Method for preparing dephenolized cottonseed protein antibacterial peptide by enzyme method

Abstract

The invention discloses a method for preparing dephenolized cottonseed protein antibacterial peptide through an enzyme method, and belongs to the technical field of cottonseed protein antibacterial peptide preparation. According to the invention, cottonseed protein is used as a raw material, a composite hydrolase system is used for hydrolyzing the cottonseed protein under a certain condition, and the hydrolysate is purified through two-stage membrane separation, affinity chromatography and HPLC (High Performance Liquid Chromatography), so that the cottonseed protein antibacterial peptide with relatively strong antibacterial activity is finally obtained. The cottonseed protein antibacterial peptide is high in practicability, and the obtained cottonseed protein antibacterial peptide is high in antibacterial property, is a broad-spectrum antibacterial peptide, can inhibit negative bacteria including pathogenic escherichia coli and gram-positive bacteria, is a functional antibacterial peptide and has a good application prospect. The method can be widely applied to the fields of medicine, food, agriculture and the like, a feasible solution is provided for replacing traditional antibiotics, and remarkable economic and environmental benefits are achieved for promoting high-value utilization of cottonseed protein and reducing resource waste.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and functional peptide preparation, and particularly relates to a method for preparing antibacterial peptides from defatted cottonseed protein by enzymatic hydrolysis. Background Art

[0002] Antibacterial peptides are a class of small polypeptides with broad-spectrum antibacterial activity. Compared with traditional antibiotics, they have the characteristics of broad-spectrum antibacterial property, unique action mechanism, and high safety. At present, antibacterial peptides have important application values in the fields of medicine, food industry, agriculture, etc. However, their large-scale production is still limited by problems such as high raw material costs and complex extraction processes, and there is an urgent need to develop economic and efficient preparation methods.

[0003] Currently, the main methods for preparing active peptides include: enzymatic hydrolysis method, microbial fermentation method, chemical synthesis method, genetic engineering method, and physical-assisted extraction method, etc. Among them, the technology of preparing antibacterial peptides by enzymatic method has become the core process in the field of bioactive peptides due to its significant advantages. This method can be carried out efficiently under mild physiological conditions (such as neutral pH, 40 - 50 °C), avoiding the destruction of the peptide chain structure in the high-temperature or strong acid-base environment in traditional chemical synthesis, and retaining the natural activity of antibacterial peptides to the greatest extent. By precisely selecting the type of protease (such as trypsin, alkaline protease, etc.) and combining with a stepwise enzymatic hydrolysis strategy, the molecular weight distribution of the product can be precisely regulated.

[0004] Cottonseed protein is the main by-product after cottonseed oil extraction. Traditionally, it has been mostly used for feed, but its high protein content (30% - 50%) and unique amino acid composition make it an ideal raw material for preparing antibacterial peptides. Compared with other plant or animal proteins, cottonseed protein is rich in resources, low in cost, has a suitable amino acid composition, and the current defatting process is mature. Modern defatting technologies (such as solvent extraction, microbial fermentation) can effectively remove gossypol, ensuring the biological safety of the product, and using cottonseed by-products to produce high-value-added antibacterial peptides conforms to the sustainable development concept of resource recycling.

[0005] The limitations of existing enzymatic hydrolysis technology are as follows: (1) The protease selection criteria are single, mainly relying on enzyme-substrate specificity parameters, while ignoring the unique amino acid sequence characteristics of cottonseed protein (such as high glutamic acid / arginine content); (2) The lack of dynamic control means, the enzyme activity ratio cannot be adjusted in real time according to the product inhibition effect during the hydrolysis process, resulting in a 40% decrease in the efficiency of late enzymatic hydrolysis; (3) The integration of auxiliary technologies is low, and the synergistic mechanism of physical field enhancement (such as ultrasound) and enzymatic hydrolysis reaction has not yet been clarified. Experimental data show that although intermittent ultrasound can increase the degree of hydrolysis by 12%, continuous action will cause the β-folding content of the enzyme molecule to increase by 9%, which in turn reduces the catalytic activity. In addition, the proportion of active peptides with a molecular weight of less than 3 kDa in the final product is usually less than 55%, while in vitro experiments have confirmed that the antibacterial efficiency of peptides in this range against Staphylococcus aureus is 2.3 times that of 3-5 kDa peptides.

[0006] In the separation and purification stage, the existing technology faces a double dilemma: on the one hand, the ultrafiltration membrane is seriously contaminated, and the polysaccharide-peptide complex in the cottonseed protein hydrolysate will cause the 3 kDa ultrafiltration membrane flux to drop by 65% ​​within 2 hours; on the other hand, the chromatographic medium is not selective enough, and the conventional hydrophobic chromatographic medium has a binding capacity of only 2-3 mg / mL for antimicrobial peptides containing 4-6 hydrophobic residues, forcing the purification process to require multiple steps in series (such as ion exchange-gel filtration-reverse phase chromatography), which directly leads to a 2.8-fold increase in the cost of the final product. More importantly, the existing process route lacks a deep analysis of the structure-activity relationship of active peptides, and it is impossible to establish a quantitative correlation model between molecular weight distribution and antibacterial potency, resulting in a large degree of blindness in the purification process.

[0007] The root causes of the above technical problems are: the synergistic mechanism of structural modification and enzyme cleavage site exposure has not been established in the raw material pretreatment stage; the enzymatic hydrolysis process lacks a multi-scale (molecule-mesoscopic-system) precise control strategy; the separation and purification system has insufficient accuracy in identifying the molecular characteristics of the target product. To overcome these obstacles, three key scientific problems need to be solved: how to construct a non-destructive dephenolization system to maintain the flexibility of the protein structure; how to design a time-controlled composite enzymatic hydrolysis network to increase the yield of the target peptide; and how to develop a biomimetic recognition medium to achieve efficient capture of active components. Existing technologies have not made breakthrough progress on these core issues, which has seriously restricted the industrialization process of cottonseed protein antimicrobial peptides. Summary of the invention

[0008] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0009] In order to achieve these purposes and other advantages according to the present invention, a method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method is provided, which comprises the following steps: Step 1. Crush and sieve cottonseed meal to obtain cottonseed meal particles. Mix the cottonseed meal particles with a 0.8% sodium bicarbonate solution by a material-liquid ratio of 1:10. After microwave treatment, oscillation, and suction filtration, defatted cottonseed meal is obtained. Step 2. Mix the defatted cottonseed meal with phosphate buffer solution by a mass ratio of 1:12. Add a composite hydrolase system accounting for 2.5% of the substrate mass. The composite hydrolase system consists of Bacillus subtilis alkaline protease, Aspergillus oryzae neutral protease, and Bacillus sp. keratinase, with an enzyme activity unit ratio of 5:3:2. Treat it in a coupled field with alternating ultrasound and microwave to obtain a hydrolysate. Step 3. Subject the hydrolysate to cross-flow filtration through a ceramic membrane to retain components with a molecular weight cut-off of more than 10 kDa. Subsequently, perform secondary filtration through a hollow fiber ultrafiltration membrane to retain components with a molecular weight cut-off of more than 3 kDa to obtain an ultrafiltrate. Step 4. Inject the ultrafiltrate into an agarose gel chromatography column modified with Aeromonas hydrophila outer membrane protein at a flow rate of 2 mL / min for purification to obtain an active component. Step 5. Purify the active component by semi-preparative high-performance liquid chromatography equipped with an evaporative light scattering detector to obtain an extract, and then obtain the product after freeze-drying the extract.

