Parameters of flow in porous alluvial aquifers evaluated by tracers

Dye tracer tests, DNA biotracer, alluvial aquifer Marche, multiwell tests

Microbial dynamics in rearing trials of Hermetia illucens larvae fed coffee silverskin and microalgae

In the present study, Hermetia illucens larvae were reared on a main rearing substrate composed of a coffee roasting byproduct (coffee silverskin, Cs) enriched with microalgae (Schizochytrium limacinum or Isochrysis galbana) at various substitution levels. The microbial diversity of the rearing substrates, larvae, and frass (excrement from the larvae mixed with the substrate residue) were studied by the combination of microbial culturing on various growth media and metataxonomic analysis (Illumina sequencing). High counts of total mesophilic aerobes, bacterial spores, presumptive lactic acid bacteria, coagulase-positive cocci, and eumycetes were detected. Enterobacteriaceae counts were low in the rearing diets, whereas higher counts of this microbial family were observed in the larvae and frass. The microbiota of the rearing substrates was characterized by the presence of lactic acid bacteria, including the genera Lactobacillus, Leuconostoc and Weissella. The microbiota of the H. illucens larvae fed Cs was characterized by the dominance of Paenibacillus. H. illucens fed diets containing I. galbana were characterized by the presence of Enterococcus, Lysinibacillus, Morganella, and Paenibacillus, depending on the algae inclusion level, while H. illucens fed diets containing S. limacinum were characterized by high relative abundances of Brevundimonas, Enterococcus, Paracoccus, and Paenibacillus, depending on the algae inclusion level. Brevundimonas and Alcaligenes dominated in the frass from larvae fed I. galbana; the predominance of Brevundimonas was also observed in the frass from larvae fed Schyzochitrium-enriched diets. Based on the results of the present study, an effect of algae nutrient bioactive substances (e.g. polysaccharides, high-unsaturated fatty acids, taurine, carotenoids) on the relative abundance of some of the bacterial taxa detected in larvae may be hypothesized, thus opening new intriguing perspectives for the control of the entomopathogenic species and foodborne human pathogens potentially occurring in edible insects. Further studies are needed to support this hypothesis. Finally, new information on the microbial diversity occurring in insect frass was also obtained

Investigation of the Dominant Microbiota in Ready-to-Eat Grasshoppers and Mealworms and Quantification of Carbapenem Resistance Genes by qPCR

In this study, 30 samples of processed edible mealworms (Tenebrio molitor L.) and 30 samples of grasshoppers (Locusta migratoria migratorioides) were obtained from producers located in Europe (Belgium and the Netherlands) and Asia (Thailand) and subjected to PCR-DGGE analyses. The PCR-DGGE analyses showed that species in the genus Staphylococcus were predominant in the samples of mealworms from Belgium and grasshoppers from the Netherlands; species in the genus Bacillus were detected in the samples of mealworms and grasshoppers from Thailand. Moreover, Weissella cibaria/confusa/spp. was found in grasshoppers from Belgium. Since data concerning the role of novel foods such as edible insects in the dissemination of carbapenem resistance are currently lacking, the quantification of five carbapenemase encoding genes (blaNDM−1, blaVIM, blaGES, blaOXA−48, and blaKPC) by qPCR was also carried out in all the samples under study. The genes coding for GES and KPC were not detected in the analyzed samples. A very low frequency of blaOXA−48 (3%) and blaNDM−1 (10%) genes was detected among mealworms. In contrast, grasshoppers were characterized by a high incidence of the genes for OXA-48 and NDM-1, accounting for 57 and 27% of the overall grasshopper samples, respectively. The blaVIM gene was detected exclusively in two grasshopper samples from Thailand, showing only 7% positivity. The analysis of variance showed that all the effects (producers, species, and producers × species) were statistically significant for blaNDM−1, whereas for blaOXA−48 and blaVIM, no significant effects were detected for the same source of variation. Further studies are necessary to assess the possible role of edible insects as reservoirs for the resistance to carbapenems and to understand the correlation with the insect microbiota. Furthermore, an intensified surveillance plan examining the occurrence of carbapenemase encoding genes in the food chain and in environmental compartments is needed for a proper risk assessment. In such a context, the appropriate use of antimicrobials represents the main preventive action that should always be applied

Microbial aspects related to edible insects and development of new insect-based food products

