A process for extracting antimicrobial peptide components from marine organisms

Abstract

The invention relates to the technical field of antimicrobial peptide extraction, and specifically discloses an extraction process for antimicrobial peptide components of marine organisms, comprising the following steps: step S1, defatting treatment; step S2, crude extraction; step S3, electric field modification; step S4, enzymatic hydrolysis; step S5, fine purification; in the technical scheme of the present invention, a buffer solution is added during the crude extraction process, and shear-assisted heat treatment is performed at the same time; an acid value regulating liquid and hydroxytyrosol are added during the electric field modification process; and functionalized silica gel is added during the fine purification process to adsorb the enzymatic hydrolyzate; through the synergistic effect of the above-mentioned steps, the purity and yield of the antimicrobial peptide components are improved.

Description

Technical Field

[0001] The present application relates to the technical field of antimicrobial peptide extraction, and more specifically, to a process for extracting antimicrobial peptide components from marine organisms. Background Art

[0002] Antimicrobial peptides are a class of polypeptide substances with a molecular weight of less than 7000, which have unique broad-spectrum antibacterial, anti-tumor and antiviral capabilities. Among them, insect antimicrobial peptides are immune factors produced by insects to resist adverse external factors. They have the characteristics of small molecular weight, high thermal stability, strong antibacterial properties, and no drug resistance in pathogenic bacteria. They are a class of good antimicrobial substances.

[0003] Large yellow croaker is a marine fish widely distributed in coastal and offshore waters with sandy and muddy bottoms. It is rich in protein and is an optimal raw material for extracting antimicrobial peptides.

[0004] At present, the commonly used antimicrobial peptide extraction methods include acid extraction, alkaline extraction and bioenzyme extraction to obtain crude extracts, and then use membrane separation technology to obtain high-purity antimicrobial peptides. Among them, the reaction conditions of the bioenzyme method are milder and the degree of hydrolysis is higher, which has little damage to amino acids and has been widely used in the crude extraction and processing of polypeptides. However, biological proteases are expensive, not only consuming a large amount of protease, but also taking a long time for enzymolysis and high production costs. In addition, the C18 stationary phase commonly used in existing membrane separation technology cannot remove impurity particles well due to its single separation mechanism, resulting in a decrease in the purity and yield of the final purified antimicrobial peptides. Summary of the invention

[0005] In order to solve the problems mentioned in the above background technology, the present application provides a process for extracting antimicrobial peptide components from marine organisms.

[0006] A process for extracting antimicrobial peptide components from marine organisms comprises the following steps:

[0007] Step S1, defatting: descaling and removing the internal organs of fresh large yellow croaker, cutting into pieces, washing with tap water for 3-5 times, and defatting to obtain defatted fish meat;

[0008] Step S2, crude extraction: adding defatted fish meat to a buffer solution, subjecting the defatted fish meat to a shearing treatment for 15-20 minutes and maintaining the temperature at 52-56° C., and ultrafiltration and centrifugation to obtain a crude protein extract, wherein the mass ratio of the buffer solution to the defatted fish meat is 20-30:1;

[0009] Step S3, electric field modification: adding hydroxytyrosol to the crude protein extract, then adding an acid value adjusting solution to adjust the pH value to 1-3, pumping the acid-adjusted crude protein extract to a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified solution, wherein the mass ratio of hydroxytyrosol to the crude protein extract is 0.06-0.1:25;

[0010] Step S4, enzymolysis: adding a pH regulator to the high-voltage pulse electric field modified solution, stirring evenly, and then adding trypsin, and digesting overnight at 36-38° C. to obtain an enzymolysis solution, wherein the mass ratio of the pH regulator, the high-voltage pulse electric field modified solution, and the trypsin is 0.07-0.08:20:6-8;

[0011] Step S5, fine purification: ultrasonically disperse the functionalized silica gel in the enzymatic hydrolysate, stir at room temperature for 2-3 hours, filter to obtain a polypeptide adsorbate, wash the polypeptide adsorbate with a desorption solution to obtain a polypeptide fine extract, and subject the polypeptide fine extract to adsorption-desalting and vacuum concentration to obtain a marine antimicrobial peptide component, wherein the mass ratio of the functionalized silica gel to the enzymatic hydrolysate is 2.2-2.4:1.

[0012] Preferably, in step S1, the defatting process includes: placing the chopped defatted fish meat in a 10-20% (w / v) sodium carbonate solution for 8-10 hours, with a material-liquid mass ratio of 1:20-30 and a temperature of 4-6°C, washing with deionized water until neutral, and then placing it in a 20-30% (v / v) isopropanol solution for 12-16 hours, with a material-liquid mass ratio of 1:20-40 and a temperature of 4-6°C, and washing with deionized water 3-5 times.

[0013] Preferably, in step S2, the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, dithiothreitol and sodium carbonate in a mass ratio of 62-74:18-22:0.6-1.0:0.04-0.06:2-4.

[0014] Preferably, in step S2, the molecular weight cutoff of the ultrafiltration membrane is 5 kDa.

[0015] Preferably, in step S3, the flow rate is set to 6-8 mL / min, the electric field strength is set to 15-18 kV / cm, and the number of pulses is set to 8 during the high-voltage pulse electric field treatment.

[0016] Preferably, in step S4, the pH adjuster is a 0.05-0.07 M ammonium carbonate aqueous solution.

[0017] Preferably, in step S5, the adsorption-desalting is to filter the polypeptide extract through a 150Da ultrafiltration membrane, and the desorption liquid is a sodium dihydrogen phosphate aqueous solution with a pH of 7.6-8.2.

[0018] Preferably, the method for preparing the functionalized silica gel comprises the following steps:

[0019] Step A1, adding silica gel to a hydrochloric acid solution, heating to 35-45°C, stirring for 8-10h, washing to neutrality, and vacuum drying to obtain acidified silica gel; ultrasonically dispersing the acidified silica gel in anhydrous toluene, adding triethylamine and 3-chloropropyltrimethoxysilane while stirring under nitrogen protection, heating to reflux, stirring for reaction for 18-24h, washing, and drying to obtain chlorine-substituted silica gel, wherein the mass ratio of silica gel to hydrochloric acid solution is 30-40:220-260, the mass fraction of hydrochloric acid solution is 20-30%, and the mass ratio of acidified silica gel, anhydrous toluene, triethylamine and 3-chloropropyltrimethoxysilane is 12-16:52-56:0.24-0.32:14-20. In the above reaction process, triethylamine is used as a catalyst, and the acidified silica gel and 3-chloropropyltrimethoxysilane undergo a condensation reaction to obtain chlorine-substituted silica gel;

[0020] Step A2, ultrasonically dispersing chlorine-substituted silica gel in anhydrous toluene, adding 4-trifluoromethyl-o-phenylenediamine under nitrogen protection, heating to reflux, stirring and reacting for 42-48 hours, washing and drying after the reaction, to obtain phenyl silica gel, wherein the mass ratio of chlorine-substituted silica gel, anhydrous toluene and 4-trifluoromethyl-o-phenylenediamine is 12-14:55-65:8.4-11.2, and during the above reaction, the active chlorine atoms on the chlorine-substituted silica gel and the nitrogen atoms on the 4-trifluoromethyl-o-phenylenediamine undergo nucleophilic substitution reaction to obtain phenyl silica gel;

[0021] Step A3, ultrasonically dispersing phenyl silica gel in anhydrous dichloromethane, adjusting the pH to 9-10, adding a mixed solution a of octadecenyl succinic anhydride and anhydrous dichloromethane dropwise while stirring, and controlling the dropping to be completed within 30 minutes. After the dropping is completed, the temperature is raised to 74-80° C., and the stirring reaction is continued for 6-10 hours. The pH is adjusted to neutral, and the mixture is rotary evaporated, washed, and dried to obtain carboxylated silica gel, wherein the mass ratio of phenyl silica gel, anhydrous dichloromethane and mixed solution a is 4-6:22-26:10, and the mass ratio of octadecenyl succinic anhydride to anhydrous dichloromethane in the mixed solution a is 0.8-1.2:10. In the above reaction process, under alkaline conditions, the amino groups on the phenyl silica gel undergo a ring-opening esterification reaction with the octadecenyl succinic anhydride to obtain carboxylated silica gel;

