showed outstanding effects and greater antioxidant potential.

[0085] Example 4:

[0086] Peptide synthesis and antioxidant activity verification

[0087] The FL6 (FDWDRL) and FF8 (FEGPPFKF) peptides were synthesized using solid-phase synthesis with a purity >95%. The effects of the synthesized peptides on cytotoxicity and cell viability were tested using the CCK-8 kit.

[0088] Figure 8 As a result of synthetic peptide cytotoxicity, Figure 9 The results are as follows: the improvement results of synthetic peptides on H2O2 damaged MC3T3-E1 cells at concentrations of 12.5-200 μM; Figure 8 and Figure 9 As shown, the synthetic polypeptide had no obvious toxic effect on cells in the range of 12.5-100 μM (p < 0.05), and had a significant improvement effect on the H2O2-induced MC3T3-E1 oxidative damage model, achieving an unexpected technical effect.

[0089] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention should fall within the scope of protection of the present invention.

Claims

1. A sturgeon ovary antioxidant peptide, characterized in that: It includes two polypeptides, whose amino acid sequences are: Phe-Asp-Trp-Asp-Arg-Leu; Phe-Glu-Gly-Pro-Pro-Phe-Lys-Phe.

2. A method for preparing the sturgeon ovary antioxidant peptide according to claim 1, characterized in that: The specific steps are as follows: (1) The ovaries of sturgeons were cleaned, drained, and minced to obtain a puree; and the crude protein content of the sturgeon ovaries was determined by the Kjeldahl method; (2) adding distilled water to the minced material in step (1), ultrasonically treating the mixture to obtain a mixed solution, adjusting the pH of the mixed solution, and then adding alkaline protease to perform enzymatic hydrolysis in a constant temperature water bath. After enzymatic hydrolysis, the mixture is subjected to enzyme inactivation treatment, cooled to room temperature, centrifuged, and the supernatant is vacuum freeze-dried to obtain a powdered sturgeon ovary enzymatic hydrolysate; (3) Ultrafiltration: Using an ultrafiltration membrane to fractionate the powdered sturgeon ovary protein hydrolysate obtained in (2) to obtain components, and freeze-drying the components to obtain freeze-dried components; (4) Sephadex G-25 gel column purification: purify the freeze-dried components in step (3), elute with distilled water, collect the eluate, and freeze-dry to obtain the freeze-dried components; (5) Preparative reversed-phase liquid chromatography column purification: The freeze-dried components in (4) are further purified. After purification, the components are collected by linear gradient elution using an eluent, and freeze-dried to finally obtain sturgeon ovary antioxidant peptides.

3. The method for preparing a sturgeon ovary antioxidant peptide according to claim 2, characterized in that: In step (2), the ratio of the minced material to distilled water is 1 g:3 mL; the pH of the mixed solution is adjusted to 10.0 using 1 mol / L NaOH.

4. The method for preparing a sturgeon ovary antioxidant peptide according to claim 2, characterized in that: In step (2), the ultrasonic treatment was performed by placing the sample in a 37°C water bath, adjusting the triple-frequency ultrasonic wave to 25 / 50 / 75 Hz, the ultrasonic power to 240 W, the ultrasonic mode to 10 s on and 10 s off, and the ultrasonic time to 30 min.

5. The method for preparing a sturgeon ovary antioxidant peptide according to claim 2, characterized in that: In step (2), alkaline protease is added at 4000 U / g for enzymatic hydrolysis according to the crude protein content in the minced material determined by Kjeldahl nitrogen determination.

6. The method for preparing a sturgeon ovary antioxidant peptide according to claim 2, characterized in that: In step (2), the conditions for enzymatic hydrolysis in a constant temperature water bath are: temperature 50°C, and enzymatic hydrolysis time 240 min.

7. The method for preparing a sturgeon ovary antioxidant peptide according to claim 2, characterized in that: In step (2), the enzyme inactivation treatment conditions are: enzyme inactivation at 95°C for 15 min; and centrifugation conditions are 10,000 r / min for 10-15 min.

8. The method for preparing a sturgeon ovary antioxidant peptide according to claim 2, characterized in that: In step (3), the molecular weight cut-off of the ultrafiltration membrane for graded ultrafiltration is 100 kDa, 10 kDa and 3 kDa.

9. The method for preparing a sturgeon ovary antioxidant peptide according to claim 2, characterized in that: In step (4), the filler used for gel column chromatography is Sephadex G-25, and the chromatography conditions are as follows: column: Sephadex G-25 gel column, column size: 1.6 cm × 60 cm, column temperature: room temperature, flow rate: 1 mL / min, sample concentration: 25 mg / mL, injection volume: 2.0-5.0 mL, detection wavelength: 280 nm, eluent: distilled water.

10. The method for preparing a sturgeon ovary antioxidant peptide according to claim 2, characterized in that: In step (5), a preparative reversed-phase liquid chromatography column with a column size of 21.2 cm × 250 mm was used; methanol and deionized water were selected as the mobile phase, and the elution parameters were: 0.01-20 min, 10% methanol; 20-20.01 min, 50% methanol; 20.01-40 min: 50% methanol; 40-40.01 min: 10% methanol; 40.01-45 min: 10% methanol; column temperature: room temperature, flow rate: 5 mL / min, sample concentration: 200 mg / mL, injection volume: 1.5 mL, detection wavelength: 280 nm.

Images