## 3 Results and Discussion
### Microbiological Quality of Freeze-Dried Edible Insects Larvae  
Changes in the total viable count (TVC), spore-forming bacteria, and total fungi count of fresh and freeze-dried _H. illucens_ and _T. molitor_ larvae are presented in Figure 5. The initial number of TVC on the fresh larvae is 7.3-7.7 log CFU/g, and it is comparable to the literature data [18; 25]. For both insect species, after the utilization of PEF and freeze drying, a decrease in TVC is observed, in the range of 6.3-6.5 and 6.2-7.0 for _H. illucens_ and _T. molitor_, respectively. All processed samples had TVC values lower than the advised limit of 7.0 log CFU/g for edible insects for food purposes [18]. In turn, according to the Commission Implementing Regulation (EU) 2022/169 of 8 February 2022, the TVC limit for _T. molitor_ larvae should be below 5.0 log CFU/g; nevertheless, all freeze-dried insects exceeded this limit. Figure 4: FTIR spectra of freeze-dried untreated (0 kJ/kg) and PEF-treated (5 and 20 kJ/kg) (**a**) _H. illucens_ and (**b**) _T. molitor_ larvae. Figure 5: Microbiological quality of fresh, and freeze-dried untreated (0 kJ/kg) and PEF-treated (5 and 20 kJ/kg) (**a**) _H. illucens_ and (**b**) _T. molitor_ larvae. The same letters above columns for each group of microorganisms denote the same homogeneous groups, which do not differ statistically (\(p<0.05\)). Aerobic spore-forming bacteria like _Bacillus_ sp. might be frequently found in fresh and proceeded edible insects. These bacteria are generally present in soil and dust [11], and are a serious problem for food products since they can produce resistant endospores which are difficult to eliminate using thermal processing [11; 12]. Treatment with PEF and freeze-drying of insects may limit the growth of spore-forming bacteria. The application of these treatments caused a reduction in the number of microorganisms by 1.0-1.5 log cycles for fresh and dried larvae (Figure 5a,b). A more effective impact of PEF application in lowering the number of these microorganisms was noted for _H. illucens_ larvae (from 4.7 to 4.3 log CFU/g) than for _T. molitor_ larvae. What is interesting is that for _T. molitor_ larvae, a dose of intake energy had no impact on a reduction in the number of these bacteria. The obtained results might be explained by a higher resistance of bacteria spores to PEF application, and thus treatment only with PEF could be applied rather for a pasteurization than for a sterilization effect [65], due to the possibility of microorganisms exhibiting recovery after PEF treatment [66]. The initial number of total fungi count in the fresh insects was approximately 5.6 log CFU/g (Figure 5a,b). A decrease in the number of fungi was observed in the studied insects after the freeze drying. A slightly higher decrease was noted for _H. illucens_ larvae by 2.3-2.6 log cycles than for _T. molitor_ larvae (1.9-2.3 log cycles). With reference to the PEF application, the use of a different dose of energy (5 and 20 kJ/kg) did not change the number of fungi probably due to their morphological structure, as well as the cell wall composition [67; 68]. Moreover, for _T. molitor_ larvae (Figure 3b), the microbial load was higher than would seem to be a result of the forming of fungi spores [69]. According to the Commission Implementing Regulation (EU) 2022/169 of 8 February 2022, the number of total fungi count for freeze-dried _T. molitor_ larvae should be lower than 2.0 log CFU/g; meanwhile, in the current study, all freeze-dried insects exceeded this limit. To reduce the microbiological hazards, raw materials should be processed before consumption [27; 28]. Various processing methods are used for this purpose, and one of them, freeze drying, is used in this study. During the freezing and freeze-drying process, formed ice crystals cause disruptions in microorganisms' cellular structure, loss of cell viability, and then their death [70; 71]. Furthermore, PEF might be used to inactivate some microorganisms.