Cluj Vet J 2024, vol 29, issue 4 http://clujveterinaryjournal.ro Article Nutritional Value, Microbiological Safety, and Mycotoxin Risk of Black Soldier Fly Larvae: Implications for Dog Nutrition Claudiu-Nicusor Ionica1, Sorana Daina1,* Andrei-Radu Szakacs1, Smaranda Craciun2, Romelia Pop3 and Adrian Macri1 1 Department of Animal Nutrition, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veteri- nary Medicine of Cluj–Napoca, Manastur Street, 400372 Cluj-Napoca, Romania; e-mail: claudiu-nicusor.ion- ica@usamvcluj.ro(C.N.I.); sorana.matei@usamvcluj.ro (S.D.); andrei.szakacs@usamvcluj.ro; adrian.macri@usamvcluj.ro (A.M.) 2 Department of Microbiology, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine of Cluj–Napoca, Manastur Street, 400372 Cluj-Napoca, Romania; e-mail: smaranda.craciun@stu- dent.usamvcluj.ro (S.C.) 3 Department of Pathology, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Med- icine of Cluj–Napoca, Manastur Street, 400372 Cluj-Napoca, Romania; e-mail: romelia.pop@usamvcluj.ro (R.P.) * Correspondence: sorana.matei@usamvcluj.ro Abstract: This study comprehensively evaluates the crude chemical composition, microbiological dynamics, and mycotoxin contam- ination in Hermetia illucens larvae (Black Soldier Fly Larvae, BSFL), aiming to assess their suitability as a sustainable protein source. Proximate analysis revealed a high protein content (43.22%), along with significant fat levels (19.99%) and moderate fiber content (12.05%), predominantly chitin. Mycotoxin analysis indicated safe levels of aflatoxin B1 (1.29 µg/kg) and deoxynivalenol (6.0 µg/kg), with undetectable levels of ochratoxin A, ensuring compliance with feed safety standards. Microbiological assessments across devel- opmental stages identified a progressive increase in microbial load, in adults. The predominant microbial species included Entero- coccus spp., Klebsiella aerogenes, and Escherichia coli. Thermal treatment via microwave drying significantly reduced microbial contam- ination, although Enterococcus spp. remained detectable post-treatment. These findings highlight BSFL's potential as a nutritionally valuable ingredient in animal feed, particularly due to their high protein and fat content. However, further refinement of microbial decontamination strategies is necessary to enhance safety, ensuring their optimal use in food and feed applications. Keywords: insect, dog, nutrition, analysis 1. Introduction The global demand for sustainable and high-quality protein sources is increasing, driven by population growth, environmental concerns, and the need to reduce reliance on conventional livestock production. Insects, particularly Black Soldier Fly Larvae (BSFL), have emerged as a promising alternative protein source for both animal feed and human consumption due to their high nutritional value and low environmental footprint [1,2]. BSFL are rich in protein, essential fatty acids, and micronutrients, making them an attractive candidate for various applications [3], including pet food [4]. In particular, in- sect-based ingredients are gaining attention in the pet food industry, where dog nutrition is being re-examined through the lens of sustainability and alternative protein sources [5]. The Black Soldier Fly (Hermetia illucens) is a non-pest species of Diptera native to tropical and temperate regions worldwide [6]. It has gained significant attention for its unique ability to convert organic waste into valuable biomass, reducing environmental waste while producing nutrient-dense larvae [7]. The life cycle of H. illucens is divided into sev- eral key stages: egg, larva, pupa, and adult, with each stage playing a vital role in its bio- logical and ecological success [8]. The use of Black Soldier Fly Larvae in dog nutrition is increasingly being explored due to the high protein content and essential fatty acids comparable to traditional protein Received: 03.10.2024 Accepted: 24.10.2024 Published: 31.12.2024 DOI: 10.52331/v29i4842 Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses /by/4.0/). mailto:claudiu-nicusor.ionica@usamvcluj.ro(C.N.I.) mailto:claudiu-nicusor.ionica@usamvcluj.ro(C.N.I.) mailto:andrei.szakacs@usamvcluj.ro mailto:smaranda.craciun@student.usamvcluj.ro mailto:smaranda.craciun@student.usamvcluj.ro mailto:romelia.pop@usamvcluj.ro Cluj Vet J 2024, vol 29, issue 4 10 of 38 sources like chicken or fish. BSFL offers additional benefits, including being rich in medium-chain fatty acids, such as lauric acid, which can promote immune function and improve skin and coat health in dogs [9]. As dogs require high-quality protein for muscle maintenance and overall health, BSFL represents a nu- tritionally balanced and environmentally sustainable ingredient that could reduce the ecological footprint of pet food production. Moreover, BSFLs are hypoallergenic, making them a potential solution for dogs with food sensitivities to common proteins like beef or poultry [10]. However, for BSFL to be integrated into dog food, microbiological safety is a critical concern. Insects, like BSFL, naturally harbor microbes, which can include both beneficial bacteria and harmful pathogens such as Salmonella spp. [11] and Escherichia coli spp. [12]. Ensuring that BSFL-derived products are microbiologically safe is essential for preventing health risks in pets [13]. Various rearing conditions and substrates, like wheat bran, can influence microbial loads [14]. By evaluating microbial populations at different developmental stages, from larvae to adults, and using thermal treatments to reduce microbial contamination in BSFL powder, this study aims to ensure that BSFL meets the stringent safety standards required for dog food ingredients. Additionally, the presence of myco- toxins, which can pose a risk to pet health, is assessed to ensure compliance with pet food safety guidelines. This study focuses on BSFL reared on wheat bran, exploring their chemical composition, microbial dynamics, and mycotoxin contamination to assess their suitability as a safe and nutritious ingredient for dog food. By integrating the nutritional benefits and addressing safety concerns, this research supports the potential of BSFL as a sustainable, eco-friendly solution for the pet food industry. 