[0022] Step A4, under nitrogen protection, add carboxylated silica gel, hydroxypropyl end-capped polysiloxane and p-toluenesulfonic acid to anhydrous toluene, stir evenly, heat to 82-86°C, stir and react for 1-2h, and after the reaction, rotary evaporate, wash and dry to obtain functionalized silica gel, wherein the mass ratio of carboxylated silica gel, hydroxypropyl end-capped polysiloxane, p-toluenesulfonic acid and anhydrous toluene is 3-4:1.4-1.8:0.04-0.05:50-60. In the above reaction process, with anhydrous toluene as solvent and p-toluenesulfonic acid as catalyst, the carboxylated silica gel and hydroxypropyl end-capped polysiloxane undergo esterification reaction to obtain functionalized silica gel.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) In the technical solution of the present invention, a buffer solution is added during the crude extraction process. The buffer solution contains glycerol, sodium chloride and dithiothreitol. Glycerol can reduce the polarity of the solution, thereby preventing hydrophobic aggregation of proteins and improving the stability of proteins. Sodium chloride can increase the surface charge of protein molecules, thereby enhancing the interaction between protein molecules and water molecules and increasing the solubility of proteins. DTT can cleave disulfide bonds in protein molecules and make the structure of proteins looser, thereby increasing their solubility in aqueous solutions. At the same time, shear-assisted heat treatment is combined with moderate external force and heat provided by heat treatment, which improves the extraction efficiency of fish protein without destroying the structure of fish protein, so that the antimicrobial peptide components obtained subsequently have higher biological activity.

[0025] (2) In the technical solution of the present invention, an acid value regulating liquid and hydroxytyrosol are added during the electric field modification process. The acid value regulating liquid can not only reduce the activity of DTT and prevent DTT from possibly destroying the spatial structure of the protein and causing protein denaturation, but also promote the chemical reaction between hydroxytyrosol and DTT, thereby removing excess DTT. At the same time, in combination with the high-voltage pulse electric field treatment, the hydrogen bond interaction between fish protein molecules is destroyed, the structure unfolds, the protein aggregates are disaggregated and evenly dispersed in the solution, more enzyme action sites are exposed, the utilization rate of the enzyme is increased, and the enzymatic hydrolysis rate is improved.

[0026] (3) In the technical scheme of the present invention, in order to improve the extraction rate of the antimicrobial peptide component and reduce the desalting performance at the same time, in the fine purification process, the present application first uses functionalized silica gel to adsorb the polypeptide component in the enzymatic hydrolyzate, then washes it with a desorption solution, and finally, desalts it to obtain the antimicrobial peptide component. The functionalized silica gel contains flexible long-chain alkanes, benzene rings and trifluoromethyl groups. The flexible long-chain alkanes are hydrophobic and can entangle with the long-chain molecules on the polypeptide chain, thereby improving the extraction rate of the antimicrobial peptide component. The presence of the benzene ring and the low-polarity trifluoromethyl group reduces the polarity of the prepared functionalized silica gel, thereby further improving the extraction rate of the antimicrobial peptide component. DETAILED DESCRIPTION

[0027] In order to make the implementation methods of the present application easier to understand, the present application will be described in detail below in conjunction with specific examples. These examples are only for illustration and are not limited to the scope of application of the present application.

[0028] The contents of the main raw materials and their components used in the examples and comparative examples are as follows:

[0029] The silica gel is C18 chromatographic filler produced by Rizhao Kepno New Materials Co., Ltd., with a CAS number of 112926-00-8 and an average particle size of 40-60µm. The hydroxypropyl-terminated polysiloxane is produced by Shanghai MacLean Biochemical Technology Co., Ltd., with a CAS number of 104780-66-7.

[0030] The present application is further described in detail below in conjunction with embodiments and comparative examples.

[0031] Preparation Examples 1-3 and Comparative Preparation Examples 1-3 provide methods for preparing functionalized silica gel.

[0032] Preparation Example 1

[0033] This preparation example provides a method for preparing functionalized silica gel, comprising the following steps:

[0034] Step A1, adding silica gel to a 20% hydrochloric acid solution, heating to 35°C, controlling the rotation speed to 400rpm, stirring for 8h, washing with deionized water until neutral, and vacuum drying at 50°C to constant weight to obtain acidified silica gel; ultrasonically dispersing the acidified silica gel in anhydrous toluene until uniform, controlling the ultrasonic frequency to 40kHz, the ultrasonic power to 600w, and ultrasonication for 30min, under nitrogen protection, controlling the rotation speed to 500rpm, adding triethylamine and 3-chloropropyltrimethoxysilane while stirring, heating to reflux, stirring and reacting for 18h, washing with anhydrous ethanol and deionized water three times in sequence, and drying at 50°C to constant weight to obtain chlorine-substituted silica gel, wherein the mass ratio of silica gel to the 20% hydrochloric acid solution is 30:220, and the mass ratio of acidified silica gel, anhydrous toluene, triethylamine and 3-chloropropyltrimethoxysilane is 12:52:0.24:14;

[0035] Step A2, ultrasonically dispersing chlorine-substituted silica gel in anhydrous toluene, controlling the ultrasonic frequency to 35kHz, the ultrasonic power to 550w, ultrasonicating for 20min, adding 4-trifluoromethyl-o-phenylenediamine under nitrogen protection, heating to reflux, stirring and reacting for 42h, after the reaction, washing with anhydrous ethanol and deionized water for 3 times in sequence, drying at 55°C to constant weight, to obtain phenyl silica gel, wherein the mass ratio of chlorine-substituted silica gel, anhydrous toluene and 4-trifluoromethyl-o-phenylenediamine is 12:55:8.4;

[0036] Step A3, ultrasonically dispersing phenyl silica gel in anhydrous dichloromethane, controlling the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, ultrasonication for 25min, adjusting the pH to 9 with 0.6M sodium hydroxide aqueous solution, controlling the rotation speed to 600rpm, maintaining the rotation speed unchanged, adding a mixed solution a of octadecenyl succinic anhydride and anhydrous dichloromethane dropwise while stirring, controlling the dripping to be completed within 30min, after the dripping is completed, heating to 74°C, continuing to stir and react for 6h, adjusting the pH to neutral with 0.6M hydrochloric acid, controlling the rotary evaporation temperature to 32°C to remove anhydrous dichloromethane, washing with anhydrous ethanol and deionized water three times in sequence, and then drying at 60°C to constant weight to obtain carboxylated silica gel, wherein the mass ratio of phenyl silica gel, anhydrous dichloromethane and mixed solution a is 4:22:10, and the mass ratio of octadecenyl succinic anhydride and anhydrous dichloromethane in the mixed solution a is 0.8:10;

[0037] Step A4, under nitrogen protection, add carboxylated silica gel, hydroxypropyl end-capped polysiloxane and p-toluenesulfonic acid to anhydrous toluene, control the rotation speed to 700 rpm, stir for 30 minutes until uniform, heat to 82°C, stir and react for 1 hour. After the reaction, control the rotary evaporation temperature to 62°C to remove anhydrous toluene, wash with anhydrous ethanol and deionized water for 3 times in sequence, and dry at 65°C to constant weight to obtain functionalized silica gel, wherein the mass ratio of carboxylated silica gel, hydroxypropyl end-capped polysiloxane, p-toluenesulfonic acid and anhydrous toluene is 3:1.4:0.04:50.

