Antibacterial peptides from black soldier fly (Hermetia illucens) larvae: mode of action and characterization | Scientific Reports

Nov 03, 2024

Scientific Reports volume 14, Article number: 26469 (2024) Cite this article

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Antibacterial peptides from black soldier fly larvae extract were prepared using Flash column chromatography. Three out of five fractions (F2, F3 and F4) showed antibacterial activity against Listeria monocytogenes DMST 17303 with a minimum inhibitory concentration (MIC) of 1 mM, followed by Salmonella enterica Enteritidis DMST 15679 and Escherichia coli O157:H7 DMST 12743 with a MIC ranging from 4 to 8 mM. Due to the higher yield, F2 and F3 were further analyzed on their mode of action against L. monocytogenes DMST 17303. Both fractions, particularly F2, exerted antibacterial activity through inducing bacterial cell membrane disintegration and interaction with intracellular compounds including fatty acids, proteins, and nucleic acids. The F3 did not show significant hemolytic activity up to 4 mM, while F2 showed lower than 5% hemolysis up to 8 mM. Time-to-kill analysis revealed that F2 was more effective and exerted a sustainable killing effect after 2 and 4 h, depending on the concentration of 1 and 2×MIC, respectively, while the F3 at 2×MIC could completely kill the test bacteria within 24 h. Among the identified peptides in the fractions, those with charged, either positively or negatively, and moderate hydrophobicity of ranging 6.68–15.70, namely CGPPRQGPFPR, HLEEELK, LEEAEERAD, TEELEEAKKK, and KGNSELEEAKKK, are potential antimicrobial peptides.

Due to concerns about antibiotics, particularly drug resistant strains, research is increasingly focused on finding alternatives. To that end, antimicrobial peptides (AMPs), as a part of a natural immune system of creatures which offer broad spectrum of activities, have increasingly been taken into consideration. Insects are well-reputed for their AMP content over other organisms. This correlates with their living area, in which such compounds make them compatible with their adverse habitat. A collection of 324 insect-derived AMPs, which have been identified so far, is the highest proportion among animals, indicating their great potency as a substitute for synthetic antibiotic1,2.

Black soldier fly (BSF) is one of the most excellent sources of AMPs among insects. Several BSF-derived AMPs such as defensins including defensin like peptides (DLP1-4), Hidefensin-1, Hill-BB (C6571, C16634, C46948 and C7985), cecropins including cecropin 1, cecropin-like peptides (CLP1-3) and Trx-stomoxynZH1a, Attacins (HI-attacin), Sarcotoxin including 1, 2a, 2b and 3, and so on, have been identified2. It has been reported that 50 encoding AMP genes are also available in the BSF genome, composing the largest AMP number among individual insects3. This implies that there are still such compounds yet to be discovered.

The mechanisms by which an AMP acts against bacteria can be varied by cell membrane modifications targeting intracellular components, such as nucleic acids, lipids or proteins. Those with the capacity to act on multiple targets are in favor due to their higher effectiveness, particularly against drug-resistant bacteria4. The majority of the above-mentioned AMPs are reported to be active at the cell membrane. For instance, defensin, a cationic Cys-rich AMP, can react with the anionic components on bacterial cell membrane, causing membrane destruction and the release of intracellular components5. Two cecropins from the BSF, namely, Hill-Cec1 and Hill-Cec10, showed antibacterial activity through a membrane permeabilizing mechanism on Pseudomonas aeruginosa ATCC 90271. The antibacterial effect of Attacins has been reported to induce cell membrane permeabilizing6. In addition, AMPs might have the ability to penetrate into the microbial cell and interact with intracellular compounds and cause cell death. The intracellular modifications can be monitored by several methods, in which Fourier Transform Infrared (FTIR) spectroscopy, by detecting the functional groups of the compounds including lipids, proteins and nucleic acids at the infrared region of 3000 − 2800, 1700 − 1500 and 1300 –1100 cm−1, respectively, can clearly illustrate the modifications. Synchrotron-based FTIR, which provides a higher signal/noise ratio, showed higher efficiency over traditional FTIR, particularly in a small area of bacterial cells7,8.

