A glycosylated solid-phase extraction packing material and its application in the enrichment and purification of insect-derived antimicrobial peptides

Abstract

This invention discloses a glycosyl solid-phase extraction packing material and its application in the enrichment and purification of insect-derived antimicrobial peptides, belonging to the field of biomaterials technology. The invention includes a packing material preparation process: the core antigen structure of lipopolysaccharide is activated at the end groups in an aqueous phase and polymerized to form a glycan polymer. This glycan polymer is then chemically coupled to the surface of polystyrene microspheres, serving as the packing material to achieve specific adsorption of antimicrobial peptides from biological samples. This invention can rapidly and selectively adsorb antimicrobial peptides from the filtrate of lyophilized insect powder and release them under mild elution conditions. It has the advantages of simple operation, continuous operation, and large-scale separation and purification.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically, it relates to a glycosylated solid-phase extraction packing material and its application in the enrichment and purification of insect-derived antimicrobial peptides. Background Technology

[0002] The overuse of antibiotics has made bacterial resistance an increasingly serious global public health issue. A significant portion of antibiotic overuse cases stems from the addition of antibiotics to animal feed. To reduce the risk of disease in poultry and livestock, farmers often add antibiotics to their feed, a large portion of which spills into natural water environments. This not only causes serious environmental problems but also contributes to antibiotic resistance in humans, leading to a year-on-year decline in the effectiveness of clinical antibiotic treatments. Insect-derived antimicrobial peptides have attracted considerable attention as potential alternatives to antibiotics. Due to their unique growth environment, insects' immune systems spontaneously produce antimicrobial peptides to cope with high concentrations of harmful substances. Furthermore, these insects (e.g., fruit flies, black soldier flies) are also used to process municipal solid waste, stably producing antimicrobial peptide products with broad-spectrum antibacterial activity (e.g., cephalosporins and insect defensins) while digesting and decomposing kitchen waste.

[0003] To achieve high added value and comprehensive utilization of insect-derived antimicrobial peptides, it is essential to first solve the problem of efficient separation and purification processes for antimicrobial peptides. This is because the dried powder raw material of insect larvae contains not only antimicrobial peptides but also a large number of contaminants such as miscellaneous proteins, genetic material, and cell debris. These are potential allergens and endotoxins, and must be strictly removed to ensure the safety of the antimicrobial peptides.

[0004] Antimicrobial peptides, as polypeptide products with a molecular weight range of 4K to 6K, require multiple chromatographic steps for traditional separation and purification, including C18 reversed-phase chromatography, ion-exchange chromatography, and gel permeation chromatography. Gel permeation chromatography, based on the principle of size exclusion, is the rate-limiting step, significantly reducing the production efficiency of antimicrobial peptides. Furthermore, reversed-phase high-performance liquid chromatography (HPLC) separation requires the use of large amounts of organic solvents, including acetonitrile and methanol, which not only poses certain safety risks but also requires environmentally compliant facilities with organic solvent handling qualifications, undoubtedly increasing production costs.

[0005] For methods of enriching and purifying antimicrobial peptides, existing technologies have related applications. For example, patent application number 2019109738273 discloses a method for extracting antimicrobial peptides from mussel processing waste using ultrasonic homogenization, which includes the following steps: a) pretreatment; b) crude extraction; c) solid-phase extraction: dissolving the crude extract in trifluoroacetic acid solution and centrifuging to obtain supernatant B, passing the supernatant through a solid-phase extraction column 1-2 times, and then eluting sequentially with trifluoroacetic acid solution and acetonitrile solution, collecting the acetonitrile eluent. The crude antimicrobial peptide A was obtained by freeze-drying; the concentration of trifluoroacetic acid solution was 0.008-0.012 wt%, the concentration of acetonitrile solution was 35-45 wt%, and the solid phase extraction column was packed with C18 packing material; d) Gel column separation: the crude antimicrobial peptide A was dissolved in water, centrifuged to obtain supernatant C, and then separated and purified in a gel column. The elution peaks were collected and combined and freeze-dried to obtain crude antimicrobial peptide B. The gel column was a Sephadex G-10 gel column, and the eluent was deionized water; e) Chromatographic column separation. For example, in patent application number 200710200375.2, entitled "Separation Method of Housefly Secretory Antimicrobial Peptides and Its Products and Applications," the extraction method for antimicrobial peptides involves solid-phase extraction. Specifically, the crude antimicrobial peptide extract after dialysis is loaded onto a Sep-pak C18 Cartridge solid-phase extraction column, and eluted stepwise with 5 ml of 0.05% trifluoroacetic acid solution containing 0%–80% methanol at a flow rate of 1 ml / min. Each eluent fraction is freeze-dried to remove methanol and trifluoroacetic acid, dissolved in ultrapure water, and its antimicrobial activity is tested. The active fraction is collected, concentrated, freeze-dried, dissolved in ultrapure water, centrifuged at 12000 rpm for 10 min, and the supernatant is collected and stored at -20°C for later use. As can be seen from the methods in the above applications, the solid-phase extraction used in the separation stage of the extract in the prior art requires the use of organic solvents as the mobile phase, which not only poses environmental pollution problems but also makes the overall operation process quite complex.

