May 17, 2018

Antimicrobial properties

Scientific papers regarding black soldier fly larvae antimicrobial properties:

The conducted feeding trials have shown the enormous potential of using Hermetia illucens in organic waste management. The material reduction of human faeces was 51.3 %, while the corresponding WBC was 9 %.
The inactivation of Salmonella Senftenberg and the two types of Salmonella Typhimurium (the widespread DT 178 and the antibiotic multiresistant DT 104) was found to be accelerated in the BSF larvae treatment, while the reduction in the concentration of S. Dublin was as rapid as in the control.

Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named
cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein
with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gramnegative bacterium Escherichia coli, fungus Rhizoctonia solani KhuÈn (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens.

The black soldier y Hermetia illucens is used for the bioconversion of organic waste into feed for livestock and aquaculture, and is economically among the most important farmed insects in the world. The larvae can be fed on agricultural waste and even liquid manure, resulting in highly unpredictable pathogen levels and dietary conditions. Here we show that H. illucens larvae express a remarkably
expanded spectrum of antimicrobial peptides (AMPs), many of which are induced by feeding on a diet containing high bacterial loads. The addition of sulfonated lignin, cellulose, chitin, brewer’s grains or sunower oil revealed the diet-dependent expression proles of AMPs in the larvae. The highest number of AMPs and the highest levels of AMP expression were induced by feeding larvae on diets supplemented
with protein or sunower oil. Strikingly, the diet-dependent expression of AMPs translated into diet dependent proles of inhibitory activities against a spectrum of bacteria, providing an intriguing example for the emerging eld of nutritional immunology. We postulate that the ne-tuned expression of the expanded AMP repertoire mediates the adaptation of the gut microbiota to the digestion of unusual diets, and this feature could facilitate the use of H. illucens for the bioconversion of organic waste.

The antibacterial effects of larval extract from Hermetia illucens, commonly known as the black soldier fly, have been demonstrated in vitro. In this study, gas chromatography–mass spectrometry analysis identified the active compound within this larval extract as hexanedioic acid. The antibacterial effects of hexanedioic acid were investigated in mice infected with Klebsiella pneumoniae. After administration of hexanedioic acid, infected mice showed decreased lung bacterial loads and lower rates of body weight loss compared to those in the infection‐only control group. Based on lung bacterial loads, oral hexanedioic acid treatment showed better protection than intraperitoneal treatment. Histopathology confirmed that daily administration of hexanedioic acid for 10 days showed zero toxicity to the kidneys or livers of mice. Therefore, hexanedioic acid may be a novel antibacterial agent.

Methicillin-resistant Staphylococcus aureus (MRSA), are the most frequent cause of sepsis, which urgently demanding new drugs for treating infection. Two homologous insect CSαβ peptides-DLP2 and DLP4 from Hermetia illucens were firstly expressed in Pichia pastoris, with the yields of 873.5 and 801.3 mg/l, respectively. DLP2 and DLP4 displayed potent antimicrobial activity against Gram-positive
bacteria especially MRSA and had greater potency, faster killing, and a longer postantibiotic effect than vancomycin. A 30-d serial passage of MRSA in the presence of DLP2/DLP4 failed to produce resistant mutants. Macromolecular synthesis showed that DLP2/DLP4 inhibited multi-macromolecular synthesis especially for RNA. Flow cytometry and electron microscopy results showed that the cell cycle was
arrested at R-phase; the cytoplasmic membrane and cell wall were broken by DLP2/DLP4; mesosomelike structures were observed in MRSA. At the doses of 3‒7.5 mg/kg DLP2 or DLP4, the survival of mice challenged with MRSA were 80‒100%. DLP2 and DLP4 reduced the bacterial translocation burden over 95% in spleen and kidneys; reduced serum pro-inflammatory cytokines levels; promoted
anti-inflammatory cytokines levels; and ameliorated lung and spleen injury. These data suggest that DLP2 and DLP4 may be excellent candidates for novel antimicrobial peptides against staphylococcal infections.

