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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 

 

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https://doi.org/10.3390/microorganisms10020319
https://doi.org/10.3390/ani10040682
https://doi.org/10.3390/ani9040182
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