A method for separating and purifying a polypeptide with antioxidant function, a polypeptide and its application

Abstract

The present application provides a method for isolating and purifying a polypeptide with antioxidant function, a polypeptide, and its preparation and application. The method comprises the following steps: S1, scrubbing animal bones with a multi-brush roller staggered scrubbing device; S2, rinsing, enzymatic hydrolysis, and ultrafiltration after scrubbing to produce a bone peptide solution; S3, mixing the bone peptide solution, chlorogenic acid-grafted chitosan, and transglutaminase, reacting, inactivating the enzyme, and dialyzing. After dialysis, the mixture is dried to produce an antioxidant polypeptide; the mass ratio of the chlorogenic acid-grafted chitosan to transglutaminase is 1:0.2-0.3; and the mass-to-volume ratio of the chlorogenic acid-grafted chitosan to the bone peptide solution is 1g:50mL-100mL. The functional polypeptide produced using the method of the present application has excellent antioxidant function.

Description

Technical Field

[0001] The present application relates to the technical field of polypeptide separation, and in particular to a method for separating and purifying a polypeptide with antioxidant function, the polypeptide and its application. Background Art

[0002] Peptides are compounds with a molecular structure intermediate between amino acids and proteins, which give proteins certain physiological functions. In human life, peptides are digested and absorbed better than free amino acids and have a distinct internal transport system. Certain short peptides not only provide essential nutrients for human growth and development, but also have the potential to prevent and treat diseases and regulate human functions. These biologically active peptides are referred to as bioactive peptides.

[0003] Commonly used bioactive peptides in related technologies include animal-derived antioxidant peptides, which have strong efficacy in inhibiting the peroxidation of biomacromolecules and scavenging free radicals in the body. Antioxidant peptides are valued for their small molecular weight, ease of absorption, and high activity, and hold great promise in pharmaceuticals, cosmetics, health supplements, and food and feed additives.

[0004] In addition, the molecular structure of polypeptides is relatively unstable and needs to be improved during use; at the same time, the antioxidant capacity of polypeptide molecules extracted by related technologies is relatively poor. Summary of the Invention

[0005] The present application is made in view of the above-mentioned problems, and its purpose is to provide a method for separating and purifying polypeptides with antioxidant function, wherein the polypeptides prepared by this method have strong antioxidant capacity.

[0006] The first aspect of the present application provides a method for separating and purifying a polypeptide having an antioxidant function, comprising the following steps:

[0007] S1. Brush the animal bones using a multi-brush roller staggered brushing device;

[0008] S2, after scrubbing, rinsing, enzymatic hydrolysis, and ultrafiltration are performed to obtain bone peptide solution;

[0009] S3, mixing the bone peptide solution, chlorogenic acid grafted chitosan and transglutaminase, reacting, inactivating the enzyme, dialyzing, and drying after the dialysis is completed to obtain an antioxidant functional polypeptide;

[0010] The mass ratio of the chlorogenic acid grafted chitosan and transglutaminase is 1:0.2-0.3;

[0011] The mass volume ratio of the chlorogenic acid grafted chitosan and the bone peptide liquid is 1g:50mL~100mL.

[0012] According to one of the technical solutions of the present application, at least the following beneficial effects are achieved:

[0013] The animal bones are first scrubbed with a multi-brush roller staggered scrubbing device and then rinsed. This process eliminates the need to manually remove residual meat and grease, saving effort and increasing efficiency. The cleaning effect is also more stable, and the higher the purity of the raw materials, the less impurities are produced after enzymatic hydrolysis.

[0014] After inorganic membrane filtration, ultrafiltration membrane filtration is used to remove unnecessary macromolecular components and retain the required target components; the optimized enzymatic hydrolysis process allows the release of target molecules to the maximum extent, with high yield and high effective ingredients.

[0015] Chitosan molecules are first modified with chlorogenic acid, and then, under the catalysis of transglutaminase, animal bone peptides are linked to the amino groups on the chitosan molecules, thereby achieving the modification of bone peptides;

[0016] The present application forms a protective barrier for the polypeptide active substance and improves the stability of the polypeptide substance by adding chlorogenic acid grafted chitosan to the polypeptide extract, thereby preparing an antioxidant functional polypeptide with excellent performance.