[0038] Preparation Example 2

[0039] This preparation example provides a method for preparing functionalized silica gel, comprising the following steps:

[0040] Step A1, adding silica gel to a 25% hydrochloric acid solution, controlling the speed to 500 rpm, heating to 40°C, stirring for 9 hours, washing to neutrality, and vacuum drying at 55°C to obtain acidified silica gel; ultrasonically dispersing the acidified silica gel in anhydrous toluene until uniform, controlling the ultrasonic frequency to 35 kHz, the ultrasonic power to 550 w, and ultrasonication for 35 minutes, under nitrogen protection, controlling the speed to 600 rpm, adding triethylamine and 3-chloropropyltrimethoxysilane while stirring, heating to reflux, stirring and reacting for 21 hours, washing with anhydrous ethanol and deionized water for 4 times in sequence, and drying at 55°C to constant weight to obtain chlorine-substituted silica gel, wherein the mass ratio of silica gel to the 25% hydrochloric acid solution is 35:240, and the mass ratio of acidified silica gel, anhydrous toluene, triethylamine and 3-chloropropyltrimethoxysilane is 14:54:0.28:17;

[0041] Step A2, ultrasonically dispersing chlorine-substituted silica gel in anhydrous toluene, controlling the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, and ultrasonicating for 30min, under nitrogen protection, adding 4-trifluoromethyl-o-phenylenediamine, heating to reflux, controlling the rotation speed to 700rpm, stirring and reacting for 45h, after the reaction is completed, washing with anhydrous ethanol and deionized water for 4 times in sequence, and drying at 60°C to constant weight to obtain phenyl silica gel, wherein the mass ratio of chlorine-substituted silica gel, anhydrous toluene and 4-trifluoromethyl-o-phenylenediamine is 13:60:9.8;

[0042] Step A3, ultrasonically dispersing phenyl silica gel in anhydrous dichloromethane, controlling the ultrasonic frequency to 35kHz, the ultrasonic power to 550w, ultrasonication for 30min, adjusting the pH to 9.5 with 0.8M sodium hydroxide aqueous solution, controlling the rotation speed to 650rpm, adding a mixed solution a of octadecenyl succinic anhydride and anhydrous dichloromethane dropwise while stirring, and controlling the dripping to be completed within 30min. After the dripping is completed, the temperature is raised to 76°C, and the stirring reaction is continued for 8h. The pH is adjusted to neutral with 0.8M hydrochloric acid, and the rotary evaporation temperature is controlled to be 34°C to remove the anhydrous dichloromethane, and the anhydrous dichloromethane is washed with anhydrous ethanol and deionized water for 4 times in sequence, and dried at 65°C to constant weight to obtain carboxylated silica gel, wherein the mass ratio of phenyl silica gel, anhydrous dichloromethane and mixed solution a is 5:24:10, and the mass ratio of octadecenyl succinic anhydride and anhydrous dichloromethane in the mixed solution a is 1.0:10;

[0043] Step A4, under nitrogen protection, add carboxylated silica gel, hydroxypropyl-terminated polysiloxane and p-toluenesulfonic acid to anhydrous toluene, control the rotation speed to 750 rpm, stir for 25 min until uniform, heat to 84°C, stir and react for 1.5 h. After the reaction, control the rotary evaporation temperature to 64°C to remove anhydrous toluene, wash with anhydrous ethanol and deionized water for 4 times in sequence, and dry at 70°C to constant weight to obtain functionalized silica gel, wherein the mass ratio of carboxylated silica gel, hydroxypropyl-terminated polysiloxane, p-toluenesulfonic acid and anhydrous toluene is 3.5:1.6:0.045:55.

[0044] Preparation Example 3

[0045] This preparation example provides a method for preparing functionalized silica gel, comprising the following steps:

[0046] Step A1, adding silica gel to a 30% hydrochloric acid solution, heating to 45°C, stirring for 10 hours, washing to neutrality, and vacuum drying at 60°C to obtain acidified silica gel; ultrasonically dispersing the acidified silica gel in anhydrous toluene until uniform, controlling the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, and the ultrasonic for 40 minutes, under nitrogen protection, controlling the speed to 700rpm, adding triethylamine and 3-chloropropyltrimethoxysilane while stirring, heating to reflux, stirring and reacting for 24 hours, washing with anhydrous ethanol and deionized water for 5 times in sequence, and drying at 60°C to constant weight to obtain chlorine-substituted silica gel, wherein the mass ratio of silica gel to the 30% hydrochloric acid solution is 40:260, and the mass ratio of acidified silica gel, anhydrous toluene, triethylamine and 3-chloropropyltrimethoxysilane is 16:56:0.32:20;

[0047] Step A2, ultrasonically dispersing chlorine-substituted silica gel in anhydrous toluene, controlling the ultrasonic frequency to 40kHz, the ultrasonic power to 600w, and the ultrasonic treatment for 35min, adding 4-trifluoromethyl-o-phenylenediamine under nitrogen protection, raising the temperature to reflux, stirring and reacting for 48h, and after the reaction, washing with anhydrous ethanol and deionized water for 5 times in sequence, and drying at 60°C to constant weight to obtain phenyl silica gel, wherein the mass ratio of chlorine-substituted silica gel, anhydrous toluene and 4-trifluoromethyl-o-phenylenediamine is 14:65:11.2;

[0048] Step A3, ultrasonically dispersing phenyl silica gel in anhydrous dichloromethane, controlling the ultrasonic frequency to 35kHz, the ultrasonic power to 550w, ultrasonication for 40min, adjusting the pH to 10 with 1.0M sodium hydroxide aqueous solution, adding a mixed solution a of octadecenyl succinic anhydride and anhydrous dichloromethane dropwise while stirring, and controlling the dripping to be completed within 30min. After the dripping is completed, the temperature is raised to 80°C, and the stirring reaction is continued for 10h, and the pH is adjusted to neutral with 1.0M hydrochloric acid, and the rotary evaporation temperature is controlled to be 36°C to remove the anhydrous dichloromethane, and the mixture is washed with anhydrous ethanol and deionized water for 5 times in sequence, and dried at 70°C to constant weight to obtain carboxylated silica gel, wherein the mass ratio of phenyl silica gel, anhydrous dichloromethane and mixed solution a is 6:26:10, and the mass ratio of octadecenyl succinic anhydride and anhydrous dichloromethane in the mixed solution a is 1.2:10;

[0049] Step A4, under nitrogen protection, add carboxylated silica gel, hydroxypropyl-terminated polysiloxane and p-toluenesulfonic acid to anhydrous toluene, stir evenly, heat to 86°C, stir and react for 2 hours. After the reaction, control the rotary evaporation temperature to 66°C to remove anhydrous toluene, wash with anhydrous ethanol and deionized water for 5 times in sequence, and dry at 75°C to constant weight to obtain functionalized silica gel, wherein the mass ratio of carboxylated silica gel, hydroxypropyl-terminated polysiloxane, p-toluenesulfonic acid and anhydrous toluene is 4:1.8:0.05:60.