This study aimed to explore the antibacterial activities of peptides extracted from the larvae of BSFs and its fractions along with their characterization and mode of action. The corresponding peptides to the antibacterial activity will also be identified and analyzed, so that novel AMP derived from BSFs would be obtained.

The BSFL extract was subjected to fractionation using Flash column chromatography, by which 5 fractions (F1-F5) were yielded (supplementary file). The F2, F3 and F4 with the yield of 130, 34 and 6 mM (L-leucine equivalent), respectively, showed the greatest antibacterial activity against Listeria monocytogenes DMST 17303 with a MIC of 1 mM or 131.18 µg/mL for each fraction. They also inhibited Salmonella enterica serovar Enteritidis DMST 15679 and Escherichia coli O157:H7 DMST 12743 with higher MIC values (Table 1). Other fractions did not exhibit any inhibitory activity. Choi et al.9 prepared BSFL extract using various solvents, among which those extracted with methanol showed greater antibacterial activity against Klebsiella pneumoniae, Neisseria gonorrhoeae and Shigella sonnei with MIC of 44.75, 43.98 and 43.96 mg/mL, respectively, which was less potent than our study. BSFL extract prepared using 20% acetic acid inhibited growth of various Salmonella species including S. Enteritidis, S. enterica serovar Pullorum and S. enterica serovar Typhimurium, as well as E.coli and Staphylococcus aureus with the MIC of 100 ~ 200 mg/mL10. Aqueous extract of Wohlfahrtia nuba larvae showed antibacterial activities against Methicillin-sensitive S. aureus (MSSA) ATCC 29213, Methicillin-resistant S. aureus (MRSA) ATCC BAA-1680, P. aeruginosa, E. coli and S. typhi with the MIC of 12.5 mg/mL for the first three and 25 mg/mL for the last two bacteria11. Such activities were also observed in fractions preared from the extracts of honeycomb moth Galleria mellonella12, BSFL5, bee Hylaeus signatus13, cockroach Periplaneta americana14, tasar silkworm Antheraea mylitta15, among others. In addition, the attacin from H. illucens displayed antimicrobial activity against P. aeruginosa at 500 µg/mL16. The common antimicrobial agents used in food industry, such as benzoic acid and sorbic acid, can be applied to food products with the permitted levels of 0.1% (1000 µg/mL) and 0.2% (2000 µg/mL), respectively17. Thus, the MIC value of approximately 1000 µg/mL (1 mM) found in the BSFL fraction could be considered as a potential for food application. Generally AMPs contain cationic residues, by which the peptides can interact with anionic compartments on the bacterial cell membrane and induce cell lysis. However, some anionic peptides have been reported to exert antibacterial activity through interacting with cell membrane by forming a salt bridge using metal ions like Zn[2+ 18. The active fractions including F2 and F3 were used to conduct further analysis on their mode of action. Due to the lower yield of peptide, the F4 was disregarded for further analysis.

Both F2 and F3 at various concentrations exhibited bactericidal effects (Fig. 1). F2 drastically reduced viable cell counts of L. monocytogenes by 6.5 logs within 3 and 2 h at 1× and 2×MIC, respectively. The bactericidal effect of F2 remained for up to 24 h. The fraction F3 exhibited different time-kill kinetics. At the concentration of 2×MIC, growth of L. monocytogenes gradually reduced with time and complete inhibition was observed at 24 h. At lower concentration of 1×MIC, an approximate 2-log reduction in the bacterial growth was observed after 8 h. However, L. monocytogenes began to grow thereafter up to 24 h and reached 6 log, indicating an unsustainable bactericidal effect. Such an effect was also observed in AMPs from larvae of Protaetia brevitarsis Lewis against S. aureus CMCC(B) 26003, in which the bacterial growth was observed after 10 h of peptides exposure19. The killing curve of flesh fly Wohlfahrtia nuba extract at the concentration of 1×MIC revealed its growth inhibition effect of about 0.8, 0.5, 0.55, 0.45 and 0.7 log CFU/mL against MSSA, MRSA, P. aeruginosa, E. coli and Salmonella typhi within 24 h compared to the control11. The F2 from BSFL obtained in our study appeared to be a potent fraction for controlling the growth of L. monocytogenes when compared to extracts obtained from other insects that have been previously reported. Differences in time-kill kinetics also implied various types of AMP presenting in the BSFL extract.