[0017] According to some embodiments of the present application, the animal bone includes bovine bone.

[0018] According to some embodiments of the present application, the scrubbing further includes an acid leaching treatment.

[0019] According to some embodiments of the present application, hydrochloric acid is used for acid leaching.

[0020] According to some embodiments of the present application, the mass fraction of the hydrochloric acid is 0.5%~1.5%.

[0021] According to some embodiments of the present application, the acid leaching time is 4 hours to 6 hours.

[0022] According to some embodiments of the present application, the acid leaching treatment is followed by rinsing.

[0023] According to some embodiments of the present application, the flushing is high-pressure flushing.

[0024] According to some embodiments of the present application, the bone paste is obtained by crushing after the flushing is completed.

[0025] According to some embodiments of the present application, the enzymatic hydrolysis is a two-step enzymatic hydrolysis process, wherein:

[0026] Neutral protease is used in the first step of enzymatic hydrolysis;

[0027] In the second step of enzymatic hydrolysis, trypsin and flavor protease are used.

[0028] According to some embodiments of the present application, the activity unit of the neutral protease is 300,000 U / g to 600,000 U / g.

[0029] According to some embodiments of the present application, the activity unit of the trypsin is 300,000 U / g to 500,000 U / g.

[0030] According to some embodiments of the present application, the activity unit of the flavor protease is 300,000 U / g to 600,000 U / g.

[0031] According to some embodiments of the present application, the temperature of the enzymatic hydrolysis is 50°C to 60°C.

[0032] According to some embodiments of the present application, the mass ratio of the neutral protease to bone mud is 1~2:1000.

[0033] According to some embodiments of the present application, the mass ratio of trypsin to bone mud is 4~6:1000.

[0034] According to some embodiments of the present application, the mass ratio of the flavor protease to bone paste is 1-2:1000.

[0035] According to some embodiments of the present application, the temperature of the first enzymatic hydrolysis step is 50°C to 60°C.

[0036] According to some embodiments of the present application, the time for the first enzymatic hydrolysis step is 1 h to 3 h.

[0037] According to some embodiments of the present application, the temperature of the second step enzymatic hydrolysis is 50°C to 60°C.

[0038] According to some embodiments of the present application, the time for the second step enzymatic hydrolysis is 1 hour to 3 hours.

[0039] According to some embodiments of the present application, the enzyme is inactivated after the enzymatic hydrolysis is completed.

[0040] According to some embodiments of the present application, the temperature of the enzyme inactivation treatment is 95°C to 105°C.

[0041] According to some embodiments of the present application, the time for the enzyme inactivation treatment is 10 min to 15 min.

[0042] According to some embodiments of the present application, the enzyme inactivation treatment is followed by microfiltration.

[0043] According to some embodiments of the present application, the pore size of the filter membrane selected for microfiltration is 0.2 μm~0.3 μm.

[0044] According to some embodiments of the present application, the molecular weight cut-off of the ultrafiltration is 2000 Da.

[0045] According to some embodiments of the present application, the method for preparing chlorogenic acid-grafted chitosan comprises the following steps:

[0046] Mixing chitosan, acetic acid, chlorogenic acid and vitamin C to prepare a first mixture;

[0047] The first mixture and hydrogen peroxide solution are mixed and reacted, dialyzed, and then freeze-dried.

[0048] Vitamin C reacts with hydrogen peroxide to generate hydroxyl radicals, which capture hydrogen atoms from chitosan molecules to form chitosan macromolecular free radicals. Chlorogenic acid molecules accept chitosan macromolecular free radicals to form chlorogenic acid-grafted chitosan.

[0049] According to some embodiments of the present application, the mass ratio of chitosan to chlorogenic acid is 1:0.5~1.

[0050] According to some embodiments of the present application, the mass ratio of chitosan to vitamin C is 1:0.05~0.2.

[0051] According to some embodiments of the present application, water is added during the preparation of the first mixture.

[0052] According to some embodiments of the present application, acetic acid and water are mixed to form an acetic acid solution.

[0053] According to some embodiments of the present application, the mass fraction of the acetic acid solution is 1%~3%.

[0054] According to some embodiments of the present application, the mass volume ratio of the chitosan and acetic acid solution is 1 g:50 mL~100 mL.