[0006] Therefore, there is an urgent need to develop an environmentally friendly and efficient method for the separation and purification of insect-derived antimicrobial peptides. Summary of the Invention

[0007] In view of the above-mentioned problems existing in the prior art, one of the objectives of the present invention is to provide a simpler and more efficient antimicrobial peptide separation and purification technology. Another objective of the present invention is to provide a green separation and purification process using an all-aqueous solution without the use of organic solvent mobile phase. A further objective of the present invention is to provide a solid-phase extraction separation method that can be achieved through simple specific adsorption and gentle elution without the use of expensive high-performance liquid chromatography equipment.

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] A first aspect of the present invention provides a glycosyl solid-phase extraction packing material, the preparation method of which includes the following steps:

[0010] 1) Based on the interaction between antimicrobial peptides and bacterial lipopolysaccharides, design corresponding glycoantigen molecular structures, disassemble the lipopolysaccharide structure, and use the combination of core glyco fragments to replace the complete bacterial lipopolysaccharide structure;

[0011] 2) By using an aqueous unprotected glycosylation reaction, unsaturated bonds are directly introduced at the reduction end of the sugar chain to prepare sugar monomers, and then sugar polymers are prepared using sugar monomers. The sugar polymers are then chemically coupled and modified onto the surface of polystyrene microspheres.

[0012] 3) Mix polystyrene microspheres modified with different sugar chain polymers as a filler for glycosyl solid phase extraction.

[0013] As a preferred embodiment of the present invention, the method of chemically coupling and modifying the sugar polymer on the surface of polystyrene microspheres in step 2) is as follows: the terminal NHS ester of the sugar polymer is activated, and then reacted with the surface-aminated polystyrene microspheres.

[0014] In a preferred embodiment of the present invention, the diameter of the surface-aminated polystyrene microspheres is 1-150 μm, more preferably 10-50 μm.

[0015] In a preferred embodiment of the present invention, the glycoantigen structure includes the following A1-A5 structures:

[0016]

[0017] As a preferred embodiment of the present invention, the general structural formula of the glycan polymer is as follows: Where n = 15 to 100.

[0018] In a preferred embodiment of the present invention, step 2) includes the following steps:

[0019] The sugar monomer (0.81 mmol), RAFT reagent (2.174 mg, 0.0081 mmol), and azobisisobutyronitrile (0.44 mg) were dissolved in dimethyl sulfoxide / water (1 mL / 1 mL), degassed with nitrogen for 2 hours, and reacted at 70 °C under anaerobic conditions for 24 hours. The mixture was purified by dialysis (molecular weight cutoff 3500) and freeze-dried to obtain the sugar polymers.

[0020] The above-mentioned glycan polymer was activated with terminal NHS esters: 0.1 g of the glycan polymer was dissolved in 2.0 mL of dimethyl sulfoxide, 3 mg of N-hydroxysuccinimide and 6 mg of 1,3-dicyclohexylcarbodiimide were added, and 5 μL of pyridine were added. The mixture was stirred at room temperature for 24 hours under nitrogen protection, purified by dialysis (molecular weight cutoff 3500), and freeze-dried to obtain the glycan polymer with terminal NHS esters activated.