The antibacterial activity of the extracts of whole black soldier fly larvae (Hermetia illucens) was evaluated and was tested against six strains of plant pathogens. The extracts were prepared by homogenization after mixing grinded larvae with 0.01% acetic acid at 4 °C for 12 hr. Two methods, agar well diffusion and growth curve assay, were used to study the antibacterial activity against these pathogens. In our study, the antibacterial properties of the extracts were demonstrated by growth inhibition of all six tested bacterial pathogens. Inhibition zone assay and fluorescence assay confirmed that larval extracts have significant antibacterial activity against bacterial pathogens. The data provide further evidence that larval extracts play a role in the defense against microorganisms.

Eumelanin type pigments are synthesized at all the stages of the life cycle of the fly Hermetia illucens: in the larvae, pre-pupae, pupae and adult flies (dead flies). The greatest content of melanin was recorded in the cuticles. Melanin was present not only in the cuticle, hence it remained in the cuticle after the emergence of the adult fly. It was also found in the insect body in a complex with lipids. In pupae, it is mostly lauric acid that was associated with melanin. Its proportion in the melanin-chitosan complex was 80%. The isolated melanin-chitosan complex of adult flies showed a wide range of antibacterial activity, inhibiting the growth of 21 out of the 25 of the test cultures. The melanin-chitosan complex of empty pupal membranes and alcohol suspension of pupal melanin inhibited twice as smaller number of test cultures and the above activity was absolutely in the pupal chitosan. The largest zone of growth inhibition was recorded with respect to Aspergillus niger, Candida albicans, salmonella, and Staphylococcus aureus. An alcohol suspension of pupal melanin inhibited the growth of 10 test cultures. In this case the greatest activity was shown in relation to Mycobacterium B5 and Acinetobacter sp. 1182.

The aim of this study was to evaluate the effect of full-fat insect meals fed ‘on top’ to broiler chickens on their performance and the microbiota composition in the gastrointestinal tract. A total of 1850 day-old Ross 308 females were used in a set of four independent experiments. The insects Gryllodes sigillatus, Shelfordella lateralis, Gryllus assimilis, Tenebrio molitor and Hermetia illucens were applied in amounts that varied from 0.05 to 0.2%. In general, the application of insect meals to the diets of broilers did not affect their growth performance over the experimental period. However, the 0.2% additions of T. molitor and H. illucens increased feed intake at days 15–35 (P = 0.011) and the entire period of feeding (days 1–35; P = 0.018) (Experiment 3). Moreover, in Experiment 4 the supplementation of 0.2% of
S. lateralis improved body weight gain (days 11–21 and 1–21), feed intake (days 1–10 and 1–21) and feed conversion ratio (days 1–21). The addition of insect meals reduced the pH value of digesta in the crop (Experiments 1 and 2) and in the caeca (Experiment 2). Supplementation with H. illucens caused the most significant effect on the microbiota populations in the crop, ileum and caeca (Experiment 3). However, at the higher levels of S. lateralis addition to the diets of broilers, the counts of selected microbiota in the crop and ileum increased (Experiment 4). These results indicate that the application of the insect full-fat meals in relatively small amounts can affect the microbiota composition in the gastrointestinal tract of broiler chickens.

Larvae of black soldier fly, Hermetia illucens, are scavengers who live in extremely unpleasant and harsh environments, such as manure and compost, populated by bacteria and fungi. Carcasses of dead animals and rotting plants could be degraded by larvae of black soldier fly. These biological characteristics suggest that the larvae of black soldier fly are rich in antimicrobial peptides (AMPs) and other substances which possess the activity to fight against resistant bacterial strains. Wound healing is a complex process consisting of four overlapping but strictly defined phases: hemostasis, inflammatory phase, proliferative phase and the remodelling phase (maturation and epithelialization). All phases of wound healing are dependent on the secretion of a variety of cellular compounds such as growth factors, chemokines, cytokines, proteinases, and extracellular matrix proteins. In vitro process of wound healing could be monitored using these different cellular compounds as molecular markers. Focus of this research is enrichment of extracts from H. illucens larvae in order to obtain purified AMPs and other antibiotic compounds and in vitro monitoring of the impact of extracts from H. illucens larvae on bacterial growth and cytotoxicity of human cells. Extracts showed high capacity of inhibition of bacterial growth, especially species Pseudomonas fluorescens. Moreover, majority of extracts which inhibited bacterial growth did not show citotoxic effect on human cells.

