Hermetia illucens antibacterial peptide, extraction method and application thereof

Abstract

The application provides a hermetia illucens antibacterial peptide and an extraction method and application thereof. The extraction method of the hermetia illucens antibacterial peptide comprises the following steps: performing leaching on defatted hermetia illucens larvae powder to obtain a hermetia illucens larvae crude protein extract; performing dialysis on the hermetia illucens larvae crude protein extract, and then performing ultrafiltration to obtain an antibacterial peptide crude extract; and finally performing nickel column purification on the antibacterial peptide crude extract to obtain the hermetia illucens antibacterial peptide. The obtained hermetia illucens antibacterial peptide has good killing or inhibiting effects on escherichia coli and staphylococcus aureus.

Description

Technical Field

[0001] This invention relates to the field of black soldier fly antimicrobial peptide technology, and in particular to a black soldier fly antimicrobial peptide, its extraction method, and its application. Background Technology

[0002] Antibiotics have been used to treat diseases caused by bacteria for decades. However, due to increased global antibiotic use, inappropriate use of antibiotics in clinical applications, and overuse during reproduction, antibiotic resistance has gradually become a global problem. Furthermore, antibiotics are difficult to completely degrade, and antibiotic residues in the environment can impact ecosystems. Animals carry 40%–90% antibiotic residues, potentially posing a threat to human health along the food chain.

[0003] The emergence of Gram-positive and Gram-negative bacteria resistant to conventional antibiotics has led to increased attention on antimicrobial peptides as potential treatment methods. Antimicrobial peptides are positively charged polypeptides encoded by genes, synthesized by ribosomes, and possessing diverse biological activities; they are an important component of an organism's innate immunity. Since the first antimicrobial peptide—cephalosporin—was induced, isolated, and purified from the pupa of the silkworm *Cyclophorus* by Swedish scientist Boman in 1972, more than 3,250 antimicrobial peptides have been discovered and purified from plants, animals, insects, birds, and even humans.

[0004] Insect antimicrobial peptides are a class of small-molecule bioactive peptides present in the hemolymph of insects after immunization. They are characterized by small molecular weight, strong thermal stability, good water solubility, broad antimicrobial spectrum, non-immunogenicity, and abundant material sources; they only act on prokaryotic cells and diseased eukaryotic cells, and have no effect on normal eukaryotic cells, making them highly favored in the field of medical research.

[0005] Current research on insect antimicrobial peptides mainly focuses on their antibacterial, antifungal, antiviral, tumor cell proliferation inhibition, and antiparasitic functions. The insects studied include Lepidoptera, Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, and Odonata. The most studied antimicrobial peptides come from Hymenoptera (50% of the studied insects), totaling 30 species; Diptera (17%); Coleoptera (13%); Lepidoptera (10%); Hemiptera (5%); Lepidoptera (3%); and Odonata (2%). Among Hymenoptera, wasps are the most studied, totaling 18 species (62% of the studied Hymenoptera insects), followed by bees (24%) and ants (14%). Antimicrobial peptides are typically isolated from larvae (24%), followed by venom (22%), adults (19%), synthetic peptides (18%), pupae (11%), genetic studies (4%), and finally culture media (2%). Among dipteran insects, the most studied genus *Fly* to date is *Drosophila*.

[0006] Black soldier flies (S. spp.) belong to the family Brachyceridae in the order Diptera. They are a widely distributed resource insect worldwide. Black soldier fly larvae are approximately 2-3 times the length of housefly larvae. Their habitat is similar to that of houseflies, both being saprophytic and feeding on a wide range of sources, including animal feces, decaying organic matter, and plant waste. Studies have shown that when black soldier fly larvae concentrate on feeding on feces, they not only reduce fecal accumulation and odor but also decrease the levels of E. coli and Salmonella in the feces, effectively controlling housefly populations. Therefore, we preliminarily infer that black soldier flies must possess a more powerful immune system than houseflies and cockroaches to achieve this function. Black soldier flies are larger than houseflies at all developmental stages; unlike adult houseflies, adult black soldier flies do not feed, die after mating and laying eggs, and do not pollute the environment or transmit diseases. Therefore, black soldier flies are excellent materials for researching antimicrobial peptides and are good materials for drug development.