[0021] As a preferred embodiment of the present invention, step 3) is specifically implemented as follows:

[0022] 10 mg of NHS ester-activated glycan polymer was dissolved in 10 mL of Tris-hydrochloric acid (pH 8.5) buffer solution. 5 mL of commercially available surface-aminated polystyrene microspheres (10-50 μm) were added and stirred at room temperature for 24 hours. After the surface glycan polymer modification was completed, the mixture was washed three times with PBS buffer to obtain the glycosyl solid-phase extraction packing material.

[0023] A second aspect of the invention provides the application of glycosylated solid-phase extraction packing material in the preparation of solid-phase extraction columns.

[0024] A third aspect of the present invention provides a solid-phase extraction column, comprising the aforementioned glycosyl solid-phase extraction packing material.

[0025] The fourth aspect of the present invention provides a method for enriching and purifying insect-derived antimicrobial peptides using glycosylated solid-phase extraction packing, comprising: pretreating insect larvae freeze-dried powder, adsorbing and enriching the antimicrobial peptides using glycosylated solid-phase extraction packing, and then collecting the purified antimicrobial peptides by elution.

[0026] As a preferred embodiment of the present invention, the method for enriching and purifying insect-derived antimicrobial peptides includes the following steps:

[0027] 1) Take the freeze-dried powder of insect larvae, dissolve the larvae powder in PBS buffer solution, add it to an ultrafiltration centrifuge tube, and obtain the filtrate;

[0028] 2) Add the filtrate to a solid-phase extraction column packed with glycosyl solid-phase extraction packing material, let it stand at room temperature, and wash with PBS buffer.

[0029] 3) Change the pH of the mobile phase to acidic and elute the extraction column after step 2). Adjust the pH of the obtained antimicrobial peptide eluent to 7.4, freeze-dry, and obtain the purified antimicrobial peptide product.

[0030] In a preferred embodiment of the present invention, in the purification step, in step 1), an ultrafiltration centrifuge tube with a molecular weight cutoff of 5K to 20KDa is added and centrifuged at 4 degrees Celsius and 8000 to 15000 rpm for 5 to 20 minutes.

[0031] More preferably, the molecular weight cutoff is 8–12 kDa.

[0032] In a preferred embodiment of the present invention, the time for standing at room temperature in step 2) is 5 to 60 minutes.

[0033] In a preferred embodiment of the present invention, the pH value of the mobile phase is changed to 3-6 in step 3).

[0034] The specific purification process is as follows: Larval powder is dissolved in PBS buffer solution and passed through an ultrafiltration centrifuge tube. The filtrate is then purified using a solid-phase extraction column packed with glycosylated solid-phase extraction packing material, with PBS buffer solution as the mobile phase. The lectin proteins contained in the insect cells are retained by the ultrafiltration tube, while lower molecular weight antimicrobial peptides can pass through the ultrafiltration membrane into the filtrate. Under the conditions of a buffer mobile phase at pH 7.4, the glycosylated solid-phase extraction column specifically adsorbs antimicrobial peptides, while other impurities elute directly. Changing the pH of the mobile phase to acidic weakens the affinity between the glycans and the antimicrobial peptides, achieving elution. Finally, the pH of the resulting antimicrobial peptide eluent is adjusted to 7.4, and after lyophilization, the purified antimicrobial peptide product is obtained. The entire separation and extraction process does not require the use of organic solvents or high-performance liquid chromatography (HPLC) equipment.

[0035] As a preferred embodiment of the present invention, insects from which the method of the present invention can be used to extract antimicrobial peptides include fly larvae, black soldier flies, or grain worms.

[0036] Beneficial effects:

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] (1) The glycosylated solid-phase extraction packing of the present invention firstly designs the corresponding glycoantigen molecular structure based on the interaction between antimicrobial peptides and bacterial lipopolysaccharides, disassembles the complex lipopolysaccharide structure, and uses the combination of core sugar fragments to replace the complete bacterial lipopolysaccharide structure; it then uses an aqueous phase unprotected glycosylation reaction to directly introduce unsaturated bonds at the reduction end of the sugar chain to prepare sugar monomers; at the same time, it mixes and uses polystyrene microspheres modified with different sugar chain polymers as the stationary phase of the packing, thereby providing a broad-spectrum antimicrobial peptide adsorption effect.