[0010] Preferably, in the method for enzymatically preparing antibacterial peptides from defatted cottonseed protein, in Step 1, the microwave treatment, oscillation, and suction filtration are specifically as follows: S1.1. Place the mixture of cottonseed meal particles and sodium bicarbonate solution in a closed microwave reactor. Set the microwave power density to 15 W / g of the material. Perform pulsed-mode microwave irradiation for 10 s and then stop for 5 s of intermittent treatment. Repeat the cycle 6 times, with a total irradiation time of 3 min. S1.2. Transfer the microwave-treated mixture to a thermostatic oscillator. Set the oscillation frequency to 250 rpm and the amplitude to 25 mm. Perform oscillatory defatting under the condition of maintaining 45 ± 0.3 °C by a PID temperature control module. Automatically switch the forward and reverse rotation directions of the drive motor every 5 min, with a total oscillation duration of 35 min. S1.3. Introduce the oscillated mixture into a Buchner funnel for two-stage suction filtration. The first stage is suction filtered at a vacuum degree of -0.08 MPa for 8 min, and the second stage is suction filtered at a vacuum degree of -0.09 MPa for 5 min. The filter cake is washed twice by countercurrent with a 0.8% sodium bicarbonate solution at 4 °C, with the amount of washing liquid used each time being 1.5 times the mass of the filter cake. After washing, the filter cake is frozen to -20 °C at a cooling rate of 5 °C / min under nitrogen atmosphere protection to form a solidified defatted cottonseed meal with a porous skeleton structure.

[0011] Preferably, in the method for enzymatically preparing antibacterial peptides from defatted cottonseed protein, in Step S1.1, the peak temperature of the material during intermittent microwave treatment is 62 - 65 °C, and the temperature difference between material layers is ≤ 3 °C through pulse control. In step S1.2, the forward and reverse switching oscillation makes the movement trajectory of the material form an 8-shaped eddy current. The observation by high-speed camera shows that the material dispersion is increased to 0.82 - 0.85. In step S1.3, the filter cloth for two-stage vacuum filtration is made of polytetrafluoroethylene-coated glass fiber, with a pore size distribution of 5 - 10 μm and a porosity of 78 - 82%.

[0012] Preferably, in the method for enzymatically preparing antibacterial peptides from defatted cottonseed protein, in step two, the enzyme activity unit of subtilisin alkaline protease is determined by the Folin - phenol method. Using 1% casein as the substrate, after reacting for 10 min at pH 9.0 and 40 °C, trichloroacetic acid is immediately added to terminate the reaction; the enzyme activity unit of aspergillus oryzae neutral protease is determined using 2% hemoglobin as the substrate, and after reacting for 15 min at pH 7.0 and 50 °C, the reaction is terminated by the ammonium sulfate precipitation method; bacillus keratinase needs to be pre-activated in a citric acid buffer solution at pH 6.5 and 45 °C for 20 min, and the mass ratio of the activated keratinase to alkaline protease is (2.8 - 3.2):5. In step two, the complex hydrolase system is added to the reaction system in two stages: in the first stage, 60% of the total enzyme system amount of subtilisin alkaline protease is first added and treated in an ultrasonic - microwave coupling field for 90 min; in the second stage, the remaining 40% of subtilisin alkaline protease and aspergillus oryzae neutral protease and the activated bacillus keratinase are added, and the treatment continues in the ultrasonic - microwave coupling field for 90 min. The dynamic enzyme activity ratio of the complex hydrolase system in the reaction system is monitored and regulated by an online pH - ORP sensor. When the redox potential value drops to - 120 mV, a 0.1 mol / L sodium hydroxide solution is automatically added to maintain the pH in the range of 8.3 - 8.7.

[0013] Preferably, in the method for enzymatically preparing antibacterial peptides from defatted cottonseed protein, in step two, the coupling field treatment of alternately applying ultrasound and microwave is specifically as follows: Step S2.1: Place the reaction system containing the complex hydrolase system in a closed reactor equipped with a rotating bracket. First, start the microwave radiation stage, set the microwave frequency at 2450 MHz and the power density at 8 W / g of the reaction system, and control the material tray to rotate at a constant speed of 6 rpm. Step S2.2: After continuously treating for 30 s to raise the material temperature to 50 ± 2 °C, switch to the ultrasonic treatment stage, turn off the microwave and start a 28 kHz ultrasonic generator, set the power density at 3.8 W / cm², and work in an intermittent emission mode for 60 s, where it pauses for 5 s after working for 15 s each time. At the same time, 10 °C cooling water is introduced into the reactor jacket through an external circulation pump. Step S2.3: Repeat the above microwave-ultrasound alternating treatment cycle 20 times, with a total duration of 1800 s. After each cycle, detect the temperature of the material. When the temperature exceeds 55 °C, automatically extend the cooling water injection time to 10 s; Step S2.4: In the last three cycles, adjust the microwave power density to 10 W / g, and at the same time adjust the ultrasound working cycle to continuous emission for 45 s; Step S2.5: After the treatment, transfer the reaction system to an ice-water bath for rapid quenching, and reduce the temperature to below 25 °C within 2 min to obtain the hydrolyzate.

[0014] Preferably, in the method for enzymatically preparing antibacterial peptides from defatted cottonseed protein, step three is specifically as follows: Step S3.1: Preheat the hydrolyzate to 50 ± 2 °C and then pump it into a tubular ceramic membrane module. The ceramic membrane is made of α-aluminum oxide, with an effective pore size of 30 nm, an operating pressure of 0.25 MPa, and a cross-flow velocity of 3.0 m / s. The permeate collected in the initial 10 min is returned to the raw material tank for circulation filtration. After the transmembrane pressure difference stabilizes at 0.18 MPa, collect the filtrate; Step S3.2: Cool the ceramic membrane filtrate to 25 ± 1 °C through a plate heat exchanger, adjust the pH to 6.8 - 7.2, and then enter a hollow fiber ultrafiltration membrane system. The ultrafiltration membrane is made of polyethersulfone, with a cut-off molecular weight of 3 kDa, an operating pressure of 0.12 MPa, and is operated in a pulsed backwashing mode. Perform 2 s of reverse flushing every 15 min of filtration, with a flushing pressure of 0.30 Mpa, and collect the ultrafiltrate; Among them, the two-stage membrane separation process is monitored by the combined use of an on-line turbidity sensor and an ultraviolet absorption detector. When the A280 value of the filtrate exceeds 0.85, the membrane cleaning program is automatically triggered, and an in-situ cleaning is performed using a mixed cleaning solution of 0.1 mol / L sodium hydroxide and 0.5% sodium dodecyl sulfate.

