## 3 Results
### Evaluation of Microbial Oil and Biomass Safety  
The safety of products derived from microorganisms cultured in industrial oil-based media is an important issue, especially in the context of their use in food applications. Indeed, this is a critical point when considering the future potential of using oleaginous yeasts, such as _Y. lipolytica_, for oil production and inclusion in the human diet, and from a broader food safety perspective. The issue of safety has already been addressed in [PERSON] et al. (2024), where the same yeast strain was used and a similar carbon source was used in the medium for obtaining the microbial oil [20]. Additionally, in the present study, microbial quality was evaluated.  
In the study by [PERSON] et al. 2024 [20], analysis of the PAH content (Table 6) showed that microbial oil obtained by culturing _Y. lipolytica_ in post-frying rapeseed oil medium (as in our study) met the requirements for oils and fats marketed to end consumers or used as food ingredients in relation to maximum levels of process contaminants, such as benzo(a)pyrene and the sum of benzo(a)pyrene (B[a]P), benzo(a)anthracene (B[a]A), benzo(b)fluoranthene (B[k]F) and chrysene (Chry). As defined in Regulation (EU) 2023/915 (Commission Regulation (EU) 2023/915 of 25 April 2023), the maximum level for B[a]A is 2 ug/kg. The B[a]A content in the microbial oil analysed was 0.61 ug/kg. The guideline indicated in the same regulation for the sum of the four PAHs--B[a]P, Chry, B[b]F and B[a]A, which should not exceed 10 ug/kg oil, was also met. The sum of the four PAHs was 4.12 ug/kg for SCO obtained from post-frying rapeseed oil medium. In the case of hexane residue content, it was 0.0007 mg/kg, and according to European Union regulations, its permissible content in fats and oils should not exceed 1 mg/kg [4; 20; 57; 58].  
To provide microbiological safety and to eliminate metabolic activity, the collected _Y. lipolytica_ yeast biomass was heat-inactivated. Samples were autoclaved at 121 \({}^{\circ}\)C for 15 min  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Biological Material** & **Yeast Biomass \({}^{*}\)** & **Microbial Oil** & \multicolumn{2}{c}{**European**} & **Reference** \\ \hline Sum of PAHs: benzo(a) & & & & \\ pyrene, benz(a) anthracene, & & & & \\ benzo(b) fluoranthene and & n.a. & 4.12 & 10.0 & [20] \\ chrysene [\(\mu\)g/kg] & & & & \\ Benzo(a) pyrene [ug/kg] & n.a. & 0.61 & 2.0 & [20] \\ Hexane residues (mg/kg) & n.a. & 0.0007 & 1.0 & [20] \\ Heavy metals & 0 & 0 & 0.1 & [20] \\ Total bacterial counts & & & & \\
[CFU/g] & 5.8 \(\times\) 10\({}^{1}\) & 1.4 \(\times\) 10\({}^{1}\) & \(\leq\)5 \(\times\) 10\({}^{3}\) & \\ Number of yeast & & & & \\ counts[CFU/g] & 0 & 0 & \(\leq\)1 \(\times\) 10\({}^{2}\) & \\ \hline \hline \end{tabular} \({}^{*}\) heat inactivated biomass 121 \({}^{\circ}\)C 15 min. n.a.—not analysed.  
\end{table}
Table 6: Assessment of the safety of biomass and microbial oil obtained from _Y. lipolytica_ yeast.  
following standard sterilisation conditions. In the context of a microbiological assessment of _Y. lipolytica_ yeast biomass, total bacterial count and the counts of yeast were evaluated. The experiment did not maintain 100% sterility of the conditions as would be observed under industrial conditions, and still the bacterial count in the yeast biomass and microbial oil did not exceed 100 CFU/g and it did not contain viable yeast cells. In light of these findings and the available evidence, the resulting microbial product cannot be considered unsafe or unsuitable for food use.