Background/Objectives: Hermetia illucens larvae can efficiently convert agri-food residues into high-protein biomass for animal feed and nutrient-rich frass for soil amendment. However, the potential spread of carbapenem resistance genes (CRGs), which confer resistance to last-resort carbapenem antibiotics, and Enterobacteriaceae, common carriers of these genes and opportunistic pathogens, raises important safety concerns. This study aimed to assess the influence of different agri-food-based diets on Enterobacteriaceae loads and the CRG occurrence during the bioconversion process. Methods: Four experimental diets were formulated from agri-food residues and anaerobic digestate: Diet 1 (peas and chickpea waste), Diet 2 (peas and wheat waste), Diet 3 (onion and wheat waste), and Diet 4 (wheat waste and digestate). Enterobacteriaceae were quantified by viable counts, while five CRGs (bla_KPC, bla_NDM, bla_OXA-48, bla_VIM, and bla_GES) were detected and quantified using quantitative PCRs (qPCRs). Analyses were performed on individual substrates, formulated diets, larvae (before and after bioconversion), and frass. Results: Plant-based diets sustained moderate Enterobacteriaceae loads. In contrast, the digestate-based diet led to a significant increase in Enterobacteriaceae in both the frass and mature larvae. CRGs were detected only in legume-based diets: bla_VIM and bla_GES were found in both mature larvae and frass, while bla_OXA-48 and bla_KPC were found exclusively in either larvae or frass. No CRGs were detected in onion- or digestate-based diets nor in young larvae or diet inputs. Conclusions: The findings suggest that the diet composition may influence the proliferation of Enterobacteriaceae and the persistence of CRGs. Careful substrate selection and process monitoring are essential to minimize antimicrobial resistance risks in insect-based bioconversion systems.

Aureobasidium pullulans is a polyextremotolerant yeast-like fungus increasingly recognized for its role in food ecosystems and its emerging potential in flavour development and nutrient modulation. However, systematic evaluations of autochthonous grape-associated populations integrating technological performance and safety-related traits remain limited. This study provides a broad phenotypic screening of 70 isolates from Maraština grapes (Dalmatia, Croatia), applying an integrated functional screening approach to link enzymatic potential, environmental resilience, and food safety. Most isolates displayed multiple hydrolytic enzymes, with widespread cellulase, pectinase, xylanase, esterase, and protease activities. Several isolates showed very high enzymatic indices, supporting their potential for plant-derived substrate transformation, aroma release, and food processing applications. β-glucosidase and urease activities were common, while amylase was limited. Ecological screening confirmed robust adaptability to salinity, osmotic stress, and wide pH ranges. Notably, 31% of isolates demonstrated phosphate solubilization capacity, indicating a possible contribution to mineral bioavailability and nutritional enhancement. Safety screening revealed decarboxylation of selected amino acids, while two isolates lacked detectable activity, highlighting them as candidates for further safety evaluation. Overall, this work establishes a framework for selecting A. pullulans isolates for next-generation, flavour-oriented and nutritionally enhanced food applications, supporting sustainable bioprocessing and future industrial validation.

Background: Hermetia illucens larvae provide a sustainable bioconversion pathway that transforms agro-industrial residues into protein- and nutrient-dense biomass and frass, suitable for animal feed and soil amendment, respectively. Nevertheless, the potential spread of antibiotic resistance (AR) genes and pathogenic microorganisms poses biosafety concerns. This study examined the impact of four residue-based diet formulations; peas and chickpea (D1), peas and wheat (D2), onion and wheat (D3), and wheat with digestate (D4), on microbial safety during the bioconversion process. Methods: Enterococcus spp. (viable counts), Salmonella spp. (presence/absence), and 13 AR genes associated with resistance to tetracyclines, macrolide-lincosamide-streptogramin B, β-lactams, vancomycin, and aminoglycosides were quantified in single substrates, diets, larvae, and frass using qPCR. Results: Principal component analysis revealed diet-driven AR gene profiles. D1 lowered the levels of the greatest number of tested AR genes, particularly erm(B), tetracycline, and β-lactam genes in frass, as well as tet(O) and vanB in mature larvae. In contrast, D2 increased the AR gene levels in frass. All diets except D4 eliminated Salmonella spp. Enterococcus spp. loads varied by diet and larval stage, with D2 reducing counts in frass. Conclusions: Diet composition directly shapes microbial dynamics and AR gene dissemination, indicating that legume-based substrates may enhance biosafety in bioconversion systems.