[0015] Preferably, in the method for enzymatically preparing antibacterial peptides from defatted cottonseed protein, the preparation of Aeromonas hydrophila outer membrane protein-modified agarose gel includes the following steps: Step a: Treat Aeromonas hydrophila with an ultrasonic crusher, take the supernatant after centrifugation, and obtain the outer membrane protein through ammonium sulfate fractional precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography purification in sequence. The purity is detected by SDS-PAGE to be ≥95%; Step b: Swell the agarose gel CL-6B carrier with 0.5 mol / L NaHCO3 buffer solution (pH 8.3) for 24 h, then mix it with a 10 mg / mL outer membrane protein solution at a volume ratio of 1:3, add a carbodiimide coupling agent with a final concentration of 5 mmol / L, and react under low-speed stirring conditions at 4 °C for 18 h; Step c: After the reaction is completed, the unbound sites are blocked with a pH 8.0 Tris-HCl buffer containing 0.1 mol / L ethanolamine for 2 h, and then washed alternately three times with a pH 7.4 buffer containing 0.5 mol / L NaCl and pure water, and finally stored in a PBS buffer containing 0.02% sodium azide to obtain the product; Among them, the protein coupling efficiency is ≥82%, the ligand density is 9.2 - 9.8 mg protein / mL gel, the upper limit of column pressure tolerance is 1.5 MPa, and the ligand shedding rate is <3% after 50 adsorption-elution cycles.

[0016] Preferably, for the method for enzymatically preparing defatted cottonseed protein antibacterial peptide, the chromatographic column purification process is specifically as follows: Step S4.1: The Aeromonas hydrophila outer membrane protein-modified agarose gel is packed into a chromatographic column at a constant pressure of 0.5 MPa, the column bed height is 45 cm, and after packing, it is washed by flowing through with a pH 7.4 PBS buffer containing 0.05% Triton X-100 at 3 times the column volume; Step S4.2: An equilibration buffer containing 0.15 mol / L NaCl is added to the chromatographic column, and equilibrated at a flow rate of 1.2 mL / min until the conductivity is stable at 12.5 ± 0.3 ms / cm, then sample loading is carried out, and the sample loading amount is controlled not to exceed 78% of the column dynamic loading capacity. During sample loading, the UV280 absorption value is monitored, and sample loading is terminated when the breakthrough peak reaches 5% of the baseline; Step S4.3: Gradient elution adopts a double-gradient mode: in the first stage, the NaCl concentration is increased at a rate of 0.8% B / min to 0.22 mol / L, and in the second stage, the histidine concentration is increased at a rate of 1.2% B / min to 12 mmol / L. The eluate is collected synchronously and sterilized with a 0.22 μm filter membrane; The screening criteria for the active component are: SDS-PAGE detection shows that the intensity ratio of the 14 - 18 kDa characteristic band is ≥65%, the retention time of reverse-phase chromatography deviates from the standard product by ≤±0.25 min, and the antibacterial activity titer is ≥3200 IU / mg.

[0017] Preferably, for the method for enzymatically preparing defatted cottonseed protein antibacterial peptide, the semi-preparative high-performance liquid chromatography purification in step five meets the following separation conditions: a) Use a C18 chromatographic column (250×10 mm, 5 μm) grafted with polyethylene glycol chains on the surface, and the column temperature is maintained at 27 ± 0.3 °C through a two-stage semiconductor temperature control system; b) Mobile phase A is an aqueous solution of pH 4.5 containing 0.05 mol / L ammonium formate, and mobile phase B is a mixed solution of acetonitrile-methanol containing 0.05 mol / L ammonium formate (acetonitrile:methanol = 7:3 v / v); c) The gradient program is set as follows: maintain 15% B from 0 - 2 min, increase to 38% B non-linearly in 2 - 15 min (gradient slope is 0.8% B / min in the first stage and 2.2% B / min in the second stage), and maintain for 15 - 18 min, then increase to 45% B at a rate of 5% B / min within 18 - 20 min.

[0018] Preferably, in the method for enzymatically preparing antibacterial peptides from defatted cottonseed protein, when the proportion of mobile phase B < 30%, the pressure is set at 3.2 bar, and when the proportion of B ≥ 30%, the pressure linearly increases to 4.5 bar, and the evaporation temperature is synchronously adjusted from 75 °C to 82 °C; Before injection, the active component is premixed with mobile phase A at a volume ratio of 1:3, filtered through a 0.22 μm PVDF membrane, the injection volume is controlled at 500 μL, and the flow rate is 1.8 mL / min; The detector signal slope dS / dt > 5 mV / s, an ion peak with a molecular weight of 1482 ± 3 Da is detected online by mass spectrometry, and the conductivity fluctuation is ≤ ±8% within the retention time window of 17.5 - 18.5 min; the collected fraction is immediately bubbled with nitrogen to remove the organic solvent, and the residual acetonitrile content is controlled < 0.1% (detected by GC).

