Preparation method of polypeptide with antibacterial and antioxidant functions

Abstract

The invention relates to the technical field of polypeptides, and discloses a preparation method of a polypeptide with antibacterial and antioxidant functions. Polypeptide is extracted from tuna bones, quaternized chitosan containing alkenyl and an imidazole group are grafted to cysteine, modified cysteine containing alkenyl imidazole is obtained, then the modified cysteine containing alkenyl imidazole and ferulic acid are grafted to the carboxyl terminal and the amino terminal of the polypeptide respectively, and the polypeptide with the antibacterial and antioxidant functions is obtained. Quaternary ammonium salt contained in the polypeptide destroys cell membranes through electrostatic attraction with bacterial cell membranes, so that cell death is caused, and an antibacterial effect is achieved; hydrogen atoms released in alkenyl can be combined with free radicals, so that the antioxidant function of the polypeptide is improved; chitosan is combined with nucleic acid of bacteria, normal synthesis of protein is inhibited, and the sterilization effect is achieved; the imidazolyl group enables ergosterol in a fungal cell membrane not to be synthesized, so that fungi die; ferulic acid can be combined with phospholipid of a biological membrane, free radical damage is reduced, and therefore the anti-oxidation effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a method for preparing a polypeptide with antibacterial and antioxidant functions. Background Art

[0002] Peptides are a type of biological molecule composed of amino acid residues connected by peptide bonds. They are also intermediate products of protein hydrolysis and are divided into biologically active peptides and artificially synthesized peptides. Peptides are widely used in drug development, clinical treatment, food, beauty and skin care, etc. With the development of peptide synthesis technology and the increase in demand for peptides, the requirements for the antibacterial and antioxidant functions of peptides are getting higher and higher. For example, patent CN110981953B discloses a peptide and the application of peptides and a composition containing peptides, which have good anticoagulant activity, but their antibacterial and antioxidant functions need to be improved. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] In view of the deficiencies of the prior art, the present invention provides a method for preparing a polypeptide with antibacterial and antioxidant functions. The polypeptide prepared by the method has good antibacterial and antioxidant functions.

[0005] (II) Technical solution

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing a polypeptide with antibacterial and antioxidant functions, the method for preparing a polypeptide with antibacterial and antioxidant functions comprising the following steps:

[0007] (1) adding 12-16 parts by weight of a washed and chopped tuna bone sample to deionized water, stirring evenly, adding 6-9 parts by weight of alkaline protease, adjusting the pH to 9-11, performing enzymolysis at 35-45° C., and after the enzymolysis is completed, inactivating the enzyme at 95-100° C. to obtain a tuna bone enzymolysis solution;

[0008] (2) adding anhydrous ethanol to 10-12 parts by weight of tuna bone hydrolysate, stirring evenly, and then standing for precipitation for 8-14 hours, filtering and rotary evaporation, and then separating by a tangential flow ultrafiltration system, collecting the filtrate and freeze-drying to obtain a polypeptide, and analyzing and identifying the amino acid sequence of the polypeptide by LC-MS, searching the fish collagen library for matching, and obtaining a polypeptide sequence of AKGQRGPPGKR;

[0009] (3) Add N-(epoxypropyl)-N-phenyl-epoxyethane methylamine and 3-bromopropylene to tetrahydrofuran solvent, stir and mix, and reflux and heat at 82-90° C. for 3-4 hours. After the reaction is completed, cool, wash and dry to obtain intermediate 1;

[0010] (4) adding 11-13 parts by weight of the intermediate 1 to deionized water, stirring to dissolve, dissolving 5-7 parts by weight of chitosan in an acetic acid aqueous solution, slowly dropping the aqueous solution of the intermediate 1 into the chitosan solution, heating under reflux at 65-75° C. for 7-9 hours, washing after the reaction, and freeze-drying to obtain alkenyl-containing quaternary ammonium chitosan;

