Microbial dynamics and vertical transmission of Escherichia coli across consecutive life stages of the black soldier fly (Hermetia illucens).

BACKGROUND: The black soldier fly (BSF, Hermetia illucens L.) is one of the most promising insects for bioconversion of organic waste, which often carry a high microbial load with potential foodborne pathogens. Although horizontal transmission (from rearing substrate to larvae) has been extensively studied, less is known about vertical transmission of microorganisms, and particularly of foodborne pathogens, across different BSF life stages. RESULTS: This study investigated the microbial dynamics and vertical transmission of Escherichia coli across different life stages (larvae, prepupae, pupae and adults) of one BSF life cycle and its associated substrate (chicken feed) and frass, based on a combination of general microbial counts (based on culture-dependent techniques) and the bacterial community composition (based on 16S rRNA gene sequencing). Multiple interactions between the microbiota of the substrate, frass and BSF larvae were affirmed. The larvae showed relative consistency among both the microbial counts and bacterial community composition. Diversification of the bacterial communities started during the pupal stage, while most notable changes of the microbial counts and bacterial community compositions occurred during metamorphosis to adults. Furthermore, vertical transmission of E. coli was investigated after substrate inoculation with approximately 7.0 log cfu/g of kanamycin-resistant E. coli, and monitoring E. coli counts from larval to adult stage. Although the frass still contained substantial levels of E. coli (> 4.5 log cfu/g) and E. coli was taken up by the larvae, limited vertical transmission of E. coli was observed with a decreasing trend until the prepupal stage. E. coli counts were below the detection limit (1.0 log cfu/g) for all BSF samples from the end of the pupal stage and the adult stage. Additionally, substrate inoculation of E. coli did not have a substantial impact on the bacterial community composition of the substrate, frass or different BSF life stages. CONCLUSIONS: The fluctuating microbial counts and bacterial community composition underscored the dynamic character of the microbiota of BSF life stages. Additionally, vertical transmission throughout one BSF life cycle was not observed for E. coli. Hence, these findings paved the way for future case studies on vertical transmission of foodborne pathogens across consecutive BSF life stages or other insect species.

In-depth biological characterization of two black soldier fly anti-Pseudomonas peptides reveals LPS-binding and immunomodulating effects.

As effector molecules of the innate immune system, antimicrobial peptides (AMPs) have gathered substantial interest as a potential future generation of antibiotics. Here, we demonstrate the anti-Pseudomonas activity and lipopolysaccharide (LPS)-binding ability of HC1 and HC10, two cecropin peptides from the black soldier fly (Hermetia Illucens). Both peptides are active against a wide range of Pseudomonas aeruginosa strains, including drug-resistant clinical isolates. Moreover, HC1 and HC10 can bind to lipid A, the toxic center of LPS and reduce the LPS-induced nitric oxide and cytokine production in murine macrophage cells. This suggests that the peptide-LPS binding can also lower the strong inflammatory response associated with P. aeruginosa infections. As the activity of AMPs is often influenced by the presence of salts, we studied the LPS-binding activity of HC1 and HC10 in physiological salt concentrations, revealing a strong decrease in activity. Our research confirmed the early potential of HC1 and HC10 as starting points for anti-Pseudomonas drugs, as well as the need for structural or formulation optimization before further preclinical development can be considered. IMPORTANCE The high mortality and morbidity associated with Pseudomonas aeruginosa infections remain an ongoing challenge in clinical practice that requires urgent action. P. aeruginosa mostly infects immunocompromised individuals, and its prevalence is especially high in urgent care hospital settings. Lipopolysaccharides (LPSs) are outer membrane structures that are responsible for inducing the innate immune cascade upon infection. P. aeruginosa LPS can cause local excessive inflammation, or spread systemically throughout the body, leading to multi-organ failure and septic shock. As antimicrobial resistance rates in P. aeruginosa infections are rising, the research and development of new antimicrobial agents remain indispensable. Especially, antimicrobials that can both kill the bacteria themselves and neutralize their toxins are of great interest in P. aeruginosa research to develop as the next generation of drugs.

Effect of heat treatment on microbiological safety of supermarket food waste as substrate for black soldier fly larvae (Hermetia illucens).