La FAO ha dichiarato che la popolazione umana è in continua crescita e raggiungerà i 9 miliardi nel 2050, provocando un aumento della domanda di cibo, in particolare di fonti proteiche, che si tradurrà in effetti negativi per l’ambiente. Una possibile soluzione potrebbe essere rappresentata dal consumo di insetti edibili: inannzitutto, per la sostenibilità ambientale legata al loro allevamento, ma anche per il loro contenuto in aminoacidi essenziali, acidi grassi insaturi, minerali, vitamine e fibre. Nel 2015, gli insetti sono stati classificati come “novel food” con il Regolamento No 2015/2283. Nello stesso anno, l’EFSA ha sottolineato la necessità di ottenere maggiori informazioni sugli insetti per definirne il rischio microbiologico e chimico. In questo contesto la presente tesi di Dottorato ha avuto lo scopo di studiare il microbiota associato agli insetti edibili per ottenere informaziori riguardo un loro possibile consumo alimentare. Inoltre, data la loro capacità di agire come serbatoio di geni trasferibili di antibiotico resistenza, che possono essere anche portati da patogeni, parte della ricerca è stata focalizzata sulla presenza di tali geni in campioni di insetto. In breve, è stata riscontrata la presenza di microorganismi commensali, agenti di deterioramento e patogeni ed anche di geni trasferibili di antibiotico resistenza. I risultati ottenuti hanno evidenziato l’influenza di vari fattori tra cui specie di insetto, substrato di crescita, condizioni di allevamento. Infine, per superare lo scetticismo dei consumatori riguardo agli insetti edibili, sono stati ottenuti prodotti in cui la presenza degli insetti è stata mascherata. I campioni prodotti sono stati sottoposti ad analisi microbiologiche, tecnologiche e sensorali per valutarne l’applicabilità. I risultati hanno sottolineato la presenza di batteri sporigeni e l’effetto delle diverse specie di insetto sui parametri tecnologici e sull’accettabilità dei consumatori.FAO declared that human population is worldwide increasingly growing and in 2050 it will reach 9 billion people. The consequent increase in food demand, and in particular, in proteins will cause several negative effects on the environment due to the intensive animal farming. In this scenario, a potential solution is represented by edible insects. First of all, their rearing is characterized by a higher environmental sustainability than livestock. Moreover, they are a nutritious food especially in terms of essential amino acids, unsaturated fatty acids, minerals, vitamins and fibre. In the European Union, insects were defined as “novel food” by the Regulation (EU) No 2015/2283. In the same year, EFSA requested further research to better assess microbiological and chemical risks related to edible insects. In this context, the present PhD. thesis was aimed to investigate about edible insects microbiota to collect information about their feasibility as food. Furthermore, since they can act as reservoir of transferable antibiotic resistance genes which can even be carried by pathogens, part of the research was focused on the occurrence of such genes in edible insects samples. Briefly, results showed the presence of commensal, spoilage and potential pathogen agents and the occurrence of transferable resistance genes. Interestingly, data highlighted the influence of insect species, growth substrate, rearing and environmental conditions. Finally, to overcome consumers scepticism generated by insects consumption as food, the development of insect-based food products where insects are invisible was assessed. To get information about the feasibility of these products, microbiological, technological and sensory analysis were performed. In a few words, the presence of spore-forming bacteria was detected in each bread and in the rusks. Moreover a different insect powder effect on the technological parameters of bread and on the consumers appreciation was observed

The potential of edible insects: microbiological features [Il potenziale degli insetti edibili: aspetti microbiologici]

Il crescente aumento della popolazione mondiale implicherà un aumento della richiesta di fonti proteiche convenzionali (carne) con conseguenti ricadute negati ve sull’ambiente. In questo contesto, gli insetti edibili potrebbero rappresentare una fonte proteica alternati va grazie al loro elevato apporto in nutrienti e al basso impatt o ambientale. Al fi ne di garanti re la sicurezza d’uso degli insetti edibili, è necessaria una approfondita valutazione del microbiota ad essi correlato. Dagli studi fi nora condotti emerge una elevata biodiversità microbica verosimilmente correlata alla specie di insett o, allo stadio di sviluppo ed alle tecniche di processamento e conservazione. Sono pertanto auspicabili ulteriori ricerche al fi ne di chiarire aspetti legati alla sicurezza d’uso in relazione alla presenza di potenziali microrganismi patogeni. Inoltre, recenti studi condotti su insetti edibili in commercio hanno rilevato la presenza di geni di resistenza agli anti bioti ci più comunemente uti lizzati in ambito clinico

A glimpse into the microbiota of marketed ready-to-eat crickets (Acheta domesticus)

The present study was aimed to get an insight into the bacterial biota of ready-to-eat small crickets (Acheta domesticus) already marketed in the European Union. 16S rRNA gene of the DNAs extracted from thirty-two samples of ready-to-eat crickets commercialized by 4 European Union producers located in Austria, Belgium, France and the Netherlands (2 batches per producer) was analyzed by Polymerase Chain Reaction–Denaturing Gradient Gel Electrophoresis (PCR–DGGE). The species belonging to the genera Hespellia, Ruminococcus and Clostridium were detected in samples from Austria, while those from genera Lysobacter, Staphylococcus and Clostridium were detected in samples from Belgium. Moreover, samples from France were characterized by Staphylococcus, Pseudomonas, and Hydrogenophilus genera. Finally, the genera Staphylococcus, Hydrogenophilus, Clostridium and Ruminococcus were identified in the samples produced in the Netherlands. When insects are intended for commercialization, rearing, processing and handling could affect the presence of the occurring microbial species. Hence, to assure a safe product, the need for a full standardization of production technologies, including feed supply as well as rearing and processing practices, is recommended

Occurrence of transferable antibiotic resistances in commercialized ready-to-eat mealworms (Tenebrio molitor L.)