Time-killing kinetics of F2 and F3 against L. monocytogenes DMST 17303 at 1×MIC and 2×MIC concentrations. All data are expressed as the mean values of triplicate ± standard deviation.

The red signals from the PI probe indicated the presence of damaged cells (Fig. 2). In contrast, the control showed all green fluorescence for the live cells stained by SYSTO-9 (Fig. 2). This indicated that both fractions induced cell membrane disintegration. Bacterial cell membrane disruption is a general mechanism of action of AMPs, by which bacteria are less capable to develop resistance compared to conventional antibiotics with specific targets20. Such a mechanism has been reported in cecropin type AMPs from BSF, Hill-Cec1 and Hill-Cec10, against P. aeruginosa and, hymenoptaecin (an AMP from bumblebees) against E. coli1,21. The results indicated that F2 induced a greater effect as less live cells (green spots) were observed.

Confocal fluorescence microscopic images of untreated L. monocytogenes DMST 17303 and treated with F2 and F3 at 1×MIC. Bar = 10 μm.

Modifications on the surface of L. monocytogenes before and after exposure to the fractions F2 and F3 were monitored using SEM. Based in SEM, smooth and intact surfaces can be seen in untreated cells (Fig. 3A-B), while F2 and F3 induced cell rupture (red arrows) and morphological changes as flattened cells as indicated by the white arrow (Fig. 3C-F). Such morphological modifications in L. monocytogenes have been reported by a 10-kDa peptide from Bacillus pumilus SF214, a de novo designed peptide zp80, and bacteriocins from Bacillus coagulans CGMCC 995122,23,24. Morphological changes have also been observed in E. coli exposed to a Gly-rich AMP, hymenoptaecin from bees, Bombus pascuorum and Bombus terrestris, which was reported to be associated with cell membrane disintegration and cytoplasm leakage caused by the peptide21. The SEM results confirmed the membrane disruptive mechanism of the two fractions from the BSFL extract.

Scanning electron microscopy images of L. monocytogenes DMST 17303, (A, B) Untreated (control), (C, D) treated with F2 at 1×MIC, (E, F) treated with F3 at 1×MIC. A, C, E, Bar = 1 μm and magnification 10,000x; B, D, F, Bar = 200 nm and magnification 20,000x.

Modifications of L. monocytogenes DMST 17,303 upon the fractions exposure at the molecular level were monitored by Synchrotron-based Fourier-transform infrared (SR-FTIR, Fig. 4). Major peaks corresponding to cellular fatty acids, proteins and a mixed region including fatty acids, proteins and phosphate-containing compounds like nucleic acids, DNA and RNA at the regions of 3000 − 2800, 1700 − 1500, 1500 − 1200, 1200 –900 cm−125, respectively, were obtained. Distinct modifications have been observed in secondary derivate of spectra at the regions of 2963, 2925, 2853, 1656, 1398, 1160 and 1030 cm−1 (Fig. 4B-C). Peak shifts at 2963, 2925 and 2853 cm−1 reflected -CH stretching of membrane fatty acids, while those at 1656 cm−1 suggested changes in proteins, particularly in the α-helix structure. All together along with a higher intensity at 1400 cm−1, representing the symmetric stretching of membrane fatty acids and COO− group of amino acids26, indicated cell membrane disruption and intracellular protein conformational changes caused by F2 and F3, which was in accordance with the CLSM results. It was also observed that both fractions induced structural modification of nucleic acids as indicated by a higher intensity at the PO specified region of 1160 and 1030 cm−1.