[0050] Comparative Preparation Example 1

[0051] This comparative preparation example provides a method for preparing functionalized silica gel, comprising the following steps:

[0052] Step A1, adding silica gel to a 20% hydrochloric acid solution, heating to 35°C, controlling the rotation speed to 400rpm, stirring for 8h, washing with deionized water until neutral, and vacuum drying at 50°C to constant weight to obtain acidified silica gel; ultrasonically dispersing the acidified silica gel in anhydrous toluene until uniform, controlling the ultrasonic frequency to 40kHz, the ultrasonic power to 600w, and ultrasonication for 30min, under nitrogen protection, controlling the rotation speed to 500rpm, adding triethylamine and 3-chloropropyltrimethoxysilane while stirring, heating to reflux, stirring and reacting for 18h, washing with anhydrous ethanol and deionized water three times in sequence, and drying at 50°C to constant weight to obtain chlorine-substituted silica gel, wherein the mass ratio of silica gel to the 20% hydrochloric acid solution is 30:220, and the mass ratio of acidified silica gel, anhydrous toluene, triethylamine and 3-chloropropyltrimethoxysilane is 12:52:0.24:14;

[0053] Step A2, ultrasonically dispersing chlorine-substituted silica gel in anhydrous toluene, controlling the ultrasonic frequency to 35 kHz, the ultrasonic power to 550 w, ultrasonicating for 20 min, adding 1,1,1-trifluoropropane-2-amine under nitrogen protection, heating to reflux, stirring and reacting for 42 h, after the reaction is completed, washing with anhydrous ethanol and deionized water for 3 times in sequence, and drying at 55° C. to constant weight to obtain phenyl silica gel, wherein the mass ratio of chlorine-substituted silica gel, anhydrous toluene and 1,1,1-trifluoropropane-2-amine is 12:55:8.4;

[0054] Step A3, ultrasonically dispersing phenyl silica gel in anhydrous dichloromethane, controlling the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, ultrasonication for 25min, adjusting the pH to 9 with 0.6M sodium hydroxide aqueous solution, controlling the rotation speed to 600rpm, maintaining the rotation speed unchanged, adding a mixed solution a of octadecenyl succinic anhydride and anhydrous dichloromethane dropwise while stirring, controlling the dripping to be completed within 30min, after the dripping is completed, heating to 74°C, continuing to stir and react for 6h, adjusting the pH to neutral with 0.6M hydrochloric acid, controlling the rotary evaporation temperature to 32°C to remove anhydrous dichloromethane, washing with anhydrous ethanol and deionized water three times in sequence, and then drying at 60°C to constant weight to obtain carboxylated silica gel, wherein the mass ratio of phenyl silica gel, anhydrous dichloromethane and mixed solution a is 4:22:10, and the mass ratio of octadecenyl succinic anhydride and anhydrous dichloromethane in the mixed solution a is 0.8:10;

[0055] Step A4, under nitrogen protection, add carboxylated silica gel, hydroxypropyl end-capped polysiloxane and p-toluenesulfonic acid to anhydrous toluene, control the rotation speed to 700 rpm, stir for 30 minutes until uniform, heat to 82°C, stir and react for 1 hour. After the reaction, control the rotary evaporation temperature to 62°C to remove anhydrous toluene, wash with anhydrous ethanol and deionized water for 3 times in sequence, and dry at 65°C to constant weight to obtain functionalized silica gel, wherein the mass ratio of carboxylated silica gel, hydroxypropyl end-capped polysiloxane, p-toluenesulfonic acid and anhydrous toluene is 3:1.4:0.04:50.

[0056] Comparative Preparation Example 2

[0057] This comparative preparation example provides a method for preparing functionalized silica gel, comprising the following steps:

[0058] Step A1, adding silica gel to a 20% hydrochloric acid solution, heating to 35°C, controlling the rotation speed to 400rpm, stirring for 8h, washing with deionized water until neutral, and vacuum drying at 50°C to constant weight to obtain acidified silica gel; ultrasonically dispersing the acidified silica gel in anhydrous toluene until uniform, controlling the ultrasonic frequency to 40kHz, the ultrasonic power to 600w, and ultrasonication for 30min, under nitrogen protection, controlling the rotation speed to 500rpm, adding triethylamine and 3-chloropropyltrimethoxysilane while stirring, heating to reflux, stirring and reacting for 18h, washing with anhydrous ethanol and deionized water three times in sequence, and drying at 50°C to constant weight to obtain chlorine-substituted silica gel, wherein the mass ratio of silica gel to the 20% hydrochloric acid solution is 30:220, and the mass ratio of acidified silica gel, anhydrous toluene, triethylamine and 3-chloropropyltrimethoxysilane is 12:52:0.24:14;

[0059] Step A2, ultrasonically dispersing chlorine-substituted silica gel in anhydrous toluene, controlling the ultrasonic frequency to 35kHz, the ultrasonic power to 550w, ultrasonicating for 20min, adding o-phenylenediamine under nitrogen protection, heating to reflux, stirring and reacting for 42h, after the reaction, washing with anhydrous ethanol and deionized water for 3 times in sequence, drying at 55°C to constant weight, to obtain phenyl silica gel, wherein the mass ratio of chlorine-substituted silica gel, anhydrous toluene and o-phenylenediamine is 12:55:8.4;

[0060] Step A3, ultrasonically dispersing phenyl silica gel in anhydrous dichloromethane, controlling the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, ultrasonication for 25min, adjusting the pH to 9 with 0.6M sodium hydroxide aqueous solution, controlling the rotation speed to 600rpm, maintaining the rotation speed unchanged, adding a mixed solution a of octadecenyl succinic anhydride and anhydrous dichloromethane dropwise while stirring, controlling the dripping to be completed within 30min, after the dripping is completed, heating to 74°C, continuing to stir and react for 6h, adjusting the pH to neutral with 0.6M hydrochloric acid, controlling the rotary evaporation temperature to 32°C to remove anhydrous dichloromethane, washing with anhydrous ethanol and deionized water three times in sequence, and then drying at 60°C to constant weight to obtain carboxylated silica gel, wherein the mass ratio of phenyl silica gel, anhydrous dichloromethane and mixed solution a is 4:22:10, and the mass ratio of octadecenyl succinic anhydride and anhydrous dichloromethane in the mixed solution a is 0.8:10;

[0061] Step A4, under nitrogen protection, add carboxylated silica gel, hydroxypropyl end-capped polysiloxane and p-toluenesulfonic acid to anhydrous toluene, control the rotation speed to 700 rpm, stir for 30 minutes until uniform, heat to 82°C, stir and react for 1 hour. After the reaction, control the rotary evaporation temperature to 62°C to remove anhydrous toluene, wash with anhydrous ethanol and deionized water for 3 times in sequence, and dry at 65°C to constant weight to obtain functionalized silica gel, wherein the mass ratio of carboxylated silica gel, hydroxypropyl end-capped polysiloxane, p-toluenesulfonic acid and anhydrous toluene is 3:1.4:0.04:50.

[0062] Comparative Preparation Example 3

[0063] This comparative preparation example provides a method for preparing functionalized silica gel, comprising the following steps:

[0064] Step A1, adding silica gel to a 20% hydrochloric acid solution, heating to 35°C, controlling the rotation speed to 400rpm, stirring for 8h, washing with deionized water until neutral, and vacuum drying at 50°C to constant weight to obtain acidified silica gel; ultrasonically dispersing the acidified silica gel in anhydrous toluene until uniform, controlling the ultrasonic frequency to 40kHz, the ultrasonic power to 600w, and ultrasonication for 30min, under nitrogen protection, controlling the rotation speed to 500rpm, adding triethylamine and 3-chloropropyltrimethoxysilane while stirring, heating to reflux, stirring and reacting for 18h, washing with anhydrous ethanol and deionized water three times in sequence, and drying at 50°C to constant weight to obtain chlorine-substituted silica gel, wherein the mass ratio of silica gel to the 20% hydrochloric acid solution is 30:220, and the mass ratio of acidified silica gel, anhydrous toluene, triethylamine and 3-chloropropyltrimethoxysilane is 12:52:0.24:14;

[0065] Step A2, ultrasonically dispersing chlorine-substituted silica gel in anhydrous toluene, controlling the ultrasonic frequency to 35kHz, the ultrasonic power to 550w, ultrasonicating for 20min, adding 4-trifluoromethyl-o-phenylenediamine under nitrogen protection, heating to reflux, stirring and reacting for 42h, after the reaction, washing with anhydrous ethanol and deionized water for 3 times in sequence, drying at 55°C to constant weight, to obtain phenyl silica gel, wherein the mass ratio of chlorine-substituted silica gel, anhydrous toluene and 4-trifluoromethyl-o-phenylenediamine is 12:55:8.4;