[0007] In recent years, the proportion of aquaculture in China has gradually increased. However, high-density culture conditions and overuse of antibiotics have led to antibiotic resistance in many infectious diseases and pathogens. Therefore, antimicrobial peptides may become a novel feed additive to improve this situation. Studies have shown the application of antimicrobial peptides in aquaculture. Henry et al. and Nogales-Merida et al. explored several important insect feeds used in fish nutrition and their potential to replace certain components in several fish feeds, including mealworms, silkworms, houseflies, mosquitoes, and black soldier flies. Black soldier fly larvae feed (BSFLM) can replace fishmeal in the feeds of Nile tilapia, Siberian sturgeon, turbot, rainbow trout, Atlantic salmon, swordfish, yellow catfish, African catfish, and European sea bass. Other studies have shown that black soldier fly larvae feed (BSFLM) has potential immunostimulatory properties, such as increasing serum lysozyme activity in yellow catfish and upregulating cytokine genes in the intestinal tissue of marron (Cherax cainii). In addition, BSFLM can enhance the antioxidant capacity of swordfish by increasing catalase activity, reducing malondialdehyde levels in sea bass serum, and reducing malondialdehyde levels in sea bass.

[0008] However, there is currently little information on the extraction and application of antimicrobial peptides from black soldier flies. Therefore, it is necessary to provide a method for extracting antimicrobial peptides from black soldier flies. Summary of the Invention

[0009] This invention provides a black soldier fly antimicrobial peptide, its extraction method, and its application, in order to solve or at least partially solve the defects existing in the prior art.

[0010] In a first aspect, the present invention provides a method for extracting antimicrobial peptides from black soldier fly larvae, comprising the following steps:

[0011] Add defatted black soldier fly larvae powder to EDTA buffer, extract, filter, and collect the supernatant;

[0012] Add ammonium sulfate to the supernatant, stir until the ammonium sulfate dissolves, centrifuge, discard the supernatant, and collect the solid, which is the crude protein extract of black soldier fly larvae;

[0013] The crude protein extract from black soldier fly larvae was dialyzed using a 1-2 kD dialysis bag to obtain the dialyzed crude protein.

[0014] The crude protein after dialysis was added into a 9-11 kD ultrafiltration tube, centrifuged, and the bottom liquid of the ultrafiltration tube was collected and freeze-dried under vacuum to obtain crude antimicrobial peptide extract.

[0015] The crude antimicrobial peptide extract was added to 15–25 mM imidazole buffer, reconstituted, and then added to a nickel column. A gradient elution method was used to elute impurities with 45–55 mM imidazole buffer and 90–110 mM imidazole buffer, followed by elution of the target protein with 240–256 mM imidazole buffer. The eluent was collected and freeze-dried under vacuum to obtain the black soldier fly antimicrobial peptide.

[0016] Preferably, the method for extracting the antimicrobial peptides from black soldier fly involves adding defatted black soldier fly larvae powder to EDTA buffer, extracting it under ultrasonic power of 45-55W for 10-15 hours, filtering, and collecting the supernatant.

[0017] Preferably, in the method for extracting the antimicrobial peptides from black soldier fly larvae, defatted black soldier fly larvae powder is added to EDTA buffer to make the material-to-liquid ratio (6-8):1.

[0018] Preferably, the method for extracting the antimicrobial peptides from black soldier fly larvae involves adding ammonium sulfate to the supernatant, stirring until the ammonium sulfate dissolves, centrifuging at 9000–11000 r / min for 10–20 min, discarding the supernatant, and collecting the solid, which is the crude protein extract from black soldier fly larvae.

[0019] Preferably, in the method for extracting the black soldier fly antimicrobial peptide, ammonium sulfate is added to the supernatant to make its mass concentration 60-80%.

[0020] Preferably, the method for extracting the antimicrobial peptides from black soldier fly involves adding the dialyzed crude protein into a 9-11 kD ultrafiltration tube, centrifuging at 4000-6000 r / min for 1-2 h, collecting the bottom liquid of the ultrafiltration tube, and freeze-drying it under vacuum to obtain the crude extract of antimicrobial peptides.

[0021] Preferably, the method for extracting the antimicrobial peptides from black soldier fly involves adding the crude antimicrobial peptide extract to 15–25 mM Mildazole buffer, reconstituted, filtering through a filter membrane with a pore size of 0.2–0.25 μm, and then adding it to a nickel column.

[0022] Preferably, the method for extracting the antimicrobial peptides from black soldier fly larvae involves dialyzing the crude protein extract from black soldier fly larvae at 1–7°C using a 1–2 kD dialysis bag for 20–30 hours to obtain the dialyzed crude protein.

[0023] Secondly, the present invention also provides a black soldier fly antimicrobial peptide, which is extracted using the extraction method described above.