[0011] (5) adding quaternized chitosan containing alkenyl groups to tetrahydrofuran solvent, stirring and dissolving, adding cysteine ​​and p-toluenesulfonic acid catalyst thereto, reacting at 60-80° C. for 2-3 h, filtering, washing and drying after the reaction to obtain intermediate 2;

[0012] (6) adding 7-10 parts by weight of the intermediate 2, 13-15 parts by weight of 4-vinyl imidazole, and 0.3-0.6 parts by weight of benzoin dimethyl ether photoinitiator to N,N-dimethylformamide solvent, irradiating with 365 nm ultraviolet light at 28-32° C., centrifuging after completion, washing and drying to obtain vinyl imidazole-modified cysteine;

[0013] (7) Adding a polypeptide with a sequence of AKGQRGPPGKR to an N,N-dimethylformamide solvent, stirring to dissolve, adding alkenyl imidazole-modified cysteine ​​and N,N-diisopropylethylamine catalyst, passing nitrogen gas for protection, reacting at 20-30° C. for 2.5-4.5 hours, washing and drying after the reaction to obtain an alkenyl imidazole-modified cysteine ​​modified polypeptide;

[0014] (8) Add ferulic acid and benzotriazole to an anhydrous ether solvent, stir to dissolve, cool to -5-0°C in an ice bath, slowly dropwise add a dicyclohexylcarbodiimide ether solution thereto, and after the addition is complete, keep the temperature to react for 2-4 hours, raise the temperature to 15-25°C, and continue the reaction for 14-15 hours. After the reaction is complete, filter, cool and crystallize to obtain benzotriazole-activated ferulic acid;

[0015] (9) Add 8-12 parts by weight of benzotriazole-activated ferulic acid to N-methylpyrrolidone solvent, cool to 0-5°C in an ice bath, add 9-11 parts by weight of cysteine-modified polypeptide containing alkenyl imidazole and 0.2-0.4 parts by weight of triethylamine catalyst, and keep the reaction warm for 2.5-3 hours. After the reaction is completed, add acetone solvent, centrifuge at a speed of 8000-10000 rpm for 8-10 minutes, remove the supernatant after standing, and freeze-dry the precipitate to obtain a polypeptide with antibacterial and antioxidant functions.

[0016] Preferably, the enzyme inactivation time in step (1) is 8-12 minutes.

[0017] Preferably, the mass fraction of ethanol in step (2) is 70-85%.

[0018] Preferably, in step (3), the mass ratio of N-(epoxypropyl)-N-phenyl-epoxyethylene methylamine to 3-bromopropylene is 1:0.8-0.84.

[0019] Preferably, the mass fraction of the acetic acid aqueous solution in step (4) is 2-4%.

[0020] Preferably, in step (5), the mass ratio of quaternized alkenyl chitosan, cysteine ​​and p-toluenesulfonic acid is 1:0.6-0.8:0.025-0.03.

[0021] Preferably, the ultraviolet light irradiation time in step (6) is 2.5-3.5 hours.

[0022] Preferably, in step (7), the mass ratio of the polypeptide having the sequence AKGQRGPPGKR, the cysteine ​​modified with alkenyl imidazole, and N,N-diisopropylethylamine is 1:0.8-1:0.05-0.07.

[0023] Preferably, in step (8), the mass ratio of ferulic acid, benzotriazole and dicyclohexylcarbodiimide is 1:0.85-0.9:0.06-0.08.

[0024] Preferably, the temperature of the refrigerated centrifugation in step (9) is -15 to -10°C.

[0025] (III) Beneficial technical effects

[0026] The invention extracts a polypeptide with a sequence of AKGQRGPPGKR from tuna bones, grafts quaternized chitosan containing alkenyl groups and imidazole groups onto cysteine ​​to obtain alkenyl imidazole-modified cysteine, and then grafts alkenyl imidazole-modified cysteine ​​and ferulic acid onto the carboxyl end and amino end of the polypeptide respectively to obtain a polypeptide with antibacterial and antioxidant functions.

