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.

Meta-analysis of larvae of the black soldier fly (Hermetia illucens) microbiota based on 16S rRNA gene amplicon sequencing.

Black soldier fly larvae (BSFL) belong to the most widely reared insects as an alternative protein source at industrial scale. Bacteria in the larval gut can provide benefits for the animal, though some bacteria can also be pathogenic for the insect. Accurate characterization of the BSFL microbiota is important for the production of BSFL in terms of yield and microbiological safety. In this study, 16S ribosomal RNA gene sequence data sets from 11 studies were re-analysed to gain better insights in the BSFL gut microbiota, potential factors that influence their composition, and differences between the gut and the whole larvae microbiota. A core gut microbiota was found consisting of members of Enterococcus, Klebsiella, Morganella, Providencia, and Scrofimicrobium. Further, the factors 'Study', 'Age' and 'Feed' (i.e. rearing substrate of the larvae) significantly affected the microbiota gut composition. When compared to whole larvae, a significantly lower diversity was found for gut samples, suggesting that the larvae harboured additional microbes on their cuticle or in the insect body. Universal choices in insect sample type, primer selection and bio-informatics analysis pipeline can strengthen future meta-analyses and improve our understanding of the BSFL gut microbiota towards the optimization of insect rearing conditions and substrates.

Overcoming Technical and Market Barriers to Enable Sustainable Large-Scale Production and Consumption of Insect Proteins in Europe: A SUSINCHAIN Perspective.

The expected global population growth to 9.7 billion people in 2050 and the significant change in global dietary patterns require an increase in global food production by about 60%. The protein supply for feed and food is most critical and requires an extension in protein sources. Edible insects can upgrade low-grade side streams of food production into high-quality protein, amino acids and vitamins in a very efficient way. Insects are considered to be the "missing link" in the food chain of a circular and sustainable economy. Insects and insect-derived products have entered the European market since first being acknowledged as a valuable protein source for feed and food production in around 2010. However, today, scaling up the insect value chain in Europe is progressing at a relatively slow pace. The mission of SUSINCHAIN (SUStainable INsect CHAIN)-a four-year project which has received funding from the European Commission-is to contribute to novel protein provision for feed and food in Europe by overcoming the remaining barriers for increasing the economic viability of the insect value chain and opening markets by combining forces in a comprehensive multi-actor consortium. The overall project objective is to test, pilot and demonstrate recently developed technologies, products and processes, to realize a shift up to Technology Readiness Level 6 or higher. In addition to these crucial activities, the project engages with stakeholders in the insect protein supply chain for feed and food by living labs and workshops. These actions provide the necessary knowledge and data for actors in the insect value chain to decrease the cost price of insect products, process insects more efficiently and market insect protein applications in animal feed and regular human diets that are safe and sustainable. This paves the way for further upscaling and commercialization of the European insect sector.

In Vitro Evaluation of Antimicrobial Peptides from the Black Soldier Fly (Hermetia Illucens) against a Selection of Human Pathogens.

Antimicrobial peptides (AMPs) are being explored as alternatives to traditional antibiotics to combat the rising antimicrobial resistance. Insects have proven to be a valuable source of new, potent AMPs with large structural diversity. For example, the black soldier fly has one of the largest AMP repertoires ever recorded in insects. Currently, however, this AMP collection has not yet undergone antimicrobial evaluation or in-depth in vitro characterization. This study evaluated the activity of a library of 36 black soldier fly AMPs against a panel of human pathogens (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus fumigatus) and a human cell line (MRC5-SV2). The activity profile of two cecropins (Hill-Cec1 and Hill-Cec10) with potent Gram-negative activity, was further explored by characterizing their hemolysis, time-to-kill kinetics, membrane-permeabilization properties, and anti-biofilm activity. Hill-Cec1 and Hill-Cec10 also showed high activity against other bacterial species, including Klebsiella pneumoniae and multi-drug resistant P. aeruginosa. Both AMPs are bactericidal and have a rapid onset of action with membrane-permeabilizing effects. Hill-Cec1 and Hill-Cec10 were also able to prevent P. aeruginosa biofilm formation, but no relevant effect was seen on biofilm eradication. Overall, Hill-Cec1 and Hill-Cec10 are promising leads for new antimicrobial development to treat critical infections caused by Gram-negative pathogens such as P. aeruginosa. IMPORTANCE With the ever growing antimicrobial resistance, finding new candidates for antimicrobial drug development is indispensable. Antimicrobial peptides have steadily gained attention as alternatives for conventional antibiotics, due to some highly desirable characteristics, such as their low propensity for resistance development. With this article, we aim to upgrade the knowledge on the activity of black soldier fly antimicrobial peptides and their potential as future therapeutics. To achieve this, we have evaluated for the first time a library of 36 synthetically produced peptides from the black soldier fly against a range of human pathogens and a human cell line. Two selected peptides have undergone additional testing to characterize their antimicrobial profile against P. aeruginosa, a clinically important Gram-negative pathogen with a high established resistance. Overall, this research has contributed to the search for new peptide drug leads to combat the rising antimicrobial resistance.

