## 4 Discussion  
### Differences in Bacterial Inactivation  
In this study, we aimed to examine the effect of PEF on whey protein content and bacterial inactivation. The results were compared to traditional pasteurization techniques used in the food industry. PEF results on whey protein content obtained in this study were comparable with those reported in previous publications [57, 58]. It has been reported that, while achieving significant bacterial inactivation by PEF, it has minimal to no effect on whey protein content, compared to control samples [57]. We were able to confirm this, as can be seen in Figures 5 and 7. However, a report published on how PEF affects the structural properties of whey protein isolates contradicts this opinion [59]. It has been reported that the PEF intensity and the number of pulses play a major role in the structural changes of whey protein isolates [59]. In this study, we did not test PEF's effect on the structural properties of WP, so we cannot draw any conclusions regarding this. It has been widely suggested that increasing the electric field strength and the number of pulses has a significant effect on bacterial inactivation [23, 57, 60, 61, 62]; we were able to achieve similar results in this study, as can be seen in Figures 3 and 4. The results in Table 1 show that the effect of PEF differs for individual species of bacteria. A more significant decrease in coliform numbers was achieved, and other bacteria might be more resistant to PEF because of the differences in bacterial morphology. It has been shown that bacterial shape, size, and cell wall thickness have significant effects on the efficiency of PEF [63]. Larger bacteria are more sensitive to PEF treatment because of the higher transmembrane potential induced during treatment. Moreover, bacteria with thicker cell walls are less sensitive to PEF-induced inactivation [63]. A wide variety of Gram-positive and Gram-negative bacterial species are found in raw milk, resulting in a different distribution between the total number of inactivated bacteria and coliforms [64]. Gram-negative bacteria seem to be more sensitive to electric pulses than Gram-positive bacteria. This difference most probably is related to differences in the composition and thickness of the bacterial cell wall [65, 66, 67]. Gram-negative bacteria have a thin layer of peptidoglycan and an outer membrane containing lipopolysaccharide, as opposed to Gram-positive. According to reports published previously, Gram-negative bacteria are more effective in performing electro-transformation than Gram-positive [68]. This is likely because of the bacterial cell envelope and cell wall, which reduces the amount of exogenous DNA entering the cell. According to articles published so far, bacterial inactivation by PEF occurs due to the breakdown of the cell membrane, affected by high-intensity electric fields [69, 70, 71, 72, 73]. Strong electric pulses permeate cells, causing damage to the cell membrane due to transmembrane potential. When this potential exceeds a critical value, the membrane becomes permeable to the outside medium [69, 70, 71, 72, 73]. The damage can be reversible or irreversible and depends on the electric field strength, duration, and number of pulses. Irreversible electroporation leads to damage to the cell membrane, which causes dielectric breakdown and the release of cell content [74, 75, 76, 77]. The thick peptidoglycan layer might protect cells from lysis by preventing cell leakage from the cell. It has been shown that bacteria can modify peptidoglycan backbones to acquire resistance against antimicrobials [78]. Gram-negative and Gram-positive bacteria, and even different strains within these categories, react differently to pH, temperature, PEF, and different combinations of the described parameters [79, 80].