[0027] The tertiary amine in N-(epoxypropyl)-N-phenyl-epoxyethylene methylamine undergoes a quaternization reaction with 3-propylene bromide, and an epoxy group and an alkenyl group are introduced to obtain an intermediate 1. The epoxy group in the intermediate 1 reacts with an amino group in chitosan to obtain an alkenyl-containing quaternized chitosan. The hydroxyl group in the alkenyl-containing quaternized chitosan undergoes an esterification reaction with a carboxyl group in cysteine, and a thiol group and an amino group are introduced to obtain an intermediate 2. The thiol group in the intermediate 2 undergoes a click reaction with 4-vinyl imidazole, and an imidazole group is introduced to obtain an alkenyl imidazole-modified cysteine. The amino group in the alkenyl imidazole-modified cysteine ​​undergoes an amidation reaction with a carboxyl end in a polypeptide to form a peptide bond, and an alkenyl imidazole-modified cysteine ​​modified polypeptide is obtained. The carboxyl group in ferulic acid undergoes a condensation reaction with the triazole ring in benzotriazole to activate the carboxyl group and obtain benzotriazole-activated ferulic acid. The benzotriazole-activated ferulic acid is then grafted onto the amino end of an alkenyl imidazole-modified cysteine-modified polypeptide to obtain a polypeptide with antibacterial and antioxidant functions.

[0028] The positive charge carried by the quaternary ammonium salt molecule can electrostatically attract the negative charge in the bacterial cell membrane, destroy the cell membrane, leak the cell contents, and cause cell death, thereby playing an antibacterial effect; the carbon-hydrogen bond in the olefin group can be broken to release hydrogen atoms, which can combine with free radicals to form more stable molecules, thereby improving the antioxidant function of the polypeptide; chitosan, as a polycationic polymer, can combine with bacterial nucleic acids such as DNA and RNA, affecting the replication and transcription of nucleic acids. Chitosan can also interact with proteins inside bacteria to inhibit the normal synthesis of bacterial proteins, thereby playing a bactericidal effect; the imidazole group inhibits enzyme activity, making it impossible to synthesize ergosterol in the fungal cell membrane, changing the permeability of the cell membrane, causing the leakage of intracellular substances and causing the death of the fungus; the phenolic hydroxyl group in ferulic acid reduces the damage of free radicals by transferring unstable electrons to itself to form a stable structure, and can also combine with the phospholipids of the biological membrane to reduce free radical damage by protecting the membrane lipids, thereby playing an antioxidant role. DETAILED DESCRIPTION

[0029] Example 1

[0030] (1) adding 12 parts by weight of a washed and chopped tuna bone sample to deionized water, stirring evenly, adding 6 parts by weight of alkaline protease, adjusting the pH to 9, performing enzymolysis at 35° C., and after the enzymolysis is completed, performing enzyme inactivation at 95° C. for 8 minutes to obtain a tuna bone enzymatic hydrolyzate;

[0031] (2) adding anhydrous ethanol to 10 parts by weight of tuna bone hydrolysate, stirring evenly and then standing for precipitation for 8 hours, wherein the mass fraction of ethanol is 70%, filtering and rotary evaporation, and then separating by a tangential flow ultrafiltration system, collecting the filtrate and freeze-drying to obtain a polypeptide, and analyzing and identifying the amino acid sequence of the polypeptide by LC-MS, and searching the fish collagen library for matching, and obtaining a polypeptide sequence of AKGQRGPPGKR;

[0032] (3) Adding N-(epoxypropyl)-N-phenyl-epoxyethane methylamine and 3-bromopropylene to tetrahydrofuran solvent, wherein the mass ratio of N-(epoxypropyl)-N-phenyl-epoxyethane methylamine to 3-bromopropylene is 1:0.8, stirring and mixing, and reflux heating at 82° C. for 3 h. After the reaction is completed, cooling, washing and drying to obtain intermediate 1;