Microbial profile during fermentation and aerobic stability of ensiled mixtures of maize stover and banana pseudostem in South Ethiopia.

AIMS: This study evaluated pH reduction and microbial growth during fermentation of maize stover (MS) mixed with banana pseudostem (BPS) under South Ethiopian conditions. MATERIALS AND RESULTS: The MS and BPS were chopped and mixed into six treatments (T): 80% BPS plus 20% DMS (T1), 70% BPS plus 30% DMS (T2), 40% BPS plus 60% FMS (fresh MS) (T3), 20% BPS plus 80% FMS (T4), 100% FMS (T5), and 95% BPS plus 5% molasses (T6). At 0, 7, 14, 30, 60, and 90 days, pH and dry matter were determined. Microbiological quality was assessed using plate counts and Illumina MiSeq sequencing. On day 60 and 90, aerobic stability was investigated. The results showed a significant reduction in pH in all mixtures, except in T1 and T2. Lactic acid bacteria counts reached a maximum in all treatments within 14 days. Sequencing showed marked changes in dominant bacteria, such as Buttiauxella and Acinetobacter to Lactobacillus and Bifidobacterium. CONCLUSIONS: The fresh MS and BPS mixtures and fresh maize showed significant pH reduction and dominance of desirable microbial groups. SIGNIFICANCE AND IMPACT OF THE STUDY: The study enables year-round livestock feed supplementation to boost milk and meat production in South Ethiopia.

Potential of Fermentation and Vacuum Packaging Followed by Chilling to Preserve Black Soldier Fly Larvae (Hermetia illucens).

Black soldier fly larvae (Hermetia illucens) are currently reared at an industrial scale, mainly as a feed ingredient. The logistic chain not only involves the production of larvae, but also stabilisation, storage, and transport. The aim of this work was to study fermentation and vacuum packaging of larvae as potential preservation technologies. For fermentation, blanched larvae were pulverised into a paste, and a starter culture, NaCl, and glucose were added. The mixture was fermented for 7 days at 35 °C and then stored for 14 days at 4 °C and pH and microbial counts were monitored. Vacuum packaging was applied to living, blanched and frozen larvae. After packaging, they were stored for 6-10 days at several temperatures and gas composition, survival (living larvae) and microbial counts (killed larvae) were recorded. Fermentation allows storage of pulverised larvae, but points to consider are a rapid pH reduction and the presence of bacterial endospores. Vacuum packaging did not bring added value over cooling alone. This was the case for all types of larvae investigated. Vacuum packaging is not considered as a valuable preservation technology to pursue for storage and transport of black soldier fly larvae.

Isolation and Identification of Dominant Bacteria From Black Soldier Fly Larvae (Hermetia illucens) Envisaging Practical Applications.

This study aimed to establish a representative strain collection of dominant aerobic bacteria from black soldier fly larvae (Hermetia illucens, BSFL). The larvae were fed either chicken feed or fiber-rich substrates to obtain a collection of BSFL-associated microorganisms. Via an approach based on only considering the highest serial dilutions of BSFL extract (to select for the most abundant strains), a total of 172 bacteria were isolated. Identification of these isolates revealed that all bacteria belonged to either the Proteobacteria (66.3%), the Firmicutes (30.2%), the Bacteroidetes (2.9%) or the Actinobacteria (0.6%). Twelve genera were collected, with the most abundantly present ones (i.e., minimally present in at least three rearing cycles) being Enterococcus (29.1%), Escherichia (22.1%), Klebsiella (19.8%), Providencia (11.6%), Enterobacter (7.6%), and Morganella (4.1%). Our collection of dominant bacteria reflects largely the bacterial profiles of BSFL already described in literature with respect to the most important phyla and genera in the gut, but some differences can be noticed depending on substrate, biotic and abiotic factors. Furthermore, this bacterial collection will be the starting point to improve in vitro digestion models for BSFL, to develop mock communities and to find symbionts that can be added during rearing cycles to enhance the larval performances, after functional characterization of the isolates, for instance with respect to enzymatic potential.