Average original SR-FTIR spectra and average of the second derivative spectra of L. monocytogenes DMST 17303 without peptide treatment (control) and cells treated with F2 and F3 at 1×MIC. (A) Average original FTIR spectra (3800 –900 cm−1), (B) average second derivative spectra of fatty acid regions (3000 –2800 cm−1), (C) protein, nucleic acid and other carbohydrate regions (1800 –950 cm−1). Triplicate experiments were conducted and a total of 150 spectra were averaged.

The PCA revealed a clear segregation of spectra of the control and the treated cells with the variation of 81% for PC-1 (Fig. 5A). The major variations were recorded at the Amide I region of the PC-1, indicating protein conformational changes induced by both fractions. In addition, F3 and F2-treated groups were separated in the PC-2 with a variation of 4%. The PC-2 plot explained the changes of fatty acids (2923, 2940 and 2852 cm−1), proteins (1693, 1658 and 1536 cm−1) and nucleic acids (1173 cm−1). Abaecin, an AMP from bumblebees, has been reported to exert activity against E. coli through its interaction with ribosomes and interfering protein biosynthesis21. The results revealed that both fractions involved peptides that can cause bacterial cell death through various deteriorative effects on membrane and intracellular components, in which F2 appeared to exert more severe effect as greater modifications were observed in the secondary derivative at the region of 1488 and 1029 cm−1, corresponding to proteins and nucleic acids (Fig. 5). The severe effect of F2 was also indicated by CLSM as more dead cells were spotted.

PCA analysis of the SR-FTIR spectra and average of the second derivative spectra of L. monocytogenes DMST 17303 without peptide treatment (control) and cells treated with F2 and F3 at 1×MIC. (A) PCA of 2D score plot and (B) PCA loading plot.

Thirteen peptides (3 from F2 and 10 from F3) with the de novo score > 65 and homology to BSF proteins were identified (Table 2). The identified peptides with diverse molecular mass ranging from 643 to 1771 Da and composed of 5–16 amino acid residues were revealed. These peptides were synthesized and their antimicrobial activities were tested against E. coli O157:H7 DMST 12743, Salmonella enteritidis DMST 15679 and Listeria monocytogenes DMST 17303. It can be observed that F2 and F3 exhibited greater activity against L. monocytogenes compared to the individual peptides. This can be due to the synergic effect of several peptides in the fractions. Rahnamaeian et al.21 reported that the AMP abaecin had no effect on E. coli up to 200 µM, and hymenoptaecin can act against the bacteria with bactericidal IC50 of 3.01 µM, when they were applied separately. However, a combination exposure of 20 µM abaecin reduced the bactericidal IC50 of hymenoptaecin to 0.63 µM. The authors observed that hymenoptaecin, an AMP with a membrane disintegrative mode of action, could facilitate penetration of abaecin and subsequently its interaction with ribosome. Therefore, a lower concentration of hymenoptaecin is sufficient to kill the defective bacteria. Such a phenomenon has also been reported for lebocin and cecropin D from silkworms, in which an application of 50 µg/mL lebocin (MIC = 800 µg/mL alone) can reduce the MIC of cecropin D from 6.25 µg/mL when applied alone to 3.13 µg/mL27.