[0033] (4) adding 11 parts by weight of intermediate 1 to deionized water, stirring and dissolving, dissolving 5 parts by weight of chitosan in an acetic acid aqueous solution, wherein the mass fraction of the acetic acid aqueous solution is 2%, slowly dropping the aqueous solution of intermediate 1 into the chitosan solution, reflux heating at 65° C. for 7 h, washing after the reaction, and freeze-drying to obtain alkenyl-containing quaternary ammonium chitosan;

[0034] (5) adding alkenyl quaternized chitosan to tetrahydrofuran solvent, stirring to dissolve, adding cysteine ​​and p-toluenesulfonic acid catalyst, wherein the mass ratio of alkenyl quaternized chitosan, cysteine ​​and p-toluenesulfonic acid is 1:0.6:0.025, reacting at 60° C. for 2 h, filtering, washing and drying after the reaction to obtain intermediate 2;

[0035] (6) adding 7 parts by weight of the intermediate 2, 13 parts by weight of 4-vinyl imidazole, and 0.3 parts by weight of benzoin dimethyl ether photoinitiator to N,N-dimethylformamide solvent, irradiating with 365 nm ultraviolet light at 28° C. for 2.5 h, centrifuging, washing, and drying to obtain vinyl imidazole-modified cysteine;

[0036] (7) Adding a polypeptide with a sequence of AKGQRGPPGKR to an N,N-dimethylformamide solvent, stirring to dissolve, adding alkenyl imidazole-modified cysteine ​​and N,N-diisopropylethylamine catalyst, wherein the mass ratio of the polypeptide with a sequence of AKGQRGPPGKR, alkenyl imidazole-modified cysteine, and N,N-diisopropylethylamine is 1:0.8:0.05, passing nitrogen protection, reacting at 20°C for 2.5 hours, and after the reaction is completed, washing and drying to obtain an alkenyl imidazole-modified cysteine ​​modified polypeptide;

[0037] (8) Add ferulic acid and benzotriazole to an anhydrous ether solvent, stir to dissolve, cool to -5°C in an ice bath, slowly dropwise add a dicyclohexylcarbodiimide ether solution thereto, wherein the mass ratio of ferulic acid, benzotriazole, and dicyclohexylcarbodiimide is 1:0.85:0.06. After the dropwise addition is complete, keep the temperature for reaction for 2 hours, raise the temperature to 15°C, and continue the reaction for 14 hours. After the reaction is completed, filter, cool, and crystallize to obtain benzotriazole-activated ferulic acid;

[0038] (9) Add 8 parts by weight of benzotriazole-activated ferulic acid to N-methylpyrrolidone solvent, cool to 0°C in an ice bath, add 9 parts by weight of cysteine-modified polypeptide containing alkenyl imidazole and 0.2 parts by weight of triethylamine catalyst, and keep the reaction warm for 2.5 hours. After the reaction is completed, add acetone solvent, centrifuge at a speed of 8000 rpm for 8 minutes, wherein the temperature of the freeze-centrifuge is -15°C, remove the supernatant after standing, and freeze-dry the precipitate to obtain a polypeptide with antibacterial and antioxidant functions.

[0039] Example 2

[0040] (1) adding 16 parts by weight of a washed and chopped tuna bone sample to deionized water, stirring evenly, adding 9 parts by weight of alkaline protease, adjusting the pH to 11, performing enzymatic hydrolysis at 45° C., and after the enzymatic hydrolysis is completed, performing enzyme inactivation at 100° C. for 12 minutes to obtain a tuna bone enzymatic hydrolyzate;

[0041] (2) adding anhydrous ethanol to 12 parts by weight of tuna bone enzymatic hydrolysate, stirring evenly and then standing for precipitation for 14 hours, wherein the mass fraction of ethanol is 85%, filtering and rotary evaporation, and then separating by a tangential flow ultrafiltration system, collecting the filtrate and freeze-drying to obtain a polypeptide, and analyzing and identifying the amino acid sequence of the polypeptide by LC-MS, and searching the fish collagen library for matching, and obtaining a polypeptide sequence of AKGQRGPPGKR;