[0019] The present invention has at least the following beneficial effects: 1) Through the integration of a five-stage process (microwave degreasing → complex enzymatic hydrolysis → two-stage membrane separation → affinity chromatography → HPLC purification), the yield of antibacterial peptides reaches 18.7% (calculated based on cottonseed meal), the purity ≥ 98.5%, the yield is increased by 40% compared with traditional methods, the MIC of the product is as low as 2.5 - 5 μg / mL, and the industrial cost is reduced by 35%, which is suitable for the efficient inhibition of Gram-positive and Gram-negative bacteria; 2) By using intermittent microwave (10 s on / 5 s off × 6 times) + forward and reverse oscillation + gradient filtration; pulsed microwave controls the temperature rise (62 - 65 °C) to avoid protein denaturation; 8-shaped eddy oscillation enhances the mass transfer efficiency, the residual fat rate of defatted cottonseed meal is 0.3%, and the protein content is 58.6%. The lipid extraction rate is increased by 17% compared with the solvent method, and the protein denaturation rate < 5%; 3) By setting the temperature difference between layers ≤ 3 °C for microwave, polytetrafluoroethylene filter cloth (porosity 82%), and freeze-curing porous framework; uniform temperature rise ensures the consistency of lipid melting, high-porosity filter cloth reduces the filtration resistance, and freezing forms a porous structure; the liquid content of the filter cake is 18%, the specific surface area for enzymatic hydrolysis is increased by 2 times, the defatting time is shortened by 40%, and the filter cloth life is extended to 200 batches; 4) The present invention preferentially cleaves hydrophobic peptide bonds through a staged composite enzyme system (with a ratio of 5:3:2) + dynamic pH regulation, where alkaline protease is used first, followed by neutral / keratinase to hydrolyze hydrophilic regions. Maintaining the pH within the optimal range for enzyme activity results in a hydrolysis degree of 68%, a proportion of small peptides with a molecular weight of 1 - 5 kDa of 73%, a 22% increase in the yield of antibacterial peptides, and an enzyme activity half-life of 4.2 h (2.3 h for a single enzyme). 5) The present invention uses an alternating ultrasonic-microwave coupling field (30 s of microwave / 60 s of ultrasound × 20 times) + rapid cooling. The thermal effect of microwave accelerates molecular movement, the cavitation effect of ultrasound destroys protein aggregation, and rapid cooling terminates enzyme activity. The enzymatic hydrolysis rate is increased by 40%, energy consumption is reduced by 35%, the retention rate of antibacterial peptide activity is 95% (64% for natural cooling), and the yield is 18.7%. 6) The present invention uses a two-stage cross-flow + pulsed backflush with a ceramic membrane (30 nm) - ultrafiltration membrane (3 kDa). The pore size sieves and classifies polypeptides, cross-flow reduces concentration polarization, and pulsed backflush delays membrane fouling. The recovery rate of polypeptides with a molecular weight of 3 - 10 kDa is 92%, the membrane life is 500 h (150 h for the traditional method), the turbidity of the filtrate is < 0.5 NTU, and A280 < 0.85. 7) The present invention uses outer membrane protein of Aeromonas hydrophila to modify agarose gel (coupling efficiency of 82%). The specific affinity between the outer membrane protein and antibacterial peptide, and the use of carbodiimide coupling to stabilize the ligand result in an adsorption capacity of 55 mg / mL of gel (28 mg / mL for Protein A medium). After 50 cycles, the ligand shedding rate is < 3%, and the retained elution activity is 95%. 8) The present invention uses dual-gradient elution (NaCl + histidine) + triple activity screening: the salt gradient removes non-specific binding, and histidine competitively elutes specifically adsorbed antibacterial peptides, increasing the purity from 62% to 91%, with a recovery rate of 85%, a proportion of characteristic bands of ≥ 65%, and an antibacterial titer of 3200 IU / mg. 9) The present invention adopts a technical scheme of polyethylene glycol-modified C18 column + non-linear gradient elution. The PEG chain enhances the retention of polar peptides, and the non-linear gradient optimizes the separation of hydrophobic differences; ultimately, a resolution Rs ≥ 1.5 and a tailing factor of 1.1 (1.8 for a common C18 column) are obtained, with a single preparation amount of 5 - 8 mg and a purity ≥ 98.5%. 10) The present invention adopts a technical scheme of pressure-temperature linkage + nitrogen desolvation. High pressure maintains the flow rate, high temperature promotes solvent evaporation, and nitrogen purging removes residual organic solvents. The final results are that the residual acetonitrile is < 0.1% (GC), the conductivity fluctuation is ≤ 8%, and the endotoxin is < 0.5 EU / mg, meeting the injection standard.

[0020] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0021] Figure 1 This is the process flow chart of the method for enzymatically preparing antibacterial peptides from defatted cottonseed protein according to the present invention; Figure 2 This is the statistical analysis result chart of the degree of hydrolysis corresponding to each enzyme of the present invention. Detailed implementation manners

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it according to the description in the specification.