Active antimicrobial peptide HI-3 was isolated and purified from the 5th instar larvae of Hermetia illucens L., and its effects on proliferation, apoptosis and migration of nasopharyngeal carcinoma (CNE2) cells were investigated. The expressions of telomerase reverse transcriptase (hTERT) in CNE2 cells were also studied in vitro to elucidate the mechanism involved in the action of HI-3 on CNE2 cells. Results showed that three fractions (HI-1, HI-2, HI-3) were isolated from the hemolymph of H. illucens larvae. After purified by RP-HPLC, only HI-3 showed the inhibitory activities to four strains of bacteria. It was also showed that HI-3 could effectively inhibit the proliferation of CNE2 cells in a dose- and time- dependent manner. Apoptosis of CNE2 cells was observed in the treatment with 160 μg/ml HI-3, and the early apoptosis rate up to 27.59%. However, no significantly inhibitory effects and apoptosis were found on human umbilical vein endothelial cells (HUV-C). Moreover, HI-3 could significantly reduce the migration ability of CNE2 cells when compared with that of the control. On the other hand, the levels of mRNA and protein of hTERT in the HI-3 treatment were all significantly lower than that of the control. Results indicated that HI-3 could inhibit the proliferation of CNE2 cells and induce the apoptosis of CNE2 cells by down-regulating the telomerase activity in CNE2 cells, while no obvious effect was occurred on HUV-C. It inferred that HI-3 is a potential anti-tumor drug with low toxicity to normal cells.

We investigate the effects of the immune function (HI titer) in broilers fed diets containing Hermetia illucens (H. illucens) peptide extract over a 40-day period. Twenty-four broiler chicks (Arbor Acres, 1 d old) were divided into four groups and fed different diets (control, 0.1%, 0.5%, and 1% H. illucens peptide extract). To evaluate HI titer, all broilers were vaccinated with H9H2 vaccine subcutaneously on the lateral thorax, according to the manufacturer’s recommendations. Similar HI titer was observed with 1% H. illucens peptide extract treatment compared to the control after 40 days (p>0.01). Groups fed 0.5% H. illucens peptide extract demonstrated the most effective immune effects (p<0.01), followed by groups fed 0.1% H. illucens peptide extract. In conclusion, using 0.1% or 0.5% H. illucens peptide extract before or after vaccination improved HI titer immune function in broilers.

To evaluate the effects of feeding a Hermetia illucens (HI) larvae meal on the different intestinal traits of hens, and to determine the toxic elements’ concentration in the insect meal and diets, 162 hens were randomly allotted to three groups. The control received a corn-soybean meal-based diet (SBM); the HI25 and HI50 groups received two diets in which the 25% and 50% of the dietary protein were replaced by the HI protein, respectively. The duodenal and jejunal villi height and villi/crypt were higher (p < 0.01) in the SBM than in the HI groups. The ileal villi height was higher (p < 0.05) in the SBM and HI25 groups than the HI50. The HI50 group exhibited a lower duodenal maltase activity. The intestinal alkaline phosphatase (IAP) activity linearly decreased in the duodenum and jejunum as the dietary insect meal inclusion increased. The HI50 group had a higher acetate and butyrate level than the SBM. The levels of cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As)in the diets and insect meal were lower than the maximum values established by the EU Commission. The 25% soybean protein replacement with Hermetia illucens larvae meal in the diet of laying hens was more suitable and closer to the optimal level than 50%.

