A culture medium for breeding hermetia illucens larvae producing high-activity antibacterial peptides, a breeding method, and an antibacterial peptide extraction method

Abstract

The application belongs to the technical field of antibacterial peptide production, and relates to a culture medium for breeding black soldier fly larvae producing high-activity antibacterial peptides, a breeding method and an antibacterial peptide extraction method. The application provides the culture medium for breeding black soldier fly larvae producing high-activity antibacterial peptides, wherein the culture medium comprises wheat bran, and the water content of the culture medium is 65-70%. The culture medium can realize rapid and effective production of high-activity antibacterial peptides by the black soldier fly larvae.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide production technology, specifically relating to a culture medium for raising black soldier fly larvae that produce highly active antimicrobial peptides, a raising method, and a method for extracting antimicrobial peptides. Background Technology

[0002] The overuse of antibiotics leads to increased drug resistance in pathogens and even fosters the emergence of "superbugs," thereby weakening the effectiveness of antibiotics. Currently, antibiotic resistance and safety have been listed by the World Health Organization as one of the threats to global public health. Since 2015, my country has issued relevant documents prohibiting the use of certain antibiotics in food animals. Furthermore, in 2020, the Ministry of Agriculture and Rural Affairs explicitly stated that enterprises should cease producing commercial feed containing growth-promoting antibiotics. The situation of "banning the use of antibiotics in feed" is very clear. Especially against the backdrop of increasingly strict restrictions on antibiotic use, finding green, safe, and efficient antibiotic alternatives has become an urgent need for the industry.

[0003] Insect-derived antimicrobial peptides are considered potential antibiotic alternatives due to their excellent antibacterial activity, stability, and safety. As a representative of insect host defense mechanisms, antimicrobial peptides are widely present in many insects and are major components of the insect host's innate immune system. Insect antimicrobial peptides are mainly composed of small molecule secondary metabolites, small proteins, and peptides, possessing defense mechanisms against pathogens such as bacteria, fungi, and viruses, and have significant potential as "antibiotic alternatives."

[0004] The first insect antimicrobial peptide was discovered in 1981. It was an inducible antimicrobial factor identified from the diapause pupa of the silkworm and was named cephalosporin. Currently, there are about 325 insect antimicrobial peptides reported in the antimicrobial peptide database (https: / / aps.unmc.edu / AP / ). Based on their amino acid characteristics, they can be divided into four categories: (1) antimicrobial peptides containing cysteine; (2) antimicrobial peptides with α-helix structure; (3) antimicrobial peptides containing proline; and (4) antimicrobial peptides containing glycine.

[0005] Among numerous insects, black soldier fly larvae contain more than 50 antimicrobial peptide genes, making it the largest family of antimicrobial peptides discovered in insects to date. Furthermore, black soldier flies have been found to possess the ability to convert waste into their own resources, surviving in environments with multiple pathogens and producing corresponding antimicrobial substances. Different types of antimicrobial peptides are expressed in black soldier fly larvae under different substrates, providing a scientific basis for black soldier fly farming and the production of highly active antimicrobial peptides. However, a method for cultivating black soldier flies to produce highly active antimicrobial peptides is still lacking. Summary of the Invention

[0006] The purpose of this invention is to provide a culture medium, a culture method, and a method for extracting antimicrobial peptides from black soldier fly larvae that produce highly active antimicrobial peptides. The culture medium described in this invention enables black soldier fly larvae to rapidly and effectively produce highly active antimicrobial peptides.

[0007] This invention provides a culture medium for raising black soldier fly larvae that produce highly active antimicrobial peptides, the culture medium comprising wheat bran, the water content of the culture medium being 65-70%.

[0008] The present invention also provides the application of the culture medium described in the above technical solution in the cultivation of black soldier fly larvae that produce highly active antimicrobial peptides.

[0009] This invention also provides a method for culturing black soldier fly larvae producing highly active antimicrobial peptides based on the culture medium described above, comprising the following steps:

[0010] Three-day-old black soldier fly larvae were cultured in the culture medium described in the above technical solution, and the feed was turned over daily during the culture process.

[0011] Preferably, the stocking density is 1 head / cm². 3 .

[0012] This invention also provides a method for extracting antimicrobial peptides from black soldier fly larvae, comprising the following steps:

[0013] Black soldier fly larvae cultured using the method described above were collected by sieving. Bacterial solution was injected into the larvae to induce antimicrobial peptides. After induction, the larvae were starved for 24 hours, and the antimicrobial peptides were extracted from the black soldier fly larvae using the hemolymph collection method.

[0014] Preferably, the collection operation is performed when the black soldier fly larvae are 8 days old.

[0015] Preferably, the sieving is performed by using an 8-mesh sieve to separate the larvae from the culture medium.