[0055] According to some embodiments of the present application, the molar concentration of the hydrogen peroxide solution is 0.5 mol / L~1 mol / L.

[0056] According to some embodiments of the present application, the mass-to-volume ratio of the chitosan and hydrogen peroxide solution is 1 g:5 mL~10 mL.

[0057] According to some embodiments of the present application, the molecular weight cut-off during dialysis during the preparation of the chlorogenic acid-grafted chitosan is 10,000 Da.

[0058] According to some embodiments of the present application, the reaction temperature in step S3 is 20°C~30°C.

[0059] The second aspect of the present application provides a polypeptide, which is prepared using the separation and purification method described in the first aspect of the present application.

[0060] The third aspect of the present application provides the use of the polypeptide prepared in the second aspect of the present application in the preparation of antioxidant active substances. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is described and illustrated below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are merely used to explain this application and are not intended to limit this application. Based on the embodiments provided in this application, all other embodiments obtained by those of ordinary skill in the art without making any creative work are within the scope of protection of this application.

[0062] Obviously, the following descriptions are merely some examples or embodiments of the present application. Those skilled in the art can apply the present application to other similar scenarios without inventive effort. Furthermore, it is also understood that, although the effort involved in such a development process may be complex and lengthy, for those skilled in the art related to the content disclosed in the present application, changes in design, manufacturing, or production based on the technical content disclosed in the present application are merely conventional technical means and should not be construed as an insufficiency of the content disclosed in the present application.

[0063] However, unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters or repeated descriptions of substantially identical structures may be omitted. This is to avoid unnecessary redundancy in the following description and to facilitate understanding by those skilled in the art. Furthermore, the following description is provided to enable those skilled in the art to fully understand this application and is not intended to limit the subject matter recited in the claims.

[0064] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0065] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0066] " range " disclosed in the present application is limited in the form of lower limit and upper limit, and given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of special range. The scope limited in this way can be to include end value or not include end value, and can be arbitrarily combined, that is, any lower limit can form a range with any upper limit combination. For example, if the scope of 60-120 and 80-110 is listed for specific parameters, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range value 1 and 2 are listed, and if the maximum range value 3,4 and 5 are listed, then the following range can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this application, unless otherwise specified, the numerical range " ab " represents the abbreviation of any real number combination between a and b, wherein a and b are all real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0067] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0068] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0069] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), indicating that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0070] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0071] Unless otherwise specified, the term "or" is used in this application to be inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied if any of the following conditions are met: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0072] The multi-brush roller staggered brushing device used in the embodiments and comparative examples of the present application uses the corresponding equipment in the embodiment of CN 202410117641.9.

[0073] Example 1

[0074] This embodiment is a method for separating and purifying a polypeptide having antioxidant function, which consists of the following steps:

[0075] S1. Brush the animal bones (cow bones) using a multi-brush roller staggered brushing device (brushing time is 30 minutes);

[0076] S2, after scrubbing, acid leaching, rinsing, and crushing to obtain bone mud;

[0077] Acid leaching is carried out by immersing the bones in a 1% hydrochloric acid solution for 5 hours; the ratio of the bones to the hydrochloric acid solution is 1 kg: 2 L;

[0078] Flushing adopts high pressure flushing, the flushing pressure is 10MPa, and the flushing time is 5min;

[0079] The bone paste was enzymatically hydrolyzed, enzyme-inactivated, microfiltered (pore size of microfiltration was 0.22 μm) and ultrafiltered (molecular weight cut-off was 2000 Da) to prepare bone peptide solution;

[0080] The enzymatic hydrolysis is a two-step enzymatic hydrolysis process, wherein:

[0081] The first step of enzymatic hydrolysis uses neutral protease (activity unit: 400,000 U / g, mass ratio of neutral protease to bone paste is 1:1000), the temperature of the first step of enzymatic hydrolysis is 55°C, the first step of enzymatic hydrolysis is performed under a neutral environment, and the first step of enzymatic hydrolysis time is 2 hours;

[0082] The second step of enzymatic hydrolysis used a complex enzyme consisting of trypsin (activity unit: 300,000 U / g, the mass ratio of trypsin to bone paste was 4:1000) and flavor protease (activity unit: 500,000 U / g, the mass ratio of flavor protease to bone paste was 1:1000), the temperature of the second step of enzymatic hydrolysis was 55°C, and the time of the second step of enzymatic hydrolysis was 2 h.