[0023] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0024] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0025] In the description of the present invention, the orientation or positional relationship indicated by terms such as "transverse", "longitudinal", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0026] <Example 1> As Figure 1 shown, the present invention provides a method for enzymatically preparing antibacterial peptides from defatted cottonseed protein, which includes the following steps: Step 1: Crush and sieve cottonseed meal to obtain cottonseed meal particles. Mix the cottonseed meal particles with a 0.8% sodium bicarbonate solution by mass concentration at a solid-liquid ratio of 1:10, place them in a closed microwave reactor, stop for 5 s of intermittent treatment after 10 s of microwave irradiation, and cycle for 6 periods. The microwave power density is 15 W / g of the material, and it is implemented in a pulse mode. The total irradiation time is 3 min. Among them, the peak temperature of the material during intermittent microwave treatment is 62 - 65 °C, and the temperature difference between layers of the material is ≤ 3 °C through pulse control; then transfer the microwave-treated mixed material to a constant temperature oscillator, set the oscillation frequency at 250 rpm and the amplitude at 25 mm, and perform oscillatory degreasing under the condition that the PID temperature control module maintains 45 ± 0.3 °C. Automatically switch the forward and reverse rotation directions of the drive motor every 5 min. The forward and reverse rotation switching oscillation makes the movement trajectory of the material form an 8-shaped eddy current. Observation by high-speed camera shows that the material dispersion is increased to 0.82 - 0.85; the total oscillation duration is 35 min. Import the oscillated mixed liquid into a Buchner funnel for two-stage suction filtration. The first stage is suction filtered at a vacuum degree of -0.08 MPa for 8 min, and the second stage is suction filtered at a vacuum degree of -0.09 MPa for 5 min. The filter cake is washed twice by countercurrent with a 0.8% sodium bicarbonate solution at 4 °C, and the amount of washing liquid used each time is 1.5 times the mass of the filter cake. After washing, the filter cake is frozen to -20 °C at a cooling rate of 5 °C / min under the protection of a nitrogen atmosphere to form a solidified degreased cottonseed meal with a porous skeleton structure. The filter cloth for two-stage suction filtration is made of polytetrafluoroethylene-coated fiberglass, with a pore size distribution of 5 - 10 μm and a porosity of 78 - 82%; Step 2: Mix the degreased cottonseed meal with a phosphate buffer solution at a mass ratio of 1:12, add a composite hydrolytic enzyme system accounting for 2.5% of the substrate mass. The composite hydrolytic enzyme system is composed of Bacillus subtilis alkaline protease, Aspergillus oryzae neutral protease, and Bacillus sp. keratinase in an enzyme activity unit ratio of 5:3:2, and perform treatment in a coupled field with alternating application of ultrasound and microwave to obtain a hydrolyzate; Screen the enzymes by measuring the degree of hydrolysis. Replace the composite hydrolytic enzyme system in Step 2 with equal amounts of Bacillus subtilis alkaline protease, Aspergillus oryzae neutral protease, and Bacillus sp. keratinase respectively, and measure the degree of hydrolysis corresponding to each enzyme. The specific results are as Figure 2 shown. It can be seen from the figure that the degree of hydrolysis of the composite hydrolytic enzyme formed by combining three enzymes is the best; Among them, for the determination of the degree of hydrolysis: Take 0.5 mL of the hydrolyzate, dilute it 10 times (to make the subsequent measured values within the range of the standard curve), take 20 μL of the diluted hydrolyzate and add 150 μL of OPA reagent, react for 2 min, measure the absorbance at 340 nm with an enzyme-labeling instrument, repeat the measurement three times, and draw a standard curve by serine concentration. Calculate the degree of hydrolysis of the sample according to the standard curve and absorbance. The calculation method of the degree of hydrolysis is shown in the following formula: DH = h / h tot × 100% In the formula, h tot is the molality of the peptide bond of the raw material protein, mmol / g (the h of cottonseed protein tot is 7.54 mmol / g); h is the molality of the hydrolyzed peptide bond of the hydrolyzed protein relative to the raw material protein, mmol / g; In step two, the enzyme activity unit of subtilisin alkaline protease is determined by the Folin-Ciocalteu method. Using 1% casein as the substrate, after reacting for 10 min at pH 9.0 and 40 °C, trichloroacetic acid is immediately added to terminate the reaction; the enzyme activity unit of aspergillus oryzae neutral protease is determined using 2% hemoglobin as the substrate, and after reacting for 15 min at pH 7.0 and 50 °C, the reaction is terminated by the ammonium sulfate precipitation method; bacillus keratinase needs to be pre-activated in a citric acid buffer solution at pH 6.5 and 45 °C for 20 min, and the mass ratio of the activated keratinase to alkaline protease is (2.8 - 3.2):5; In step two, the complex hydrolase system is added to the reaction system in two stages: in the first stage, 60% of the total enzyme system amount of subtilisin alkaline protease is added first and treated in an ultrasonic-microwave coupled field for 90 min; in the second stage, the remaining 40% of subtilisin alkaline protease and aspergillus oryzae neutral protease and the activated bacillus keratinase are added, and the treatment continues in the ultrasonic-microwave coupled field for 90 min; The dynamic enzyme activity ratio of the complex hydrolase system in the reaction system is monitored and regulated by an on-line pH-ORP sensor. When the redox potential value drops to -120 mV, 0.1 mol / L sodium hydroxide solution is automatically added to maintain the pH in the range of 8.3 - 8.7; The coupled field treatment of alternating ultrasound and microwave is specifically as follows: Step S2.1: Place the reaction system containing the complex hydrolase system in a closed reactor equipped with a rotating bracket. First, start the microwave radiation stage, set the microwave frequency to 2450 MHz and the power density to 8 W / g of the reaction system, and control the material tray to rotate at a constant speed of 6 rpm; Step S2.2: After continuously treating for 30 s to raise the material temperature to 50 ± 2 °C, switch to the ultrasonic treatment stage, turn off the microwave and start the 28 kHz ultrasonic generator, set the power density to 3.8 W / cm², and work in an intermittent emission mode for 60 s, where it pauses for 5 s after every 15 s of work. At the same time, 10 °C cooling water is introduced into the reactor jacket through an external circulation pump; Step S2.3: Repeat the above microwave-ultrasound alternating treatment cycle 20 times, with a total duration of 1800 s. Among them, the material temperature is detected after each cycle. When the temperature exceeds 55 °C, the cooling water introduction time is automatically extended to 10 s; Step S2.4: In the last three cycles, adjust the microwave power density to 10 W / g, and at the same time adjust the ultrasonic working cycle to continuous emission for 45 s; Step S2.5: After the treatment, transfer the reaction system to an ice-water bath for rapid quenching, and cool the temperature to below 25°C within 2 minutes to obtain the hydrolyzate. Step three: Specifically: Step S3.1: Preheat the hydrolyzate to 50 ± 2°C and then pump it into a tubular ceramic membrane module. The ceramic membrane is made of α-aluminum oxide with an effective pore size of 30 nm, an operating pressure of 0.25 MPa, a cross-flow velocity of 3.0 m / s. The permeate collected in the initial 10 minutes is returned to the raw material tank for circulating filtration. After the transmembrane pressure difference stabilizes at 0.18 MPa, collect the filtrate. Step S3.2: Cool the ceramic membrane filtrate to 25 ± 1°C through a plate heat exchanger, adjust the pH to 6.8 - 7.2, and then enter a hollow fiber ultrafiltration membrane system. The ultrafiltration membrane is made of polyethersulfone with a cut-off molecular weight of 3 kDa, an operating pressure of 0.12 MPa, and operates in a pulsed backwash mode. Perform a 2-s reverse wash every 15 minutes of filtration, with a wash pressure of 0.30 Mpa, and collect the ultrafiltrate. Among them, the two-stage membrane separation process is monitored by the combined use of an online turbidity sensor and an ultraviolet absorption detector. When the A280 value of the filtrate exceeds 0.85, the membrane cleaning program is automatically triggered, and an in-situ cleaning is performed using a mixed cleaning solution of 0.1 mol / L sodium hydroxide and 0.5% sodium dodecyl sulfate. Step four: Inject the ultrafiltrate into an agarose gel chromatography column modified with Aeromonas hydrophila outer membrane protein at a flow rate of 2 mL / min for purification to obtain the active component. Among them, the preparation of the agarose gel modified with Aeromonas hydrophila outer membrane protein includes the following steps: Step a: Treat Aeromonas hydrophila with an ultrasonic crusher, take the supernatant after centrifugation, and purify the outer membrane protein through ammonium sulfate fractional precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography in sequence. The purity is detected by SDS-PAGE to be ≥95%. Step b: Swell the agarose gel CL-6B carrier with 0.5 mol / L NaHCO3 buffer solution for 24 hours, then mix it with a 10 mg / mL outer membrane protein solution at a volume ratio of 1:3, add a carbodiimide coupling agent with a final concentration of 5 mmol / L, and react under low-speed stirring conditions at 4°C for 18 hours. Step c: After the reaction is completed, block the unbound sites with a pH 8.0 Tris-HCl buffer solution containing 0.1 mol / L ethanolamine for 2 hours, wash it three times alternately with a pH 7.4 buffer solution containing 0.5 mol / L NaCl and pure water, and finally store it in a PBS buffer solution containing 0.02% sodium azide to obtain it. Among them, the protein coupling efficiency is ≥82%, the ligand density is 9.2 - 9.8 mg protein / mL gel, the upper limit of column pressure tolerance is 1.5 MPa, and the ligand shedding rate is <3% after 50 adsorption-elution cycles. The specific process of column purification is as follows: Step S4.1: Pack the Aeromonas hydrophila outer membrane protein-modified agarose gel into a chromatography column at a constant pressure of 0.5 MPa, with a column bed height of 45 cm. After packing, wash it with a pH 7.4 PBS buffer containing 0.05% Triton X-100 at 3 times the column volume by flowing through. Step S4.2: Add an equilibration buffer containing 0.15 mol / L NaCl to the chromatography column and equilibrate at a flow rate of 1.2 mL / min until the conductivity is stable at 12.5 ± 0.3 ms / cm, then load the sample. Control the sample loading amount not to exceed 78% of the column dynamic loading capacity. Monitor the UV280 absorption value during sample loading and terminate sample loading when the breakthrough peak reaches 5% of the baseline. Step S4.3: Gradient elution adopts a double-gradient mode: in the first stage, increase the NaCl concentration to 0.22 mol / L at a rate of 0.8% B / min, and in the second stage, increase the histidine concentration to 12 mmol / L at a rate of 1.2% B / min. Synchronously collect the eluate and sterilize it with a 0.22 μm filter membrane. The screening criteria for active components are as follows: SDS-PAGE detection shows that the intensity ratio of the 14-18 kDa characteristic band is ≥ 65%, the retention time of reverse-phase chromatography deviates from the standard product by ≤ ±0.25 min, and the antibacterial activity titer is ≥ 3200 IU / mg.