Five peptides, namely, CGPPRQGPFPR from the F2, and HLEEELK, LEEAEERAD, TEELEEAKKK and KGNSELEEAKKK from the F3 showed antimicrobial activity against three test bacteria with a MIC of 4 mM (Table 2). These peptides can be considered as new AMPs in BSFL as they have never been reported in the Antimicrobial Peptide Database available on https://aps.unmc.edu/home and the Database of Antimicrobial Activity and Structure of Peptides available on https://dbaasp.org/home. These peptides are much smaller than previously reported AMPs in BSL, including defensins, cecropins, attacins, and sarcotoxins, which are composed of 20–50 amino acid residues2. It should be noted that peptides extracted from our study were derived from homogenate of whole BSFL, while hemolymph samples of immunized BSF by bacteria were mainly used for AMP isolation5,28,29. Our study indicated that BSFL without bacterial immunization also contained short peptides exhibiting antimicrobial activity. All the active AMPs were either positively or negatively charged. The positively charged residues can facilitate electrostatic interactions between the peptides and anion compartments in bacterial cell membrane, which can lead to destruction and subsequently cell death4. However, those with negatively charged residues can indirectly interact with bacterial cell membrane through forming salt bridges by employing metal ions such as Zn2+. In addition, the anionic antimicrobial peptides can bind to divalent ions such as Ca2+ and Mg2+ and cause a lack of the elements which are essential for bacterial growth30. The active peptides possessed hydrophobicity between 6.68 and 15.70 (Table 2). Strandberg et al.31 reported that higher hydrophobicity can lead to higher antibacterial and lower hemolytic activities in short peptides, while it is not a requirement in longer peptides (> 8 residues). In this study, the longer active peptides have a hydrophobicity of 6.68–8.39. Those beyond the range did not show activity except for CGPPRQGPFPR with the hydrophobicity of 15.70. Chen et al.32 reported that hydrophobicity has an optimum window regarding antibacterial activity, in which a higher or lower hydrophobicity can lead to lower activity. Higher hydrophobicity likely induces the self-association of peptides and hemolytic activity. However, the activity of CGPPRQGPFPR might be correlated to the presence of Pro and Cys. Pro has a part in the linear conformation of AMPs, which is appropriate for the cell permeation and Cys can induce the formation of S-S bonds to stabilize β-hairpin or sheet structure7. The result of the study indicated that the antibacterial activities of BSFL extract induced by peptides, in which hydrophobicity, charge, and amino acid type govern the activity. Since all the peptides were illustrated with random coil structures, their transformation needs to be taken into consideration. Structure modification of these peptides could be one of the promising approaches leading to novel peptides with enhanced antimicrobial activity.

Hemolysis rate is an important aspect of an antibacterial compound which reflects its safety. In this study the hemolysis rate of both fractions was dose-dependent, however, F2 exerted a remarkably lower rate compared to F3 (Fig. 6). According to Standard Practice for Assessment of Hemolytic Properties of Materials (ASTM) reported by Choi et al.33, a hemolytic rate less than 5% has been reported as a safe limit. The F2 exhibited a hemolysis rate of less than 5% at concentrations up to 8 mM, while the F3 appeared to be safe up to 4 mM. These concentrations are 8 and 4 times, respectively, higher than the respective MIC values, implying their safety for food and feed application. The higher hemolytic rate in F3 might be correlated to the presence of peptides with higher hydrophobicity (Table 2). Peptides with higher hydrophobicity can interact with the cell wall to a greater extent, resulting in higher killing capacity. Although peptides with high hydrophobicity tend to be dimer and in a α-helical folded conformation, they can be easily dissociated to monomer in a high hydrophobic environment of eukaryote cell wall mediated by bilayer phospholipids and cholesterol, which allows more cellular penetration32. Shin et al.34 observed that Leu substitution in the AMP, KWKLFKKIPKFLHLAKKF, caused an increased hemolytic effect which could be due to an increase in hydrophobicity. The peptides KLPEWRW, AERELVR and EVKLR are among those with higher proportions of hydrophobic residues (52, 42 and 40%, respectively). The presence of more peptides with higher hydrophobic residues in F3 would explain its higher hemolysis rate.

Hemolytic activity in human red blood cells. Data are the average of at least four independent experiments. Error bars represent the standard deviations. Lowercase and uppercase letters indicate a significant difference in hemolysis rate of F2 or F3 (Tukey’s test, p < 0.05), respectively. The asterisk (*) denotes a significant difference between F2 and F3 (Student’s t-test, p < 0.05) at the same peptide concentration.