[0066] Step A3, ultrasonically disperse phenyl silica gel in anhydrous dichloromethane, control the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, and ultrasonicate for 25min, adjust the pH to 9 with 0.6M sodium hydroxide aqueous solution, control the rotation speed to 600rpm, maintain the rotation speed unchanged, and drip the mixed solution a of allyl succinic anhydride and anhydrous dichloromethane while stirring, and control the dripping to be completed within 30min. After the dripping is completed, the temperature is raised to 74°C, and the stirring reaction is continued for 6h, and the pH is adjusted to neutral with 0.6M hydrochloric acid, and the rotary evaporation temperature is controlled to be 32°C to remove the anhydrous dichloromethane, and the mixture is washed with anhydrous ethanol and deionized water for 3 times in sequence, and then dried at 60°C to constant weight to obtain carboxylated silica gel, wherein the mass ratio of phenyl silica gel, anhydrous dichloromethane and mixed solution a is 4:22:10, and the mass ratio of allyl succinic anhydride and anhydrous dichloromethane in the mixed solution a is 0.8:10;

[0067] Step A4, under nitrogen protection, add carboxylated silica gel, hydroxypropyl end-capped polysiloxane and p-toluenesulfonic acid to anhydrous toluene, control the rotation speed to 700 rpm, stir for 30 minutes until uniform, heat to 82°C, stir and react for 1 hour. After the reaction, control the rotary evaporation temperature to 62°C to remove anhydrous toluene, wash with anhydrous ethanol and deionized water for 3 times in sequence, and dry at 65°C to constant weight to obtain functionalized silica gel, wherein the mass ratio of carboxylated silica gel, hydroxypropyl end-capped polysiloxane, p-toluenesulfonic acid and anhydrous toluene is 3:1.4:0.04:50.

[0068] Examples 1-3 and Comparative Examples 1-5 provide a process for extracting antimicrobial peptide components from marine organisms. Example 1

[0069] This embodiment provides a process for extracting antimicrobial peptide components from marine organisms, comprising the following steps:

[0070] Step S1, defatting treatment: after descaling and removing the internal organs of fresh large yellow croaker, chopping it into pieces, washing it with tap water for 3 times, and defatting it to obtain defatted fish meat, wherein the defatting treatment process is specifically as follows: placing the chopped defatted fish meat in a 10% (w / v) sodium carbonate solution for 8 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, washing it with deionized water until it is neutral, and then placing it in a 20% (v / v) isopropanol solution for 12 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, and washing it with deionized water for 3 times;

[0071] Step S2, crude extraction: adding defatted fish meat to a buffer solution, controlling the shear rate to be 1000 rpm, treating for 15 min until the defatted fish meat is evenly dissolved, and maintaining the temperature to be 52° C., passing through an ultrafiltration membrane with a molecular weight cutoff of 5 kD, and then centrifuging to obtain a crude protein extract, wherein the mass ratio of the buffer solution to the defatted fish meat is 20:1, and the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, dithiothreitol and sodium carbonate in a mass ratio of 62:18:0.6:0.04:2;

[0072] Step S3, electric field modification: adding hydroxytyrosol to the crude protein extract, stirring for 25 min at a controlled speed of 700 rpm until uniform, then adding 1 M acetic acid aqueous solution to adjust the pH value to 1, pumping the acid-adjusted crude protein extract to a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified liquid, wherein the mass ratio of hydroxytyrosol to the crude protein extract is 0.06:25, and the flow rate is set to 6 mL / min, the electric field strength is 15 kV / cm, and the number of pulses is 8 during the high-voltage pulse electric field treatment;

[0073] Step S4, enzymolysis: adding 0.05M ammonium carbonate aqueous solution to the high-voltage pulse electric field modified solution, stirring for 18 minutes at a speed of 800 rpm until uniform, then adding trypsin, and digesting at 36°C overnight to obtain an enzymolysis solution, wherein the mass ratio of pH regulator, high-voltage pulse electric field modified solution and trypsin is 0.07:20:6;

[0074] Step S5, fine purification: ultrasonically disperse the functionalized silica gel prepared in Preparation Example 1 in the enzymatic hydrolysate, control the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, and the ultrasonic wave for 25min. Stir for 2h at room temperature and control the speed to 500rpm. Filter to obtain a polypeptide adsorbate. Wash the polypeptide adsorbate with a sodium hydrogen phosphate aqueous solution at pH 7.6 for 4 times to obtain a polypeptide fine extract. Filter the polypeptide fine extract with a 150Da ultrafiltration membrane, and then vacuum concentrate to dryness. Control the vacuum concentration multiple to 40 times and the vacuum degree to 11Pa to obtain a marine antimicrobial peptide component, wherein the mass ratio of the functionalized silica gel to the enzymatic hydrolysate is 2.2:1. Example 2

[0075] This embodiment provides a process for extracting antimicrobial peptide components from marine organisms, comprising the following steps:

[0076] Step S1, defatting treatment: after descaling and removing the internal organs of fresh large yellow croaker, chopping it into pieces, washing it with tap water for 4 times, and defatting it to obtain defatted fish meat, wherein the defatting treatment process is specifically as follows: placing the chopped defatted fish meat in a 15% (w / v) sodium carbonate solution for 9 hours, the material-liquid mass ratio is 1:25, the temperature is 5°C, washing it with deionized water until it is neutral, and then placing it in a 25% (v / v) isopropanol solution for 14 hours, the material-liquid mass ratio is 1:30, the temperature is 5°C, and washing it with deionized water 4 times;

[0077] Step S2, crude extraction: adding defatted fish meat to a buffer solution, controlling the shear rate to be 1100 rpm, treating for 17 minutes until the defatted fish meat is evenly dissolved, and maintaining the temperature to be 54° C., passing through an ultrafiltration membrane with a molecular weight cutoff of 5 kD, and then centrifuging to obtain a crude protein extract, wherein the mass ratio of the buffer solution to the defatted fish meat is 25:1, and the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, dithiothreitol and sodium carbonate in a mass ratio of 68:20:0.8:0.05:3;

[0078] Step S3, electric field modification: adding hydroxytyrosol to the crude protein extract, stirring for 20 min at a controlled speed of 750 rpm until uniform, then adding 1.5 M acetic acid aqueous solution to adjust the pH value to 2, pumping the acid-adjusted crude protein extract to a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified liquid, wherein the mass ratio of hydroxytyrosol to the crude protein extract is 0.08:25, and the flow rate is set to 7 mL / min, the electric field strength is 16 kV / cm, and the number of pulses is 8 during the high-voltage pulse electric field treatment;

[0079] Step S4, enzymolysis: adding 0.06M ammonium carbonate aqueous solution to the high-voltage pulse electric field modified solution, stirring for 15 minutes at a speed of 900 rpm until uniform, then adding trypsin, and digesting overnight at 37°C to obtain an enzymolysis solution, wherein the mass ratio of pH regulator, high-voltage pulse electric field modified solution and trypsin is 0.075:20:7;

[0080] Step S5, fine purification: ultrasonically disperse the functionalized silica gel prepared in Preparation Example 2 in the enzymatic hydrolyzate, control the rotation speed to 600 rpm at room temperature and stir for 2.5 hours, filter to obtain a polypeptide adsorbate, wash the polypeptide adsorbate with a sodium dihydrogen phosphate aqueous solution at pH 7.9 for 5 times to obtain a polypeptide extract, filter the polypeptide extract with a 150Da ultrafiltration membrane, and then vacuum concentrate to dryness, control the vacuum concentration multiple to 45 times, and the cold vacuum degree to 12Pa to obtain a marine antimicrobial peptide component, wherein the mass ratio of the functionalized silica gel to the enzymatic hydrolyzate is 2.3:1. Example 3

[0081] This embodiment provides a process for extracting antimicrobial peptide components from marine organisms, comprising the following steps:

[0082] Step S1, defatting treatment: after descaling and removing the internal organs of fresh large yellow croaker, chopping it into pieces, washing it with tap water for 5 times, and defatting it to obtain defatted fish meat, wherein the defatting treatment process is specifically as follows: placing the chopped defatted fish meat in a 20% (w / v) sodium carbonate solution for 10 hours, the material-liquid mass ratio is 1:30, the temperature is 6°C, washing it with deionized water until it is neutral, and then placing it in a 30% (v / v) isopropanol solution for 16 hours, the material-liquid mass ratio is 1:40, the temperature is 6°C, and washing it with deionized water for 5 times;