Microbial symbionts of insects as a source of new antimicrobials: a review.

To stop the antimicrobial resistance crisis, there is an urgent need for increased investment in antimicrobial research and development. Currently, many researchers are focussing on insects and their microbiota in the search for new antimicrobials. This review summarizes recent literature dedicated to the antimicrobial screening of insect symbionts and/or their metabolites to uncover their value in early drug discovery. We summarize the main steps in the methodology used to isolate and identify active insect symbionts and have noted substantial variation among these studies. There is a clear trend in isolating insect Streptomyces bacteria, but a broad range of other symbionts has been found to be active as well. The microbiota of many insect genera and orders remains untargeted so far, which leaves much room for future research. The antimicrobial screening of insect symbionts has led to the discovery of a diverse array of new active biomolecules, mainly peptides, and polyketides. Here, we discuss 15 of these symbiont-produced compounds and their antimicrobial profile. Cyphomycin, isolated from a Streptomyces symbiont of a Cyphomyrmex fungus-growing ant, seems to be the most promising insect symbiont-derived antimicrobial so far. Overall, insect microbiota appears to be a promising search area to discover new antimicrobial drug candidates.

Impact of Heat Treatment on the Microbiological Quality of Frass Originating from Black Soldier Fly Larvae (Hermetia illucens).

Since black soldier fly larvae (BSFL, Hermetia illucens) are being produced at substantial volumes, concomitantly large amounts of the resulting by-product, called frass, are generated. This frass can potentially be applied as valuable plant fertilizer or soil improver. Since frass carries high microbial counts, potentially including foodborne pathogens, safety problems for consumers should be prevented. A heat treatment of 70 °C for 60 min is proposed to reduce harmful organisms in insect frass, based on EU regulations ((EU) No. 2021/1925). This study evaluated for the first time the impact of the proposed heat treatment on BSFL frass. This was done by applying the treatment on uninoculated frass as well as on frass inoculated with Salmonella or Clostridium perfringens at 5.0 log cfu/g. The heat treatment resulted in a reduction (maximum one log-cycle) of total viable counts and did not noticeably reduce bacterial endospores. In contrast, Enterobacteriaceae counts were reduced to below the detection limit (10 cfu/g). Heat treatment of inoculated frass resulted in absence of Salmonella in 25 g of frass and reduction of vegetative C. perfringens to below the detection limit (1 cfu/g). The proposed heat treatment appears to be appropriate to meet the microbiological regulations for insect frass.

Fermentation Versus Meat Preservatives to Extend the Shelf Life of Mealworm (Tenebrio molitor) Paste for Feed and Food Applications.

Freshly prepared pastes from blanched mealworms (Tenebrio molitor) are highly perishable and prone to microbial and chemical changes upon storage due to their high water activity, near-neutral pH, and their rich nutrient profile. Their shelf life is short unless preservation methods are used. In this study, the effects of preservatives (sodium nitrite and sodium lactate) and lactic acid fermentation (with the starter cultures Bactoferm® F-LC and Lactobacillus farciminis) on the microbiological and the chemical stability of mealworm pastes stored at 4°C were compared. During the storage experiment, the pH, water activity, color, microbial counts, and fat oxidation were monitored. In addition, the prevalence of the pathogens Bacillus cereus, Salmonella spp., and Listeria monocytogenes were studied. Microbial quality evaluation of the mealworm pastes showed that the addition of preservatives did not inhibit microbial growth during refrigerated storage, reaching the upper limits for consumption between seven and 14 days. By contrast, the acid medium (pH < 4.50) created by fermentation stabilized all microbial populations investigated, indicating that these pastes could be consumed up to (at least) 8 weeks of refrigerated storage. L. monocytogenes, Salmonella, and B. cereus were not detected in any of the samples and lipid oxidation of the samples was minimal. Altogether, this study shows that lactic acid fermentation can be used successfully to inhibit microbial growth, to maintain chemical quality, and to extend the shelf life of mealworm pastes stored at 4°C.

Silage making of maize stover and banana pseudostem under South Ethiopian conditions: evolution of pH, dry matter and microbiological profile.