2. Materials and Methods Black Soldier Fly Larvae (BSFL), adults, pupae and eggs were purchased from an industrial fa- cility located in Brasov County, Romania. The larvae were delivered dried and were subsequently ground to a fine powder in preparation for further analyses. Chemical Composition Analysis: The powder was an- alyzed for crude protein, fat, moisture, fiber, NFE (nitrogen-free extract) and ash content, following standard procedures. Mycotoxin Testing: The powder was also assessed for the presence of mycotoxins, including aflatoxins and ochratoxins, to ensure compliance with safety standards for pet food ingredients. Nutritional Value: samples were homogenized before analysis to ensure consistency. The gross chemical composition, including moisture, crude protein, crude fiber, crude fat, NFE (nitrogen-free extract) and ash content, was determined following the procedures outlined in the AOAC official methods. To analyze the chemical com- position of Black Soldier Fly larvae, specific devices and methods are employed. Dry matter content is de- termined using a drying oven, such as the Memmert Universal Oven UF. Crude protein is measured via the Kjeldahl method, utilizing a semi-automatic device like the Kjeltec 8400 Analyzer Unit. The ether extract is obtained through the Soxhlet method, using a Soxhlet apparatus. Crude ash was quantified using a muffle furnace in accordance with standard incineration methods. The Nitrogen-Free Extract (NFE) was calculated by subtracting the sum of the crude protein, ash, ether extract, and crude fiber from 100% of the dry matter, as per standard proximate analysis methods. This calculation provides an estimate of the carbohydrate con- tent. Microbiological Analysis: To evaluate the microbiological safety and monitor microbial dynamics throughout the developmental stages, a total of 8 samples were examined. One sample was taken from each key life stage of the larvae, including adults, live eggs, first and second-instar larvae, third and fourth-instar larvae, pupae, and microwave-dried larvae. Each sample was analyzed for microbial load and species com- position to determine the progression of microbial communities during larval development and post-pro- cessing treatment.: *Eggs: Purchased immediately after oviposition. *First Instar: Purchased 3 days post- hatching. *Second Instar: Purchased 8 days post-hatching. *Third Instar: Purchased 12 days post-hatching. *Fourth Instar: Purchased 18 days post-hatching. *Fifth Instar: Purchased on the day of slaughtering (22 days post-hatching). *Pupae: Purchased at 24 days post-hatching. *Adults: Purchased immediately upon emer- gence. The samples were analyzed using quantitative and qualitative methods. Quantitative analysis: The serial dilution method was employed to estimate the number of microorganisms. Initially, 0.5 g of the sample was diluted in 4.5 ml of sterile saline to obtain a 1:10 dilution. Serial dilutions (up to 10⁻⁵) were prepared similarly. From each dilution, 0.5 ml were plated on nutrient agar (Merck, Darmstadt, Germany) and incu- bated at 37°C for 24 hours. The number of colonies formed was calculated using the following formula: TGN = number of colonies x dilution factor x 1/volume plated. Qualitative analysis: For bacterial identification, samples from the final dilutions were inoculated on MacConkey agar (BioMaxima S.A., Lublin, Poland) and UriSelect medium (Bio-Rad Laboratories Inc., Hercules, CA, USA) URI chromogenic medium. Colonies that Cluj Vet J 2024, vol 29, issue 4 11 of 38 could not be identified based on morphological or cultural characteristics were further analyzed using the Vitek® 2 Compact device (BioMerieux, Marcy l’Etoile, France), which determines 64 biochemical character- istics of bacteria. Mycotoxin Analysis: for the mycotoxicological examination, the test of RIDAS- CREEN®FAST Aflatoxin, RIDASCREEN®FAST Ochratoxin A and RIDASCREEN®FAST Deoxynivalenol were used, competitive enzyme immunoassay tests for the quantitative determination of total aflatoxin, ochratoxin A and deoxynivalenol in cereals and food. The basis of the test is the antigen-antibody reaction. The measurement was performed photometrically at 450 nm using Awareness Technology Model 4300 ChroMate Microplate Reader. 