[0039] (2) The glycosyl solid-phase extraction packing of the present invention utilizes characteristic oligosaccharide fragments of lipopolysaccharide (e.g., A1→A5), introduces double bonds at the reduced end through end-group activation, and then obtains glycopolymers (e.g., GP1→GP5) with oligosaccharide antigens on the side chains and NHS active ester structures at the ends through living polymerization and further chemical reactions. The glycopolymers are then chemically coupled and modified on the surface of microspheres to prepare an extraction packing with high binding force against antimicrobial peptides, enabling efficient separation and purification in the aqueous phase. The solid-phase extraction packing prepared by the method of the present invention is a highly efficient separation and purification packing. The operation process is simple, and the use of organic solvent mobile phase is avoided, realizing the purification process in the entire aqueous phase, which is more environmentally friendly, reliable, and has a wide range of applications.

[0040] (3) The glycosyl solid-phase extraction packing material prepared in this invention is a polystyrene microsphere with a chemically modified sugar chain polymer. Because the sugars on the side chains of the polymer enhance the interaction between sugar and protein through the glycocluster effect, it has a stronger binding force with antimicrobial peptides than the brush structure of a single sugar chain, and provides more action sites, thus having a more effective adsorption effect on antimicrobial peptides, thereby improving the separation and purification efficiency of antimicrobial peptides.

[0041] (4) The present invention utilizes a glycosylated solid-phase extraction packing material to separate and purify insect-derived antimicrobial peptides. The high binding force between the packing material and the antimicrobial peptides makes separation in the aqueous phase possible. This is a simpler and more efficient antimicrobial peptide separation and purification technology. It does not require the use of organic reagents, nor does it require expensive high-performance liquid chromatography equipment. It is a solid-phase extraction method that can be achieved through simple specific adsorption and gentle elution. The operation is simple and convenient. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the structure of an antimicrobial peptide glycosyl solid-phase extraction packing material;

[0043] Figure 2 A schematic diagram of the oligosaccharide antigen molecule structure (A1→A5) that binds to the antimicrobial peptide;

[0044] Figure 3 A schematic diagram illustrating the end-group activation of oligosaccharide molecules and the preparation of glycan polymers (GP1→GP5);

[0045] Figure 4 The gel permeation chromatography characterization spectra are for the separated components 1, 2, and 3. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments.

[0047] The following embodiments are for illustrative purposes only, and the scope of protection of the present invention is not limited to these embodiments. Those skilled in the art can achieve the objectives of the present invention based on the above disclosure and the ranges of the parameters.

[0048] Example 1

[0049] I. Preparation of glycosyl solid-phase extraction packing material:

[0050] A) Preparation of glycan polymers:

[0051] 1) Prepare glycomonomers (M1, M2, and M5) containing A1, A2, and A5 antigenic structures, and glycomonomers (M3 and M4) containing A3 and A4 antigenic structures, respectively. All five glycoantigen fragment structures (A1→A5) are readily available for purchase. The A1→A5 molecular structure is shown below. Figure 2 As shown. A3 was purchased from Shanghai Huicheng Biotechnology Co., Ltd., item number: GY143; A1, A2, A4, and A5 were purchased from Sugar Lock Engineering Research Institute Co., Ltd., as customized standard products, item numbers: CA1, CA2, CA3, and CA4.