[0042] (3) Adding N-(epoxypropyl)-N-phenyl-epoxyethane methylamine and 3-bromopropylene to tetrahydrofuran solvent, wherein the mass ratio of N-(epoxypropyl)-N-phenyl-epoxyethane methylamine to 3-bromopropylene is 1:0.84, stirring and mixing, and reflux heating at 90° C. for 4 h. After the reaction is completed, cooling, washing and drying to obtain intermediate 1;

[0043] (4) adding 13 parts by weight of intermediate 1 to deionized water and stirring to dissolve, dissolving 7 parts by weight of chitosan in an acetic acid aqueous solution, wherein the mass fraction of the acetic acid aqueous solution is 4%, slowly dropping the aqueous solution of intermediate 1 into the chitosan solution, reflux heating at 75° C. for 9 h, washing after the reaction is completed, and freeze-drying to obtain quaternized chitosan containing alkenyl;

[0044] (5) adding alkenyl quaternized chitosan to tetrahydrofuran solvent, stirring to dissolve, adding cysteine ​​and p-toluenesulfonic acid catalyst, wherein the mass ratio of alkenyl quaternized chitosan, cysteine ​​and p-toluenesulfonic acid is 1:0.8:0.03, reacting at 80° C. for 3 h, filtering, washing and drying after the reaction to obtain intermediate 2;

[0045] (6) adding 10 parts by weight of the intermediate 2, 15 parts by weight of 4-vinyl imidazole, and 0.6 parts by weight of benzoin dimethyl ether photoinitiator to N,N-dimethylformamide solvent, irradiating with 365 nm ultraviolet light at 32° C. for 3.5 h, centrifuging, washing, and drying to obtain vinyl imidazole-modified cysteine;

[0046] (7) Adding a polypeptide having a sequence of AKGQRGPPGKR to an N,N-dimethylformamide solvent, stirring to dissolve, adding alkenyl imidazole-modified cysteine ​​and N,N-diisopropylethylamine catalyst, wherein the mass ratio of the polypeptide having a sequence of AKGQRGPPGKR, alkenyl imidazole-modified cysteine, and N,N-diisopropylethylamine is 1:1:0.07, passing nitrogen protection, reacting at 30°C for 4.5 hours, and after the reaction is completed, washing and drying to obtain an alkenyl imidazole-modified cysteine ​​modified polypeptide;

[0047] (8) Add ferulic acid and benzotriazole to an anhydrous ether solvent, stir to dissolve, cool to 0° C. in an ice bath, slowly dropwise add a dicyclohexylcarbodiimide ether solution, wherein the mass ratio of ferulic acid, benzotriazole, and dicyclohexylcarbodiimide is 1:0.9:0.08. After the dropwise addition is complete, keep the temperature for reaction for 4 hours, raise the temperature to 25° C., and continue the reaction for 15 hours. After the reaction is completed, filter, cool, and crystallize to obtain benzotriazole-activated ferulic acid;

[0048] (9) Add 12 parts by weight of benzotriazole-activated ferulic acid to N-methylpyrrolidone solvent, cool to 5°C in an ice bath, add 11 parts by weight of cysteine-modified polypeptide containing alkenyl imidazole and 0.4 parts by weight of triethylamine catalyst, and keep warm for 3 hours. After the reaction, add acetone solvent, centrifuge at a speed of 10,000 rpm for 10 minutes, wherein the temperature of the centrifuge is -10°C, remove the supernatant after standing, and freeze-dry the precipitate to obtain a polypeptide with antibacterial and antioxidant functions.