The extract prepared from black soldier fly larvae (BSFL) showed antibacterial activity against Listeria monocytogenes DMST 17303, Salmonella enteritidis DMST 15679 and E. coli O157:H7 DMST 12743, in which the former was the most susceptible bacterium. The C18-prepared fractions, F2 and F3, included peptides which acted against bacterial cell membrane integrity and intracellular compounds. Although both fractions exerted comparable activities, F2 had a low hemolytic rate and showed more potent and sustainable effects which inhibited growth of L. monocytogenes DMST 17303 immediately after exposure, making it a proper source of antibacterial peptides. Identified peptides from F2 and F3 revealed that charge and a moderate hydrophobicity appeared to be critical factors contributing to the antibacterial activity.

Black soldier fly Hermetia illucens larvae were obtained from Industrial Insects Pilot Production Plant (Khon Kaen University, Thailand). Three bacterial strains, including L. monocytogenes DMST 17303, S. Enteritidis DMST 15679 and E. coli O157:H7 DMST 12,743 were obtained from Department of Medical Science of Thailand (DMST). Chemicals and HPLC grade solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Type I water with resistivity of approximately 18 MΩ. was used.

The BSFLs were cleaned, lyophilized, and finely grounded prior to extraction with acidified methanol (methanol/water/acetic acid; 90/9/1; v/v/v). The extract was centrifuged at 1600×g for 10 min at 4 °C. Solvent was removed by a rotary evaporator at 60 °C. Lipid was removed by sequential extraction with chloroform three times. The water-soluble fraction was collected and stored in a refrigerator at -20 °C until use.

L. monocytogenes DMST 17303, S. Enteritidis DMST 15679 and E. coli O157:H7 DMST 12743 were purchased from the Department of Medical Sciences (DMST; Bangkok, Thailand). All bacterial strains were maintained as frozen stock culture supplemented with 30% glycerol at -80 °C in a freezer. The frozen cultures were activated in tryptic soy broth (TSB) for S. Enteritidis DMST 15,679 and E. coli O157:H7 DMST 12743 and TSB supplement with 0.6% yeast extract (TSBYE) for L. monocytogenes DMST 17303 and grown overnight at 37 °C. Subsequently, activated cells were cultured on tryptic soy agar (TSA) for the first 2 bacteria or TSA with 0.6% yeast extract for L. monocytogenes, and incubated at 37 °C for 24 h. A single colony was used as a working stock culture.

The water-soluble extract was injected into a C18 Flash column (C18AQ-15 μm, 30 × 250 mm, Interchim, Montluçon, France) equipped with Puriflash® 5.250 system. DI with 0.05% trifluoroacetic acid (v/v; TFA) and acetonitrile was added with 0.05% TFA were used as mobile phases of A and B, respectively and the elution was carried out as follows: 100% A, 0–12 min; 90%A, 12–30 min; 70%, 30–50 min; 70% A to 0% A, 50–55 min; 0% A, 55–105 min; 0% A to 100% A, 105–110 min and 100% A, 110–120 min. Eluted peptides were detected at 214 nm. All fractions were collected and lyophilized (CHRIST Gamma 2–16 LSC, Germany) for further experiments, including the antibacterial activity assay.

The antibacterial activity of fractions and synthetic peptides was evaluated using the microbroth dilution assay according to a modified Clinical Laboratory Standards Institute (CLSI) -based method. Briefly, overnight-grown cultures of L. monocytogenes DMST 17303, S. Enteritidis DMST 15679 and E. coli O157:H7 DMST 12743 were collected and diluted with a fresh Mueller-Hinton broth (MHB) or MHB with 0.6% yeast extract to attain a final concentration of approximately 1 × 106 CFU/mL. Equal volumes of the bacterial suspension were incubated with serially-diluted peptide solutions in a 96-well microtiter plate. Bacterial cells without peptides served as a negative control. After incubation for 18–24 h at 37 °C, absorbance at 600 nm was determined to evaluate bacterial growth inhibition using a microtiter plate reader (Thermo Fisher Scientific, Waltham, MA, USA). The minimum inhibitory concentration (MIC) was defined as the lowest concentration of peptide that inhibited 90% of bacterial growth. All experiments were conducted in triplicate.