The study was conducted to evaluate the microbial dynamics during silage of maize stover and banana pseudostem in the environmental conditions of southern Ethiopia. To meet this objective, microsilos containing either maize stover or banana pseudostem, both with and without molasses, were prepared. Subsequently, samples were analysed on day 0, 7, 14, 30, 60 and 90 of the fermentation process. As a result, on day 7, all treatments except banana pseudostem without molasses showed a significant reduction in pH. It was also this silage type that supported the growth of Enterobacteriaceae longer than three other silage types, i.e. until 30 days. The yeasts and moulds and the Clostridum endospore counts also showed a reducing trend in early fermentation and afterwards remained constant until day 90. Illumina MiSeq sequencing revealed that Leuconostoc, Buttiauxella species and Enterobacteriaceae were the most abundant bacteria in the initial phases of the fermentation. Later on, Buttiauxella, Lactobacillus, Weissella and Bifidobacterium species were found to be dominant. In conclusion, silage of the two crop by-products is possible under South Ethiopian conditions. For banana pseudostem, the addition of molasses is crucial for a fast fermentation, in contrast to maize. Upscaling needs to be investigated for the two by-products.

Identification of bacterial endospores and targeted detection of foodborne viruses in industrially reared insects for food.

With edible insects being increasingly produced, food safety authorities have called for the determination of microbiological challenges posed to human health. Here, we find that the bacterial endospore fraction in industrially reared mealworm and cricket samples is largely comprised of Bacillus cereus group members that can pose insect or human health risks. Hepatitis A virus, hepatitis E virus and norovirus genogroup II were not detected in the sample collection, indicating a low food safety risk from these viral pathogens.

Protein fortification with mealworm (Tenebrio molitor L.) powder: Effect on textural, microbiological, nutritional and sensory features of bread.

In the present study, inclusion of mealworm (Tenebrio molitor L.) powder into bread doughs at 5 and 10% substitution level of soft wheat (Triticum aestivum L.) flour was tested to produce protein fortified breads. The addition of mealworm powder (MP) did not negatively affect the technological features of either doughs or breads. All the tested doughs showed the same leavening ability, whereas breads containing 5% MP showed the highest specific volume and the lowest firmness. An enrichment in protein content was observed in experimental breads where the highest values for this parameter were recorded in breads containing 10% MP. Breads fortified with 10% MP also exhibited a significant increase in the content of free amino acids, and especially in the following essential amino acids: tyrosine, methionine, isoleucine, and leucine. By contrast, no differences in nutritional quality of lipids were seen between fortified and control breads. Results of sensory analyses revealed that protein fortification of bread with MP significantly affected bread texture and overall liking, as well as crust colour, depending on the substitution level. Overall, proof of concept was provided for the inclusion of MP into bread doughs started with different leavening agents (sourdough and/or baker's yeast), at 5 or 10% substitution level of soft wheat flour. Based on the Technology Readiness Level (TRL) scale, the proposed bread making technology can be situated at level 4 (validation in laboratory environment), thus suggesting that the production of breads with MP might easily be scaled up at industrial level. However, potential spoilage and safety issues that need to be further considered were highlighted.

Microbial characterisation of the edible grasshopper Ruspolia differens in raw condition after wild-harvesting in Uganda.

This research aimed at establishing the chemical intrinsic properties and the microbial quality of an edible grasshopper Ruspolia differens and the effect of its source (geographical area) in Uganda, trading point, swarming season and plucking on these parameters. The intrinsic properties of the grasshopper can support the growth of a wide variety of microorganisms. High counts of total aerobic microbes, Enterobacteriaceae, lactic acid bacteria, total aerobic spores, and yeasts and moulds were obtained. Metagenetic analyses yielded 1793 Operational Taxonomic Units (OTUs) belonging to 24 phyla. Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria were the most abundant phyla, while members of the genera Acinetobacter, Buttiauxella, Lactococcus, Staphylococcus and Undibacterium were the most abundant OTUs. A number of genera harbouring potential pathogens (Acinetobacter, Bacillus, Buttiauxella, Campylobacter, Clostridium, Staphylococcus, Pseudomonas and Neisseria) were identified. The geographical area, trading point, swarming season and plucking significantly influenced microbial counts and bacterial diversity. The high microbial counts predispose R. differens to fast microbial spoilage, while the presence of Clostridium and Campylobacter makes this grasshopper a potential source of food borne diseases. Further research should identify the specific spoilage microorganisms of R. differens and assess the characteristics of this grasshopper that support growth of food pathogens.

Real-time PCR detection and quantification of selected transferable antibiotic resistance genes in fresh edible insects from Belgium and the Netherlands.