3. Results The chemical composition analysis of Hermetia illucens larvae was conducted to evaluate their nutri- tional potential, focusing on key parameters such as water content, dry matter, crude protein, crude fat, NFE ( nitrogen-free extract), and ash content. The laboratory analysis revealed a water content of 9,70%, contrib- uting to a dry matter (DM) content of 90,30%. This dry matter was composed predominantly of organic matter (82.76% of the sample, or 91.65% of DM), while ash content, representing mineral salts, accounted for 7.54% of the sample (8.35% of DM). Protein analysis demonstrated a substantial nitrogenous content, with crude protein making up 43.22% of the sample, equivalent to 47.86% of DM. Furthermore, the fat content measured 19.99% of the total sample (22.14% of DM), underscoring the larvae’s suitability as a source of essential lipids. The fiber content of 12.05% (13.34% of dry matter), is substantial for promoting gastro-intes- tinal health in dogs. A significant portion of this fiber is represented by chitin, a natural polysaccharide found in the exoskeletons of insects. Chitin is known for its prebiotic properties, contributing to the development of beneficial gut bacteria, and promoting overall digestive health in dogs [15]. However, chitin is not as easily digestible as other fibers, meaning that while it has nutritional benefits, it must be complemented with other easily digestible ingredients to ensure a well-rounded diet. The nitrogen-free extract (NFE), at 7.50%, consists mainly of digestible carbohydrates, providing an important source of energy. Despite its nutritional value, the moderate levels of NFE and fiber indicate that this insect powder should be included as a supplementary ingredient in dog food formulations, rather than being used as a sole dietary component. Hermetia illucens larvae reared on wheat bran exhibit a high protein and fat content, along with moderate levels of ash, indi- cating a rich mineral composition, as well as moderate amounts of fiber and nitrogen-free extract (carbohy- drates). While these nutritional properties make them a promising ingredient for dog food, particularly for fulfilling protein and essential fatty acid requirements, it is recommended that the larvae powder be used as a supplementary ingredient rather than a singular, complete meal for dogs. Table 1. Values obtained from the performed determinations (chemical composition) GROSS CHEMICAL COMPOSITION % of sample % of DM Water 9,70 - Dry matter 90,30 100 Ash 7,54 8,35 Total organic matter 82,76 91,65 Protein 43,22 47,86 Fat 19,99 22,14 Fibres 12,05 13,34 NFE 7,50 8,31 NFE- nitrogen-free extract; DM-dry matter The detected concentrations of various mycotoxins in the analyzed samples are presented in Table 2. Aflatoxin B1 was measured at 1.29 µg/kg, while Deoxynivalenol was found at 6.0 µg/kg. Ochratoxin A levels were undetectable in the samples. Cluj Vet J 2024, vol 29, issue 4 12 of 38 Table 2. Mycotoxin Levels in BSF Larvae Mycotoxin Detected Level (µg/kg) Aflatoxin B1 1.29 Deoxynivalenol 6.0 Ochratoxin A 0.0 The microbial load and species diversity across different life stages and processing conditions of wheat bran-fed insects were assessed through quantitative and qualitative tests (Table 3). The highest microbial load was observed in the adults, with a concentration of 2.72 × 10⁷ CFU/mL. The alive larvae (first/second instar) showed a microbial load of 9.36 × 10⁶ CFU/mL while for the third/fourth instar, we managed to detect 1.12 × 10⁶ CFU/mL. For the alive eggs, microbial counts were found at 9.0 × 10³ CFU/mL and 1.0 × 10⁴ CFU/mL. Alive pupae expressed a microbial load of 1.32 × 10⁷ CFU/mL. Microwave-dried larvae showed lower microbial levels, with 2.1 × 10⁵ CFU/mL and 1.6 × 10⁴ CFU/mL (Table 3). The microbial load in black soldier flies increases progressively from eggs to adults, with the highest concentration observed in the adult stage (2.72 × 10⁷ CFU/mL), compared to lower levels in eggs (9.0 × 10³ to 1.0 × 10⁴ CFU/mL) and other devel- opmental stages (Figure 1). Figure 1. Quantitative test - Microbiology essay A qualitative microbiological analysis was conducted to evaluate the microbial presence at various developmental stages of Hermetia illucens (Black Soldier Fly). The results showed that in adults, the microbial species included Enterococcus spp., Proteus mirabilis, Myroides spp., and Providencia rettgeri was detected. For the eggs, Enterococcus spp. was the only isolated genus. In the first and second instar larvae cultivation showed the presence of Klebsiella aerogenes, Escherichia coli, Myroides spp., Enterococcus spp., Klebsiella pneu- moniae ssp. pneumoniae. In the third and fourth instar larvae, Escherichia. coli, Enterococcus spp., Klebsiella aer- ogenes, Myroides spp. and members of the Enterobacter cloacae complex were present. The analysis of the pupae indicated the presence of Enterococcus spp. and Klebsiella aerogenes . Furthermore, in microwave-dried larvae, only Enterococcus spp. showed any growth. These results demonstrate that the microbial diversity changes across developmental stages of Hermetia illucens. In partic- ular, larvae exhibit a more diverse microbiota compared to pupae and adults, with a predominance of En- terococcus spp. and Klebsiella spp.. Thermal treatment, such as microwave drying, reduces bacterial contam- ination significantly but does not eliminate it, as evidenced by the persistence of Enterococcus spp. in the dried larvae. 0 5000000 10000000 15000000 20000000 25000000 30000000 Microbial Load (CFU/mL)" 11500 9360000 1120000 13200000 27200000 Eggs Larvae (first/second instar) Larvae (third/fourth instar) Pupae Adults Cluj Vet J 2024, vol 29, issue 4 13 of 38 Table 3. Results of qualitative and quantitative examination of Black Soldier Fly larvae /adults/pupae/ eggs fed Product name Adults Alive eggs Alive larvae – First/Second instar Alive larvae – Third/Fo urth instar Alive pupae Microwave- dried wheat barn-fed larvae Quantitative test (TNG- CFU/mL) 10-1 - - - - - - 10-2 - 9000 - - - - 10-3 - 10000 - 11200000 - 210000 10-4 - - - - 132120000 160000 10-5 272800000 - 93600000 - - - Quality review URI 10-1 Enterococc us spp., Proteus mirabilis, Myroides spp. Entero coccus spp. Klebsiella aerogenes, E. coli,Myroides spp., Enterococcus spp. E. coli, Enterococc us spp., Klebsiella aerogenes, Myroides spp Enterococc us spp., Klebsiella aerogenes Enterococcus spp. 10-2 10-3 