[0016] Preferably, the bacterial solution comprises a Staphylococcus aureus bacterial solution, wherein the concentration of Staphylococcus aureus in the Staphylococcus aureus bacterial solution is 10. 6 ~10 7 The concentration of cfu / μL is specified, and the amount of injected bacterial solution is 10μL per animal.

[0017] The present invention also provides black soldier fly antimicrobial peptides extracted by the extraction method described in the above technical solution.

[0018] The present invention also provides the application of the antimicrobial peptide described in the above technical solution in the preparation of an agent for inhibiting Staphylococcus aureus.

[0019] This invention provides a culture medium for raising black soldier fly larvae that produce highly active antimicrobial peptides. The culture medium described in this invention enables black soldier fly larvae to rapidly and effectively produce highly active antimicrobial peptides. Experimental results show that, under a pure wheat bran substrate with 70% moisture content, at a ratio of 1 larvae / cm², [the desired growth rate is achieved]. 3 Black soldier fly larvae are cultured at a controlled density without adjusting pH or adding salt or oil. Under these conditions, larvae capable of producing highly active antimicrobial peptides can be obtained. The antimicrobial peptides obtained from the culture medium of this invention can disrupt the cell membrane structure of Staphylococcus aureus, altering cell membrane permeability; disrupt the integrity of Staphylococcus aureus cells, leading to changes in cell surface morphology; and cause cell lysis and cytoplasmic leakage in Staphylococcus aureus. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0021] Figure 1 The diagram shows the diameter of the inhibition zone under different breeding conditions provided by this invention.

[0022] Figure 2 Optical and fluorescence microscope images of Staphylococcus aureus provided for this invention;

[0023] Figure 3 Scanning electron microscope and transmission electron microscope images of Staphylococcus aureus provided for this invention. Detailed Implementation

[0024] This invention provides a culture medium for raising black soldier fly larvae that produce highly active antimicrobial peptides. The culture medium comprises wheat bran, and its moisture content is 65-70%. More preferably, the moisture content is 70%. Preferably, the wheat bran comprises flaky coarse wheat bran. The particle size of the wheat bran is preferably in the range of micrometers to millimeters. The culture medium of this invention, when used for black soldier fly larvae rearing, can relatively improve the antimicrobial activity of the black soldier fly's antimicrobial peptides.

[0025] The present invention also provides the application of the culture medium described in the above technical solution in the cultivation of black soldier fly larvae that produce highly active antimicrobial peptides.

[0026] This invention also provides a method for culturing black soldier fly larvae producing highly active antimicrobial peptides based on the culture medium described above, comprising the following steps:

[0027] Three-day-old black soldier fly larvae were cultured in the culture medium described in the above technical solution, and the feed was turned over daily during the culture process.

[0028] In this invention, the 3-day-old black soldier fly larvae are preferably obtained by hatching black soldier fly eggs. The hatching temperature is preferably 25–30°C. The black soldier flies are preferably hatched on a black soldier fly feed. The stocking density is preferably 1 larvae / cm². 3 In this invention, the preferred temperature for aquaculture is 25–30°C.

[0029] This invention also provides a method for extracting antimicrobial peptides from black soldier fly larvae, comprising the following steps:

[0030] Black soldier fly larvae cultured using the method described above are collected by sieving. Bacterial solution is injected into the larvae to induce antimicrobial peptides. After induction, the larvae are starved for 24 hours, and the antimicrobial peptides are extracted using a hemolymph collection method. In this invention, the collection operation is preferably performed when the black soldier fly larvae are 8 days old. In this invention, the sieving is preferably performed using an 8-mesh sieve to separate the larvae from the culture medium.

[0031] In this invention, the bacterial solution preferably comprises a Staphylococcus aureus bacterial solution, and the concentration of Staphylococcus aureus in the Staphylococcus aureus bacterial solution is preferably 10. 6 ~10 7 cfu / μL, more preferably 10 6 The cfu / μL concentration of the injected bacterial solution is preferably 10 μL per animal.

[0032] The present invention also provides black soldier fly antimicrobial peptides extracted by the extraction method described in the above technical solution.

[0033] This invention also provides the application of the antimicrobial peptide described in the above-mentioned technical solution in the preparation of an agent for inhibiting Staphylococcus aureus. The antimicrobial peptide of this invention can be used to treat diseases caused by Staphylococcus aureus. In this invention, the treatment method preferably includes applying the antimicrobial peptide of this invention to patients with Staphylococcus aureus diseases. The antimicrobial peptide of this invention can disrupt the cell membrane structure of Staphylococcus aureus, altering cell membrane permeability; disrupt the integrity of Staphylococcus aureus cells, leading to changes in cell surface morphology; and cause cell lysis and cytoplasmic leakage of Staphylococcus aureus.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a culture medium, a culture method, and an antimicrobial peptide extraction method for black soldier fly larvae producing highly active antimicrobial peptides provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] 1. Prepare the feeding materials: dry pure wheat bran, soybean meal, peanut meal and rapeseed meal in an oven at 60-70℃ for 3 hours before use. Adjust the moisture content to 70% before feeding black soldier flies.

