Method for improving antibacterial ability of hermetia illucens and application thereof

Abstract

The invention belongs to the technical field of biological genetic engineering, and particularly relates to a method for improving the antibacterial ability of hermetia illucens larvae. The method comprises the following steps: feeding black soldier fly larvae with a vibrio alginolyticus bacterial liquid to obtain induced black soldier fly larvae; the method comprises the following steps: cleaning and disinfecting larvae, extracting total mRNA of the larvae by adopting a Trizol method, reversely transcribing the total mRNA into cDNA, and detecting expression change of antibacterial peptide genes by adopting a qRT-PCR (quantitative reverse transcription-polymerase chain reaction) technology; meanwhile, the antibacterial effect of the antibacterial peptide in the induced hermetia illucens larvae is evaluated. The result of the embodiment shows that the vibrio alginolyticus successfully induces the expression of the antibacterial peptide gene of the hermetia illucens larvae, the optimal induction time is determined, the antibacterial ability of the larvae after induction is remarkably enhanced, and an important basis is provided for the expression of the antibacterial peptide gene of the hermetia illucens and the research on related antibacterial mechanisms.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for improving the antibacterial ability of black soldier flies and its application. Background Technology

[0002] Antimicrobial peptides ( Antimicrobial Peptides Antimicrobial peptides (AMPs) are a class of small polypeptides with broad-spectrum antimicrobial activity, typically composed of 20-60 amino acid residues, produced in vivo through induction. As an important component of the body's innate immune defense system, antimicrobial peptides not only inhibit Gram-positive and Gram-negative bacteria, but some also exhibit killing activity against fungi, viruses, and even tumor cells. Compared to traditional antibiotics, the unique membrane-targeting mechanism of antimicrobial peptides makes them less likely to induce bacterial resistance, providing a new approach to solving the increasingly serious problem of antibiotic resistance.

[0003] Black soldier fly ( Hermetia illucens L As a valuable resource insect, the black soldier fly larvae have shown great potential in the treatment of organic waste and the production of high-value insect protein. Their larvae are not only rich in nutrients such as protein and fat, but also have attracted widespread attention due to their powerful immune system and abundant antimicrobial peptide resources. Studies have shown that black soldier fly larvae contain a variety of antimicrobial peptides, among which defensins and cephalosporins are two of the most crucial and efficient antimicrobial peptide families. These antimicrobial peptides constitute an important immune defense line for black soldier flies against pathogenic microorganisms.

[0004] Therefore, there is an urgent need to provide a method that can efficiently induce the expression of antimicrobial peptide genes in black soldier fly larvae. By screening specific inducing bacteria and optimizing key induction parameters, a reliable technical path can be provided for in-depth research and resource utilization of black soldier fly antimicrobial peptides. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the antibacterial ability of black soldier fly larvae. The method provided by this invention can significantly improve the antibacterial ability of black soldier fly larvae, and provides an important foundation for the study of the expression of antimicrobial peptide genes and related antimicrobial mechanisms in black soldier fly larvae.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for improving the antibacterial ability of black soldier flies, comprising the following steps: S1, Vibrio alginolyticus and basic feed were mixed and fed to fifth-instar black soldier fly larvae to obtain induced black soldier fly larvae; S2. After cleaning and disinfecting the induced black soldier fly larvae, the surface liquid of the black soldier fly larvae is aspirated, and the extract is added to the extract solution for extraction and centrifugation. The supernatant is then treated in a water bath and centrifuged again to obtain the black soldier fly antimicrobial peptide extract solution.

[0007] Preferably, the basic feed in step S1 is a mixture of wheat bran and corn flour in a 3:2 ratio.

[0008] Preferably, the concentration of Vibrio alginolyticus in step S1 is 2 × 10⁻⁶. 8 CFU / mL.

[0009] Preferably, the ratio of Vibrio alginolyticus to the basic feed in step S1 is 1:1.

[0010] Preferably, the culture conditions for black soldier fly larvae in step S1 are: cultured at 24-27 ℃ for 72 h.

[0011] Preferably, the cleaning and disinfection process in step S2 involves rinsing with distilled water followed by disinfection with 75% ethanol.