[0083] After the enzymatic hydrolysis is completed, the enzyme is inactivated (the temperature of the enzyme inactivation treatment is 100°C and the time is 15 minutes).

[0084] S3, mixing the bone peptide solution, chlorogenic acid grafted chitosan and transglutaminase, reacting (reaction temperature: 25°C, reaction time: 5h), inactivating the enzyme (enzyme inactivation treatment temperature: 100°C, time: 15min), dialyzing (molecular weight cut-off: 10,000Da, dialysis time: 72h), and freeze-drying after dialysis to obtain the antioxidant functional peptide;

[0085] The preparation method of chlorogenic acid grafted chitosan in this embodiment includes the following steps:

[0086] Chitosan, an acetic acid aqueous solution (mass fraction of 2%, mass volume ratio of chitosan to acetic acid aqueous solution of 1 g:50 mL), chlorogenic acid, and vitamin C were mixed (mixing temperature of 25° C., mixing time of 30 min) to prepare a first mixture;

[0087] The first mixture and hydrogen peroxide solution (1 mol / L, mass volume ratio of chitosan to hydrogen peroxide solution is 1 g:8 mL) were mixed and reacted (reaction temperature was 25°C, time was 24 h), dialyzed (molecular weight cut-off was 10,000 Da, dialysis time was 72 h) and then freeze-dried.

[0088] The mass ratio of chitosan to chlorogenic acid is 1:0.8.

[0089] The mass ratio of chitosan to vitamin C is 1:0.1.

[0090] The mass ratio of chlorogenic acid grafted chitosan and transglutaminase was 1:0.28.

[0091] The mass volume ratio of chlorogenic acid grafted chitosan and bone peptide solution is 1g:80mL.

[0092] Example 2

[0093] This example is a method for separating and purifying a polypeptide having antioxidant function, which differs from Example 1 in that:

[0094] The mass ratio of chitosan to chlorogenic acid is 1:0.5.

[0095] The mass ratio of chitosan to vitamin C is 1:0.2.

[0096] The mass ratio of chlorogenic acid grafted chitosan and transglutaminase was 1:0.2.

[0097] The mass volume ratio of chlorogenic acid grafted chitosan and bone peptide solution is 1g:100mL.

[0098] Example 3

[0099] This example is a method for separating and purifying a polypeptide having antioxidant function, which differs from Example 1 in that:

[0100] The mass ratio of chitosan to chlorogenic acid is 1:1.

[0101] The mass ratio of chitosan to vitamin C is 1:0.05.

[0102] The mass ratio of chlorogenic acid grafted chitosan and transglutaminase was 1:0.3.

[0103] The mass volume ratio of chlorogenic acid grafted chitosan and bone peptide solution is 1g:100mL.

[0104] Example 4

[0105] This example is a method for separating and purifying a polypeptide having antioxidant function, which differs from Example 1 in that:

[0106] The mass ratio of chitosan to chlorogenic acid is 1:0.6.

[0107] The mass ratio of chitosan to vitamin C is 1:0.15.

[0108] The mass ratio of chlorogenic acid grafted chitosan and transglutaminase was 1:0.21.

[0109] The mass volume ratio of chlorogenic acid grafted chitosan and bone peptide solution is 1g:60mL.

[0110] Example 5

[0111] This example is a method for separating and purifying a polypeptide having antioxidant function, which differs from Example 1 in that:

[0112] The mass ratio of chitosan to chlorogenic acid is 1:0.5.

[0113] The mass ratio of chitosan to vitamin C is 1:0.05.

[0114] The mass ratio of chlorogenic acid grafted chitosan and transglutaminase was 1:0.2.

[0115] The mass volume ratio of chlorogenic acid grafted chitosan and bone peptide solution is 1g:50mL.