[0027] Step Five: Purify the active component by semi-preparative high-performance liquid chromatography equipped with an evaporative light scattering detector to obtain an extract, and then obtain the product after freeze-drying the extract. The semi-preparative high-performance liquid chromatography purification meets the following separation conditions: a) Use a C18 chromatographic column grafted with polyethylene glycol chains on the surface, and maintain the column temperature at 27 ± 0.3 °C through a two-stage semiconductor temperature control system. b) Mobile phase A is an aqueous solution of pH 4.5 containing 0.05 mol / L ammonium formate, and mobile phase B is a mixed solution of acetonitrile-methanol containing 0.05 mol / L ammonium formate. c) The gradient program is set as follows: maintain 15% B for 0 - 2 min, increase it to 38% B in a non-linear gradient within 2 - 15 min and maintain it for 15 - 18 min, and then increase it to 45% B at a rate of 5% B / min within 18 - 20 min. When the proportion of mobile phase B < 30%, set the pressure to 3.2 bar, and when the proportion of B ≥ 30%, linearly increase the pressure to 4.5 bar, and synchronously adjust the evaporation temperature from 75 °C to 82 °C. Before injection, premix the active component with mobile phase A at a volume ratio of 1:3, filter it through a 0.22 μm PVDF membrane, control the injection volume to 500 μL, and the flow rate to 1.8 mL / min. The slope of the detector signal dS / dt > 5 mV / s, an ion peak with a molecular weight of 1482 ± 3 Da is detected online by mass spectrometry, and the conductivity fluctuation is ≤ ±8% within the retention time window of 17.5 - 18.5 min; <Comparative Example 1> On the basis of Example 1, the following modifications are made: In step two, the complex hydrolase system is replaced with a single subtilisin alkaline protease (the enzyme activity unit is equivalent to the total enzyme activity in Example 1), the ultrasonic-microwave coupling field treatment is cancelled, and it is changed to a constant temperature water bath oscillation (250 rpm) treatment at 50 °C for 180 min. The remaining steps are exactly the same as those in Example 1.

[0028] <Comparative Example 2> On the basis of Example 1, the following modifications are made: In step three, the two-stage membrane separation (ceramic membrane + ultrafiltration membrane) is cancelled, and only traditional centrifugation (8000×g, 20 min) combined with a common ultrafiltration membrane (made of polyethersulfone, with a cut-off molecular weight of 3 kDa and no pulsed backwashing) is used. The remaining steps are exactly the same as those in Example 1. The collected fraction is immediately bubbled with nitrogen to remove the organic solvent, and the residual acetonitrile content is controlled < 0.1%.

[0029] <Comparative Example 3> On the basis of Example 1, the following modifications are made: In step one, the forward and reverse oscillation is cancelled (changed to one-way oscillation, with other parameters unchanged), and the remaining steps are the same as those in Example 1.

[0030] <Comparative Example 4> On the basis of Example 1, the following modifications are made: In step four, an unmodified agarose gel (ordinary hydrophobic chromatography medium) is used instead of the chromatography column modified with Aeromonas hydrophila outer membrane protein, and the remaining steps are the same as those in Example 1.

[0031] <Comparative Example 5> On the basis of Example 1, the following modifications are made: In step five, the pressure-temperature linkage control of HPLC purification is cancelled (fixed pressure 3.2 bar, temperature 75 °C), and nitrogen stripping is not used. The remaining steps are the same as those in Example 1.

[0032] <Comparative Example 6> On the basis of Example 1, the following modifications are made: In step two, the dynamic pH regulation is cancelled (fixed pH 8.5, without adding sodium hydroxide), and the remaining steps are the same as those in Example 1.

[0033] <Comparative Example 7> On the basis of Example 1, the following modifications are made: In Step 1, the freeze-curing treatment under nitrogen protection is cancelled, and the defatted cottonseed meal is vacuum-dried at room temperature (25°C) instead. The remaining steps are the same as those in Example 1.

[0034] <Comparative Example 8> On the basis of Example 1, the following modifications are made: In Step 2, all of the composite hydrolase system is added at once (canceling the staged addition). The remaining steps are the same as those in Example 1.

[0035] <Comparative Example 9> On the basis of Example 1, the following modifications are made: In Step 2, the parameter adjustment of the ultrasonic-microwave alternating treatment is cancelled (maintaining a microwave power of 8 W / g and an ultrasonic cycle of 15 s on / 5 s off throughout). The remaining steps are the same as those in Example 1.

[0036] Comprehensive comparison (as shown in Table 1) and analysis of the three core performance indicators of the antibacterial peptide yield, purity, and antibacterial activity titer (MIC) for Example 1 and Comparative Examples 1-9: Table 1 Performance Index Statistics As can be seen from the data in Table 1, the antibacterial peptide yields of all comparative examples are significantly lower than that of Example 1 (with a maximum decrease of 49.7%). Among them, Comparative Example 1 (single enzymatic hydrolysis) and Comparative Example 7 (without porous structure) have the largest decreases, indicating that composite enzymatic hydrolysis and porous framework pretreatment are the cores for improving the yield. Due to membrane fouling problems, the yield of Comparative Example 2 (traditional membrane separation) decreased by 24-28%, highlighting the necessity of two-stage membrane separation.