[0037] 2. Initially, black soldier fly eggs are hatched in small incubators. After 3 days, they are separated into different incubators and reared under different conditions (as shown in ①~⑤) until they reach 8 days old. During the rearing process, the feed is turned over daily until the larvae reach 8 days old.

[0038] ① The pH values ​​of pure wheat bran as the substrate were adjusted to 3, 5, 7, 9, and 11 respectively, for feeding black soldier fly larvae (larval density of 0.5 larvae / cm³). 3 When the insects are 8 days old, use an 8-mesh sieve to separate them from the sand, and take 100 insects for later use.

[0039] ②During the breeding process, black soldier fly larvae were fed with pure wheat bran, pure wheat bran + soybean meal (1:1), pure wheat bran + peanut meal (1:1), and pure wheat bran + rapeseed meal (1:1) respectively (larval density 0.5 larvae / cm³). 3 In this invention, the above treatments are marked as A, B, C and D respectively. When the insects are 8 days old, they are separated from the sand using an 8-mesh sieve, and 100 insects are taken for use.

[0040] ③ Adjust the feed moisture content to 50%, 60%, 70%, and 80% respectively for feeding black soldier fly larvae (substrate is pure wheat bran, larvae density is 0.5 larvae / cm³). 3 When the insects are 8 days old, use an 8-mesh sieve to separate them from the sand, and take 100 insects for later use.

[0041] ④ Add salt (0%, 1%, 2%, and 3%) or soybean oil (0%, 2%, 4%, and 6%) to pure wheat bran to feed black soldier fly larvae. When they are 8 days old, use an 8-mesh sieve to separate the larvae from the sand and take 100 larvae for later use.

[0042] ⑤ Adjust the insect rearing density to 0.25 individuals / cm². 3 0.5 heads / cm 3 0.75 heads / cm 3 1 head / cm 3 Feed black soldier fly larvae (with pure wheat bran as the substrate), and when they are 8 days old, use an 8-mesh sieve to separate the larvae from the sand, and take 100 larvae for later use.

[0043] 3. Rinse the larvae separated from ① to ⑤ using a 10% concentration. 6 Induction was achieved by injecting 10 μL of Staphylococcus aureus bacterial solution (cfu / μL) into black soldier flies using a microsyringe.

[0044] 4. After induction, starve for 24 hours (starvation treatment means placing the isolated black soldier fly larvae in a ventilated glass container and not adding any feed), and extract the black soldier fly antimicrobial peptides using the hemolymph collection method to obtain the black soldier fly larvae antimicrobial peptides.

[0045] 5. The inhibition zone was determined using the filter paper disc method. The diameter of the inhibition zone was used to evaluate the antibacterial activity and inhibition rate, thereby determining the optimal feed pH, optimal feed ratio, optimal moisture content, optimal insect rearing density, and optimal salt / soybean oil addition amount.

[0046] 6. To further determine the optimal conditions for preparing black soldier fly antimicrobial peptides to achieve good antibacterial effects, the antimicrobial properties of the black soldier fly antimicrobial peptides were studied. Optical electron microscopy, fluorescence microscopy, scanning electron microscopy, and transmission electron microscopy were used to observe the effects of the antimicrobial peptides at concentrations of 36.97 μg / mL, 73.94 μg / mL (MIC), and 2MIC on the cell membrane and surface morphology of Staphylococcus aureus (MIC concentration was obtained by twofold dilution).

[0047] Experimental results

[0048] This embodiment judges the antibacterial activity of the antimicrobial peptides produced by black soldier flies by comparing the diameter of the inhibition zone. The results are shown in Table 1 and... Figure 1 As shown. The results showed that, after induction, pH did not affect the activity of antimicrobial peptides produced by black soldier fly larvae cultured under different pH conditions. Among black soldier fly larvae cultured with different feed ratios, those cultured with pure wheat bran showed the highest antimicrobial peptide activity after induction. Among black soldier fly larvae cultured with different water contents, those cultured with 70% water content showed the highest antimicrobial peptide activity after induction. Among black soldier fly larvae cultured with different salinity contents, those cultured without added salt showed the highest antimicrobial peptide activity after induction. Among black soldier fly larvae cultured with different oil contents, those cultured without added oil showed the highest antimicrobial peptide activity after induction (since there was no significant difference between 0% and 2% addition, 0% addition was chosen to save costs). Among black soldier fly larvae cultured at different densities, after induction, 1 larvae / cm² was found to be the optimal concentration. 3 Cultured black soldier fly larvae produce the most active antimicrobial peptides.