MAC 10-1 Providenci a rettgeri 0 E. coli, Klebsiella pneumoniae ssp. Pneumoniae E. coli, Enterobact er cloacae complex 0 0 10-2 10-3 4. Discussion The gross chemical composition of Hermetia illucens larvae in our study aligns with previously re- ported findings, particularly for dry matter content, supporting the consistency of the larvae’s moisture re- tention and nutrient concentration [3]. Our protein analysis showed levels comparable to those cited in the literature, reinforcing the larvae’s role as a protein-rich resource for both animal and human nutrition [16]. The stable protein content, even with minor substrate variations, suggests that wheat bran is a viable alter- native for maintaining nutritional value in pet diets. In addition to protein, the larvae powder is rich in lipids and minerals, while providing moderate amounts of fiber and carbohydrates. Although the measured fat and ash content were slightly below typical ranges [3, 20], they still confirm the larvae’s potential as a significant lipid source with a valuable mineral profile, particularly for calcium and phosphorus, which are essential for canine health [17]. Moreover, the presence of chitin, known for its prebiotic properties, highlights the potential gut health benefits of incorpo- rating Hermetia illucens larvae into dog diets [18]. Moreover, Hermetia illucens larvae powder is already used as an ingredient in several commercial rec- ipes for extruded dry dog food due to its balanced nutrient profile and environmental sustainability [19]. Beyond its industrial applications, the powder could also serve as an ingredient in homemade dog feed or treats, offering pet owners a sustainable and nutrient-dense alternative [20]. However, despite the impres- sive nutritional qualities of Hermetia illucens larvae powder, it is not suited to be the sole ingredient for bal- anced canine nutrition. While it provides high levels of protein, fat, and minerals, a complete and balanced diet requires a more diverse range of nutrients that cannot be met by insect powder alone. Therefore, this ingredient should be used as part of a broader nutritional strategy to meet all of a dog's dietary needs [21]. Our study aimed to assess the quality of insect meal, taking Hermetia illucens larvae in their dried form, grinding them ourselves, and evaluating the nutritional properties of the resulting powder. Specifically, we Cluj Vet J 2024, vol 29, issue 4 14 of 38 sought to determine whether the substrate used to feed the larvae influences the nutritional composition of the insect powder and assess its potential for inclusion in dog nutrition. Through this analysis, we have contributed to understanding the versatility and limitations of Hermetia illucens powder as a functional in- gredient in canine diets. The analysis of mycotoxins in Hermetia illucens (Black Soldier Fly) larvae revealed relatively low con- tamination levels compared to the maximum allowable limits set by the European Union for dogs [22]. Spe- cifically, Aflatoxin B1 was measured at 1.29 µg/kg, Deoxynivalenol (DON) at 6.0 µg/kg, while Ochratoxin A was below detectable levels. These findings are significant as they suggest that BSF larvae raised on wheat bran present a low risk of acute mycotoxin toxicity for animals, including dogs. When compared to the max- imum admitted levels, which are 20 µg/kg for Aflatoxin B1(C, 2006), 5,000 µg/kg for Deoxynivalenol [22], and 10 µg/kg for Ochratoxin A [22], the detected concentrations in our study are far below the thresholds. This suggests that feeding BSF larvae to dogs would not likely lead to acute mycotoxicosis, a condition that typically occurs following the ingestion of high mycotoxin concentrations. However, while the acute risk is minimal, the potential for chronic exposure should not be ignored. Prolonged consumption of feed contain- ing low levels of mycotoxins could predispose dogs to long-term health issues, such as liver disease or can- cer, particularly in the case of Aflatoxin B1 [23]. Chronic aflatoxin exposure has been linked to hepatotoxicity and hepatocellular carcinoma in various animal species, including dogs, even at subclinical levels. This raises concerns about the potential cumulative effects of long-term, low-level mycotoxin ingestion in pet diets [24]. To mitigate chronic exposure to mycotoxins in animal diets, particularly for dogs, it is crucial to implement effective monitoring systems and establish clear thresholds for mycotoxin levels in feed ingredients such as Black Soldier Fly larvae. While commercial dry dog food typically shows mycotoxin contamination levels below regulatory limits, the risk of chronic exposure from consistent, low-level intake remains a concern. Dogs may be exposed daily to small quantities of mycotoxins, which, over time, could pose health risks. Regular testing of both the larvae and their substrates, as well as periodic monitoring of mycotoxins in dog feed, is essential to ensure safety [25]. Further processing methods, including thermal treatments and the use of detoxifying additives, can help significantly reduce mycotoxin concentrations. Additionally, high-quality substrate management is vital, as it directly affects the nutritional value and safety of the larvae as a feed source. Prioritizing these strategies will enhance the health and well-being of pets and livestock, minimizing the long-term risks associated with mycotoxin exposure [26]. Despite the low levels of mycotoxins detected in BSF larvae, the literature suggests that BSF larvae have a unique ability to degrade or tolerate certain contaminants, including mycotoxins. Previous studies have