A time-kill assay was performed with L. monocytogenes DMST 17303 in the presences of active fractions at the concentrations of 1×MIC and 2×MIC, and incubation at 37 °C. Aliquots of 100 µL of cell suspension were taken at 0, 1, 2, 4, 6, 8, and 24 h, then diluted with normal saline and an aliquot of 10 µL was dropped on tryptic soy agar supplemented with 0.6% yeast extract. Enumeration was carried out after 24 h of incubation at 37 °C. Data were obtained from two independent experiments performed in triplicate.

The effect of active fractions on membrane integrity was investigated by CLSM using propidium iodide (PI) and SYTO-9. L. monocytogenes DMST 17303 cells in mid-log phase in TSBYE were harvested, washed, and resuspended to a final concentration of 107 CFU/mL in PBS (pH7.4). Bacterial suspension was treated with F2 and F3 at the concentration of 1×MIC for 2 h, while the controls were carried out without peptide. Bacterial cells were added with either PI or SYTO-9 solutions at final concentrations of 5 µg/mL and 20 µg/mL, respectively. The mixture was incubated at 4 °C in the dark for 30 min. Samples were washed three times with PBS and transferred into a glass slide. Images were taken by a confocal laser scanning microscope (Nikon 90i A1R, Tokyo, Japan), with an excitation filter (488 nm, green; 538 nm, red), and an emission filter (530 nm, green; 617 nm, red).

SEM was applied to elucidate morphological changes after exposure to active fraction(s) of BSFL. Bacterial culture in the logarithmic phase was incubated with samples at the concentration of 1×MIC for 2 h at 37 °C. Untreated bacterial cells were used as a control. All samples were centrifugated at 3000×g for 10 min. Cells were fixed overnight with 2.5% (w/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 °C, followed by fixation with 1% osmium tetroxide for 2 h and washed three times with distilled water. Samples were dehydrated by a graded series of acetone (20, 40, 60, 80, and 100% for 10 min). The samples were mounted on aluminum tape (diameter 4 mm) and then attached on stubs and coated with carbon at approximately 20 nm thicknesses (Sputter Coater, Leica/EM ACE600, Wetzlar, Germany) as previously described by Su et al.35. The specimens were observed under a scanning electron microscope (Zeiss AURIGA FESEM/FIB/EDX, Jena, Germany).

The effect of active fractions on cellular components of L. monocytogenes DMST 17303 was elucidated using SR-FTIR spectroscopy. The samples were prepared for SR-FTIR microspectroscopy measurements as previously described by Pimchan et al.25. Briefly, overnight-grown culture was diluted with TSBYE broth to obtain a cell density of 108 CFU/mL. The Bacterial suspensions were treated with F2 and F3 at final concentrations of 1 × MIC at 37 °C for 2 h. Bacterial cells without peptides were used as the control. Cells were collected by centrifugation at 3,000×g for 10 min at 4 °C. Cell pellets were washed twice with normal saline and then resuspended in distill water. The resuspended cells were transferred onto an IR-transparent 2-mm-thick barium fluoride (BaF2) window. Samples were dried in a desiccator before FT-IR analysis.

SR-FTIR microspectroscopy measurements were performed at the beamline 4.1 of the Synchrotron Light Research Institute (Nakhon Ratchasima, Thailand). FT-IR spectra were acquired in transmission mode using a Bruker Vertex 70 spectrometer coupled with a Bruker Hyperion 2000 microscope (Bruker Optics Inc., Ettlin-Gen, Germany). All spectra were recorded in the region between 3000 –800 cm−1 at a 4 cm−1 spectral resolution with 64 scans. Approximately 150 spectra (50 spectra per replicate) were recorded and used for analysis. OPUS 7.5 software (Bruker Optics Ltd., Ettlingen, Germany) was used for spectral acquisition and instrument control. The signal intensity of second derivatives were calculated using Savitzky-Golay algorithms (seventeen smoothing points) and subjected to principal component analysis (PCA) using the Unscramble X 10.4 software (CAMO Software AS, Oslo, Norway).