[0012] Preferably, the ratio of the amount of extract added in step S2 to the black soldier fly larvae is 3:1; the extract is prepared by mixing 2‰ mercaptoethanol, 35 µg / mL benzyl sulfonyl fluoride PMSF and 0.05 mol / L pH 5 ammonium acetate buffer in a ratio of 2:1:97.

[0013] Preferably, the first centrifugation in step S2 is performed 4 times, at a temperature of 4 ℃, at a speed of 12000 r / min, and for a time of 15 min.

[0014] Preferably, the process of centrifugation after water bath treatment in step S2 is as follows: the supernatant is placed in a water bath at 100 °C for 5 min, followed by centrifugation at 4 °C and 4800 r / min for 30 min.

[0015] The present invention also provides the application of the above method in improving the antibacterial ability of black soldier flies, wherein the antibacterial activity is against Vibrio alginolyticus.

[0016] The beneficial effects of this invention are: This invention screened Vibrio alginolyticus as a highly efficient inducing source and determined its optimal inducing concentration to be 2 × 10⁻⁶. 8 With a concentration of CFU / mL and an optimal induction time of 72 h, the study systematically revealed the temporal expression changes of antimicrobial peptide genes in black soldier fly larvae under these conditions, filling a gap in the research on immune induction of this strain in black soldier fly larvae.

[0017] The induction method established in this invention is simple in process and has controllable cost. The crude extract of black soldier fly antimicrobial peptides obtained after induction shows obvious antibacterial effect, providing a practical basis for the large-scale extraction and application of antimicrobial peptides.

[0018] This invention not only lays the experimental foundation for elucidating the mechanism of action of black soldier fly antimicrobial peptides, but also provides key molecular biological theoretical basis and technical path for the further development and utilization of black soldier fly antimicrobial peptide resources, promoting their potential application in the fields of biomedicine and feed additives. Attached Figure Description

[0019] 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.

[0020] Figure 1 The following figures illustrate the changes in antimicrobial peptide genes and defensins in black soldier fly larvae under different induction conditions: A represents the change in DEF gene expression in black soldier flies after 24 h of Vibrio vulnificus stress; B represents the change in DEF gene expression in black soldier flies after 48 h of Vibrio vulnificus stress; C represents the change in DEF gene expression in black soldier flies after 72 h of Vibrio vulnificus stress; D represents the change in DEF gene expression in black soldier flies after 24 h of Vibrio splenificus stress; E represents the change in DEF gene expression in black soldier flies after 48 h of Vibrio splenificus stress; F represents the change in DEF gene expression in black soldier flies after 72 h of Vibrio splenificus stress; G represents the change in DEF gene expression in black soldier flies after 24 h of Vibrio alginolyticus stress; H represents the change in DEF gene expression in black soldier flies after 48 h of Vibrio alginolyticus stress; and I represents the change in DEF gene expression in black soldier flies after 72 h of Vibrio alginolyticus stress. Figure 2 The changes in the antimicrobial peptide gene cephalosporin in black soldier fly larvae under different induction conditions are shown in Figure 1. A represents the change in CEC gene expression in black soldier flies after 24 h of Vibrio vulnificus stress; B represents the change in CEC gene expression in black soldier flies after 48 h of Vibrio vulnificus stress; C represents the change in CEC gene expression in black soldier flies after 72 h of Vibrio vulnificus stress; D represents the change in CEC gene expression in black soldier flies after 24 h of Vibrio splenificus stress; E represents the change in CEC gene expression in black soldier flies after 48 h of Vibrio splenificus stress; F represents the change in CEC gene expression in black soldier flies after 72 h of Vibrio splenificus stress; G represents the change in CEC gene expression in black soldier flies after 24 h of Vibrio alginolyticus stress; H represents the change in CEC gene expression in black soldier flies after 48 h of Vibrio alginolyticus stress; and I represents the change in CEC gene expression in black soldier flies after 72 h of Vibrio alginolyticus stress. Detailed Implementation

[0021] The purpose of this invention is to provide a method for inducing the expression of antimicrobial peptide genes in black soldier fly larvae. This method screens out highly efficient inducing bacterial strains and clarifies their optimal induction concentration and time parameters.

[0022] Another objective of this invention is to obtain black soldier fly larvae antimicrobial peptides with high antibacterial activity through the above method.