highlighted that Black Soldier Fly larvae can degrade aflatoxins and other harmful compounds during digestion, reducing the risk of contamination in the final product [27]. However, the efficacy of this bioconversion varies depending on the type of mycotoxin and the concentration present in the feed substrate [28]. For instance, some research has shown that BSF larvae can significantly reduce Aflatoxin B1 levels in contaminated substrates, although complete degrada- tion may not always occur [29]. This ability to partially detoxify their feed could explain the low mycotoxin concentrations observed in our study, despite the presence of wheat bran, a substrate that can be prone to fungal contamination. Our findings align with this body of research, as the larvae's low mycotoxin levels indicate that wheat bran is a suitable substrate for BSF rearing without introducing significant risks of con- tamination. Nevertheless, further research is required to better understand the long-term effects of chronic exposure to low mycotoxin levels in both BSF larvae and the animals consuming them. For now, the results are promising in terms of the safety and sustainability of using BSF larvae as a protein source for dog nutri- tion, particularly given their low mycotoxin content and the larvae’s inherent detoxification capabilities. Our study explored the microbial load and species diversity across different developmental stages of Her- metia illucens (Black Soldier Fly) fed on wheat bran, without dissecting the larvae, pupae, or adult gut. In- stead, we aimed to observe the bacterial dynamics throughout their development in a commonly used sub- strate, assessing microbial presence on the external body surface and internal gut as total. The results high- light significant changes in both microbial load and bacterial species composition as the larvae progressed through their life cycle, as well as the effect of thermal processing (microwave drying) on bacterial contam- ination. Quantitatively, the microbial load increased progressively from eggs to adults, with the highest load detected in the adults (2.72 × 10⁷ CFU/mL at a dilution of 10⁻⁵), while the lowest levels were recorded in the eggs (9.0 × 10³ to 1.0 × 10⁴ CFU/mL). Notably, the microbial load in the larvae also varied with developmental stage; first/second instar larvae exhibited a load of 9.36 × 10⁶ CFU/mL, while the third/fourth instar larvae Cluj Vet J 2024, vol 29, issue 4 15 of 38 showed a reduced load of 1.12 × 10⁶ CFU/mL. These fluctuations in microbial load are consistent with find- ings in the literature [7], which emphasize that as insects grow, they change the microbiota due to shifts in their diet, metabolic activity, and immune system responses [30]. Qualitatively, our analysis revealed diverse microbial communities at different life stages. For example, first/second instar larvae hosted a more varied microbiota, including Klebsiella aerogenes, Escherichia coli, Myroides spp., and Enterococcus spp., while third/fourth instar larvae displayed similar species with the addition of members from the Enterobacter cloa- cae complex. This aligns with previous research [31] indicating that BSF larvae possess a dynamic microbiota, which can help degrade organic matter and enhance nutrient recycling. The decrease in microbial diversity observed as the larvae progressed to the pupal and adult stages, particularly the predominance of Enterococ- cus spp. and Klebsiella spp., has also been reported in other studies [32], suggesting that microbial diversity tends to narrow as insects undergo metamorphosis. The results highlight significant changes in both micro- bial load and bacterial species composition as the larvae progressed through their life cycle, as well as the effect of thermal processing (microwave drying) on bacterial contamination. This variation in microbial load across different developmental stages is crucial for optimizing thermal treatment methods, as certain life stages may require more stringent processing to ensure microbial safety. Practical solutions could involve tailoring the duration and intensity of heat treatment based on the larvae’s developmental stage. For in- stance, larvae in later stages, which may harbour more resilient bacterial species, could benefit from longer microwave drying times or higher temperatures to ensure a more comprehensive microbial reduction [33]. Furthermore, our study aimed to assess whether thermal treatment via microwave drying could effectively reduce bacterial contamination in BSF byproducts to make them safer for consumption, particularly in ani- mal feed. The results indicate that while microwave drying significantly reduced the microbial load—show- ing 2.1 × 10⁵ CFU/mL and 1.6 × 10⁴ CFU/—it did not eliminate microbial presence. Enterococcus spp. was still detected in dried larvae, a finding that aligns with the notion that while heat treatment is effective [34], complete sterility is difficult to achieve. These results are critical when considering the potential use of BSF larvae as a feed ingredient for dogs. Although thermal processing substantially reduces microbial contami- nation, the persistence of Enterococcus spp. is noteworthy. In the context of canine nutrition, this species, while commonly present in the environment, could pose a risk to immunocompromised dogs [34,35]. Chronic exposure to low levels of potentially pathogenic bacteria could increase the likelihood of infection or contribute to the development of other conditions in susceptible animals [36]. To mitigate this risk, future studies could explore combining microwave drying with other sterilization