[0116] Comparative Example 1

[0117] This comparative example is a method for separating and purifying a polypeptide having antioxidant function, which differs from Example 5 in that:

[0118] S3, mixing the bone peptide solution, chlorogenic acid grafted chitosan and transglutaminase, reacting (reaction temperature: 25°C, reaction time: 5h), inactivating the enzyme (enzyme inactivation treatment temperature: 100°C, time: 15min), dialyzing (molecular weight cut-off: 10,000Da, dialysis time: 72h), and cooling and drying after dialysis to obtain an antioxidant functional polypeptide;

[0119] The preparation method of chlorogenic acid grafted chitosan in this comparative example comprises the following steps:

[0120] Chitosan, an acetic acid aqueous solution (mass fraction of 2%, mass volume ratio of chitosan to acetic acid aqueous solution of 1 g:50 mL) and chlorogenic acid were mixed (mixing temperature of 25° C., mixing time of 30 min) to prepare a first mixture;

[0121] The first mixture and hydrogen peroxide solution (1 mol / L, mass volume ratio of chitosan to hydrogen peroxide solution is 1 g:8 mL) were mixed and reacted (reaction temperature was 25°C, time was 24 h), dialyzed (molecular weight cut-off was 10,000 Da, dialysis time was 72 h) and then freeze-dried.

[0122] The mass ratio of chitosan to chlorogenic acid is 1:0.5.

[0123] The mass ratio of chlorogenic acid grafted chitosan and transglutaminase was 1:0.2.

[0124] The mass volume ratio of chlorogenic acid grafted chitosan and bone peptide solution is 1g:50mL.

[0125] Comparative Example 2

[0126] This comparative example is a method for separating and purifying a polypeptide having antioxidant function, which differs from Example 5 in that:

[0127] S3, mixing the bone peptide solution, modified chitosan and transglutaminase, reacting (reaction temperature: 25°C, reaction time: 5 hours), inactivating the enzyme (enzyme inactivation treatment temperature: 100°C, reaction time: 15 minutes), dialyzing (molecular weight cut-off: 10,000 Da, dialysis time: 72 hours), and cooling and drying after dialysis to obtain an antioxidant functional polypeptide;

[0128] The preparation method of the modified chitosan in this comparative example comprises the following steps:

[0129] Chitosan, an acetic acid aqueous solution (mass fraction of 2%, mass volume ratio of chitosan to acetic acid aqueous solution of 1 g:50 mL) and vitamin C were mixed (mixing temperature of 25° C., mixing time of 30 min) to prepare a first mixture;

[0130] The first mixture and hydrogen peroxide solution (1 mol / L, mass volume ratio of chitosan to hydrogen peroxide solution is 1 g:8 mL) were mixed and reacted (reaction temperature was 25°C, time was 24 h), dialyzed (molecular weight cut-off was 10,000 Da, dialysis time was 72 h) and then freeze-dried.

[0131] The mass ratio of chitosan to vitamin C is 1:0.05.

[0132] The mass ratio of modified chitosan to transglutaminase is 1:0.2.

[0133] The mass volume ratio of modified chitosan and bone peptide solution is 1g:50mL.

[0134] Comparative Example 3

[0135] This comparative example is a method for separating and purifying a polypeptide having antioxidant function, which differs from Example 5 in that:

[0136] S3, mixing the bone peptide solution, chitosan acetic acid mixture and transglutaminase, reacting (reaction temperature: 25°C, reaction time: 5h), inactivating the enzyme (enzyme inactivation treatment temperature: 100°C, time: 15min), dialyzing (molecular weight cut-off: 10,000Da, dialysis time: 72h), and freeze-drying after dialysis to obtain an antioxidant functional polypeptide;

[0137] The preparation method of the chitosan acetic acid mixed solution in this comparative example comprises the following steps:

[0138] Chitosan and acetic acid aqueous solution (mass fraction of 2%, mass volume ratio of chitosan to acetic acid aqueous solution of 1g:50mL) were mixed (mixing temperature of 25°C, mixing time of 30min) to prepare a chitosan acetic acid mixed solution.

[0139] The mass volume ratio of chitosan and bone peptide solution is 1g:50mL.

[0140] Comparative Example 4

[0141] This comparative example is a method for separating and purifying a polypeptide having antioxidant function, which differs from Example 5 in that:

[0142] The mass ratio of chlorogenic acid grafted chitosan and transglutaminase was 1:0.1.

[0143] Comparative Example 5

[0144] This comparative example is a method for separating and purifying a polypeptide having antioxidant function, which differs from Example 5 in that:

[0145] S3. The bone peptide solution was dialyzed (molecular weight cut-off 10,000 Da, dialysis time 72 h), and after dialysis, freeze-dried to obtain antioxidant functional peptides.

