# Unconventional Extraction Methods of Oleaginous Yeast Cell Pretreatment and Disruption
## 4 Discussion  
The biomass of the yeast _Y. lipolytica_, which has the GRAS status, was used in the described experiments. Importantly, following the Commission Implementing Regulation (EU) 2019/760 of 13 May 2019, the biomass of this yeast species is authorized as a novel food and may be used as an ingredient in human nutrition [29]. It is a well-known fact that _Y. lipolytica_ garnered plenty of attention in the scientific community and constitutes a perfect platform for the biosynthesis of numerous, extremely valuable secondary metabolites with their potential application in the food, cosmetic, and pharmaceutical industries [30]. Regarding research on one of them, more precisely microbial oil--which is accumulated intracellularly--it is necessary to select and use an appropriate extraction method. In fact, extraction is one of the most commonly used methods for obtaining and purifying chemical compounds. Unconventional extraction methods not only shorten the process time and reduce energy consumption but also eliminate the use of harmful solvents and improve the quality of the final product [31]. In the current study, the following methods for the improvement of the microbial oil extraction process were applied: PEF, US, and HPP. Figure 8: _Yarrowia lipolytica_ KKP 379 yeast cells treated with (**a**) acetone; (**b**) acetone and glass beads; (**c**) Tween-80 surfactant; (**d**) Tween-80 surfactant and glass beads; (**e**) Triton X-100; (**f**) Triton X-100 and glass beads; (**g**) glass beads; (**h**) Y-PER lytic preparation; (**i**) ultrasonic treatment. Images were taken with scanning electron microscope. Comparing the extraction efficiencies of intracellular lipids from yeast cells treated with different permeabilization methods, it can be concluded that the highest efficiency was achieved by using PEF. When yeast cells are exposed to a voltage higher than their natural potential, both the cell wall and the membrane become permeable, and because of the electric current, the formation of pores on the cell surface occurs, i.e., the so-called electroporation [32]. The application of PEF for the extraction of intracellular metabolites including proteins or polysaccharides from yeast has been studied several times [33]. The use of PEF also allows some lipid molecules to pass through the pores in the membrane, and thus, PEF can be considered a good method for the permeabilization of yeast cells; in the current study, this was acknowledged both in the case of lipid extraction and the measurements of protein concentration in the remaining liquids after oil extraction experiments. In recent years, scientists have attempted to use PEF treatment for lipid extraction (Table 1). [PERSON] et al. [34] applied PEF as a pretreatment step in ethanol-hexane-mediated lipid extraction from _Saitozyma podzolica_. The authors emphasized the importance of lower conductivity on the efficiency of PEF treatment, and moreover, the biomass was not subjected to any processing such as freezing or freeze-drying. Washing steps for yeast biomass both reduced the conductivity, as well as increased the efficiency of lipid extraction by 54% [34]. [PERSON] et al. [35] also evaluated numerous cell disruption methods for the improvement of oil extraction from _Y. lipolytica_. The authors applied, e.g., high PEF, high voltage electrical discharges, US, and high-pressure homogenization. Compared to 19.8% for control samples, the authors observed an increase in the oil extraction yield to 29.4% when 20 kV/cm of electrical field strength was used. In addition, in the case of microalgae lipids, [PERSON] et al. [36] observed increased lipid extraction yield by up to 166.67% in _Chorella_ cells when the PEF treatment was applied compared to control samples without any disruption method. The authors also claimed that the too-high voltage caused a decrease in the quality of the extracted oil [36]. There were also provided experiments on PEF-assisted protein extraction. In the study of [PERSON] et al.