techniques, such as pressure- based methods (e.g., high-pressure processing), which have been shown to inactivate heat-resistant bacterial species. [37] In comparison to findings in the literature [38], our study supports the general trend that microbial load and diversity fluctuate across the life stages of BSF, influenced by both the insect’s developmental biol- ogy and the substrate composition. Additionally, the literature suggests that BSF larvae can reduce certain pathogens in their environment due to their microbial communities [39], yet some bacteria, such as Entero- coccus spp., are resilient and can persist despite thermal processing. This highlights the practical importance of refining thermal treatment methods, such as adjusting drying times or temperatures, to target resilient bacteria more effectively. Solutions such as incorporating temperature gradients—where the drying process begins at lower temperatures to avoid nutritional degradation and gradually increases to ensure bacterial inactivation—could optimize both safety and quality. Additionally, the use of microbial inhibitors (e.g., or- ganic acids) during the processing phase could further suppress bacterial survival while maintaining the larvae’s nutritional integrity. Therefore, our findings contribute to the ongoing understanding of microbial dynamics in BSF and underscore the need for further investigation into optimizing processing methods to ensure microbial safety without compromising the larvae’s nutritional integrity. These insights could inform future processing standards, where variations in microbial load across different life stages are accounted for. Establishing spe- cific protocols for each larval stage could lead to more precise drying times and temperatures, minimizing nutrient loss while maximizing microbial safety. A combination of tailored heat treatment, surface cleaning, and supplementary sterilization methods could be integrated into industrial processes to achieve the highest safety standards for BSF-derived products. 5. Conclusions Cluj Vet J 2024, vol 29, issue 4 16 of 38 Our study highlights the nutritional viability of Hermetia illucens larvae as a protein-rich, sustainable ingredient for dog nutrition. The larvae's gross composition, particularly their high protein content, aligns well with the dietary needs of most animals. Despite slight deviations in fat and ash content compared to previous research, the larvae maintain a robust nutritional profile suitable for pet diets, supporting both energy needs and bone health. The low levels of mycotoxins detected, far below the EU-admitted thresholds, indicate minimal risk for acute toxicity in dogs. However, the potential long-term effects of low-level Afla- toxin B1 exposure warrant further investigation, particularly given its link to chronic liver disease. Addi- tionally, our microbial analysis underscores the need for rigorous safety protocols in processing larvae for dog food, as Enterococcus spp. can pose a risk to immunocompromised individuals. Black soldier fly larvae reared on wheat bran demonstrate great potential as a sustainable, nutrient-dense ingredient for dog food and animal feedings in general, offering high protein content with manageable my- cotoxin contamination. While thermal processing effectively reduces bacterial load, optimizing this process for microbial safety remains crucial, especially for sensitive pets. However, while Hermetia illucens larvae powder has many desirable nutritional qualities, it cannot serve as a sole ingredient for a balanced canine diet. A complete and balanced diet for dogs requires a broader range of nutrients that cannot be provided by insect powder alone. Therefore, its role should be considered as a complementary ingredient within a more comprehensive dietary framework. Overall, our findings support the further investigation of Hermetia illucens larvae as a cost-effective, eco-friendly alternative protein source for not only pet nutrition but also for various animal feed industries. Its excellent nutritional profile, combined with its sustainable production, offers a promising solution for advancing animal feed formulations without compromising nutritional qual- ity or safety across different species." Supplementary Materials: Not applicable. Author Contributions: Conceptualization: CNI, SD, AM; Methodology: SC, ARZ; Analysis: SC, ARZ; Writing – original draft: CNI, RP; Writing – review and editing: SD, AM. All authors declare that they have read and approved the publi- cation of the manuscript in this present form Funding: The authors would like to express their sincere gratitude to the German Foundation for Environment (Deutsche Bundestiftung Umwelt - DBU) for their generous support in funding this research through the Small Grant Project - 30024/029 – 43/0, conferred on April 30, 2024. This funding has been instrumental in advancing our work and contributing to the successful completion of the project.. Institutional Review Board Statement: Not applicable. Conflicts of Interest: Not applicable References 1. Govorushko S. Global status of insects as food and feed source: A review. Trends Food Sci Technol Else-vier; 2019;91:436– 445. doi: https://doi.org/10.1016/j.tifs.2019.07.032 2. Kierończyk B, Rawski M, Mikołajczak Z, Homska N, Jankowski J, Ognik K, Józefiak A, Mazurkiewicz J, Józefiak D. Availa- ble for millions of years but discovered through the last decade: Insects as a source of nutrients and energy in animal diets. Animal Nutrition Elsevier; 2022;11:60–79. doi: https://doi.org/10.1016/j.aninu.2022.06.015 3. Barragan-Fonseca KB, Dicke M, van Loon JJA. Nutritional