[0024] Thirdly, the present invention also provides an application of the black soldier fly antimicrobial peptide prepared by the extraction method described above, or the black soldier fly antimicrobial peptide described above as an antibacterial agent.

[0025] The method for extracting antimicrobial peptides from black soldier fly larvae of the present invention has the following advantages over the prior art:

[0026] The method for extracting black soldier fly antimicrobial peptides of the present invention involves extracting defatted black soldier fly larvae powder to obtain a crude protein extract of black soldier fly larvae. This crude protein extract is then dialyzed and ultrafiltered to obtain a crude antimicrobial peptide extract. Finally, the crude antimicrobial peptide extract is purified by nickel column chromatography to obtain the black soldier fly antimicrobial peptides. The obtained black soldier fly antimicrobial peptides exhibit good killing or inhibitory effects against Escherichia coli and Staphylococcus aureus. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The size of the inhibition zone for different groups of Escherichia coli bacterial plates;

[0029] Figure 2 The size of the inhibition zone in different groups of Staphylococcus aureus bacterial plates. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0032] The following provides a detailed description of each example. It should be noted that the order of description of the embodiments below is not intended to limit the preferred order of the embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0033] This invention provides a method for extracting antimicrobial peptides from black soldier fly larvae, comprising the following steps:

[0034] S1. Add defatted black soldier fly larvae powder to EDTA buffer, extract, filter, and collect the supernatant.

[0035] S2. Add ammonium sulfate to the supernatant, stir until the ammonium sulfate dissolves, centrifuge, discard the supernatant, and collect the solid, which is the crude protein extract of black soldier fly larvae.

[0036] S3. Dialyze the crude protein extract from black soldier fly larvae using a 1-2kD dialysis bag to obtain the dialyzed crude protein.

[0037] S4. Add the dialysis crude protein into a 9-11kD ultrafiltration tube, centrifuge, collect the bottom liquid of the ultrafiltration tube, freeze dry under vacuum to obtain crude antimicrobial peptide extract.

[0038] S5. Add the crude antimicrobial peptide extract to 15-25 mM imidazole buffer, reconstitute, and then add it to a nickel column. Use a gradient elution method to elute impurities with 45-55 mM imidazole buffer and 90-110 mM imidazole buffer, and then elute the target protein with 240-256 mM imidazole buffer. Collect the eluent, freeze-dry under vacuum, and obtain the black soldier fly antimicrobial peptide.

[0039] This invention involves extracting defatted black soldier fly larvae powder to obtain a crude protein extract of black soldier fly larvae. This crude protein extract is then dialyzed and ultrafiltered to obtain a crude antimicrobial peptide extract. Finally, the crude antimicrobial peptide extract is purified using a nickel column to obtain black soldier fly antimicrobial peptides. The obtained black soldier fly antimicrobial peptides exhibit good killing or inhibitory effects against Escherichia coli and Staphylococcus aureus.

[0040] In some embodiments, defatted black soldier fly larvae powder is added to EDTA buffer and extracted with ultrasonic power of 45-55W for 10-15 hours, filtered, and the supernatant is collected.

[0041] In some embodiments, defatted black soldier fly larvae powder is added to EDTA buffer at a ratio of g / mL to 1:(6-8).

[0042] "Solid-to-liquid ratio" refers to the ratio of the mass of the solid "material" to the volume of the "liquid" used as the extraction solution (i.e., EDTA buffer). In this application, the unit of "solid-to-liquid ratio" is g / mL, which means 1g of defatted black soldier fly larvae powder is added to 6-8mL of EDTA buffer.

[0043] In some embodiments, ammonium sulfate is added to the supernatant and stirred until the ammonium sulfate dissolves. The mixture is then centrifuged at 9000–11000 r / min for 10–20 min, the supernatant is discarded, and the solid is collected, which is the crude protein extract of black soldier fly larvae.

[0044] In some embodiments, ammonium sulfate is added to the supernatant to make its mass concentration 60-80%.

[0045] In some embodiments, the crude protein after dialysis is added to a 9-11 kD ultrafiltration tube, centrifuged at 4000-6000 r / min for 1-2 h, the bottom liquid of the ultrafiltration tube is collected, and the solution is freeze-dried under vacuum to obtain crude antimicrobial peptide extract.

[0046] In some embodiments, the crude antimicrobial peptide extract is added to 15–25 mM imidazole buffer, reconstituted, filtered through a filter membrane with a pore size of 0.2–0.25 μm, and then added to a nickel column.

[0047] Specifically, the imidazole buffer in the above examples is an imidazole buffer.