[0049] Example 3

[0050] (1) adding 14 parts by weight of a washed and chopped tuna bone sample to deionized water, stirring evenly, adding 8 parts by weight of alkaline protease, adjusting the pH to 10, performing enzymatic hydrolysis at 40° C., and after the enzymatic hydrolysis is completed, performing enzyme inactivation at 97° C. for 10 min to obtain a tuna bone enzymatic hydrolyzate;

[0051] (2) adding anhydrous ethanol to 11 parts by weight of tuna bone hydrolysate, stirring evenly and then standing for precipitation for 11 hours, wherein the mass fraction of ethanol is 78%, filtering and rotary evaporation, and then separating by a tangential flow ultrafiltration system, collecting the filtrate and freeze-drying to obtain a polypeptide, and analyzing and identifying the amino acid sequence of the polypeptide by LC-MS, and searching the fish collagen library for matching, and obtaining a polypeptide sequence of AKGQRGPPGKR;

[0052] (3) Adding N-(epoxypropyl)-N-phenyl-epoxyethane methylamine and 3-bromopropylene to tetrahydrofuran solvent, wherein the mass ratio of N-(epoxypropyl)-N-phenyl-epoxyethane methylamine to 3-bromopropylene is 1:0.82, stirring and mixing, and reflux heating at 86° C. for 3.5 h. After the reaction is completed, cooling, washing and drying to obtain intermediate 1;

[0053] (4) adding 12 parts by weight of the intermediate 1 to deionized water and stirring to dissolve, dissolving 6 parts by weight of chitosan in an acetic acid aqueous solution, wherein the mass fraction of the acetic acid aqueous solution is 3%, slowly dropping the aqueous solution of the intermediate 1 into the chitosan solution, reflux heating at 70° C. for 8 h, washing after the reaction is completed, and freeze-drying to obtain quaternized chitosan containing alkenyl groups;

[0054] (5) adding alkenyl quaternized chitosan to tetrahydrofuran solvent, stirring to dissolve, adding cysteine ​​and p-toluenesulfonic acid catalysts, wherein the mass ratio of alkenyl quaternized chitosan, cysteine, and p-toluenesulfonic acid is 1:0.7:0.028, reacting at 70° C. for 2.5 h, filtering, washing, and drying after the reaction to obtain intermediate 2;

[0055] (6) adding 8 parts by weight of the intermediate 2, 14 parts by weight of 4-vinyl imidazole, and 0.4 parts by weight of benzoin dimethyl ether photoinitiator to N,N-dimethylformamide solvent, irradiating with 365 nm ultraviolet light at 30° C. for 3 h, centrifuging, washing, and drying to obtain vinyl imidazole-modified cysteine;

[0056] (7) Adding a polypeptide with a sequence of AKGQRGPPGKR to an N,N-dimethylformamide solvent, stirring to dissolve, adding alkenyl imidazole-modified cysteine ​​and N,N-diisopropylethylamine catalyst, wherein the mass ratio of the polypeptide with a sequence of AKGQRGPPGKR, alkenyl imidazole-modified cysteine, and N,N-diisopropylethylamine is 1:0.9:0.06, passing nitrogen protection, reacting at 25°C for 3.5 hours, and after the reaction is completed, washing and drying to obtain an alkenyl imidazole-modified cysteine ​​modified polypeptide;

[0057] (8) Add ferulic acid and benzotriazole to an anhydrous ether solvent, stir to dissolve, cool to -3°C in an ice bath, slowly dropwise add a dicyclohexylcarbodiimide ether solution thereto, wherein the mass ratio of ferulic acid, benzotriazole, and dicyclohexylcarbodiimide is 1:0.87:0.07. After the dropwise addition is complete, keep the temperature for reaction for 3 hours, raise the temperature to 20°C, and continue the reaction for 14.5 hours. After the reaction is completed, filter, cool, and crystallize to obtain benzotriazole-activated ferulic acid;

[0058] (9) Add 10 parts by weight of benzotriazole-activated ferulic acid to N-methylpyrrolidone solvent, cool to 2°C in an ice bath, add 10 parts by weight of cysteine-modified polypeptide containing alkenyl imidazole and 0.3 parts by weight of triethylamine catalyst, and keep the reaction warm for 2.8 hours. After the reaction is completed, add acetone solvent, centrifuge at a speed of 9000 rpm for 9 minutes, wherein the temperature of the freeze-centrifuge is -12°C, remove the supernatant after standing, and freeze-dry the precipitate to obtain a polypeptide with antibacterial and antioxidant functions.