Supermarket food waste, constituting 13% of global food waste, can be upcycled as substrate for black soldier fly larvae (BSFL) and converted into larval biomass. Since presence of food pathogens in supermarket food waste is likely, microbiological safety should be ensured when using waste as insect substrate. Heat treatment provides a suitable pre-treatment to reduce microbiological contaminations. This study investigated the effect of different temperature-time combinations on the microbiological safety of supermarket food waste as BSFL substrate. Artificial supermarket food waste without meat and fish (SFW) was inoculated with both Salmonella and Staphylococcus aureus (7.0log cfu/g) and treated at 50 and 60 °C for 10, 20 and 30 min. While 50 °C was insufficient for adequate pathogen reduction, 60 °C only required 10 min to reduce the Enterobacteriaceae and S.aureus counts to < 1.0logcfu/g and for absence of Salmonella in 25 g. Heat-treated SFW could be stored for two days at ambient temperature or refrigerated without pathogen growth. Treatment of supermarket food waste containing meat and fish at 60 °C for 10 min caused similar results as for SFW, but S.aureus persisted (2.4logcfu/g), possibly by protective effects of fat and/or proteins. Finally, BSFL rearing experiments on SFW revealed significantly higher larval mass, bioconversion efficiency and waste reduction than on Gainesville diet, with no notable differences between untreated and heat-treated SFW. Rearing BSFL on supermarket food waste is possible, and unsafe food waste can be heated to obtain safety without eliminating nutrients necessary for rearing.

Ingestion and excretion dynamics of microplastics by black soldier fly larvae and correlation with mouth opening size.

Black soldier fly (BSF) larvae (Hermetia illucens) are voracious feeders that can be reared on food waste streams originating from the food industry and retailers. Because these food waste streams are automatically being unpacked in substantial amounts, they can contain microplastics, potentially jeopardising the larvae's chemical safety when applied as compound feed ingredients. During this study, the dynamics of ingestion and excretion of microplastics by BSF larvae reared on substrates containing different contents (wMP = 0.00, 0.01, 0.10, 0.50, 1.00, 3.00%) of fluorescent blue-labelled microplastics (median size, Dv(50) = 61.5 µm) were monitored. To correlate the particle size with their uptake, larval mouth opening dimensions were measured during the rearing process. In conclusion, it appeared that ingestion of microplastics by BSF larvae depends on initial particle load, mouth size, and consequently also age. The larvae took up between 131 (wMP = 0.01%) and 4866 (wMP = 3.00%) particles leading to bioaccumulation factors (BAF) between 0.12 (wMP = 3.00%) and 1.07 (wMP = 0.01%). Larvae also appeared to excrete the microplastics, lowering the BAFs to values between 0.01 (wMP = 3.00%) and 0.54 (wMP = 0.01%).

A Production and Fractionation Protocol for Polyvinyl Chloride Microplastics.

Concerns about the presence of microplastics in the environment has increased in recent years, prompting more attention from scientists. Thorough exposure studies using artificially produced microplastics containing additives are required to assess their potentially hazardous effects. Therefore, an efficient microplastic production and fractionation protocol was established using a cryogenic grinding and wet-sieving approach. The developed cryogenic grinding method was able to produce (20-40 g/h) polyvinyl chloride (PVC) microplastics having a volume-weighted mean particle size of 391 µm and a span of 2.12. Performing a second grinding cycle on the same particles resulted in microplastics which were smaller (volume-weighted mean size = 219 µm) and had a narrower particle size distribution (span = 1.59). In addition, the microplastics were also fractionated into different particle size ranges using a vibrating wet-sieving tower. The latter technique allowed separating 10 g of PVC microplastics into seven different fractions using six sieves (Ø 200 mm) for 30 min while shaking. By using the developed method, PVC microplastics could easily be made and fractionated into desired particle-size ranges. The proposed protocol could also be adjusted to produce and fractionate microplastics of other plastics.

Optimizing the Rheological and Textural Properties of Chapatti Enriched with House Crickets (Acheta domesticus) Flour Using Hydrocolloids by an I-Optimal Design.

The fortification of food with edible insect flour can improve its nutrition profile, but also affect its techno-functional characteristics. In this study, an I-optimal design was applied to improve the rheological and textural properties of wheat flour chapatti containing 10% cricket (Acheta domesticus) flour. More specifically, the impact and optimal addition of hydrocolloids (carboxymethyl cellulose, hydroxypropyl methylcellulose, guar gum and xanthan gum) and water content were studied. For all the responses, the model and model terms were highly significant and showed the different impact of the hydrocolloids on the rheological properties. To evaluate the predictive power of the models, two sets of optimal process settings were chosen: one based on dough properties, and another on baked chapatti. For both sets, the actual responses were in the range of predicted responses for almost all properties. In addition, it was shown that using the settings based on dough properties, the actual responses were not significantly different from the control chapatti, whereas for the settings based on baked chapatti, there were differences in terms of the extensibility of both dough and chapatti. Thus, the I-optimal design is suitable to optimize the dough properties and the baked chapatti when enriching chapatti with cricket flour.