[0052] The method for synthesizing glycomonomers containing A1, A2, and A5 antigen structures is as follows: Add 0.1 g of 2-chloro-1,3-dimethylimidazoline chloride to an acetonitrile / water (2 mL / 2 mL) solution of glycoantigen (0.2 mmol) and triethylamine (0.25 mL), and stir the mixture in an ice-water bath for 1 hour. Then, add 0.2 g of sodium thiosulfate and 0.8 mL of hydrochloric acid solution, and continue stirring at room temperature for 10 hours. Next, add 0.08 mL of 2 M potassium carbonate solution, and continue stirring at room temperature for 2 hours. Then, add 0.1 mL of an aqueous solution of sodium sulfide nonahydrate (53 mg), and stir at room temperature for 1 hour. Finally, add 1 mL of an acetonitrile / water solution of N,N'-methylenebisacrylamide (0.1 g), and continue stirring at room temperature for 24 hours. After the reaction was completed, the mixture was concentrated under vacuum, dissolved in 5 mL of aqueous solution, extracted three times with chloroform, concentrated under vacuum, and separated by high performance liquid chromatography (using a C18 reversed-phase column with a gradient mobile phase of methanol / water = 5 / 95 → 50 / 50; and an Xbridge BEH Amide normal-phase column with a mobile phase of 0.05% trifluoroacetic acid acetonitrile aqueous solution with a gradient of acetonitrile / water = 95 / 5 → 10 / 90). After freeze-drying, the glycan monomers (M1, M2, and M5) were obtained.

[0053] The synthesis method of glycomonomers containing A3 and A4 antigen structures is as follows: Sodium thiosulfate (0.3 g) and 2-chloro-1,3-dimethylimidazoline chloride (0.1 g) were added to an acetonitrile / water (2 mL / 2 mL) solution of glycoantigen (0.2 mmol) and triethylamine (0.25 mL), respectively. The reaction mixture was stirred in an ice-water bath for 1.5 hours. Subsequently, an aqueous solution of sodium sulfide nonahydrate (53 mg) (0.1 mL) was added, and the mixture was stirred at room temperature for 1 hour. Finally, an aqueous solution of N,N'-methylenebisacrylamide (0.1 g) in acetonitrile (1 mL / 1 mL) was added, and the mixture was stirred at room temperature for another 24 hours. After the reaction was completed, the mixture was concentrated under vacuum, dissolved in 5 mL of aqueous solution, extracted three times with chloroform, concentrated under vacuum, and separated by high performance liquid chromatography (using a C18 reversed-phase column with a gradient mobile phase of methanol / water = 5 / 95 → 50 / 50; and an Xbridge BEH Amide normal-phase column with a mobile phase of 0.05% trifluoroacetic acid acetonitrile aqueous solution with a gradient of acetonitrile / water = 95 / 5 → 10 / 90). After freeze-drying, the glycan monomers (M3 and M4) were obtained.

[0054] B) Preparation of glycan polymers using sugar monomers, as follows:

[0055] The sugar monomer (0.81 mmol), RAFT reagent (2.174 mg, 0.0081 mmol), and azobisisobutyronitrile (0.44 mg) were dissolved in dimethyl sulfoxide / water (1 mL / 1 mL). The mixture was degassed with nitrogen for 2 hours and reacted at 70°C under anaerobic conditions for 24 hours. The resulting product was purified by dialysis (molecular weight cutoff 3500) and freeze-dried to obtain the sugar polymers (GP1→GP5). The preparation process is as follows: Figure 3 As shown.

[0056] 3) The above-mentioned glycan polymer (GP1→GP5) was activated by terminal NHS ester: 0.1 g of glycan polymer (GP1→GP5) was dissolved in 2.0 mL of dimethyl sulfoxide, 3 mg of N-hydroxysuccinimide and 6 mg of 1,3-dicyclohexylcarbodiimide were added, and 5 μL of pyridine was added. The mixture was stirred at room temperature for 24 hours under nitrogen protection, purified by dialysis (molecular weight cutoff 3500), and freeze-dried to obtain the terminal NHS ester activated glycan polymer (NHS-activated GP1→GP5).

[0057] C) Preparation of Glycosyl-Modified Packing Material: Surface-aminated polystyrene microspheres were added to NHS ester-activated glycan polymers to prepare the chromatographic packing material. Specifically, 10 mg of NHS ester-activated glycan polymer was dissolved in 10 mL of Tris-hydrochloric acid (pH 8.5) buffer solution, and 5 mL of commercially available surface-aminated polystyrene microspheres (10-50 μm) were added. The mixture was stirred at room temperature for 24 hours to complete the surface modification of the glycan polymer. After washing three times with PBS buffer, the glycosylated solid-phase extraction packing material was obtained. The structure of the packing material is shown below. Figure 1 As shown.