[0059] Comparative Example 1

[0060] This comparative example is different from Example 3 in that no alkenyl imidazole-modified cysteine ​​is added.

[0061] Comparative Example 2

[0062] The difference between this comparative example and Example 3 is that no benzotriazole-activated ferulic acid is added.

[0063] Using Staphylococcus aureus, Escherichia coli or Candida krusei as the test bacteria, 0.5 mL of the bacterial suspension of the test bacteria (concentration of 1×110 CFU / mL) was added to a sterile culture dish containing MH broth medium, and then the polypeptide sample with antibacterial and antioxidant functions was attached to the surface of the culture medium, and cultured at 35°C in a constant temperature incubator for 18 hours. After culture, the diameter of the inhibition zone was measured.

[0064] Table 1: Antimicrobial testing.

[0065]

[0066]

[0067] It can be seen from Table 1 that Examples 1-3 and Comparative Example 2 of the present invention have better antibacterial function than Comparative Example 1.

[0068] DPPH was dissolved in ethanol to prepare a solution with a concentration of 0.1 mmol / L. The polypeptide with antibacterial and antioxidant functions was added to the DPPH solution. After reacting at room temperature for 25 minutes, the change in absorbance was measured at 517 nm to calculate the DPPH free radical scavenging rate.

[0069] Ultrapure water was used to prepare 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution, mixed in a ratio of 1:1, allowed to stand at room temperature for 14 h, diluted with phosphate buffer solution to an absorbance of 0.7-0.8 at 734 nm to obtain ABTS free radical working solution, and polypeptides with antibacterial and antioxidant functions were added to the ABTS free radical working solution. After reacting at room temperature for 5 min, the absorbance change was measured at 734 nm to calculate the ABTS free radical scavenging rate.

[0070] Table 2: Free radical scavenging ability test.

[0071]

[0072]

[0073] It can be seen from Table 2 that Examples 1-3 of the present invention have better antioxidant function than Comparative Examples 1-2.