[0023] A method for inducing the expression of antimicrobial peptide genes in black soldier fly larvae, comprising the following steps: (1) Induction of black soldier fly larvae: Fifth instar black soldier fly larvae were used as the induction target, and the concentration was 2×10⁻⁶. 8 Black soldier fly larvae were fed a mixture of CFU / mL Vibrio alginolyticus and basal feed at a temperature of 24-27℃ for 72 hours.

[0024] (2) Crude extraction of antimicrobial peptides from black soldier fly larvae: After cleaning and disinfecting the induced black soldier fly larvae, extract them with the extraction solution and centrifuge. Take the supernatant, boil it in a water bath, and then centrifuge it again to obtain the concentrated extract.

[0025] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0027] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0028] Example 1: qRT-PCR detection of antimicrobial peptide genes in black soldier fly larvae induced by different bacteria. 1.1 Induction of black soldier flies The experiment used three inducing sources: Vibrio vulnificus, Vibrio alginolyticus, and Vibrio brilliantus. The strains were provided by the Microbiology Laboratory of the College of Fisheries and Life Sciences, Dalian Ocean University.

[0029] The thawed and preserved bacterial culture was inoculated into 10 mL test tubes, and 2 mL of liquid culture medium was added. The tubes were incubated at 37 °C and 160 rpm for 24 h. The cultured bacterial solution was then streaked onto solid culture medium using the streak plate method. The streaked plates were incubated at 37 °C for 24 h. Single colonies were isolated and inoculated into liquid culture medium for further activation for 24 h. The cultured bacterial solution was diluted to 2 × 10⁻⁶ with PBS buffer. 7 CFU / mL, 4×107 CFU / mL, 2×10 8 Store after CFU / mL.

[0030] Five-day-old black soldier fly larvae were fed with cultured bacteria. The five-day-old black soldier fly larvae were obtained by hatching black soldier fly eggs. The ratio of bacterial culture to feed was 1:1. The culture temperature was 24-27 ℃, and the culture time was 24 h, 48 h, and 72 h, respectively.

[0031] 1.2 Extraction of mRNA and synthesis of cDNA from black soldier fly larvae The experiment used the Trizol method to extract total mRNA from black soldier fly larvae. The specific steps are as follows: The sample, stored at -80 ℃, was ground. Removed from the freezer, 200 μL of pre-chilled Trizol lysis buffer was added to ice. The sample was then thoroughly ground on ice to lyse the cells. 800 μL of Trizol lysis buffer and 200 μL of pre-chilled chloroform were added, and the mixture was vigorously shaken for 3 min. Centrifuged at 4 ℃, 12000 rpm for 15 min. The supernatant was transferred to a new centrifuge tube, an equal volume of isopropanol was added, and the mixture was shaken well and placed at -20 ℃ for 30 min to settle. Centrifuged at 4 ℃, 12000 rpm for 15 min. The supernatant was discarded, and the precipitate was retained. The precipitate was washed twice with 1 mL of 75% ethanol, centrifuged at 4 ℃, 12000 rpm for 5 min, and the supernatant was discarded. The mRNA precipitate was dried for 5-10 min, dissolved in 20-50 μL of DEPC water, and reverse transcribed into cDNA using a reverse transcription kit (TransGen Biotech, China).

[0032] 1.3 qRT-PCR The cDNA was diluted 10-fold for PCR. The qRT-PCR assay was performed using an ABI 7500 real-time quantitative PCR instrument (Applied Biosystems, Germany). The instrument program was: 95 °C, 30 s; 95 °C, 5 s followed by 60 °C, 34 s, 40 cycles; 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s. Melting curves and standard curves were obtained after the reaction, and the ct value was then calculated. β-actin was selected as the internal reference gene.

[0033] The primer sequences used in the qRT-PCR experiment are shown in Table 1: Table 1 Primer Sequences

[0034] The examples demonstrate the changes in the antimicrobial peptide genes of black soldier fly larvae after induction with different bacterial solutions and concentrations. The experimental results are as follows: Figure 1 As shown in (AI), the relative expression level of the DEF gene is 2 × 10⁻⁶. 7 CFU / mL concentration of Vibrio vulnificus was highest 24 h after induction, due to Figure 2 (AI) indicates that the relative expression level of the CEC gene is 2×10⁻⁶. 8 The concentration of CFU / mL of Vibrio alginolyticus reached its highest level after 72 h of induction, showing a significant increase compared to the uninduced group and outperforming other experimental groups.