[0083] Step S2, crude extraction: adding defatted fish meat to a buffer solution, controlling the shear rate to 1200 rpm, for 20 min until the defatted fish meat is evenly dissolved, and maintaining the temperature to 56° C., passing through an ultrafiltration membrane with a molecular weight cutoff of 5 kD, and then centrifuging to obtain a crude protein extract, wherein the mass ratio of the buffer solution to the defatted fish meat is 30:1, and the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, dithiothreitol and sodium carbonate in a mass ratio of 74:22:1.0:0.06:4;

[0084] Step S3, electric field modification: adding hydroxytyrosol to the crude protein extract, stirring for 15 min at a controlled speed of 800 rpm until uniform, then adding 2M acetic acid aqueous solution to adjust the pH value to 3, pumping the acid-adjusted crude protein extract to a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified liquid, wherein the mass ratio of hydroxytyrosol to the crude protein extract is 0.1:25, and the flow rate is set to 8 mL / min, the electric field strength is 18 kV / cm, and the number of pulses is 8 during the high-voltage pulse electric field treatment;

[0085] Step S4, enzymolysis: adding 0.07M ammonium carbonate aqueous solution to the high-voltage pulse electric field modified solution, stirring for 12 minutes at a speed of 1000 rpm until uniform, then adding trypsin, and digesting overnight at 38°C to obtain an enzymolysis solution, wherein the mass ratio of pH regulator, high-voltage pulse electric field modified solution and trypsin is 0.08:20:8;

[0086] Step S5, fine purification: ultrasonically disperse the functionalized silica gel prepared in Preparation Example 3 in the enzymatic hydrolyzate, control the rotation speed to 700 rpm at room temperature and stir for 3 hours, filter to obtain a polypeptide adsorbate, wash the polypeptide adsorbate with a desorption solution for 6 times to obtain a polypeptide fine extract, filter the polypeptide fine extract with a 150Da ultrafiltration membrane, and then vacuum concentrate to dryness, control the vacuum concentration multiple to 50 times, and the vacuum degree to 13Pa to obtain a marine antimicrobial peptide component, wherein the mass ratio of the functionalized silica gel to the enzymatic hydrolyzate is 2.4:1.

[0087] Comparative Example 1

[0088] This comparative example provides a process for extracting antimicrobial peptide components from marine organisms, comprising the following steps:

[0089] Step S1, defatting treatment: after descaling and removing the internal organs of fresh large yellow croaker, chopping it into pieces, washing it with tap water for 3 times, and defatting it to obtain defatted fish meat, wherein the defatting treatment process is specifically as follows: placing the chopped defatted fish meat in a 10% (w / v) sodium carbonate solution for 8 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, washing it with deionized water until it is neutral, and then placing it in a 20% (v / v) isopropanol solution for 12 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, and washing it with deionized water for 3 times;

[0090] Step S2, crude extraction: adding defatted fish meat to a buffer solution, controlling the shear rate to 100 rpm, treating for 15 min until the defatted fish meat is evenly dissolved, and maintaining the temperature to 52° C., passing through an ultrafiltration membrane with a molecular weight cutoff of 5 kD, and then centrifuging to obtain a crude protein extract, wherein the mass ratio of the buffer solution to the defatted fish meat is 20:1, and the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, dithiothreitol and sodium carbonate in a mass ratio of 62:18:0.6:0.04:2;

[0091] Step S3, electric field modification: adding hydroxytyrosol to the crude protein extract, stirring for 25 min at a controlled speed of 700 rpm until uniform, then adding 1 M acetic acid aqueous solution to adjust the pH value to 1, pumping the acid-adjusted crude protein extract to a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified liquid, wherein the mass ratio of hydroxytyrosol to the crude protein extract is 0.06:25, and the flow rate is set to 6 mL / min, the electric field strength is 15 kV / cm, and the number of pulses is 8 during the high-voltage pulse electric field treatment;

[0092] Step S4, enzymolysis: adding 0.05M ammonium carbonate aqueous solution to the high-voltage pulse electric field modified solution, stirring for 18 minutes at a speed of 800 rpm until uniform, then adding trypsin, and digesting at 36°C overnight to obtain an enzymolysis solution, wherein the mass ratio of pH regulator, high-voltage pulse electric field modified solution and trypsin is 0.07:20:6;

[0093] Step S5, fine purification: ultrasonically disperse the functionalized silica gel prepared in comparative preparation example 1 in the enzymatic hydrolyzate, stir for 2 hours at room temperature with a control speed of 500 rpm, filter to obtain a polypeptide adsorbate, wash the polypeptide adsorbate with a sodium hydrogen phosphate aqueous solution at pH 7.6 for 4 times to obtain a polypeptide extract, filter the polypeptide extract with a 150Da ultrafiltration membrane, and then vacuum concentrate to dryness, control the vacuum concentration multiple to 40 times, and the vacuum degree to 11Pa to obtain a marine antimicrobial peptide component, wherein the mass ratio of the functionalized silica gel to the enzymatic hydrolyzate is 2.2:1.

[0094] Comparative Example 2

[0095] This comparative example provides a process for extracting antimicrobial peptide components from marine organisms, comprising the following steps:

[0096] Step S1, defatting treatment: after descaling and removing the internal organs of fresh large yellow croaker, chopping it into pieces, washing it with tap water for 3 times, and defatting it to obtain defatted fish meat, wherein the defatting treatment process is specifically as follows: placing the chopped defatted fish meat in a 10% (w / v) sodium carbonate solution for 8 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, washing it with deionized water until it is neutral, and then placing it in a 20% (v / v) isopropanol solution for 12 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, and washing it with deionized water for 3 times;

[0097] Step S2, crude extraction: adding defatted fish meat to a buffer solution, controlling the shear rate to be 100 rpm, treating for 15 minutes until the defatted fish meat is evenly dissolved, and maintaining the temperature to be 52° C., passing through an ultrafiltration membrane with a molecular weight cutoff of 5 kD, and then centrifuging to obtain a crude protein extract, wherein the mass ratio of the buffer solution to the defatted fish meat is 20:1, and the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, dithiothreitol and sodium carbonate in a mass ratio of 62:18:0.6:0.04:2;

[0098] Step S3, electric field modification: adding hydroxytyrosol to the crude protein extract, stirring for 25 min at a controlled speed of 700 rpm until uniform, then adding 1 M acetic acid aqueous solution to adjust the pH value to 1, pumping the acid-adjusted crude protein extract to a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified liquid, wherein the mass ratio of hydroxytyrosol to the crude protein extract is 0.06:25, and the flow rate is set to 6 mL / min, the electric field strength is 15 kV / cm, and the number of pulses is 8 during the high-voltage pulse electric field treatment;

[0099] Step S4, enzymolysis: adding 0.05M ammonium carbonate aqueous solution to the high-voltage pulse electric field modified solution, stirring for 18 minutes at a speed of 800 rpm until uniform, then adding trypsin, and digesting at 36°C overnight to obtain an enzymolysis solution, wherein the mass ratio of pH regulator, high-voltage pulse electric field modified solution and trypsin is 0.07:20:6;

[0100] Step S5, fine purification: ultrasonically disperse the functionalized silica gel prepared in comparative preparation example 2 in the enzymatic hydrolyzate, stir for 2 hours at room temperature at a speed of 500 rpm, filter to obtain a polypeptide adsorbate, wash the polypeptide adsorbate 4 times with a sodium hydrogen phosphate aqueous solution at pH 7.6 to obtain a polypeptide extract, filter the polypeptide extract through a 150Da ultrafiltration membrane, and then concentrate to dryness under vacuum, control the vacuum concentration multiple to 40 times, and the vacuum degree to 11Pa, wherein the mass ratio of the functionalized silica gel to the enzymatic hydrolyzate is 2.2:1.

