A method for improving the activity of antibacterial peptides of hermetia illucens

Abstract

The application discloses an inducing method for improving antibacterial peptide activity of black soldier fly, and belongs to the field of insect biotechnology. The method realizes efficient induction of antibacterial peptide by adding Rhodopseudomonas palustris (RP) in the 5th instar larvae of black soldier fly to reinforce the basic daily ration, and combining ultrasonic reinforcement with short-time heat shock stress treatment. Test data show that the antibacterial peptide crude extract induced by the method has an inhibition zone diameter of 40.48 mm on salmonella. The application solves the problem of large-scale production of antibacterial peptide, balances the immune cost caused by high-intensity induction through reinforcing substrate compensation mechanism, and has the advantages of strong induction activity, stable growth performance, low process cost and the like, and has application prospects in the fields of animal feed additives and biological antibacterial agents.

Description

Technical Field

[0001] This invention relates to the field of insect biotechnology, specifically to a method for synergistically inducing the enhancement of antimicrobial peptide activity and yield in black soldier fly larvae using microbial lysates, physical stress factors, and enhanced substrate compensation. Background Technology

[0002] Antimicrobial peptides, as small molecule polypeptides with broad-spectrum antimicrobial activity, are of great value in areas such as antibiotic alternatives and the development of novel antimicrobial agents. Black soldier flies, due to their rapid reproduction rate and low breeding costs, have become a high-quality biological source for the preparation of antimicrobial peptides.

[0003] However, existing induction techniques have significant drawbacks: physical needle-pricking is complex to operate, results in high larval mortality, and cannot be industrialized; single-feed supplementation methods use only one inducing factor, have fluctuating efficiency, and high-intensity immune stimulation often leads to impaired larval growth. Currently, there is a lack of a multi-dimensional synergistic induction technology that can simultaneously balance the high activity output of antimicrobial peptides with larval growth performance. Summary of the Invention

[0004] To address the aforementioned shortcomings, this invention aims to provide a method for inducing enhanced antimicrobial peptide activity in black soldier fly larvae. This method significantly improves the antimicrobial activity of antimicrobial peptides through multi-factor synergistic effects and maintains host metabolic homeostasis by enhancing substrate-compensated immune metabolic consumption.

[0005] To achieve the above technical objectives, the present invention is implemented through the following solution:

[0006] A method for inducing enhanced activity of antimicrobial peptides from black soldier flies, comprising the following steps:

[0007] (1) Inoculum treatment: Rhodopseudomonas palustris (RP) was cultured to the logarithmic growth phase to obtain Rhodopseudomonas palustris lysate;

[0008] (2) Feed preparation: The hatched black soldier fly larvae were fed an unfermented intensive basal diet until the 5th instar, then mixed with RP for 24 hours, starved for 12 hours and then transferred to subsequent induction.

[0009] (3) Synergistic induction: The larvae obtained in step (2) were subjected to ultrasonic intervention, Salmonella exposure and heat shock treatment in sequence;

[0010] (4) Larval collection: After induction under the conditions described in step (3), larvae are collected and antimicrobial peptides are extracted.

[0011] Further, the preparation method of the Rhodopseudomonas palustris lysate in step (1) is as follows: collect Rhodopseudomonas palustris cells in the logarithmic growth phase, centrifuge at 4000r for 15min, then resuspend in PBS, the concentration after resuspension is 10% (w / v), add 0.5g of lysozyme per 10ml of resuspension for enzymatic hydrolysis, incubate in a 37℃ water bath for 1h, and then perform repeated freeze-thaw cycles for lysis.

[0012] Furthermore, the specific operation of the repeated freeze-thaw cycle lysis is as follows: the enzymatically hydrolyzed bacterial solution is deep-frozen at -196°C, and then rapidly thawed in a 50°C water bath, and the cycle is repeated 5 times.

[0013] Furthermore, the unfermented fortified basal diet described in step (2) comprises, by weight percentage, 60% wheat bran, 20% soybean meal and 20% corn syrup.

[0014] Furthermore, the parameters for ultrasound intervention in step (3) are limited to: power 150W and processing time 15min.

[0015] Furthermore, the treatment conditions for Salmonella exposure in step (3) are limited to: a bacterial concentration of 10... 8 CFU / mL, soaking time 60s.

[0016] Furthermore, the procedure for heat shock treatment in step (3) is defined as follows: incubate at 35°C for 10 min, raise the temperature to 40°C and incubate for 10 min, and then maintain at 42°C for 30 min.