[0037] The purity of Example 1 reaches 98.5%, which is higher than that of Comparative Examples 1-9. The purity of Comparative Example 4 (ordinary chromatography medium) decreased by 9.1%, proving the key role of the outer membrane protein-modified chromatography column in specific purification. Due to insufficient separation efficiency, the purity of Comparative Example 5 (unoptimized HPLC) decreased by 8.9% and 6.1% respectively. Example 1 has the lowest MIC values against Gram-positive bacteria (2.5 μg / mL) and Gram-negative bacteria (3.8 μg / mL), and the best antibacterial activity. Comparative Example 1 (single enzymatic hydrolysis) has the worst activity (MIC 8.0 / 12.5 μg / mL) because the residual macromolecular peptide segments inhibit the antibacterial effect. Due to insufficient enzymatic hydrolysis, the MIC values of Comparative Example 9 (fixed parameters) and Comparative Example 6 (fixed pH) increased by 60% and 92% respectively. Example 1 synergistically combines technologies such as compound enzymatic hydrolysis, dynamic regulation, and multi-stage purification to comprehensively optimize the yield, purity, and activity titer. By setting up physical field enhancement (Comparative Example 1 / 9), the enzymatic hydrolysis efficiency can be improved and energy consumption can be reduced; by optimizing the structure (Comparative Example 3 / 7), protein denaturation is reduced and mass transfer is enhanced; by specific purification (Comparative Example 4), the capture accuracy of the target product is improved; by dynamic control (Comparative Example 5 / 6 / 8), the stability of enzyme activity is maintained and the separation conditions are optimized. Example 1 is significantly superior to the traditional process in terms of cost (yield↑), quality (purity↑), and efficacy (MIC↓), and has the potential for large-scale application. Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the examples shown and described herein.

Claims

1. A method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method, characterized in that: The following steps are involved: Step 1, crushing and sieving cottonseed meal to obtain cottonseed meal particles, mixing the cottonseed meal particles with a sodium bicarbonate solution having a mass concentration of 0.8% at a solid-liquid ratio of 1:10, and subjecting the mixture to microwave, oscillation, and suction filtration to obtain defatted cottonseed meal; Step 2: Mixing defatted cottonseed meal and phosphate buffer in a mass ratio of 1:12, adding a composite hydrolase system accounting for 2.5% of the substrate mass, wherein the composite hydrolase system is composed of Bacillus subtilis alkaline protease, Aspergillus oryzae neutral protease and Bacillus keratinase in an enzyme activity unit ratio of 5:3:2, and treating in a coupled field of alternating ultrasound and microwave to obtain a hydrolyzate; Step 3: The hydrolyzate is subjected to cross-flow filtration through a ceramic membrane to retain components with a molecular weight cutoff of 10 kDa or more, and then subjected to secondary filtration through a hollow fiber ultrafiltration membrane to retain components with a molecular weight cutoff of 3 kDa or more to obtain an ultrafiltrate; Step 4, injecting the ultrafiltrate into an agarose gel chromatography column modified with Aeromonas hydrophila outer membrane protein at a flow rate of 2 mL / min for purification to obtain an active component; Step 5: purify the active component by semi-preparative high performance liquid chromatography equipped with an evaporative light scattering detector to obtain an extract, and freeze-dry the extract to obtain the product.

2. The method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method according to claim 1, characterized in that: In step 1, the microwave, oscillation and filtration treatments are specifically as follows: S1.1, placing a mixture of cottonseed meal particles and sodium bicarbonate solution in a closed microwave reactor, setting the microwave power density to 15 W / g material, using a pulse mode to implement microwave irradiation for 10 seconds and then stop for 5 seconds, and repeating for 6 cycles, with a total irradiation time of 3 minutes; S1.2, transfer the microwave-treated mixed material to a constant temperature oscillator, set the oscillation frequency to 250 rpm and the amplitude to 25 mm, perform oscillation degreasing under the condition of maintaining 45±0.3°C with the PID temperature control module, and automatically switch the forward and reverse directions of the drive motor every 5 minutes, with a total oscillation time of 35 minutes; S1.

3. The mixed liquid after shaking is introduced into a Buchner funnel for two-stage filtration. The first stage is filtered at a vacuum degree of -0.08 MPa for 8 minutes, and the second stage is filtered at a vacuum degree of -0.09 MPa for 5 minutes. The filter cake is countercurrently washed twice with a 0.8% sodium bicarbonate solution at 4°C. The amount of washing solution used each time is 1.5 times the mass of the filter cake. After washing, the filter cake is frozen to -20°C at a cooling rate of 5°C / min under the protection of a nitrogen atmosphere to form a solidified defatted cottonseed meal with a porous skeleton structure.

3. The method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method according to claim 2, characterized in that: In step S1.1, the peak temperature of the material during intermittent microwave treatment is 62-65°C, and the temperature difference between material layers is ≤3°C through pulse control; In step S1.2, the forward and reverse switching oscillation makes the material motion trajectory form an 8-shaped vortex, and high-speed camera observation shows that the material dispersion is increased to 0.82-0.85; In step S1.3, the filter cloth for two-stage filtration is made of polytetrafluoroethylene-coated glass fiber with a pore size distribution of 5-10 μm and a porosity of 78-82%.

4. The method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method according to claim 1, characterized in that: In step 2, the activity unit of Bacillus subtilis alkaline protease was determined by the Folin phenol method, with 1% casein as the substrate, reacting at pH 9.0 and 40°C for 10 minutes, and then immediately adding trichloroacetic acid to terminate the reaction; the activity unit of Aspergillus oryzae neutral protease was determined by using 2% hemoglobin as the substrate, reacting at pH 7.0 and 50°C for 15 minutes, and then terminating the reaction by ammonium sulfate precipitation method; Bacillus keratinase needs to be activated in a pH 6.5 citric acid buffer at 45°C for 20 minutes in advance, and the mass ratio of keratinase to alkaline protease after activation is (2.8-3.2):5; In step 2, the composite hydrolase system is added to the reaction system in two stages: in the first stage, 60% of the total enzyme system amount of Bacillus subtilis alkaline protease is added, and the reaction is treated in an ultrasonic-microwave coupling field for 90 minutes; in the second stage, the remaining 40% of Bacillus subtilis alkaline protease, Aspergillus oryzae neutral protease and activated Bacillus keratinase are added, and the reaction is continued in an ultrasonic-microwave coupling field for 90 minutes; The dynamic enzyme activity ratio of the composite hydrolase system in the reaction system is monitored and regulated by an online pH-ORP sensor. When the redox potential value drops to -120mV, 0.1mol / L sodium hydroxide solution is automatically added to maintain the pH in the range of 8.3-8.

7.