[0058] II. A solid-phase extraction column was prepared using the above-mentioned packing material. The packing material in the solid-phase extraction column included five types: polystyrene microspheres with glycosylated surfaces modified with GP1, GP2, GP3, GP4, and GP5, and these five packing materials were mixed in equal proportions. This resulted in a broad-spectrum separation of antimicrobial peptides.

[0059] It should be noted that the filler materials modified with GP1, GP2, GP3, GP4, or GP5 glycan polymers formed using the method of this invention can be used alone or in combination. All can achieve highly efficient enrichment and purification.

[0060] (ii) Application of packing materials in the enrichment and separation of insect antimicrobial peptides

[0061] The solid-phase extraction column prepared in this embodiment (such as...) Figure 1 The steps for separating and purifying fly larvae are as follows:

[0062] 5g of lyophilized fly larvae powder from the Nanjing Liuhe District Kitchen Waste Treatment Center was dissolved in 10mL of PBS buffer and added to an ultrafiltration centrifuge tube with a molecular weight cutoff of 20kDa. The mixture was centrifuged at 15,000 rpm for 5 minutes at 4°C. 1mL of the lower layer was collected and added to a 2.0mL solid-phase extraction column containing polystyrene microspheres modified with glycan polymers (GP1→GP5). The column was incubated at room temperature for 60 minutes, washed with 10mL of PBS buffer, and then eluted with PBS solution at pH 6. Elution was stopped when the protein content was below 0.06mg / mL. The protein content in the eluent was determined using a Nanotrap microspectrophotometer. The pH of the eluent was adjusted to 7.4 to obtain fraction 1.

[0063] To test the bioactivity of the purified antimicrobial peptides, the antibacterial effects of isolated fraction 1 against Staphylococcus aureus and Escherichia coli were evaluated, and the results are shown in Table 1.

[0064] Table 1 Evaluation of the antibacterial effect of isolated component 1

[0065]

[0066]

[0067] The MIC determination method uses the broth dilution method: The suspensions of Staphylococcus aureus and Escherichia coli were diluted in broth to a concentration of 10

[0075] ,

[0074] , ,

[0077] ,

[0076] , , , , CFU / mL. Add the directly freeze-dried samples of the centrifuged liquid with different concentration gradients, separated component 1, and the PBS solution of tetracycline into a 96-well plate, and then add an equal volume of the diluted bacterial suspension to each well. The PBS buffer solution is used as a negative control. After culturing for 12 - 16 hours, add an aqueous solution of resazurin and continue culturing for 2 - 4 hours. Determine the MIC of each sample directly according to the color change. Pink indicates the presence of live bacteria, and purple represents the absence of live bacteria.

[0068] As can be seen from the above results, separated component 1 after the separation treatment of the present invention has a significant antibacterial effect.

[0069] Example 2

[0070] The solid-phase extraction column prepared in Example 1 was used for the separation and purification of black soldier fly, and the steps are as follows:

[0071] Take 5 g of freeze-dried black soldier fly powder, dissolve it in 10 mL of PBS buffer solution, add an ultrafiltration centrifuge tube with a molecular weight cut-off of 5 KDa, and centrifuge at 8000 rpm at 4 °C for 20 minutes. Take 1 mL of the lower-layer centrifuged liquid and add it to a pre-packed column (2.0 mL) of polystyrene microspheres modified with glycan polymers (GP1→GP5), let it stand at room temperature for 5 minutes, wash it with 10 mL of PBS buffer solution, and then elute it with a PBS solution with pH = 3. Manually collect the eluate, and stop eluting when the protein content is lower than 0.06 mg / mL. Use a Nanotrap ultra-micro spectrophotometer to measure the protein content in the eluate to obtain separated component 2. The antibacterial effect evaluation of Example 2 is shown in Table 2 below.