A simple, rapid and accurate method for the sample preparation and quantification of meso- and microplastics in food and food waste streams.

Plastics are produced and used in large quantities worldwide (e.g. as food packaging). In line with this, plastic particles are found throughout the ecosphere and in various foods. As a result, plastics are also present in energy-rich waste biomass derived from the food industry, supermarkets, restaurants, etc. These waste streams are a valuable source for biogas production but can also be used to feed insects that in turn upcycle it into new high-value biomass. In both applications, the remaining residue can be used as fertilizer. Due to the present plastic particles, these applications could pose a continued threat to the environment, and both human and animal health. Therefore, the need of determining the (micro)plastic content to assess the potential danger is rising. In this research, a closed-vessel microwave-assisted acid digestion method was developed to accurately determine meso- and microplastic contents in food (waste) matrices by solubilising this food matrix. Polyvinyl chloride (PVC) food packaging foil was used to develop the method, using a full factorial design with three parameters (nitric acid concentration (c(HNO3)), temperature (T), and time (t)). According to this model, the best practical conditions were c(HNO3) = 0.50 mol/L, T = 170 °C, and t = 5.00 min. Subsequently, the method was tested on five other plastics, namely high- and low-density polyethylene (HDPE and LDPE), polypropylene (PP), polystyrene (PS), and polyethylene terephthalate (PET), mixed with a food matrix, resulting in a mean plastic recovery of 102.2 ± 4.1%. Additionally, the polymers were not oxidised during the microwave digestion. For PVC and PS hardly any degradation was found, while HDPE, LDPE, and PP showed slight chain degradation, although without recovery loss. In conclusion, the method is an accurate approach to quantify the total meso- and microplastic content in food (waste) matrices with minimal change in their intrinsic characteristics.

Rheological Characterization of Chapatti (Roti) Enriched with Flour or Paste of House Crickets (Acheta domesticus).

Addition of edible insects to food products may improve the nutritional status but can also influence their techno-functional properties. This study investigates the impact of supplementing wheat flour by cricket flour or paste at different levels (5-15%) on the rheological and textural properties of flour, dough, and baked chapatti. Addition of freeze-dried cricket flour resulted in the highest water absorption. The storage modulus increased at higher level (10-15%) of supplementation to wheat flour indicating an increased dough consistency. Similarly, biaxial extension of the dough showed an increased resistance to extension and decreased extensibility at higher level of supplementation due to a reduced strength of the gluten network. Uniaxial extension of baked chapatti showed less extensible and harder chapatti with the addition of a higher amount of cricket flour or paste. At lower level (5%), incorporation of cricket flour resulted in chapatti with textural properties comparable to the reference. Oven dried cricket powder is suggested as the best option for incorporating in chapatti dough to improve food security in Asian Countries.

On the nitrogen content and a robust nitrogen-to-protein conversion factor of black soldier fly larvae (Hermetia illucens).

Currently, a broad discussion exists in the literature regarding insect protein analysis. At its core, main difficulties and uncertainties are the inconsistent use of analysis methods and nitrogen-to-protein conversion (kP) factors. While the Kjeldahl and Dumas methods are both used in the literature, their result represents inherently different nitrogen fractions. Thus far, no correlation between them is established for insect matrices, which is a major uncertainty. Although much effort has been made towards more accurate kP factors, calculation of these was based on merely one sample while the chemical composition varies depending on rearing conditions. Using a broad variation in black soldier fly (BSF) larvae samples in the present study, a correlation between Kjeldahl and Dumas and a robust kP factor have been established. Moreover, the nitrogen distribution of BSF samples was also assessed after accurate chitin analyses. A highly significant linear correlation existed between the results of Kjeldahl and Dumas (slope, 1.009; intercept, - 0.008; R2, 0.9997). Consequently, both methods were deemed interchangeable for BSF larvae. Using amino acid data, a practical, more accurate and robust kP factor of 4.43 was obtained. Concerning the chitin content, the average of all BSF larvae samples was 5.95 ± 0.86 g N-acetylglucosamine/100 g dry matter and no correlation with the kP factor was observed. Regarding the nitrogen distribution of the samples, it was found that the contribution of nitrogenous compounds other than protein and chitin is not only high but also prone to variation (12-30% of the total nitrogen content).