[0017] Furthermore, the method for collecting larvae in step (4) is as follows: after washing the larvae, rinse them twice with pure water, rinse with alcohol for 60 seconds, and then rinse with pure water until all the alcohol is washed away.

[0018] The present invention also provides a black soldier fly antimicrobial peptide, wherein the crude extract of the antimicrobial peptide has an inhibition zone diameter of ≥30mm against Salmonella.

[0019] The present invention also provides the application of black soldier fly antimicrobial peptides in the preparation of animal feed additives, antimicrobial agents or biopesticides.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Significantly enhanced activity: The diameter of the inhibition zone of the crude antimicrobial peptide extract against Salmonella can reach 40.48 mm, which is 12.1% higher than that of the conventional group.

[0022] 2. Standardized process: It adopts a combination of feeding and physical intervention, which is simple to operate and suitable for large-scale industrial production. Attached Figure Description

[0023] Figure 1: Flowchart of the synergistic induction process of this invention. Detailed Implementation

[0024] The present invention will be further described in detail below through specific embodiments and pharmacodynamic tests. These embodiments are intended to describe the invention more fully, but not to limit the scope of the invention in any way. Unless otherwise specified, all reagents and materials used are commercially available.

[0025] Example 1: Hatching of Black Soldier Fly Eggs and Preparation of Fermented Feed

[0026] 1. Insect eggs hatching

[0027] Fresh black soldier fly eggs were provided by Guangxi Yankun Biotechnology Co., Ltd. 20g of eggs were weighed and evenly spread on fermented bran in an incubation tray. The incubation temperature was controlled at 31±1℃, and the bran was kept moist by spraying daily. After 72 hours of incubation, a large number of larvae were observed to hatch. The first-instar larvae were collected and transferred to an incubator for further cultivation.

[0028] 2. Preparation of Fermented Feed

[0029] 2.1 Preparation of Fermented Bran

[0030] Take 10 kg of dried wheat bran and add a certain amount of tap water to bring the moisture content of the material to 55%. Add 3 × 10 7 CFU / g (wet weight) Bacillus subtilis inoculant (10 billion CFU / g viable cells). Spread the inoculated material into a 10-15cm thick layer, evenly poke several small holes to increase the oxygen contact area, and carry out aerobic fermentation at room temperature, turning the pile every 6 hours. After 24 hours of fermentation, the fermented bran is obtained and stored in a refrigerator at 4℃ for later use.

[0031] 2.2 Preparation of Fermentation Mixture

[0032] Take 6 kg of dry wheat bran and proceed according to step 2.1. Take 2 kg of soybean meal and 2 kg of solid corn syrup, mix them, and add a certain amount of tap water to bring the moisture content of the material to 55%. After the soybean meal and corn syrup have fully absorbed the moisture, mix the above-mentioned wet soybean meal-corn syrup mixture with the fermented wheat bran prepared above to obtain a mixture. Add 6 × 10⁶ kJ of water to the mixture. 7 CFU / g (based on the wet weight of the mixture) of *Lactobacillus plantarum* inoculum (10 billion CFU / g viable bacteria). After inoculation, the mixture is placed in a vacuum-sealed bag and allowed to stand at room temperature for anaerobic fermentation for 12 hours. After fermentation, the material will have a sour aroma, indicating fermented mixed feed. Store at 4°C for later use.

[0033] 2.3 Unfermented bran and unfermented mixed feed

[0034] Take 10 kg of dry bran and mix it with a certain amount of tap water to make the moisture content of the material reach 55%, and you will get non-fermented bran feed.

[0035] Mix 6 kg of dry wheat bran, 2 kg of soybean meal and 2 kg of solid corn syrup, then add a certain amount of tap water to make the material moisture content reach 55%, which is the non-fermented mixed feed.

[0036] Example 2: Optimization and Screening of Induction Conditions

[0037] Experimental Objective: This invention utilizes orthogonal experimental design combined with a quasi-level method to systematically optimize the rearing and induction conditions of black soldier fly larvae, screen and verify the optimal combination for inducing antimicrobial peptides, and achieve the goal of efficiently inducing black soldier fly larvae to synthesize antimicrobial peptides. This is specifically achieved through the following technical steps (see...). Figure 1 ):

[0038] 1. Materials and Methods

[0039] 1.1 Feeding Management and Experimental Design

[0040] The experimental factors and level design selected six key factors affecting the induction of antimicrobial peptides in black soldier fly larvae, each with four levels. Hatched black soldier fly larvae were grouped according to an orthogonal array design, based on L25(5) 6 ) An orthogonal array design was used to create 25 experimental groups, and the quasi-level method was used to test L25(5 6 An orthogonal array was used for fitting, with 100 animals per group, for a total of 25 groups, and 3 replicates per group. Feeding conditions were set according to the parameters of each group, with feed spread out to a thickness of 7cm, and feeding was carried out at 9:00 am every day.