[0074] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing a polypeptide having antibacterial and antioxidant functions, characterized in that: The method for preparing the polypeptide having antibacterial and antioxidant functions comprises the following steps: (1) adding 12-16 parts by weight of a washed and chopped tuna bone sample to deionized water, stirring evenly, adding 6-9 parts by weight of alkaline protease, adjusting the pH to 9-11, performing enzymolysis at 35-45° C., and after the enzymolysis is completed, inactivating the enzyme at 95-100° C. to obtain a tuna bone enzymolysis solution; (2) adding anhydrous ethanol to 10-12 parts by weight of tuna bone hydrolysate, stirring evenly, and then standing for precipitation for 8-14 hours, filtering and rotary evaporation, and then separating by a tangential flow ultrafiltration system, collecting the filtrate and freeze-drying to obtain a polypeptide, and analyzing and identifying the amino acid sequence of the polypeptide by LC-MS, searching the fish collagen library for matching, and obtaining a polypeptide sequence of AKGQRGPPGKR; (3) Add N-(epoxypropyl)-N-phenyl-epoxyethane methylamine and 3-bromopropylene to tetrahydrofuran solvent, stir and mix, and reflux and heat at 82-90° C. for 3-4 hours. After the reaction is completed, cool, wash and dry to obtain intermediate 1; (4) adding 11-13 parts by weight of the intermediate 1 to deionized water, stirring to dissolve, dissolving 5-7 parts by weight of chitosan in an acetic acid aqueous solution, slowly dropping the aqueous solution of the intermediate 1 into the chitosan solution, heating under reflux at 65-75° C. for 7-9 hours, washing after the reaction, and freeze-drying to obtain alkenyl-containing quaternary ammonium chitosan; (5) adding quaternized chitosan containing alkenyl groups to tetrahydrofuran solvent, stirring and dissolving, adding cysteine ​​and p-toluenesulfonic acid catalyst thereto, reacting at 60-80° C. for 2-3 h, filtering, washing and drying after the reaction to obtain intermediate 2; (6) adding 7-10 parts by weight of the intermediate 2, 13-15 parts by weight of 4-vinyl imidazole, and 0.3-0.6 parts by weight of benzoin dimethyl ether photoinitiator to N,N-dimethylformamide solvent, irradiating with 365 nm ultraviolet light at 28-32° C., centrifuging after completion, washing and drying to obtain vinyl imidazole-modified cysteine; (7) Adding a polypeptide with a sequence of AKGQRGPPGKR to an N,N-dimethylformamide solvent, stirring to dissolve, adding alkenyl imidazole-modified cysteine ​​and N,N-diisopropylethylamine catalyst, passing nitrogen gas for protection, reacting at 20-30° C. for 2.5-4.5 hours, washing and drying after the reaction to obtain an alkenyl imidazole-modified cysteine ​​modified polypeptide; (8) Add ferulic acid and benzotriazole to an anhydrous ether solvent, stir to dissolve, cool to -5-0°C in an ice bath, slowly dropwise add a dicyclohexylcarbodiimide ether solution thereto, and after the addition is complete, keep the temperature to react for 2-4 hours, raise the temperature to 15-25°C, and continue the reaction for 14-15 hours. After the reaction is complete, filter, cool and crystallize to obtain benzotriazole-activated ferulic acid; (9) Add 8-12 parts by weight of benzotriazole-activated ferulic acid to N-methylpyrrolidone solvent, cool to 0-5°C in an ice bath, add 9-11 parts by weight of cysteine-modified polypeptide containing alkenyl imidazole and 0.2-0.4 parts by weight of triethylamine catalyst, and keep the reaction warm for 2.5-3 hours. After the reaction is completed, add acetone solvent, centrifuge at a speed of 8000-10000 rpm for 8-10 minutes, remove the supernatant after standing, and freeze-dry the precipitate to obtain a polypeptide with antibacterial and antioxidant functions.

2. The method for preparing a polypeptide having antibacterial and antioxidant functions according to claim 1, characterized in that: The enzyme inactivation time in step (1) is 8-12 minutes.

3. The method for preparing a polypeptide having antibacterial and antioxidant functions according to claim 1, characterized in that: The mass fraction of ethanol in step (2) is 70-85%.

4. The method for preparing a polypeptide having antibacterial and antioxidant functions according to claim 1, characterized in that: In the step (3), the mass ratio of N-(epoxypropyl)-N-phenyl-epoxyethylene methylamine to 3-bromopropylene is 1:0.8-0.

84.

5. The method for preparing a polypeptide having antibacterial and antioxidant functions according to claim 1, characterized in that: The mass fraction of the acetic acid aqueous solution in step (4) is 2-4%.

6. The method for preparing a polypeptide having antibacterial and antioxidant functions according to claim 1, characterized in that: In the step (5), the mass ratio of alkenyl quaternized chitosan, cysteine ​​and p-toluenesulfonic acid is 1:0.6-0.8:0.025-0.

03.

7. The method for preparing a polypeptide having antibacterial and antioxidant functions according to claim 1, characterized in that: The ultraviolet light irradiation time in step (6) is 2.5-3.5 hours.

8. The method for preparing a polypeptide having antibacterial and antioxidant functions according to claim 1, characterized in that: In the step (7), the mass ratio of the polypeptide having the sequence of AKGQRGPPGKR, the cysteine ​​modified with alkenyl imidazole, and N,N-diisopropylethylamine is 1:0.8-1:0.05-0.

07.

9. The method for preparing a polypeptide having antibacterial and antioxidant functions according to claim 1, characterized in that: In the step (8), the mass ratio of ferulic acid, benzotriazole and dicyclohexylcarbodiimide is 1:0.85-0.9:0.06-0.

08.

10. The method for preparing a polypeptide having antibacterial and antioxidant functions according to claim 1, characterized in that: The temperature of the refrigerated centrifugation in step (9) is -15 to -10°C.

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