Black soldier fly (BSF) larvae and pre-pupae could be satisfactorily raised on household organic waste and used as poultry feed, offering a potential sustainable way to recycle untapped resources of waste. The present study was conducted to determine if whole (non-defatted) BSF larvae and pre-pupae raised on experimental household waste could substitute soybean meal and oil as ingredients for laying hen diets. While no significant differences in feed intake and the egg-laying rate of hens were observed throughout the experiment, egg weight and eggshell thickness were greater in the pre-pupae-fed group than in the other groups. Moreover, although diversity of the cecal microbiota was significantly higher in the pre-pupae-fed than in the control group, no significant differences in bacterial genera known to cause food poisoning were observed when comparing the treatment groups. Nonetheless, Lactobacillus and Bifidobacterium populations were significantly lower in the treatment than in the control group. Fat content in BSF was possibly related with the changes in the cecal microbiota. Hence, since BSF fat was deficient in essential fatty acids, special attention should be paid to the fat content and its fatty acid composition in the case of regular inclusion of BSF larvae and pre-pupae oil as an ingredient in poultry diets.

This study evaluated the effects of dietary insect meal from Hermetia illucens larvae on autochthonous gut microbiota of rainbow trout (Oncorhynchus mykiss). Three diets, with increasing levels of insect meal inclusion (10%, 20%, and 30%) and a control diet without insect meal were
tested in a 12-week feeding trial. To analyze the resident intestinal microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME pipeline were used. The number of reads taxonomically classified according to the Greengenes database was 1,514,155. Seventy-four Operational Taxonomic Units (OTUs) at 97% identity were identified. The core of adhered intestinal microbiota, i.e., OTUs present in at least 80% of mucosal samples and shared regardless of the diet, was constituted by three OTUs assigned to Propiobacterinae, Shewanella, and Mycoplasma genera, respectively. Fish fed the insect-based diets showed higher bacterial diversity with a reduction in Proteobacteria in comparison to fish fed the fishmeal diet. Insect-meal inclusion in the diet increased the gut abundance of Mycoplasma, which was attributed the ability to produce lactic and acetic acid as final products of its fermentation. We believe that the observed variations on the autochthonous intestinal microbiota composition of trout are principally due to the prebiotic properties of fermentable chitin.

The objective of this experiment was to test the effects of supplementation of defatted black soldier fly (Hermetia illucens) larvae (BSFL) meal in beagle dogs. A total of nine healthy female beagles (initial body weight 12.1±1.76 kg) were fed grain-based diets with three levels of BSFL meal (0, 1% or 2%) in a 42-day feeding trial. At the end of week 6 of the experiment, all dogs were intraperitoneally challenged with Escherichia coli lipopolysaccharide (LPS) at 100 μg/kg of body weight. Albumin concentration was linearly increased as increasing BSFL meal level (P<0.05). A linear increase (P<0.05) in calcium concentration was observed when increasing dietary BSFL meal. Although dietary treatments did not affect the digestibility of ether extract, the digestibility of dry matter and crude protein were linearly increased as increasing the level of BSFL meal. The concentration of tumor necrosis factor-α was linearly decreased but glutathione peroxidase (GPx) concentration was linearly increased when increasing the level of BSFL meal at 6 h after challenge (P<0.05). In addition, there were quadratic increases in concentrations of GPx and superoxide dismutase with increasing dietary BSFL meal level at 3 h after challenge (P<0.05). These findings from the present study demonstrate that BSFL meal can be supplemented in the diet to convert beneficial effects to beagle dogs indicated as improved digestibility of dry matter and crude protein and antiinflammatory and anti-oxidative capacity.