Fractions (F2 and F6) were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were dissolved in 0.1% formic acid, filtered thought a 0.22-µm filter, and separated on the AdvanceBio peptides plus column (150 mm×4.6 mm×2.7 μm, Agilent Technologies, Santa Clara, CA, USA), equipped with the Ultimate 3000 UHPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA) coupled to electrospray ionization (ESI) tandem mass spectrometer (micro TOF-Q II) (Bruker Daktinics, Bremem, Germany). Samples were eluted by 0.1% formic acid in deionized water as a solvent A, and 0.1% formic acid in acetonitrile as a solvent B at a flow rate of 0.5 mL/min. The elution was performed as follows: 0–5 min (2% B), 5–35 min (2–35% B), 35–40 min (35–95% B), 40–45 min (95% B), 45–47 min (95 − 2% B) and 47–55 min (2% B). The LC-QTOF data were acquired and processed by Compass 1.3 software (Bruker, Germany) and data were analyzed using the PEAKS Studio 10.0 software (Waterloo, ON, Canada). Peptides with de novo scores of > 65 and similar amino acids to BSFL proteins (taxid:343691) were synthesized using a solid phase synthesis and subjected to antibacterial activity evaluation. Charge and hydrophobicity of peptides were evaluated using peptide analyzing tools available at https://www.thermofisher.com/.

The hemolytic activity of samples was determined using human red blood cells (hRBC) obtained from healthy volunteers. The hRBCs were centrifuged at 1000 ×g for 5 min to collect erythrocytes and washed three times with phosphate-buffered saline (PBS) pH 7.4. An aliquot of 50 µL of the peptide solution in PBS (two-fold serial dilution) were mixed with 50 µL of 2% (v/v) hRBC suspension in PBS. The samples were incubated at 37 °C for 1 h and centrifuged at 1000 ×g for 5 min. The RBC was incubated with PBS, and 10% Triton-X-100 was also prepared as a negative and positive control, respectively. The supernatants were transferred to 96-well plates. Hemoglobin release was measured at 570 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Percent hemolysis was calculated by the following formula:

Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 23.0 (Chicago, Illinois, USA). Results were expressed as the mean ± standard deviation. The student’s t-test was used to evaluate differences between 2 samples, while differences among samples were tested by ANOVA. Differences were considered significant at p < 0.05.

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. All the original and analyzed data are available upon request from the corresponding author

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This research received funding support from Agricultural Research Development Agency (Public Organization) under a research grant number CRP6405031400. TP has received a postdoctoral fellowship from (i) Suranaree University of Technology (SUT) and (ii) the NSRF via the Program Management Units for Human Resource & Institutional Development, Research and Innovation (PMU-B) (grant number B13F660067). Additional funding from the National Research Council of Thailand (N42A650548) was greatly appreciated.

School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand

Thippawan Pimchan, Ali Hamzeh, Patcharin Siringan & Jirawat Yongsawatdigul

Synchrotron Light Research Institute, Nakhon Ratchasima, 30000, Thailand

Kanjana Thumanu

Department of Entomology, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

Yupa Hanboonsong

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T.P. carried out the investigation and wrote the main manuscript text. A.H wrote discussion and edited the manuscript. P.S. assisted on microbiological testings. K.T. helped analysis of SR-FTIR data and interpretation. Y.H. reared black solider flies for the experiment. J.Y conceptualized the work, edited the manuscript, and managed overall project. All authors reviewed the manuscript.

Correspondence to Jirawat Yongsawatdigul.

The authors declare no competing interests.

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Pimchan, T., Hamzeh, A., Siringan, P. et al. Antibacterial peptides from black soldier fly (Hermetia illucens) larvae: mode of action and characterization. Sci Rep 14, 26469 (2024). https://doi.org/10.1038/s41598-024-73766-1

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Received: 23 April 2024

Accepted: 20 September 2024

Published: 02 November 2024

DOI: https://doi.org/10.1038/s41598-024-73766-1

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