[0041] The experiment was conducted according to the induction parameters of each group in the orthogonal array: that is, the animals were fed according to the type of diet, and Rhodopseudomonas palustris was added after the animals reached 5 years of age. They were starved for 12 hours, and then each group underwent ultrasonic treatment, Salmonella induction, heat induction, and 8 hours of nutritional support. After nutritional support, they were starved for 16 hours to end the induction process.

[0042]

[0043]

[0044] 1.2 Cleaning and Collection of Insects

[0045] After induction, the larvae were cleaned and disinfected. The solution was used to submerge the larvae. After washing, the larvae were rinsed twice with pure water, rinsed with alcohol for 60 seconds, and then rinsed with pure water until all the alcohol was washed off. The larvae were then placed in 50ml test tubes and quick-frozen in liquid nitrogen, and then ground in a mortar and pestle.

[0046] 1.3 Extraction of antimicrobial peptides

[0047] The ground black soldier fly larvae were added to the extraction solution at a ratio of 1:5 (w / v). The extraction solution consisted of 10% acetic acid (v / v, analytical grade) + 0.01M Na2EDTA. The mixture was shaken for 3 min, sonicated at 250W for 20 min, centrifuged at 3220g for 25 min, and the supernatant was collected. An equal volume of chloroform was added, the mixture was shaken for 10 min, and then allowed to stand in ice water for 30 min. The aqueous phase was collected and filtered through a 0.22μm filter membrane to obtain the crude extract.

[0048] 1.4 Measurement of inhibition zones and screening of optimal combinations

[0049] Antibacterial activity was determined using the Oxford cup method, with Salmonella (10⁻⁶) as the target. 9 Using CFU / mL as the indicator bacteria, the sample was spread onto LB solid medium, placed in Oxford cups, and 200 μL of crude extract was added. After incubation at 37°C for 24 h, the diameter of the inhibition zone was measured (accurate to 0.1 mm). Each group was divided into three replicates. The order of importance and significance of factors were determined by range analysis and variance analysis to screen for the theoretically optimal combination.

[0050] 1.5 Data Processing and Statistical Analysis

[0051] SPSS 25.0 was used for general linear model analysis or independent samples t-test analysis, and Duncan's method was used for multiple comparisons. Results are expressed as mean and standard error of mean (SEM). P < 0.05 was considered statistically significant, and 0.05 ≤ P < 0.10 was considered to indicate a significant trend.

[0052] 2. Experimental Results

[0053] 2.1 Range Analysis and Determination of Optimal Combination in Orthogonal Experiments

[0054] Range analysis was performed on the orthogonal experimental data, and the results and range distribution are shown in Tables 3 and 4. Based on the R values ​​calculated in Table 4, the influence of each factor on the diameter of the inhibition zone of the antimicrobial peptide was as follows: Factor 4 (R=4.19) > Factor 5 (R=2.92) > Factor 1 (R=2.31) = Factor 6 (R=2.31) > Factor 2 (R=1.62) > Factor 3 (R=1.26).

[0055] Based on the ki values ​​in Table 4, the optimal level is determined, and the maximum mean value of each dimension is extracted as the preferred level.

[0056] Factor 1 (diet type): k3 had the highest mean (32.79), with an optimal level of 3;

[0057] Factor 2 (amount of Rhodopseudomonas palustris added): k2 mean (32.28) was the highest, with level 2 being the preferred level;

[0058] Factor 3 (ultrasound processing parameters): k3 mean value (31.98) is the highest, so level 3 is preferred;

[0059] Factor 4 (Salmonella usage): k3 mean (33.24) is the highest, with the preferred level being 3;

[0060] Factor 5 (thermal induction time): k3 has the highest mean value (32.87), with level 3 being the preferred level;

[0061] Factor 6 (Nutritional Support Formula): k1 mean (32.90) is the highest, with an optimal level of 1.

[0062] Based on the above range mean, the theoretically optimal multi-factor combination is: A3B2C3D3E3F1.