[0048] In some embodiments, crude protein extract from black soldier fly larvae is dialyzed at 1–7°C using a 1–2 kD dialysis bag for 20–30 h to obtain the dialyzed crude protein.

[0049] Specifically, the crude protein extract from black soldier fly larvae was dialyzed at 1–7°C using a 1–2 kD dialysis bag for 20–30 hours, with the buffer changed three times during the process, to obtain the dialyzed crude protein. Specifically, the dialysis bag was placed in EDTA buffer for dialysis.

[0050] Based on the same inventive concept, the present invention also provides a black soldier fly antimicrobial peptide, which is extracted using the above-described extraction method.

[0051] Based on the same inventive concept, the present invention also provides the application of the black soldier fly antimicrobial peptide prepared by the above extraction method or the above black soldier fly antimicrobial peptide as an antibacterial agent.

[0052] The black soldier fly antimicrobial peptide of the present invention has a good killing or inhibitory effect on Escherichia coli and Staphylococcus aureus.

[0053] The following specific embodiments further illustrate the black soldier fly antimicrobial peptides, their extraction methods, and applications. This section further illustrates the content of the present invention in conjunction with specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0054] In the following examples, defatted black soldier fly larvae powder was purchased from Biosource Biotechnology (Shenzhen) Co., Ltd. The imidazole buffer solution was prepared from imidazole, which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0055] Example 1

[0056] This application provides a method for extracting antimicrobial peptides from black soldier fly larvae, comprising the following steps:

[0057] S1. Add 10g of defatted black soldier fly larvae powder to EDTA buffer to make a material-to-liquid ratio of 1:7 (1g:7mL), and extract under ultrasonic power of 50W for 12h. After extraction, filter with 8 layers of gauze, centrifuge at 8000r / min for 15min, and collect the supernatant.

[0058] S2. Add ammonium sulfate to the supernatant of S1 to make its mass concentration 70%, stir until the ammonium sulfate dissolves, let it stand at 4℃ for 4 hours, then centrifuge at 10000r / min for 15 minutes, discard the supernatant, and collect the solid, which is the crude protein extract of black soldier fly larvae.

[0059] S3. The crude protein extract of black soldier fly larvae in S2 was dialyzed at 4°C for 24 hours using a 1kD dialysis bag (the dialysis bag was placed in EDTA buffer) to obtain the dialyzed crude protein.

[0060] S4. Add the crude protein after dialysis in S3 to a 10kD ultrafiltration tube, centrifuge at 5000r / min for 1h, collect the bottom liquid of the ultrafiltration tube, freeze dry under vacuum to obtain crude antimicrobial peptide extract.

[0061] S5. Add the crude antimicrobial peptide extract from S4 to 20 mM imidazole buffer, reconstitute, filter through a 0.22 μm pore membrane, and add to a cleaned nickel column. Wash with 50 mM and 100 mM imidazole buffer to remove contaminating proteins. Use a Coomassie G250 to check if the contaminating proteins have been washed away. If the Coomassie G250 does not change color, the contaminating proteins have been washed away. Add 250 mM imidazole buffer to elute the target protein. Start collecting the target protein and monitor the collection with a Coomassie G250. After collection, wash the Ni column with 400 mM imidazole buffer (do not collect this eluent). Perform vacuum freeze-drying on the collected sample to obtain the black soldier fly antimicrobial peptide.

[0062] Performance testing

[0063] Escherichia coli and Staphylococcus aureus were formulated into OD. 600 A bacterial suspension of 0.1 g was inoculated at a volume ratio of 5% into LB solid medium at 45°C. After shaking well, the solution was poured into sterile agar plates. Once the medium solidified, sterile paper discs with a diameter of 6 mm were placed into the inoculated plates. Each plate contained: ① negative control group (ultrapure water); ② positive control group (1% gentamicin); ③ flow-through solution group; ④ 50 mM imidazole elution group; ⑤ 100 mM imidazole elution group; ⑥ 250 mM imidazole elution group; ⑦ 400 mM imidazole elution group; ⑧ stock solution group. The sample volume was 10 μL (experimental group concentration 100 g / L). The plates were labeled and incubated at 37°C for 24 hours. The size of the inhibition zone was then observed and measured. The results are as follows. Figures 1-2 As shown.

[0064] The stock solution group is the crude antimicrobial peptide extract obtained in step S4 of Example 1, and the 250mM imidazole eluent group is the imidazole eluent containing black soldier fly antimicrobial peptides in step S5 of Example 1.