[0101] Comparative Example 3

[0102] This comparative example provides a process for extracting antimicrobial peptide components from marine organisms, comprising the following steps:

[0103] Step S1, defatting treatment: after descaling and removing the internal organs of fresh large yellow croaker, chopping it into pieces, washing it with tap water for 3 times, and defatting it to obtain defatted fish meat, wherein the defatting treatment process is specifically as follows: placing the chopped defatted fish meat in a 10% (w / v) sodium carbonate solution for 8 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, washing it with deionized water until it is neutral, and then placing it in a 20% (v / v) isopropanol solution for 12 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, and washing it with deionized water for 3 times;

[0104] Step S2, crude extraction: adding defatted fish meat to a buffer solution, controlling the shear rate to 100 rpm, treating for 15 min until the defatted fish meat is evenly dissolved, and maintaining the temperature to 52° C., passing through an ultrafiltration membrane with a molecular weight cutoff of 5 kD, and then centrifuging to obtain a crude protein extract, wherein the mass ratio of the buffer solution to the defatted fish meat is 20:1, and the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, dithiothreitol and sodium carbonate in a mass ratio of 62:18:0.6:0.04:2;

[0105] Step S3, electric field modification: adding hydroxytyrosol to the crude protein extract, stirring for 25 min at a controlled speed of 700 rpm until uniform, then adding 1 M acetic acid aqueous solution to adjust the pH value to 1, pumping the acid-adjusted crude protein extract to a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified liquid, wherein the mass ratio of hydroxytyrosol to the crude protein extract is 0.06:25, and the flow rate is set to 6 mL / min, the electric field strength is 15 kV / cm, and the number of pulses is 8 during the high-voltage pulse electric field treatment;

[0106] Step S4, enzymolysis: adding 0.05M ammonium carbonate aqueous solution to the high-voltage pulse electric field modified solution, stirring for 18 minutes at a speed of 800 rpm until uniform, then adding trypsin, and digesting at 36°C overnight to obtain an enzymolysis solution, wherein the mass ratio of pH regulator, high-voltage pulse electric field modified solution and trypsin is 0.07:20:6;

[0107] Step S5, fine purification: ultrasonically disperse the functionalized silica gel prepared in comparative preparation example 3 in the enzymatic hydrolyzate, stir for 2 hours at room temperature with a control speed of 500 rpm, filter to obtain a polypeptide adsorbate, wash the polypeptide adsorbate with a sodium hydrogen phosphate aqueous solution at pH 7.6 for 4 times to obtain a polypeptide extract, filter the polypeptide extract with a 150Da ultrafiltration membrane, and then vacuum concentrate to dryness, control the vacuum concentration multiple to 40 times, and the vacuum degree to 11Pa to obtain a marine antimicrobial peptide component, wherein the mass ratio of the functionalized silica gel to the enzymatic hydrolyzate is 2.2:1.

[0108] Comparative Example 4

[0109] This comparative example provides a process for extracting antimicrobial peptide components from marine organisms, comprising the following steps:

[0110] Step S1, defatting treatment: after descaling and removing the internal organs of fresh large yellow croaker, chopping it into pieces, washing it with tap water for 3 times, and defatting it to obtain defatted fish meat, wherein the defatting treatment process is specifically as follows: placing the chopped defatted fish meat in a 10% (w / v) sodium carbonate solution for 8 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, washing it with deionized water until it is neutral, and then placing it in a 20% (v / v) isopropanol solution for 12 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, and washing it with deionized water for 3 times;

[0111] Step S2, crude extraction: adding defatted fish meat to a buffer solution, controlling the shear rate to be 100 rpm, treating for 15 minutes until the defatted fish meat is evenly dissolved, and maintaining the temperature to be 52° C., passing through an ultrafiltration membrane with a molecular weight cutoff of 5 kD, and then centrifuging to obtain a crude protein extract, wherein the mass ratio of the buffer solution to the defatted fish meat is 20:1, and the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, threitol and sodium carbonate in a mass ratio of 62:18:0.6:0.04:2;

[0112] Step S3, electric field modification: adding hydroxytyrosol to the crude protein extract, stirring for 25 min at a controlled speed of 700 rpm until uniform, then adding 1 M acetic acid aqueous solution to adjust the pH value to 1, pumping the acid-adjusted crude protein extract to a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified liquid, wherein the mass ratio of hydroxytyrosol to the crude protein extract is 0.06:25, and the flow rate is set to 6 mL / min, the electric field strength is 15 kV / cm, and the number of pulses is 8 during the high-voltage pulse electric field treatment;

[0113] Step S4, enzymolysis: adding 0.05M ammonium carbonate aqueous solution to the high-voltage pulse electric field modified solution, stirring for 18 minutes at a speed of 800 rpm until uniform, then adding trypsin, and digesting at 36°C overnight to obtain an enzymolysis solution, wherein the mass ratio of pH regulator, high-voltage pulse electric field modified solution and trypsin is 0.07:20:6;

[0114] Step S5, fine purification: ultrasonically disperse the functionalized silica gel prepared in Preparation Example 1 in the enzymatic hydrolyzate, stir for 2 hours at room temperature at a speed of 500 rpm, filter to obtain a polypeptide extract, filter the polypeptide extract through a 150Da ultrafiltration membrane, and then vacuum concentrate to dryness, control the vacuum concentration multiple to 40 times, and the vacuum degree to 11Pa to obtain a marine antimicrobial peptide component, wherein the mass ratio of the functionalized silica gel to the enzymatic hydrolyzate is 2.2:1.

[0115] Comparative Example 5

[0116] This comparative example provides a process for extracting antimicrobial peptide components from marine organisms, comprising the following steps:

[0117] Step S1, defatting treatment: after descaling and removing the internal organs of fresh large yellow croaker, chopping it into pieces, washing it with tap water for 3 times, and defatting it to obtain defatted fish meat, wherein the defatting treatment process is specifically as follows: placing the chopped defatted fish meat in a 10% (w / v) sodium carbonate solution for 8 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, washing it with deionized water until it is neutral, and then placing it in a 20% (v / v) isopropanol solution for 12 hours, the material-liquid mass ratio is 1:20, the temperature is 4°C, and washing it with deionized water for 3 times;

[0118] Step S2, crude extraction: adding defatted fish meat to a buffer solution, controlling the shear rate to be 100 rpm, treating for 15 minutes until the defatted fish meat is evenly dissolved, and maintaining the temperature to be 52° C., passing through an ultrafiltration membrane with a molecular weight cutoff of 5 kD, and then centrifuging to obtain a crude protein extract, wherein the mass ratio of the buffer solution to the defatted fish meat is 20:1, and the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, dithiothreitol and sodium carbonate in a mass ratio of 62:18:0.6:0.04:2;

[0119] Step S3, electric field modification: deionized water is added to the crude protein extract, and the mixture is stirred at a speed of 700 rpm for 25 min until uniform, and then a 1 M acetic acid aqueous solution is added to adjust the pH value to 1, and the acid-adjusted crude protein extract is pumped into a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified liquid, wherein the mass ratio of deionized water to the crude protein extract is 0.06:25, and the flow rate is set to 6 mL / min, the electric field strength is 15 kV / cm, and the number of pulses is 8 during the high-voltage pulse electric field treatment;

[0120] Step S4, enzymolysis: adding 0.05M ammonium carbonate aqueous solution to the high-voltage pulse electric field modified solution, stirring for 18 minutes at a speed of 800 rpm until uniform, then adding trypsin, and digesting at 36°C overnight to obtain an enzymolysis solution, wherein the mass ratio of pH regulator, high-voltage pulse electric field modified solution and trypsin is 0.07:20:6;

[0121] Step S5, fine purification: ultrasonically disperse the functionalized silica gel prepared in Preparation Example 1 in the enzymatic hydrolyzate, stir for 2 hours at room temperature with a control speed of 500 rpm, filter to obtain a polypeptide adsorbate, wash the polypeptide adsorbate 4 times with a sodium hydrogen phosphate aqueous solution at pH 7.6 to obtain a polypeptide extract, filter the polypeptide extract through a 150Da ultrafiltration membrane, and then vacuum concentrate to dryness, control the vacuum concentration multiple to 40 times, and the vacuum degree to 11Pa to obtain a marine antimicrobial peptide component, wherein the mass ratio of the functionalized silica gel to the enzymatic hydrolyzate is 2.2:1.