[0035] Example 2: Antibacterial effect of antimicrobial peptides induced by different bacteria in black soldier fly larvae Black soldier fly larvae were collected after induction, rinsed repeatedly with distilled water, and disinfected with 75% ethanol. The surface liquid of the larvae was blotted dry, and they were placed in centrifuge tubes. Extraction buffer (1:3) (prepared by mixing 2‰ mercaptoethanol, 35 µg / mL benzyl sulfonyl fluoride PMSF, and 0.05 mol / L pH=5 ammonium acetate buffer in a 2:1:97 ratio) was added and homogenized thoroughly in a homogenizer. The mixture was then centrifuged (4 ℃, 12000 r / min) for 15 min, repeated 3 times. The resulting supernatants were mixed and placed in a 100 ℃ water bath for 5 min, followed by centrifugation (4 ℃, 4800 r / min) for 30 min to remove denatured proteins. The supernatant, free of oil layer, was collected using a pipette tip and stored at -20 ℃ for later use.

[0036] Staphylococcus aureus was used as an indicator bacterium, and the inhibition zone experiment was conducted using the perforation method. The inhibition ability was evaluated by measuring the diameter of the inhibition zone.

[0037] This embodiment uses the comparison of inhibition zone diameter to determine the antimicrobial activity of antimicrobial peptides produced by black soldier flies, and the results are shown in Table 2. The results show that the antimicrobial peptide activity of black soldier fly larvae induced with different bacterial solutions and concentrations increases with the induction time, and compared with other induction conditions, Vibrio alginolyticus induction for 72 h has the best antimicrobial effect.

[0038] Table 2. Results of inhibition zone diameter under different induction conditions

[0039] Note: A: Vibrio brilliance; B: Vibrio vulnificus; C: Vibrio alginolyticus; 1:2×10 7 CFU / mL; 2: 4 × 10 7 CFU / mL; 3: 2×10 8 CFU / mL.

[0040] 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 improving the antibacterial ability of black soldier fly larvae, characterized in that, Includes the following steps: S1, Vibrio alginolyticus and basic feed were mixed and fed to fifth-instar black soldier fly larvae to obtain induced black soldier fly larvae; S2. After cleaning and disinfecting the induced black soldier fly larvae, the surface liquid of the black soldier fly larvae is aspirated, and the extract is added to the extract solution for extraction and centrifugation. The supernatant is then treated in a water bath and centrifuged again to obtain the black soldier fly antimicrobial peptide extract solution.

2. The method according to claim 1, characterized in that, The basic feed mentioned in step S1 is a mixture of wheat bran and corn flour in a 3:2 ratio.

3. The method according to claim 1, characterized in that, The concentration of Vibrio alginolyticus in step S1 is 2 × 10⁻⁶. 8 CFU / mL.

4. The method according to claim 1, characterized in that, In step S1, the ratio of the mixed liquid of Vibrio alginolyticus and the basic feed is 1:

1.

5. The method according to claim 1, characterized in that, The culture conditions for black soldier fly larvae in step S1 are: culture at 24-27℃ for 72 h.

6. The method according to claim 1, characterized in that, The cleaning and disinfection process in step S2 involves rinsing with distilled water followed by disinfection with 75% ethanol.

7. The method according to claim 1, characterized in that, In step S2, the ratio of the amount of extract added to the black soldier fly larvae is 3:1; the extract is prepared by mixing 2‰ mercaptoethanol, 35 µg / mL benzyl sulfonyl fluoride PMSF and 0.05 mol / L, pH 5 ammonium acetate buffer in a ratio of 2:1:

97.

8. The method according to claim 1, characterized in that, The first centrifugation in step S2 is performed 4 times, at a temperature of 4°C, at a speed of 12000 r / min, and for 15 min.

9. The method according to claim 1, characterized in that, The process of centrifugation after water bath treatment in step S2 is as follows: the supernatant is placed in a water bath at 100 °C for 5 min, followed by centrifugation at 4 °C and 4800 r / min for 30 min.

10. The application of the method according to claim 1 in improving the antibacterial ability of black soldier fly larvae, characterized in that, The antibacterial agent is against Vibrio alginolyticus.

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