The occurrence of antibiotic resistance genes in foodstuffs involves a human health risk. Fresh edible insects present an emerging source of human food but they were not yet assessed in a quantitative way for antibiotic resistances as a matter of food safety. In this study, a real-time quantitative PCR assessment was optimised to detect and quantify relevant transferable antibiotic resistance genes [tet(O, K, M, S) and erm(B)] in edible insects. Subsequently, the technology was applied on 30 fresh insect samples, including two mealworm species and two cricket species from different production batches and rearing companies in Belgium and the Netherlands. The sampling periods and the post-harvest treatments applied were also taken into account. Results showed that mealworms contained, on average, higher numbers of tet(K), tet(M), and tet(S) genes than crickets, but tet(O) was almost uniquely present in crickets. The erm(B) gene was only detected in one mealworm sample and the tet(K) gene showed higher abundances in samples originating from the Netherlands than in samples from Belgium. A large difference in antibiotic resistance profile was revealed between mealworms and crickets, but not between different mealworm species or cricket species. Species-specific microbiomes and insect feed may have contributed to this distinction. Interestingly, important correlations between the presence of some tet genes and the microbiota previously encountered in the investigated edible insects were uncovered. While a geographical distribution was observed for the tet(K) gene, post-harvest treatments and sampling period were not shown to have a significant influence on the occurrence of the antibiotic resistance genes considered. In conclusion, insects may carry considerable amounts of antibiotic resistance genes, but the health risk in terms of antibiotic resistances is comparable to other food matrices.

Microbial Dynamics during Industrial Rearing, Processing, and Storage of Tropical House Crickets (Gryllodes sigillatus) for Human Consumption.

In this study, the microbiota during industrial rearing, processing, and storage of the edible tropical house cricket, Gryllodessigillatus, was investigated. To this end, we analyzed samples from the cricket feed, obtained before feeding as well as from the cages, and from the crickets during rearing, after harvest, and after processing into frozen, oven-dried, and smoked and oven-dried (smoked/dried) end products. Although the feed contained lower microbial numbers than the crickets, both were dominated by the same species-level operational taxonomic units, as determined by Illumina MiSeq sequencing. They corresponded, among others, to members of Porphyromonadaceae, Fusobacterium, Parabacteroides, and Erwinia The harvested crickets contained high microbial numbers, but none of the investigated food pathogens Salmonella spp., Listeria monocytogenes, Bacillus cereus, or coagulase-positive staphylococci. However, some possible mycotoxin-producing fungi were isolated from the crickets. A postharvest heat treatment, shortly boiling the crickets, reduced microbial numbers, but an endospore load of 2.4 log CFU/g remained. After processing, an increase in microbial counts was observed for the dried and smoked/dried crickets. Additionally, in the smoked/dried crickets, a high abundance of a Bacillus sp. was observed. Considering the possible occurrence of food-pathogenic species from this genus, it is advised to apply a heat treatment which is sufficient to eliminate spores. Nevertheless, the microbial numbers remained constant over a 6-month storage period, whether frozen (frozen end product) or at ambient temperature (oven-dried and smoked/dried end products).IMPORTANCE The need for sustainable protein sources has led to the emergence of a new food sector, producing and processing edible insects into foods. However, insight into the microbial quality of this new food and into the microbial dynamics during rearing, processing, and storage of edible insects is still limited. Samples monitored for their microbiota were obtained in this study from an industrial rearing and processing cycle. The results lead first to the identification of process steps which are critical for microbial food safety. Second, they can be used in the construction of a Hazard Analysis and Critical Control Points (HACCP) plan and of a Novel Food dossier, which is required in Europe for edible insects. Finally, they confirm the shelf-life period which was determined by the rearer.

Microbial Community Dynamics during Rearing of Black Soldier Fly Larvae (Hermetia illucens) and Impact on Exploitation Potential.

The need to increase sustainability in agriculture, to ensure food security for the future generations, is leading to the emergence of industrial rearing facilities for insects. One promising species being industrially reared as an alternative protein source for animal feed and as a raw material for the chemical industry is the black soldier fly (Hermetia illucens). However, scientific knowledge toward the optimization of the productivity for this insect is scarce. One knowledge gap concerns the impact of the microbial community associated with H. illucens on the performance and health of this insect. In this review, the first steps in the characterization of the microbiota in H. illucens and the analysis of substrate-dependent dynamics in its composition are summarized and discussed. Furthermore, this review zooms in on the interactions between microorganisms and the insect during H. illucens development. Finally, attention is paid to how the microbiome research can lead to alternative valorization strategies for H. illucens, such as (i) the manipulation of the microbiota to optimize insect biomass production and (ii) the exploitation of the H. illucens-microbiota interplay for the discovery of new enzymes and novel antimicrobial strategies based on H. illucens immunity using either the whole organism or its molecules. The next decade promises to be extremely interesting for this research field and will see an emergence of the microbiological optimization of H. illucens as a sustainable insect for industrial rearing and the exploitation of its microbiome for novel biotechnological applications.