[0065] Preparation of Escherichia coli culture: Escherichia coli was inoculated into LB liquid medium (10g peptone, 5g yeast extract, 10g sodium chloride, 1L deionized water) and incubated in the dark at 37℃ and 200r / min for 24h. After dilution with sterile water, the OD was measured using a spectrophotometer. 600 The bacterial solution was 0.1.

[0066] Preparation of Staphylococcus aureus culture: Staphylococcus aureus was inoculated into LB liquid medium (10g peptone, 5g yeast extract, 10g sodium chloride, 1L deionized water) and incubated in the dark at 37℃ and 200r / min for 24h. After dilution with sterile water, the OD value was measured using a spectrophotometer. 600 The bacterial solution was 0.1.

[0067] Figure 1 The size of the inhibition zone for different groups of E. coli bacterial culture plates. Figure 2 The size of the inhibition zone in different groups of Staphylococcus aureus bacterial plates.

[0068] Tables 1 and 2 show the inhibition rates of different groups against Escherichia coli and Staphylococcus aureus.

[0069] Table 1 - Escherichia coli inhibition rate

[0070] Group 1 2 3 4 5 6 7 8 Antibacterial rate <![CDATA[0 d ]]> <![CDATA[78.04±0.004 a ]]> <![CDATA[0 d ]]> <![CDATA[0 d ]]> <![CDATA[0 d ]]> <![CDATA[66.39%±0.015 b ]]> <![CDATA[0 d ]]> <![CDATA[43.64%±0.031 c ]]>

[0071] Table 2 - Staphylococcus aureus inhibition rate

[0072] Group 1 2 3 4 5 6 7 8 Antibacterial rate <![CDATA[0 b ]]> <![CDATA[36.73±0.033 a ]]> <![CDATA[0 b ]]> <![CDATA[0 b ]]> <![CDATA[0 b ]]> <![CDATA[31.76%±0.061 a ]]> <![CDATA[0 b ]]> <![CDATA[30.38±0.140 a ]]>

[0073] Note: Inhibition rate = (Diameter of inhibition zone in treatment group - Diameter of inhibition zone in control group) / Diameter of inhibition zone in treatment group × 100%

[0074] As shown in Tables 1 and 2, the crude black soldier fly antimicrobial peptides extracted in this invention exhibited inhibition rates of 43.64% and 30.38% against Escherichia coli and Staphylococcus aureus, respectively. After Ni-column purification, the inhibition rates against Escherichia coli and Staphylococcus aureus were 66.39% and 31.76%, respectively. These results demonstrate that the black soldier fly antimicrobial peptides extracted and purified in this invention have good killing or inhibitory effects on Escherichia coli and Staphylococcus aureus.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a black soldier fly antibacterial peptide in the preparation of an antibacterial agent; the black soldier fly antibacterial peptide has antibacterial effects on Escherichia coli and Staphylococcus aureus. The extraction method of the black soldier fly antibacterial peptide comprises the following steps: S1. 10 g of defatted black soldier fly larvae powder is added to EDTA buffer to make the solid-liquid ratio 1 g:7 mL, and is extracted under ultrasonic power of 50 W for 12 h. After extraction, 8 layers of gauze are used for filtration, and centrifugation is performed at 8000 r / min for 15 min, and the supernatant is collected; S2. Ammonium sulfate is added to the supernatant of S1 to make the mass concentration 70%, and is stirred until the ammonium sulfate is dissolved. After standing at 4°C for 4 h, centrifugation is performed at 10000 r / min for 15 min, and the supernatant is discarded, and the solid is collected, which is the crude protein extract of black soldier fly larvae; S3. The crude protein extract of black soldier fly larvae in S2 is placed in a 1kD dialysis bag, and the dialysis bag is placed in EDTA buffer and dialyzed at 4°C for 24 h to obtain the dialyzed crude protein; S4. The dialyzed crude protein in S3 is added to a 10kD ultrafiltration tube, and centrifugation is performed at 5000 r / min for 1 h, and the bottom liquid of the ultrafiltration tube is collected, and vacuum freeze-drying is performed to obtain the crude antibacterial peptide extract; S5. The crude antibacterial peptide extract in S4 is added to 20mM imidazole buffer, is redissolved, is filtered with a filter membrane with a pore size of 0.22μm, is added to a cleaned nickel column, and is eluted with 50mM imidazole buffer and 100mM imidazole buffer to remove impurities, and then is eluted with 250mM imidazole buffer to obtain the target protein, and the eluate is collected, and vacuum freeze-drying is performed to obtain the black soldier fly antibacterial peptide.

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