[0049] Table 1 Results of inhibition zone diameter under different culture conditions

[0050]

[0051]

[0052] Based on the above, the optimal conditions determined by this invention are: under a pure wheat bran substrate with a moisture content of 70%, at a ratio of 1 head / cm². 3When black soldier fly larvae are cultured at the appropriate insect density without adjusting the pH or adding salt or oil, larvae with the highest antimicrobial peptide activity can be obtained.

[0053] Table 2 shows the physical characteristics of black soldier fly larvae and the diameter of their antimicrobial peptides under optimal conditions:

[0054] Table 2. Physical characteristics and antimicrobial peptide diameter data of black soldier fly larvae cultured under optimal conditions.

[0055] Average weight per worm 0.064g crude protein content 36.76％ Crude fat content 9.50％ Moisture content 79.97％ Diameter of the inhibition zone 18.17mm

[0056] The antimicrobial peptides of black soldier fly larvae cultured under optimal conditions are shown in the following results:

[0057] Figure 2 These are optical and fluorescence micrographs of Staphylococcus aureus. A, B, and C are optical micrographs, while D, E, and F are fluorescence micrographs. A and D are Staphylococcus aureus (control), B and E are Staphylococcus aureus treated with MIC antimicrobial peptides, and C and F are Staphylococcus aureus treated with 2MIC antimicrobial peptides. The antimicrobial peptides disrupt the cell membrane, allowing PI dye to enter the bacterial cells, resulting in red fluorescence.

[0058] Staphylococcus aureus treated with different concentrations of antimicrobial peptides emitted red fluorescence under green laser light, indicating that the antimicrobial peptides can disrupt the cell membrane structure of Staphylococcus aureus, altering cell membrane permeability and allowing PI dye to enter the bacterial cell and bind to nucleic acids, resulting in red fluorescence under green laser light.

[0059] Figure 3 Images of Staphylococcus aureus are shown in scanning electron microscope (SEM) and transmission electron microscope (TEM) images. In the images, A, B, and C are SEM images, and D, E, and F are TEM images. A and D are the original images of Staphylococcus aureus (controls). B and E are Staphylococcus aureus treated with MIC antimicrobial peptides, and C and F are Staphylococcus aureus treated with 2MIC antimicrobial peptides.

[0060] Effects of antimicrobial peptides on the surface morphology of Staphylococcus aureus: Scanning electron microscopy revealed that Staphylococcus aureus treated with MIC concentrations of antimicrobial peptides exhibited wrinkling of the surface and cell adhesion. These destructive phenomena were more severe in cells treated with a 2MIC concentration. The results indicate that antimicrobial peptides can disrupt the integrity of Staphylococcus aureus cells, leading to changes in cell surface morphology.

[0061] Effects of antimicrobial peptides on intracellular microscopic changes in Staphylococcus aureus cells: Untreated Staphylococcus aureus cells showed a uniform cytoplasmic appearance with clear cell walls and membranes. However, some Staphylococcus aureus cells treated with antimicrobial peptides had ruptured cell walls, and some cell fragments could be seen in the surrounding environment. The results indicate that antimicrobial peptides can induce cell lysis and cytoplasmic efflux in Staphylococcus aureus.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting highly active black soldier fly antimicrobial peptides that inhibit Staphylococcus aureus, characterized in that, Includes the following steps: Three-day-old black soldier fly larvae were cultured in a substrate, which was turned daily during the rearing process. The substrate was pure wheat bran with a moisture content of 70%, and contained 0% salt and 0% soybean oil. The stocking density was 1 larvae / cm². 3 ; When the black soldier fly larvae were 8 days old, the cultured larvae were collected by screening. A bacterial solution was injected into the larvae to induce antimicrobial peptides. After induction, the larvae were starved for 24 hours, and the antimicrobial peptides were extracted using the hemolymph collection method. The bacterial solution was a Staphylococcus aureus solution with a Staphylococcus aureus concentration of 10%. 6 ~10 7 The concentration of cfu / μL is specified, and the amount of injected bacterial solution is 10μL per animal.

2. The extraction method according to claim 1, characterized in that, The sieving process involves using an 8-mesh sieve to separate the larvae from the culture medium.

3. The black soldier fly antimicrobial peptide extracted by the extraction method according to claim 1 or 2.

4. The use of the black soldier fly antimicrobial peptide according to claim 3 in the preparation of an agent for inhibiting Staphylococcus aureus.

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