[0063] Mapping this theoretical matrix to specific physical and biochemical processes establishes the standardized synergistic induction procedure of this invention: The basal feeding uses an unfermented fortified diet (bran:soybean meal = 3:1:1, 60% bran, 20% soybean meal, 20% corn syrup), and when the animals reach 5 years of age, a 10% nutrient solution is added to the diet. 8 The bacteria were treated with CFU / g feed lysate of Rhodopseudomonas palustris for 24 h, followed by starvation for 12 h for induction, and then subjected to sonication at 150 W for 15 min and 10 min for 10 hours. 8 CFU / mL Salmonella immersion for 60 s, heat induction: 35℃ (10 min) → 40℃ (10 min) → 42℃ (30 min), without nutrient support. This process model achieved a significant upregulation of antimicrobial peptide expression while maintaining host survival.

[0064]

[0065]

[0066] 2.2 Analysis of Variance for Orthogonal Experiments

[0067] Range analysis directly reflects the absolute influence of each factor on the index. To further isolate the interference of random errors and clarify the statistical significance and underlying mechanism of the multi-factor synergistic induction model of this invention, analysis of variance (ANOVA) was performed on the orthogonal experimental results. The specific results are shown in Table 5. As shown in Table 5, the ANOVA indicates that factor 4 (Salmonella dosage) significantly affects the antimicrobial peptide activity of black soldier fly larvae (P<0.05). 8 A CFU / mL concentration of Salmonella can effectively penetrate the physical barriers of the black soldier fly's gut or epidermis, be specifically recognized by host pattern recognition receptors (PRRs), and then efficiently activate the Toll or Imd immune signaling cascade pathways, mediating the massive transcription and translation of downstream effector molecules (antimicrobial peptides).

[0068] Data synchronization showed that the individual statistical differences among factors 5, 2, 6, 1, and 3 did not reach a highly significant level (P>0.05). However, in the combined induction of this invention, moderate ultrasound and stepwise heat stress are designed to increase the membrane permeability of the epithelial cells on the surface or intestinal lamina of black soldier fly larvae, thereby reducing the physical resistance to the entry of factors 4 (Salmonella) and 2 (Rhodopseudomonas palustris lysate) into target cells and shortening the latency period of the immune response. In addition, under high-intensity stimulation solely dependent on pathogens (factor 4), the amino acid reserves in the larvae are drastically depleted for the synthesis of antimicrobial peptides.

[0069] In summary, this invention constructs not a linear superposition of single factors, but a nonlinear synergistic system of ultrasonic physical intervention and pathogen activation. Analysis of variance confirmed the absolute effectiveness of the core activation factor (Salmonella), while range analysis, combined with the aforementioned compensatory mechanism, ultimately identified the optimal process combination A3B2C3D3E3F1. This combination, while ensuring high-level antimicrobial peptide output, theoretically avoids the technical bottleneck of larval biomass loss and was incorporated as a standard induction procedure into subsequent embodiments for in vivo scale-up verification.

[0070]

[0071] 3. Experimental Conclusions

[0072] This embodiment systematically verified the regulatory weights of multi-factor intervention on the antimicrobial peptide synthesis efficacy of black soldier fly larvae through orthogonal experiments and range and variance analyses, and established the optimal synergistic induction process. Specific conclusions are as follows:

[0073] (1) The optimal parameter model for multi-factor synergistic induction was locked.

[0074] The study established a standardized procedure for high-throughput expression of antimicrobial peptides: a basal diet of unfermented fortified food (bran:soybean meal = 3:1:1, 60% bran, 20% soybean meal, 20% corn syrup) was used, and at 5 years of age, a 10% fortified diet was added. 8 The bacteria were treated with CFU / g feed lysate of Rhodopseudomonas palustris for 24 h, followed by starvation for 12 h for induction, and then subjected to sonication at 150 W for 15 min and 10 min for 10 hours. 8 CFU / mL Salmonella immersion for 60 seconds, heat induction: 35℃ (10 min) → 40℃ (10 min) → 42℃ (30 min), without nutritional support.

[0075] (2) The signal cascade amplification mechanism induced by ultrasound physical intervention and immunogen was revealed.

[0076] Statistical results confirmed that exposure to specific concentrations of pathogens (Salmonella) constitutes the main factor triggering humoral immunity in black soldier flies, directly mediating the efficient transcription of antimicrobial peptides. Ultrasound and heat stress upregulated the barrier permeability of the larval surface and intestinal epithelium, amplified the host's recognition efficiency of external microbial elicitors, shortened the latency of the immune response, and achieved a synergistic enhancement of antimicrobial peptide synthesis signals.