Enhancing the specificity of chitin determinations through glucosamine analysis via ultra-performance LC-MS.

As chitin is gaining an increased attention as feedstock for industry, quantification thereof is becoming increasingly important. While gravimetric procedures are long, not specific and highly labour-intensive, acidic hydrolysis of chitin into glucosamine followed by quantification of the latter is more performant. Even though several quantification procedures for the determination of chitin can be found in the literature, they give inconsistent results and their accuracy was not assessed due to the lack of certified analytical standards. Therefore, in the present study, commercially available chitin from practical grade was characterised in detail, allowing the assessment of method accuracy. The procedure for the hydrolysis of chitin into glucosamine and subsequent quantification via UPLC-MS was investigated in detail as well. Using 9-fluorenylmethyl chloroformate (FMOC-Cl) as derivatisation reagent, glucosamine was quantified using reversed-phase chromatography. For the chitin hydrolysis, the highest glucosamine recovery was obtained with 8.0 M HCl for 2 h at 100 °C. The entire procedure for chitin quantification, including the hydrolysis, was characterised by high interday and intraday precision and accuracy. The specificity of the procedure was assessed as well by analysing different mixtures of cellulose and chitin. Chitin recoveries from these analyses ranged from 98.8 to 105.8% while no signal was observed for 100% cellulose, indicating the high specificity of the procedure. It was also concluded that the procedure is much faster and less labour-intensive compared to the gravimetric procedure.

Protein films from black soldier fly (Hermetia illucens, Diptera: Stratiomyidae) prepupae: effect of protein solubility and mild crosslinking.

BACKGROUND: This work evaluated the performances of protein-based bioplastics obtained from black soldier fly (Hermetia illucens) prepupae. Protein films were synthesized by film casting, using both the whole proteins and their soluble fraction at pH = 10. The effects of glycerol as a plasticizer and of citric acid as a mild crosslinker on film properties were also evaluated. RESULTS: Films obtained using the soluble protein fraction were the strongest, as well as the most homogeneous and transparent ones. Protein mild crosslinking improved film tensile properties, especially in films obtained with the whole protein fraction. Non-crosslinked samples had a high affinity with water while crosslinking almost eliminated the ability of films to absorb water. All protein-based films proved to be effective barriers to red light (transmittance less than 2%). CONCLUSIONS: Bioplastics derived from black soldier fly prepupae may find applications in the agricultural sector (biodegradable pots, mulching films, utensils) and deserve to be tested for food and non-food packaging. © 2021 Society of Chemical Industry.

Affordable Processing of Edible Orthopterans Provides a Highly Nutritive Source of Food Ingredients.

Edible orthopterans (grasshoppers, crickets, and locusts) are major delicacies, especially across sub-Saharan Africa. Their promotion as food ingredients is increasingly gaining momentum. This study evaluates the nutritional profiles of three widely consumed orthopterans: Gryllus bimaculatus, Locusta migratoria, and Schistocerca gregaria after blanching and oven-drying. All three species had high protein (65.3, 54.2, and 61.4% on a dry matter (DM) basis for G. bimaculatus, L. migratoria, and S. gregaria, respectively) and fat contents. Oleic (22.9-40.8%) and palmitic (26.1-43.0%) were the two most abundant fatty acids. All essential amino acids (in mg/100 g protein) were present, with glutamic acid (120-131), alanine (90.2-123), and leucine (82.3-84.6) being the most abundant. The minerals (in mg/100 g dry matter) potassium (796-1309) and phosphorus (697-968) were moderately high, and iron (4.60-7.31), zinc (12.7-24.9), manganese (0.40-7.15), and copper (1.20-4.86) were also observed in the samples. Vitamin B12 contents were high (0.22-1.35 µg/100 g dry matter). Our findings demonstrate that the excellent nutritional profile of the three processed insects could serve as promising alternative ingredients for improving food and nutritional security.

Effect of heat processing on the nutrient composition, colour, and volatile odour compounds of the long-horned grasshopper Ruspolia differens serville.