[0146] The antioxidant test method is as follows:

[0147] 1. Grouping and Dosing of Zebrafish Embryos

[0148] 4 hpf zebrafish embryos were randomly divided into a blank control group (no hydrogen peroxide added), a H2O2 induction group (5 mmol / L), and an experimental group (the antioxidant functional polypeptides prepared in Examples and Comparative Examples, with a hydrogen peroxide concentration of 5 mmol / L), with 50 embryos in each group.

[0149] Eight hours after fertilization, zebrafish embryos were incubated with a drug solution prepared in fish culture water for 1 hour, and then the hydrogen peroxide concentration was controlled to 5 mM and incubated for 24 hours, and then the solution was changed to fish culture water for feeding.

[0150] 2. Zebrafish embryo survival rate

[0151] At 48 hours, the embryo hatching rate was calculated and the heartbeat was maintained.

[0152] Survival rate = (hatched embryos / total number of fertilized eggs) × 100%, and the number of heartbeats within 1 minute was calculated; the experiment was conducted on the third day of feeding.

[0153] 3. ROS analysis in zebrafish embryos

[0154] Select the hatched fry and place them in a 24-well plate;

[0155] For DCFH-DA incubation, remove excess water from the well plate and replace with DCFH-DA with a final concentration of 500 μM and incubate in an incubator for 30 min;

[0156] After the DCFH-DA incubation was completed, the cells were washed twice with PBS.

[0157] Detection method: Fluorescence was detected by fluorescence microscopy with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0158] The survival rate and ROS analysis results are shown in Table 1.

[0159]

[0160] Under normal physiological conditions, ROS levels in zebrafish are low and can be cleared by the antioxidant defense system. However, ROS are primarily generated in the mitochondria. Under oxidative stress, increased ROS production exceeds the clearance capacity of the antioxidant defense system, damaging mitochondrial DNA and impairing mitochondrial biogenesis. Oxidative stress disrupts the balance between reactive oxygen species (ROS) levels and the antioxidant system's capacity, affecting survival.

[0161] However, the ROS content in the examples of the present application is relatively low, indicating that the polypeptide in the present application improves the antioxidant capacity of zebrafish, thereby improving the survival rate of zebrafish.

[0162] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for separating and purifying a polypeptide having antioxidant function, characterized in that: The following steps are involved: S1. Brush the animal bones using a multi-brush roller staggered brushing device; S2, after scrubbing, rinsing, enzymatic hydrolysis, and ultrafiltration are performed to obtain bone peptide solution; S3, mixing the bone peptide solution, chlorogenic acid grafted chitosan and transglutaminase, reacting, inactivating the enzyme, dialyzing, and drying after the dialysis is completed to obtain an antioxidant functional polypeptide; The mass ratio of the chlorogenic acid grafted chitosan and transglutaminase is 1:0.2-0.3; The mass volume ratio of the chlorogenic acid grafted chitosan and bone peptide solution is 1g:50mL~100mL; The preparation method of chlorogenic acid grafted chitosan comprises the following steps: Mixing chitosan, acetic acid, chlorogenic acid and vitamin C to prepare a first mixture; mixing the first mixture with a hydrogen peroxide solution, reacting the mixture, dialyzing the mixture, and then freeze-drying the mixture; The reaction temperature in step S3 is 20°C to 30°C.

2. The separation and purification method according to claim 1, wherein The enzymatic hydrolysis is a two-step enzymatic hydrolysis process, wherein: Neutral protease is used in the first step of enzymatic hydrolysis; In the second step of enzymatic hydrolysis, trypsin and flavor protease are used.

3. The separation and purification method according to claim 1, wherein The temperature of the enzymatic hydrolysis is 50°C to 60°C.

4. The separation and purification method according to claim 1, wherein The molecular weight cut-off of the ultrafiltration was 2000 Da.

5. The separation and purification method according to claim 1, wherein The mass ratio of chitosan to chlorogenic acid is 1:0.5~1; The mass ratio of the chitosan to vitamin C is 1:0.05-0.

2.

6. The separation and purification method according to claim 1, wherein The molecular weight cut-off during dialysis during the preparation of the chlorogenic acid-grafted chitosan is 10,000 Da.

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