[0072] Table 2 Antibacterial effect evaluation of separated component 2

[0073] <00​​​​​​​​​Take 5g of commercially available dried powder of *Polygonum multiflorum* (a type of Chinese medicinal herb), dissolve it in 10mL of PBS buffer, and add it to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kDa. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Take 1mL of the lower layer and add it to a 2.0mL solid-phase extraction column containing polystyrene microspheres modified with glycan polymers (GP1→GP5). Let it stand at room temperature for 10 minutes, wash with 10mL of PBS buffer, and then elute with PBS solution at pH=5. Stop elution when the protein content is below 0.06mg / mL. Measure the protein content in the eluent using a Nanotrap ultra-micro spectrophotometer. Adjust the pH of the eluent to 7.4 to obtain fraction 3. The antibacterial effect of fraction 3 is evaluated as shown in Table 3 below.

[0078] To test the bioactivity of the purified antimicrobial peptides, the antibacterial effects of isolated fraction 3 against Staphylococcus aureus and Escherichia coli were evaluated, and the results are shown in Table 3.

[0079] Table 3 Evaluation of the antibacterial effect of separated component 3

[0080]

[0081] As can be seen from the above results, the separated component 3 after the separation treatment of the present invention has a significant antibacterial effect. Figure 4 The gel permeation chromatography characterization spectra of components 1, 2, and 3 are shown in Table 4. As can be seen from the results, components 1, 2, and 3 of this invention all possess high purity.

Claims

1. A glycosyl solid-phase extraction packing material, characterized in that, The preparation method includes the following steps: 1) Based on the interaction between antimicrobial peptides and bacterial lipopolysaccharides, design corresponding glycoantigen molecular structures, and replace the complete bacterial lipopolysaccharide structure by dissecting the lipopolysaccharide structure and using the combination of core sugar fragments. 2) By using an aqueous unprotected glycosylation reaction, unsaturated bonds are directly introduced at the reduction end of the sugar chain to prepare sugar monomers, and then sugar polymers are prepared using sugar monomers. The sugar polymers are then chemically coupled and modified onto the surface of polystyrene microspheres. 3) Mix polystyrene microspheres modified with different sugar chain polymers as a filler for glycosyl solid phase extraction.

2. The glycosyl solid-phase extraction packing material according to claim 1, characterized in that: In step 2), the method of chemically coupling and modifying the sugar polymer onto the surface of polystyrene microspheres is as follows: the terminal NHS ester of the sugar polymer is activated, and then reacted with the surface-aminated polystyrene microspheres.

3. The glycosyl solid-phase extraction packing material according to claim 1 or 2, characterized in that, The polystyrene microspheres with surface aminated modification have a diameter of 1–150 μm.

4. The glycosyl solid-phase extraction packing material according to claim 1 or 2, characterized in that: The general structural formula of glycan polymers is shown below: Where n = 15 to 100.

5. The application of the glycosyl solid-phase extraction packing material according to any one of claims 1-4 in the preparation of solid-phase extraction columns.

6. A solid-phase extraction column, characterized in that, Includes the glycosyl solid-phase extraction packing material of claim 1.

7. A method for enriching and purifying insect-derived antimicrobial peptides using the glycosyl solid-phase extraction packing material described in claim 1, characterized in that, Includes the following steps: After pretreatment of freeze-dried insect larvae powder, the antimicrobial peptides were adsorbed and enriched using the glycosylated solid-phase extraction packing material, and then collected by elution to obtain purified antimicrobial peptides.

8. The method for enriching and purifying insect-derived antimicrobial peptides according to claim 7, characterized in that, Includes the following steps: 1) Take the freeze-dried powder of insect larvae, dissolve the larvae powder in PBS buffer solution, add it to an ultrafiltration centrifuge tube, and obtain the filtrate; 2) Add the filtrate to a solid-phase extraction column packed with glycosylated solid-phase extraction packing material, let it stand at room temperature, and wash with PBS buffer. 3) Change the pH of the mobile phase to acidic and elute the extraction column after step 2). Adjust the pH of the obtained antimicrobial peptide eluent to 7.4, freeze-dry, and obtain the purified antimicrobial peptide product.

9. The method for enriching and purifying insect-derived antimicrobial peptides according to claim 8, characterized in that, In step 3), change the pH of the mobile phase to 3-6.

10. The method for enriching and purifying insect-derived antimicrobial peptides according to claim 8, characterized in that, The insects include fly larvae, black soldier flies, or grain worms.

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