Background: Insects, such as Hermetia illucens larvae, are rich in chitin and proteins, and represent a suitable feed ingredient replacement for animals. However, little is known about the effect of administering H. Illucens larvae on intestinal microbiota, bacterial metabolite profiles, and mucosal immune status in animals. This study aimed to investigate the effects of administering H. illucens larvae on colonic microbiota and bacterial metabolites production in finishing pigs. Seventy-two crossbred (Duroc × Landrace × Large White) female pigs (initial body weight, 76.0 ± 0.
52 kg) were randomly allocated to three different dietary treatments: a control diet (Control group) and two diets corresponding to 4% (H1 group) and 8% (H2 group) H. illucens larvae inclusion levels, respectively. Each treatment consisted of eight pens (replicates), with three pigs per pen. After 46 days of feeding, eight pigs per treatment (n = 8) were slaughtered, and the colonic digesta and mucosa were collected for microbial composition and microbial fermentation products, and genes expression analyses.

Results: The results showed that the H1 diet significantly increased the abundance of Lactobacillus, Pseudobutyrivibrio, Roseburia, and Faecalibacterium compared with those in the control group (P < 0.05), with a decrease in the abundance of Streptococcus. The numbers of Lactobacillus, Roseburia, and Clostridium cluster XIVa were significantly greater in the H1 group than in the control group (P < 0.05). Meanwhile, H2 diet increased the number of Clostridium cluster XIVa compared with the control group (P < 0.05). For colonic metabolites, total short chain fatty acids, butyrate, and isobutyrate concentrations were significantly higher in the H1 group than those in the control group (P < 0.05); the H1 treatment caused a striking decrease in protein fermentation compared with the control group, as the concentrations of total amines, cadaverine, tryptamine, phenol, p-cresol, and skatole were significantly lower (P < 0.05). Additionally, H2 diet also increased butyrate concentration compared with control group (P < 0.05), while decreased the concentrations of phenol, p-cresol, and skatole (P < 0.05). Pigs in the H1 group down-regulated the expression of TLR-4 and pro-inflammatory cytokines (IFN-γ) compared with pigs in the control group (P < 0.05), and up-regulated anti-inflammatory cytokine (IL-10) and intestinal barrier genes (ZO-1, occludin, and mucin-1). H2 diet up-regulated the expression of ZO-1 compared with control group (P < 0.05). Furthermore, the changes in the colonic mucosal gene expression were associated with changes in the bacterial composition and their metabolites.

Conclusions: Collectively, dietary inclusion of Hermetia illucens larvae may enhance mucosal immune homeostasis of pigs via altering bacterial composition and their metabolites. These findings provide a new perspective on insect meal as a sustainable protein source rich in nutrient ingredients for swine.

The gut microbiota of insects contains a wide range of organisms that protect them against the attack of pathogens by releasing various types of bioactive compounds. In the present study, we report the isolation and identification of the fungus Chrysosporium multifidum as a component of the microbiota from the larval gut of Hermetia illucens. Extract from the broth culture of C. multifidum showed moderate activity on a strain of methicillin resistant Staphylococcus aureus (MRSA). The bioguided isolation of the extract resulted in the characterization of six α-pyrone derivatives (1-6) and one diketopiperazines (7), among them 5,6-dihydro-4-methoxy-6-(1-oxopentyl)-2H-pyran-2-one (4) showed the best activity (IC50 = 11.4 ± 0.7 µg/ml and MIC = 62.5 μg/ml).

The aim of this study was to evaluate the in vitro antibacterial activity of
Black soldier fly (BSF) larva extract. The BSF larva was extracted using methanol and then tested for antibacterial activity using agar diffusion method (zone growth inhibition). The antibacterial activity was conducted against Salmonella sp. and Escherichia coli, two important bacterial strains
in poultry, using six dilution levels (10 mg/ml, 20 mg/ml, 40 mg/ml, 80 mg/ml, 160 mg/ml and 320 mg/ml). All the results were subjected analyze
using t-test method. Based on the diameter of the inhibition zone, the BSF larva extract has a strong (P<0.05) antibacterial activity against Salmonella sp. and E. coli when the concentration used 320 mg/ml. In addition, BSF larva extract also contain high amount of lauric acid (49.18%), a saturated fatty acid that has been proven to proposes as antibacterial agent. Therefore, it could be concluded that the BSF larva extract could be used as a candidate for antibacterial substances.