value of the black soldier fly (Hermetia illucens L.) and its suita- bility as animal feed–a review. J Insects Food Feed Wageningen Academic Publish-ers; 2017;3(2):105–120. doi: https://doi.org/10.3920/JIFF2016.0055 4. Penazzi L, Schiavone A, Russo N, Nery J, Valle E, Madrid J, Martinez S, Hernandez F, Pagani E, Ala U. In vivo and in vitro digestibility of an extruded complete dog food containing black soldier fly (Hermetia illucens) larvae meal as protein source. Front Vet Sci Frontiers Media SA; 2021;8:653411. doi: https://doi.org/10.3389/fvets.2021.653411 5. Higa JE, Ruby MB, Rozin P. Americans’ acceptance of black soldier fly larvae as food for themselves, their dogs, and farmed animals. Food Qual Prefer Elsevier; 2021;90:104119. doi: https://doi.org/10.1016/j.foodqual.2020.104119 6. Kim J-G, Choi Y-C, Choi J-Y, Kim W-T, Jeong G-S, Park K-H, Hwang S-J. Ecology of the black soldier fly, Hermetia illucens (Diptera: Stratmyidae) in Korea. Korean journal of applied entomology Korean Society of Applied Entomology; 2008;47(4):337–343. 7. De Smet J, Wynants E, Cos P, Van Campenhout L. Microbial community dynamics during rearing of black soldier fly larvae (Hermetia illucens) and impact on exploitation potential. Appl Environ Micro-biol Am Soc Microbiol; 2018;84(9):e02722-17. doi: https://doi.org/10.1128/AEM.02722-17 8. Boakye-Yiadom KA, Ilari A, Duca D. Greenhouse gas emissions and life cycle assessment on the black soldier fly (Hermetia illucens L.). Sustainability MDPI; 2022;14(16):10456. https://doi.org/10.1016/j.tifs.2019.07.032 https://doi.org/10.1016/j.aninu.2022.06.015 file:///Users/robert/Downloads/cvj/JIFF2016.0055 https://doi.org/10.3389/fvets.2021.653411 https://doi.org/10.1016/j.foodqual.2020.104119 https://doi.org/10.1128/AEM.02722-17 Cluj Vet J 2024, vol 29, issue 4 17 of 38 9. Sutton A, Costa ND. The role of black soldier fly larval protein and fat in companion-animal nutrition: challenges and opportunities from an industry perspective. Anim Prod Sci CSIRO Publishing; 2023; doi: https://doi.org/10.1071/AN23080 10. Kotob G, Sluczanowski N, Siddiqui SA, Tome NM, Dalim M, van der Raad P, Aarts K, Paul A. Potential application of black soldier fly fats in canine and feline diet formulations: A review of literature. J Asia Pac Entomol Elsevier; 2022;25(4):101994. doi: https://doi.org/10.1016/j.aspen.2022.101994 11. De Smet J, Vandeweyer D, Van Moll L, Lachi D, Van Campenhout L. Dynamics of Salmonella inoculated during rearing of black soldier fly larvae (Hermetia illucens). Food Research International Elsevier; 2021;149:110692. doi: https://doi.org/10.1016/j.foodres.2021.110692 12. Van Looveren N, IJdema F, van der Heijden N, Van Der Borght M, Vandeweyer D. Microbial dynamics and vertical trans- mission of Escherichia coli across consecutive life stages of the black soldier fly (Hermetia illucens). Anim Microbiome Springer; 2024;6(1):29. doi: https://doi.org/10.1186/s42523-024-00317-4 13. Gold M, Von Allmen F, Zurbrügg C, Zhang J, Mathys A. Identification of bacteria in two food waste black soldier fly larvae rearing residues. Front Microbiol Frontiers Media SA; 2020;11:582867. doi: https://doi.org/10.3389/fmicb.2020.582867 14. Schreven SJJ, de Vries H, Hermes GDA, Zeni G, Smidt H, Dicke M, Van Loon JJA. Black soldier fly larvae influence internal and substrate bacterial community composition depending on substrate type and larval density. Appl Environ Microbiol Am Soc Microbiol; 2022;88(10):e00084-22. doi: https://doi.org/10.1128/aem.00084-22 15. Lopez-Santamarina A, Mondragon A del C, Lamas A, Miranda JM, Franco CM, Cepeda A. Ani-mal-origin prebiotics based on chitin: An alternative for the future? a critical review. Foods MDPI; 2020;9(6):782. doi: https://doi.org/10.3390/foods9060782 16. Diener S, Zurbrügg C, Tockner K. Conversion of organic material by black soldier fly larvae: establishing optimal feeding rates. Waste management & research Sage Publications Sage UK: London, England; 2009;27(6):603–610. doi: https://doi.org/10.1177/0734242X091038 17. Spranghers T, Ottoboni M, Klootwijk C, Ovyn A, Deboosere S, De Meulenaer B, Michiels J, Eeckhout M, De Clercq P, De Smet S. Nutritional composition of black soldier fly (Hermetia illucens) prepupae reared on different organic waste substrates. J Sci Food Agric Wiley Online Library; 2017;97(8):2594–2600. doi: https://doi.org/10.1002/jsfa.8081 18. Biasato I, Colombino E, Luna A, Capucchio MT. Insects and gut health in food-producing animals. Environmental effects on gut health in production animals Wageningen Academic; 2024. p. 365–399. doi: https://doi.org/10.3920/9789004695467_017ISBN:900469546X 19. Bosch G, Vervoort JJM, Hendriks WH. In vitro digestibility and fermentability of selected insects for dog foods. Anim Feed Sci Technol Elsevier; 2016;221:174–184. 20. Pinney J, Costa-Font M. A Model for Consumer Acceptance of Insect-Based Dog Foods among Adult UK Dog Owners. Animals MDPI; 2024;14(7):1021. doi: https://doi.org/10.3390/ani14071021 21. FEDIAF. Nutritional Guidelines for Pet Food for Cats and Dogs. FEDIAF. 2021. Available from: https://fediaf.orghttps://fe- diaf.org [accessed Sep 30, 2024] 22. C E. Commission Recommandation 2006/576. Official Journal of the European Union OJ L 2006;229:7–9. 