Heat processing is commonly used to prepare edible insects for consumption. This study aimed at determining the effect of boiling and subsequent oven roasting on Ruspolia differens' nutrient composition, colour and odor compounds. Boiling leads to: a significant increase in protein and decrease in fat content on a dry matter basis; a minimal influence on its amino and fatty acids profile; a significant reduction in its ash content due to leaching of phosphorus, potassium and sodium; a significant increase in iron, zinc, copper, manganese and calcium content; and a fivefold reduction in the amount of vitamin B12. Roasting leads to a relative increase in the amount of calcium and trace mineral elements but doesn't affect other nutrients. Roasting results into a more uniform colour intensity when green and brown polymorphs are roasted together. Lipid oxidation is responsible for the colour and aroma of heat processed R. differens.

Nutrient composition of the long-horned grasshopper Ruspolia differens Serville: Effect of swarming season and sourcing geographical area.

This research investigates the effect of swarming season and sourcing geographical area on the nutrient composition of R. differens. On a dry matter basis, protein (34.2-45.8%) and fat (42.2-54.3%) are the major macro-nutrients. Leucine (80. 9-88.5), lysine (54.0-69.8) and valine (59.2-61.8) are the most abundant essential amino acids (EAA) (mg/g protein). Tryptophan is the most limiting EAA. Its EAA composition is like the children and adults' reference protein. Its fat is highly unsaturated (63.5-64.7). Oleic (38.4-42.7), linoleic (19.0-23.0) and palmitic (26.6-27.8) fatty acids (FA) (g/100 g total FA) are the most abundant. Geographical area significantly influences its mineral content within seasons. Season significantly influences its fat, protein, ash and mineral content but not its AAs' and FAs' content. Both factors significantly influence its vitamin B12 content (0.73-1.35 µg/100 g).

Ultra-high-performance liquid chromatography coupled to quadrupole orbitrap high-resolution mass spectrometry for multi-residue screening of pesticides, (veterinary) drugs and mycotoxins in edible insects.

A generic extraction and UHPLC-Q-Orbitrap™-HRMS method was developed for four insect species (mealworm, grasshopper, house cricket and black soldier fly) analyzing a large spectrum of organic chemical contaminants, including pesticides (n = 25), (veterinary) drugs (n = 29), and mycotoxins (n = 23). To prove the method as 'fit-for-purpose', a successful validation was performed, both qualitatively, by determining the screening detection limit (SDL), selectivity and specificity, as well as semi-quantitatively, by assessing the within-day precision (relative standard deviation (RSD)) and recovery. For both the mealworm, grasshopper, house cricket and black soldier fly, 64, 61, 59 and 62 compounds were detected at the respective SDL levels (1-100 µg kg-1), predominantly below existing maximum residue limits for other edible matrices. Mean recoveries ranged between 70% and 120% and RSD-values were in line with European regulations (CD 2002/657/EC; SANCO). Finally, the potential of the screening methodology was demonstrated on real insect samples, revealing minor to no contamination.

Influence of Freeze-Drying and Oven-Drying Post Blanching on the Nutrient Composition of the Edible Insect Ruspolia differens.

The longhorn grasshopper, Ruspolia differens (Serville), plays an important role as a food source across Sub-Saharan Africa, where it is consumed as a delicacy in both rural and urban areas. The effect of two drying methods (freeze-drying and oven-drying), employed after blanching, on the proximate, fatty acid and mineral composition of the two most common morphs was determined. Ruspolia differens grasshoppers were harvested in Uganda and Kenya from wild swarms during the rainy periods of November-December 2016. Based on cuticular coloration, we identified three morphs, green, brown and purple, which occurred at a ratio of 65:33:2, respectively. Results indicated that these insects have a high lipid content of 36%, as well as significant protein levels ranging between 33% and 46% dry matter. Oleic acid (44%) and palmitic acid (28%) were the two most abundant fatty acids; while the presence of arachidonic acid (0.6%) and docosahexaenoic acid (0.21%) suggests that Ruspolia differens is also a source of polyunsaturated fatty acids. The observed amino acid profile showed similar trends in all morphs, and all essential amino acids were present. Calcium (896-1035 mg/100 g), potassium (779-816 mg/100 g) and phosphorus (652-685 mg/100 g) were quite high among the minerals. The presence of the trace elements iron (217-220 mg/100 g), zinc (14.2-14.6 mg/100 g), manganese (7.4-8.3 mg/100 g) and copper (1.66 mg/100 g) suggests that inclusion of these grasshoppers in human diets may aid in combatting micronutrient deficiencies. Oven-drying Ruspolia differens delivered the same nutritional quality as freeze-drying. Hence, both drying approaches can be adequately used to formulate insect-based food products without noticeable nutritional changes.