[0077] Example 3: Verification of the optimal combination and product analysis

[0078] Experimental objective: This embodiment performs three independent replicates to verify the optimal inducing combination screened in Example 2, with the uninduced group as a blank control, to detect antibacterial activity, total protein concentration, and small peptide concentration, in order to evaluate the stability and effectiveness of the inducing combination.

[0079] 1. Test Methods

[0080] 1.1 Feeding and Experimental Design

[0081] Induction treatment was performed according to the optimal induction combination selected in Example 2. Fifth-instar black soldier fly larvae reared in Example 1 were divided into two groups:

[0082] Optimal induction group: Physical and chemical induction treatments were carried out under the best combination of conditions, with 100 larvae in each group and 6 replicates.

[0083] Blank control group: Except for no physical or chemical induction treatment (only an equal amount of sterile water was used to replace the chemical inducer, and no physical treatment was performed), the other feeding conditions were exactly the same as those of the optimal induction group, and 6 replicates were set up.

[0084] The larvae were cultured according to the method in Example 2. After the culture was completed, the crude extract of antimicrobial peptides was prepared according to the method in Example 2.

[0085] 1.2 Determination of total protein concentration

[0086] The total soluble protein concentration in the crude extract was determined using the Bradford Protein Assay Kit with bovine serum albumin (BSA) as the standard. The results are shown in Table 6.

[0087] 1.3 Determination of small peptide concentration

[0088] The content of small peptides was determined using a trichloroacetic acid (TCA) precipitation method combined with the Folin-Ciocalteu method. The extracted antimicrobial peptide solution was mixed with an equal volume of 20% trichloroacetic acid, allowed to stand at 4°C for 30 min, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The content of small peptides was determined using a BCA kit (White Shark), following the instructions.

[0089] 2. Test Results

[0090] The results of independent repeated validation of the optimal induction group and the uninduced blank group, and the quantitative detection results of its antibacterial activity, total protein and small peptide concentration are shown in Table 6.

[0091]

[0092] 2.1 Quantitative evaluation of the antimicrobial titer of antimicrobial peptides

[0093] Table 6 shows that after treatment with the ultrasonic physical intervention-immunogen induction multi-factor synergistic induction program constructed in this invention, the diameter of the inhibition zone of the crude extract of antimicrobial peptides from black soldier fly larvae against indicator bacteria (Salmonella) reached 40.48 mm. Compared with the uninduced control group (36.11 mm), this index showed a significant increase, improving by 12.1%. This validation result indicates that exogenous induction not only increases the total protein expression but also significantly promotes the enrichment ratio of small peptides. The multi-factor intervention of this invention can effectively break through the baseline of basal immune expression in the uninduced state and significantly upregulate antimicrobial peptide products by synergistically activating the host defense response.

[0094] 2.2 Specific expression and enrichment of small peptide components

[0095] The absolute concentration and relative proportion of small peptides are quantitative indicators for evaluating the targeted synthesis efficiency of antimicrobial peptides. Table 6 shows that the optimal induction group had a small peptide concentration of 348.79 μg / mL, an increase of 24.9% compared to the uninduced control group (279.21 μg / mL); simultaneously, the proportion of small peptides in total soluble protein increased from 33.04% to 39.21%. This change in component proportion confirms that the multi-factor synergistic intervention program of this invention did not induce the generalized expression of non-specific proteins in larvae, but rather specifically activated the host humoral immune network, directly mediating the targeted transcription and enrichment of downstream small-molecule antimicrobial peptides.

[0096] 2.3 Quantitative verification of the compensatory mechanism for immune metabolic consumption

[0097] In a single high-intensity pathogen provocation model, excessive transcription of antimicrobial peptides competes with the host's basal metabolic components for amino acids and ATP substrates, leading to a decrease in the concentration of soluble total protein in larvae. Table 6 shows that the total protein concentration in the induced group was 889.63 μg / mL, with no concentration decay. This data confirms that a fortified basal diet (bran, soybean meal, and corn syrup matrix) upregulates available substrate flux, effectively offsetting the metabolic resource depletion induced by the immune cascade response, and mediating the efficient expression of antimicrobial peptides while maintaining host-targeted metabolic homeostasis.

[0098] 3. Experimental Conclusions

[0099] This embodiment uses independent repeated experiments to conduct in vivo validation and quantitative analysis of the product of the multi-factor synergistic induction model selected by orthogonal screening, further confirming the process stability and underlying biological mechanism of this technical solution. The specific conclusions are as follows:

[0100] (1) The process stability and efficacy enhancement effects of the synergistic induction model were verified.