The present study aimed to assess the occurrence of transferable determinants conferring resistance to tetracyclines, macrolide-lincosamide-streptogramin B, vancomycin, beta-lactams, and aminoglycosides in 40 samples of commercialized edible mealworms (Tenebrio molitor L.) purchased from European Union (EU) and non-EU producers. A high prevalence of tet(K) was observed in all of the samples assayed, with percentages of PCR-based positivity that ranged from 80% (samples from Thailand) to 100% (samples from the Netherlands, Belgium and France). For macrolides, erm(B) prevailed, being detected in 57.5% of the samples assayed, whereas erm(A) and erm(C) were detected with lower frequencies. Genes for resistance to vancomycin were only detected in samples produced in France and Belgium, with 90% and 10% of the samples being positive for vanA, respectively. Beta-lactamase genes were found with low occurrence, whereas the gene aac-aph, conferring high resistance to aminoglycosides, was found in 40% of the samples produced in the Netherlands and Belgium and 20% of the samples produced in Thailand. The results of Principal Coordinate Analysis and Principal Component Analysis depicted a clean separation of the samples collected from the four producers based on the distribution of the 12 AR determinants considered. Given the growing interest on the use of mealworms as a novel protein source, AR detection frequencies found in the present study suggest further investigation into the use of antibiotics during rearing of this insect species and more extensive studies focused on the factors that can affect the diffusion of transferable ARs in the production chain. Until such studies are completed, prudent use of antibiotics during rearing of edible insects is recommended

Flow parameters in porous alluvial aquifers evaluated by multiple tracers

Dye tracer tests are a good technique useful to determine the hydrogeological parameters of aquifers, especially in alluvial plains, where the heterogeneity of the lithology is a key factor leading the groundwater circulation. In the last decade, new tracers have been developed and introduced in the hydrological and geological context, including DNA tracers, that are used in the same way as the traditional tracers. In this study, some experiments were performed in a porous aquifer with the aim of comparing the behavior of different tracers for the evaluation of aquifer's parameters, and of testing the suitability of a DNA tracer in this geological context. Single well and multiwell tests were performed during the winter and spring 2017, involving one piezometer and 4 wells. The tracer test results highlighted faster zones of flow in the alluvial plain, being located at different depths, whose levels are characterized by gravel and sand layers. A maximum groundwater velocity of around 170 cm/day was revealed by the single well tests, whereas the main direction of flow was along the watercourse axis, as resulted by the multi well tracer test. The overall results obtained confirmed that fluorescent tracers are a very valuable and reliable tool for the determination of groundwater velocity, groundwater flow direction and dispersion parameters, even in cases of multi-layered aquifers, whereas DNA tracers are very helpful in the determination of faster circuits and vertical flow even in multi-layered aquifers

Bread enriched with cricket powder (Acheta domesticus): A technological, microbiological and nutritional evaluation

Recently, the high nutritional value of edible insects attracted the attention of researchers and food industry for their potential use in foods with enhanced nutritional characteristics. In this study cricket (Acheta domesticus) powder was added to wheat flour to obtain bread with enhanced nutritional value. Bread loaves were obtained from doughs produced using different blends of wheat flour and cricket powder added in amounts of 10 or 30% (based on wheat flour) and baker's yeast and/or sourdough. Raw materials, dough and breads were subjected to technological, microbiological, chemical and sensory analyses. Overall, a negative linear correlation between the amount of added cricket powder and the dough technological parameters was seen. However, compared to control breads produced with the sole wheat flour, breads containing cricket powder showed a higher nutritional profile in terms of fatty acid composition, high protein content and occurrence of essential amino acids. Finally, bread enriched with 10% cricket powder showed a discrete global liking by untrained panelists. Data overall collected highlighted a good suitability of cricket powder for the production of enriched bread. Of note, the presence of spore-forming bacteria in cricket-based bread loaves, thus highlighting potential safety issues to be deepened. Industrial relevance: The present study demonstrated that edible insects powder can successfully be included in leavened baked goods to enhance their protein content. The present research also responds to the growing awareness of consumers towards innovative and wholesome leavened goods, proving that edible insects can constitute a novel source of innovative ingredients to be used in bread making

Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR

The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source