[0122] Performance Testing

[0123] The purity and extraction rate of the marine antimicrobial peptide components extracted by each extraction process in Examples 1-3 and Comparative Examples 1-5 were detected by liquid chromatography. The detection conditions of the high performance liquid chromatography are as follows: analytical RP-HPLC System, ODS-C18, mobile phase A: ultrapure water (containing 0.1% TFA), mobile phase B: 80% acetonitrile (containing 0.1% TFA), flow rate: 1.0 mL / min, column temperature: 21 ° C, detector: DAD, detection wavelength: 214 nm.

[0124] Table 1 Purity and yield of marine antimicrobial peptide components obtained in Examples 1-3 and Comparative Examples 1-5

[0125]

[0126] It can be seen from Table 1 that, compared with Comparative Examples 1-5, the antimicrobial peptides obtained using the extraction process of the marine biological antimicrobial peptide components in Examples 1-3 have higher purity and extraction rate.

[0127] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A process for extracting antimicrobial peptide components from marine organisms, characterized in that: The following steps are involved: Step S1, defatting: descaling and removing the internal organs of fresh large yellow croaker, cutting into pieces, washing with tap water for 3-5 times, and defatting to obtain defatted fish meat; Step S2, crude extraction: adding defatted fish meat into a buffer solution, subjecting it to shear treatment for 15-20 minutes while maintaining the temperature at 52-56° C., and ultrafiltration and centrifugation to obtain a crude protein extract; Step S3, electric field modification: adding hydroxytyrosol to the crude protein extract, stirring evenly, then adding an acid value adjusting solution to adjust the pH value to 1-3, pumping the acid-adjusted crude protein extract to a high-voltage pulse electric field treatment chamber for high-voltage pulse electric field treatment to obtain a high-voltage pulse electric field modified solution; Step S4, enzymatic hydrolysis: adding a pH regulator to the high voltage pulse electric field modified solution, stirring evenly, then adding trypsin, and digesting at 36-38° C. overnight to obtain an enzymatic hydrolyzate; Step S5, fine purification: ultrasonically disperse the functionalized silica gel in the enzymatic solution, stir at room temperature for 2-3 hours, filter to obtain a polypeptide adsorbate, wash the polypeptide adsorbate with a desorption solution to obtain a polypeptide fine extract, and perform adsorption-desalting and vacuum concentration on the polypeptide fine extract to obtain a marine antimicrobial peptide component; The functionalized silica gel is firstly acidified with silica gel to obtain acidified silica gel, then subjected to condensation reaction with 3-chloropropyltrimethoxysilane to obtain chlorine-substituted silica gel, then subjected to nucleophilic substitution reaction with 4-trifluoromethyl o-phenylenediamine to obtain phenyl silica gel, then subjected to ring-opening esterification reaction with octadecene succinic anhydride to obtain carboxylated silica gel, and then subjected to esterification reaction with hydroxypropyl-terminated polysiloxane to obtain the functionalized silica gel; In step S2, the buffer solution is prepared by mixing deionized water, glycerol, sodium chloride, dithiothreitol and sodium carbonate in a mass ratio of 62-74:18-22:0.6-1.0:0.04-0.06:2-4.

2. The extraction process of marine antimicrobial peptide components according to claim 1, characterized in that: In the step S1, the defatting process includes: placing the chopped defatted fish meat in a 10-20% (w / v) sodium carbonate solution for 8-10 hours, the material-liquid mass ratio is 1:20-30, the temperature is 4-6°C, washing with deionized water until neutral, and then placing it in a 20-30% (v / v) isopropanol solution for 12-16 hours, the material-liquid mass ratio is 1:20-40, the temperature is 4-6°C, and washing with deionized water 3-5 times.

3. The extraction process of marine antimicrobial peptide components according to claim 1, characterized in that: In step S3, the acid value adjusting liquid is a 1-2M acetic acid aqueous solution, and the flow rate is set to 6-8mL / min, the electric field strength is set to 15-18kV / cm, and the number of pulses is set to 8 during the high-voltage pulse electric field treatment.

4. The extraction process of marine antimicrobial peptide components according to claim 1, characterized in that: In the step S4, the pH regulator is a 0.05-0.07 M aqueous solution of ammonium carbonate. In the step S5, the mass ratio of the functionalized silica gel to the enzymatic hydrolysate is 2.2-2.4:

1.

5. The extraction process of marine antimicrobial peptide components according to claim 1, characterized in that: The method for preparing the functionalized silica gel comprises the following steps: Step A1, adding silica gel to 20-30% hydrochloric acid by mass fraction, heating to 35-45° C., stirring for 8-10 hours, washing to neutrality, and vacuum drying to obtain acidified silica gel; ultrasonically dispersing the acidified silica gel in anhydrous toluene, adding triethylamine and 3-chloropropyltrimethoxysilane while stirring under nitrogen protection, heating to reflux, stirring and reacting for 18-24 hours, washing, and drying to obtain chlorine-substituted silica gel; Step A2, ultrasonically dispersing chlorine-substituted silica gel in anhydrous toluene, adding 4-trifluoromethyl-o-phenylenediamine under nitrogen protection, heating to reflux, stirring and reacting for 42-48 hours, washing and drying after the reaction to obtain phenyl silica gel; Step A3, ultrasonically dispersing phenyl silica gel in anhydrous dichloromethane, adjusting the pH to 9-10, adding a mixed solution a of octadecenyl succinic anhydride and anhydrous dichloromethane dropwise while stirring, and controlling the dropping to be completed within 30 minutes. After the dropping is completed, the temperature is raised to 74-80° C., and the stirring reaction is continued for 6-10 hours. The pH is adjusted to neutral, rotary evaporation is performed, washing, and drying are performed to obtain carboxylated silica gel; Step A4: under nitrogen protection, add carboxylated silica gel, hydroxypropyl-terminated polysiloxane and p-toluenesulfonic acid into anhydrous toluene, stir evenly, heat to 82-86° C., stir to react for 1-2 hours, and after the reaction is completed, rotary evaporate, wash and dry to obtain functionalized silica gel.

6. The extraction process of marine antimicrobial peptide components according to claim 5, characterized in that: In the step A1, the mass ratio of silica gel to 20-30% hydrochloric acid is 30-40:220-260, and the mass ratio of acidified silica gel, anhydrous toluene, triethylamine and 3-chloropropyltrimethoxysilane is 12-16:52-56:0.24-0.32:14-20.

7. The extraction process of marine antimicrobial peptide components according to claim 5, characterized in that: In the step A2, the mass ratio of chlorine-substituted silica gel, anhydrous toluene and 4-trifluoromethyl-o-phenylenediamine is 12-14:55-65:8.4-11.

2.

8. The extraction process of marine antimicrobial peptide components according to claim 5, characterized in that: In the step A3, the mass ratio of phenyl silica gel, anhydrous dichloromethane and mixed solution a is 4-6:22-26:10, and in the mixed solution a, the mass ratio of octadecenyl succinic anhydride and anhydrous dichloromethane is 0.8-1.2:

10.

9. The extraction process of marine antimicrobial peptide components according to claim 5, characterized in that: In the step A4, the mass ratio of carboxylated silica gel, hydroxypropyl-terminated polysiloxane, p-toluenesulfonic acid and anhydrous toluene is 3-4: 1.4-1.8: 0.04-0.05: 50-60.

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