[0101] Experimental data show that the combined induction program constructed in this invention can significantly upregulate the basal immune expression level of black soldier fly larvae. In in vivo independent replication validation, the antimicrobial peptide crude extract in the treatment group showed a significant increase in antibacterial effect against indicator bacteria (Salmonella), confirming the feasibility of the synergistic intervention system under scale-up preparation conditions and the stability of target product expression.

[0102] (2) The specific expression and enrichment of effector molecules were confirmed.

[0103] Product composition analysis showed that the concentration of small peptides and their relative proportion in total soluble protein were significantly upregulated in the induced group. This change in component ratio confirms that the multi-factor combined intervention program of the present invention did not induce the generalized expression of non-specific stress proteins in the host, but rather specifically activated the humoral immune signaling cascade pathway of the black soldier fly, directly mediating the large-scale transcription and enrichment of effector molecules such as antimicrobial peptides.

Claims

1. A method for inducing enhanced activity of antimicrobial peptides from black soldier fly larvae, characterized in that, Includes the following steps: (1) Inoculum treatment: Rhodopseudomonas palustris was cultured to the logarithmic growth phase to obtain Rhodopseudomonas palustris lysate; (2) Feed preparation: The hatched black soldier fly larvae were fed an unfermented intensive basal diet until they reached a suitable age, and then treated with Rhodopseudomonas swampus. After starvation, they were transferred to subsequent induction. (3) Synergistic induction: The larvae obtained in step (2) were subjected to ultrasonic intervention, Salmonella exposure and heat shock treatment in sequence; (4) Larval collection: After induction under the conditions described in step (3), larvae are collected and antimicrobial peptides are extracted.

2. The method for inducing enhanced activity of antimicrobial peptides in black soldier fly larvae according to claim 1, characterized in that: The preparation method of Rhodopseudomonas palustris lysate in step (1) is as follows: collect Rhodopseudomonas palustris cells in the logarithmic growth phase, centrifuge at 4000r for 15min, then resuspend in PBS, the concentration after resuspending is 10% (w / v), add 0.5g of lysozyme per 10ml of resuspending solution for enzymatic hydrolysis, incubate in a 37℃ water bath for 1h, and then perform repeated freeze-thaw cycles for lysis.

3. The method for inducing enhanced activity of antimicrobial peptides in black soldier fly larvae according to claim 2, characterized in that: The specific operation of the repeated freeze-thaw cycle lysis is as follows: the enzymatically hydrolyzed bacterial solution is placed in a -196°C environment for deep freezing, and then placed in a 50°C water bath for rapid thawing, and the cycle is repeated 5 times.

4. The method for inducing enhanced activity of antimicrobial peptides in black soldier fly larvae according to claim 1, characterized in that: The unfermented fortified basal diet described in step (2) comprises, by weight percentage, 60% wheat bran, 20% soybean meal and 20% corn syrup.

5. The method for inducing enhanced activity of antimicrobial peptides in black soldier fly larvae according to claim 1, characterized in that: In step (3), the parameters for ultrasound intervention are limited to: power 150W and processing time 15min.

6. The method for inducing enhanced activity of antimicrobial peptides in black soldier fly larvae according to claim 1, characterized in that: The treatment conditions for Salmonella exposure in step (3) are limited to: bacterial concentration of 10... 8 CFU / mL, soaking time 60s.

7. The method for inducing enhanced activity of antimicrobial peptides in black soldier fly larvae according to claim 1, characterized in that: The procedure for heat shock treatment in step (3) is as follows: incubate at 35°C for 10 min, raise the temperature to 40°C and incubate for 10 min, and then maintain at 42°C for 30 min.

8. The method for inducing enhanced activity of antimicrobial peptides in black soldier fly larvae according to claim 1, characterized in that: The method for collecting larvae in step (4) is as follows: after washing the larvae, rinse them twice with pure water, rinse with alcohol for 60 seconds, and then rinse with pure water until all the alcohol is washed away.

9. A black soldier fly antimicrobial peptide induced by the method according to any one of claims 1-8, characterized in that: Its crude extract of antimicrobial peptides has an inhibition zone diameter of ≥30mm against Salmonella.

10. The application of the black soldier fly antimicrobial peptide according to claim 9 in the preparation of animal feed additives, antibacterial agents or biopesticides.

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