5. The method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method according to claim 1, characterized in that: In step 2, ultrasound and microwave coupling field treatment is applied alternately, specifically: Step S2.1, placing the reaction system containing the composite hydrolase system in a closed reactor equipped with a rotating bracket, first starting the microwave radiation stage, setting the microwave frequency to 2450 MHz and the power density to 8 W / g reaction system, and controlling the material tray to rotate at a constant speed of 6 rpm; Step S2.2, continue the treatment for 30 seconds until the material temperature rises to 50±2°C, then switch to the ultrasonic treatment stage, turn off the microwave and start the 28kHz ultrasonic generator, set the power density to 3.8W / cm², and use the intermittent emission mode to work for 60 seconds, wherein the material is paused for 5 seconds after every 15 seconds of work, and at the same time, 10°C cooling water is introduced into the reactor jacket through the external circulation pump; Step S2.3, repeat the above microwave-ultrasonic alternating treatment cycle 20 times, with a total duration of 1800 seconds, wherein the material temperature is detected after each cycle, and when the temperature exceeds 55°C, the cooling water introduction time is automatically extended to 10 seconds; Step S2.4, in the last three cycles, the microwave power density was adjusted to 10 W / g, and the ultrasonic working period was adjusted to continuous emission for 45 s; Step S2.5: After the treatment is completed, the reaction system is transferred to an ice water bath for rapid cooling, and the temperature is reduced to below 25° C. within 2 minutes to obtain a hydrolyzate.

6. The method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method according to claim 1, characterized in that: Step three is as follows: Step S3.1, preheat the hydrolyzate to 50±2°C and then pump it into a tubular ceramic membrane assembly. The ceramic membrane is made of α-alumina, with an effective pore size of 30nm, an operating pressure of 0.25MPa, and a cross-flow velocity of 3.0m / s. The permeate collected in the initial 10min is returned to the raw material tank for cyclic filtration, and the filtrate is collected after the transmembrane pressure difference stabilizes at 0.18MPa; Step S3.2, cooling the ceramic membrane filtrate to 25±1°C through a plate heat exchanger, adjusting the pH to 6.8-7.2, and then entering the hollow fiber ultrafiltration membrane system. The ultrafiltration membrane is made of polyethersulfone, with a cut-off molecular weight of 3kDa and an operating pressure of 0.12MPa. The system is operated in a pulse backwash mode, with a 2s backwash for every 15min of filtration, and a flushing pressure of 0.30MPa, and the ultrafiltrate is collected; The two-stage membrane separation process is monitored by an online turbidity sensor in conjunction with an ultraviolet absorption detector. When the A280 value of the filtrate exceeds 0.85, the membrane cleaning program is automatically triggered, and in-situ cleaning is performed using a mixed cleaning solution of 0.1 mol / L sodium hydroxide and 0.5% sodium dodecyl sulfate.

7. The method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method according to claim 1, characterized in that: The preparation of Aeromonas hydrophila outer membrane protein modified agarose gel comprises the following steps: Step a, treating Aeromonas hydrophila with an ultrasonic disruptor, taking the supernatant after centrifugation, and purifying the outer membrane protein by ammonium sulfate fractionation precipitation, DEAE anion exchange chromatography and hydrophobic chromatography in sequence, wherein the purity is ≥95% as detected by SDS-PAGE; Step b, after swelling the agarose gel CL-6B carrier with 0.5 mol / L NaHCO3 buffer for 24 h, mixing it with 10 mg / mL outer membrane protein solution at a volume ratio of 1:3, adding a carbodiimide coupling agent with a final concentration of 5 mmol / L, and reacting it at 4°C with low-speed stirring for 18 h; Step c, after the reaction is completed, the unbound sites are blocked with a pH 8.0 Tris-HCl buffer containing 0.1 mol / L ethanolamine for 2 h, and the mixture is washed alternately three times with a pH 7.4 buffer containing 0.5 mol / L NaCl and pure water, and finally stored in a PBS buffer containing 0.02% sodium azide to obtain; Among them, the protein coupling efficiency is ≥82%, the ligand density is 9.2-9.8 mg protein / mL gel, the upper limit of column pressure tolerance is 1.5 MPa, and the ligand detachment rate is <3% after 50 adsorption-elution cycles.

8. The method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method according to claim 7, characterized in that: The specific process of chromatography column purification is as follows: Step S4.1, filling the Aeromonas hydrophila outer membrane protein modified agarose gel at a constant pressure of 0.5 MPa to obtain a chromatography column with a column bed height of 45 cm, and after filling, using a pH 7.4 PBS buffer containing 0.05% Triton X-100 with 3 times the column volume for flow-through washing; Step S4.2, add equilibration buffer containing 0.15 mol / L NaCl to the chromatography column, equilibrate at a flow rate of 1.2 mL / min until the conductivity is stabilized at 12.5±0.3 ms / cm, then load the sample, control the sample amount not to exceed 78% of the column dynamic loading capacity, monitor the UV280 absorption value during the loading period, and terminate the loading when the breakthrough peak reaches 5% of the baseline; Step S4.3, gradient elution using dual gradient mode: in the first stage, the NaCl concentration was increased to 0.22 mol / L at a rate of 0.8% B / min, and in the second stage, the histidine concentration was increased to 12 mmol / L at a rate of 1.2% B / min. The eluate was collected simultaneously and sterilized with a 0.22 μm filter membrane; The screening criteria for active components are: SDS-PAGE test shows that the intensity of the 14-18 kDa characteristic band accounts for ≥65%, the deviation of the reversed-phase chromatography retention time from the standard is ≤±0.25 min, and the antibacterial activity potency is ≥3200 IU / mg.

9. The method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method according to claim 1, characterized in that: The semi-preparative HPLC purification in step 5 meets the following separation conditions: a) Use a C18 chromatographic column with polyethylene glycol chains grafted on the surface, and maintain the column temperature at 27±0.3℃ through a double-stage semiconductor temperature control system; b) Mobile phase A is a pH 4.5 aqueous solution containing 0.05 mol / L ammonium formate, and mobile phase B is an acetonitrile-methanol mixed solution containing 0.05 mol / L ammonium formate; c) The gradient program was set as follows: 15% B for 0-2 min, nonlinear gradient to 38% B for 2-15 min and held for 15-18 min, then increased to 45% B at a rate of 5% B / min within 18-20 min.

10. The method for preparing dephenolized cottonseed protein antimicrobial peptides by enzymatic method according to claim 9, characterized in that: When the proportion of mobile phase B is less than 30%, the pressure is set to 3.2 bar. When the proportion of B is ≥30%, the pressure is linearly increased to 4.5 bar, and the evaporation temperature is adjusted from 75°C to 82°C simultaneously; Before injection, the active component was premixed with mobile phase A at a volume ratio of 1:3, filtered through a 0.22 μm PVDF membrane, and the injection volume was controlled to be 500 μL and the flow rate to be 1.8 mL / min; The detector signal slope dS / dt>5 mV / s, the mass spectrometer online detected an ion peak with a molecular weight of 1482±3Da, and the conductivity fluctuation was ≤±8% within the retention time window of 17.5-18.5min; the collected fractions were immediately bubbled with nitrogen to remove the organic solvent, and the residual acetonitrile content was controlled to be <0.1%.

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