23. Macías-Montes A, Rial-Berriel C, Acosta-Dacal A, Henríquez-Hernández LA, Almeida-González M, Rodríguez-Hernández Á, Zumbado M, Boada LD, Zaccaroni A, Luzardo OP. Risk assessment of the exposure to mycotoxins in dogs and cats through the consumption of commercial dry food. Science of The Total Environment Elsevier; 2020;708:134592. doi: https://doi.org/10.1016/j.scitotenv.2019.134592 24. Martínez-Martínez L, Valdivia-Flores AG, Guerrero-Barrera AL, Quezada-Tristán T, Rangel-Muñoz EJ, Ortiz-Martínez R. Toxic effect of aflatoxins in dogs fed contaminated commercial dry feed: a review. Toxins (Basel) MDPI; 2021;13(1):65. doi: https://doi.org/10.3390/toxins13010065 25. Yang, L., Yang, L., Cai, Y., Luo, Y., Wang, H., Wang, L.,Chen J., Liu X., Wu Y.,Qin Y.,Wu Z., Liu, N. Natural mycotoxin contamination in dog food: a review on toxicity and detoxification methods. Ecotoxicology and Environmental Safety Elsevier 2023. , 257, 114948, doi:https://doi.org/10.1016/j.ecoenv.2023.114948 26. Agriopoulou, S., Stamatelopoulou, E., Varzakas, T. Advances in occurrence, importance, and mycotoxin control strategies: Prevention and detoxification in foods. Foods MDPI 2020. , 9(2), 137, doi:https://doi.org/10.3390/foods9020137 27. Gold M, Niermans K, Jooste F, Stanford L, Uwamahoro F, Wanja M, Veldkamp T, Sanderson A, Nunes VDS, Mathys A. Conversion of mycotoxin-contaminated maize by black soldier fly larvae into feed and fertilizer. J Insects Food Feed Wageningen Academic; 2023;1(aop):1–14. doi: https://doi.org/10.1163/23524588-00001006 28. Camenzuli L, Van Dam R, De Rijk T, Andriessen R, Van Schelt J, Van der Fels-Klerx HJ. Tolerance and excretion of the mycotoxins aflatoxin B1, zearalenone, deoxynivalenol, and ochratoxin A by Alphitobi-us diaperinus and Hermetia illucens from contaminated substrates. Toxins (Basel) MDPI; 2018;10(2):91. doi: https://doi.org/10.3390/toxins10020091 29. Niermans K, Hoek-van den Hil EF, van der Fels-Klerx HJ, van Loon JJA. The role of larvae of black soldier fly and house fly and of feed substrate microbes in biotransformation of aflatoxin B1. Ecotoxicol Environ Saf Elsevier; 2024;279:116449. doi: https://doi.org/10.1016/j.ecoenv.2024.116449 30. Querejeta M, Hervé V, Perdereau E, Marchal L, Herniou EA, Boyer S, Giron D. Changes in bacterial community structure across the different life stages of black soldier fly (Hermetia illucens). Microb Ecol Springer; 2023;86(2):1254–1267. doi: https://doi.org/10.1007/s00248-022-02146-x 31. Gorrens E, Van Moll L, Frooninckx L, De Smet J, Van Campenhout L. Isolation and identification of dominant bacteria from black soldier fly larvae (Hermetia illucens) envisaging practical applications. Front Microbiol Frontiers Media SA; 2021;12:665546. doi: https://doi.org/10.3389/fmicb.2021.665546 https://doi.org/10.1071/AN23080 https://doi.org/10.1016/j.aspen.2022.101994 https://doi.org/10.1016/j.foodres.2021.110692 https://doi.org/10.1186/s42523-024-00317-4 https://doi.org/10.3389/fmicb.2020.582867 https://doi.org/10.1128/aem.00084-22 https://doi.org/10.3390/foods9060782 https://doi.org/10.1177/0734242X091038 https://doi.org/10.1002/jsfa.8081 https://doi.org/10.3920/9789004695467_017ISBN:900469546X https://doi.org/10.3390/ani14071021 https://doi.org/10.1016/j.scitotenv.2019.134592 https://doi.org/10.3390/toxins13010065 https://doi.org/10.1016/j.ecoenv.2023.114948 https://doi.org/10.3390/foods9020137 https://doi.org/10.1163/23524588-00001006 https://doi.org/10.3390/toxins10020091 https://doi.org/10.1016/j.ecoenv.2024.116449 https://doi.org/10.1007/s00248-022-02146-x https://doi.org/10.3389/fmicb.2021.665546 Cluj Vet J 2024, vol 29, issue 4 18 of 38 32. Klüber P, Müller S, Schmidt J, Zorn H, Rühl M. Isolation of bacterial and fungal microbiota associated with Hermetia illucens larvae reveals novel insights into entomopathogenicity. Microorganisms MDPI; 2022;10(2):319. doi: https://doi.org/10.3390/microorganisms10020319 33. Campbell, M., Ortuño, J., Stratakos, A. C., Linton, M., Corcionivoschi, N., Elliott, T.,Koidis A., Theodoridou, K. Impact of thermal and high-pressure treatments on the microbiological quality and in vitro digestibility of black soldier fly (Hermetia illucens) larvae. Animals MDPI ,2020. 10(4), 682 doi:https://doi.org/10.3390/ani10040682 34. Larouche J, Deschamps M-H, Saucier L, Lebeuf Y, Doyen A, Vandenberg GW. Effects of killing methods on lipid oxidation, colour and microbial load of black soldier fly (Hermetia illucens) larvae. Animals MDPI; 2019;9(4):182. doi: https://doi.org/10.3390/ani9040182 35. Wood MW, Lepold A, Tesfamichael D, Lasarev MR. Risk factors for enterococcal bacteriuria in dogs: A retrospective study. J Vet Intern Med Wiley Online Library; 2020;34(6):2447–2453. doi: https://doi.org/10.1111/jvim.15916 36. Harvey B, Tarrant J, McClosky M, Nathanson O, Cole S. Enterococcus spp. meningoencephalitis, ventriculitis, and hy- pophysitis in a dog. J Am Anim Hosp Assoc American Animal Hospital Association; 2021;57(6):290–293. doi: https://doi.org/10.5326/JAAHA-MS-7112 37. Van Looveren, N., Vandeweyer, D., & Van Campenhout, L. (2021). Impact of heat treatment on the microbiological quality of frass originating from black soldier fly larvae (Hermetia illucens). Insects, 13(1), 22. 38. Li X, Mei C, Luo X, Wulamu D, Zhan S, Huang Y, Yang H. Dynamics of the intestinal bacterial community in black soldier fly larval guts and its influence on insect growth and development. Insect Sci Wiley Online Library; 2023;30(4):947–963. doi: https://doi.org/10.1111/1744-7917.13095s 39. Erickson MC, Islam M, Sheppard C, Liao J, Doyle MP. Reduction of Escherichia coli O157: H7 and Salmonella enterica serovar Enteritidis in chicken manure by larvae of the black soldier fly. J Food Prot Elsevier; 2004;67(4):685–690. doi: https://doi.org/10.4315/0362-028X-67.4.685 https://doi.org/10.3390/microorganisms10020319 https://doi.org/10.3390/ani10040682 https://doi.org/10.3390/ani9040182 https://doi.org/10.1111/jvim.15916 https://doi.org/10.5326/JAAHA-MS-7112 https://doi.org/10.1111/1744-7917.13095 https://doi.org/10.4315/0362-028X-67.4.685