## 4 Discussion  
PEF processing can result in different levels of structural damage in heterogeneous material such as plant tissue. [PERSON] and [PERSON] (2018) demonstrated that the distribution of permeabilized cells was non-homogenous across a one-cell-thick layer of abaxial onion epidermal tissue if treated with PEF at an electric field strength below the critical value (\(<\)0.33 kV/cm). While it has been well evidenced that irreversible cell damage of onion epidermal tissues usually occurs at an electric field strength greater  
Figure 6: Total ion chromatogram of the headspace of the sodium phosphate buffer (0.01 M, pH 7) following 6 h of steeping with a PEF-treated onion bulb, obtained using solid-phase microextraction gas chromatography–mass spectrometry (SPME-GC-MS). Target peaks of interest are identified and labelled. Note that 1-propanethiol co-eluted with the broad air peak (\(\approx\)3.2–5.2 min), quantified with an extracted ion (\(m\)/\(z\) 76). Figure 7: Extracted ion peak area of dipropyl disulfide (DPDS) (\(m\)/\(z\) 150) detected in the headspace of the sodium phosphate buffer over the course of 6 h of steeping with untreated and PEF-treated onion buls. Bars represent mean \(\pm\) standard error (\(n=3\)). Significant differences (\(p<0.05\)) between the means are indicated with different letters. than 0.33 kV/cm [9; 15], these major structural changes may not be the case for all onion cells across the fleshy scale layers when PEF was used to treat a whole, intact bulb. In this study, validation of PEF efficiency to achieve irreversible and uniform disruption of cellular tissues was achieved using a combination of simple, yet reliable measurements, namely cell viability staining using tetrazolium salt and microscopic evaluation using cryo-SEM. Based on the findings, to achieve uniform and high levels of cellular damage and complete structural changes, PEF conditions should be designed to employ a higher electric field strength (1.2 kV/cm) at fewer pulses, to result in low energy input (6 kJ/kg), and using an onion orientation with the root end of the bulb facing to the high voltage electrode. The purplish red-colored parts in the dried coats and outer parts of the fleshy scale-leaves of the bulbs of the red onion are attributed to the anthocyanin pigments such as cyanidin-3-glucoside, cyanidin-3-laminaribioside, cyanidin-3-malonyl-glucose, and cyanidin-3-malonyl-laminaribioside [21]. In the present study, changes in the onion anthocyanins due to PEF were not measured directly; however, the results clearly demonstrated that PEF-treated onions had reduced the purplish-red colored pigments distributed in the outermost fleshy scale (Figure 3), indicating that PEF led to a loss of anthocyanins, possibly due to leaching or degradation. Changes in the color and stability of anthocyanins have been known to be affected by pH, temperature, light, oxygen, metal ions, and chemical association with other compounds [22]. However, the actual mechanism to explain whether PEF has direct involvement in causing the chemical degradation or instability of anthocyanins in onion cellular tissues is not the focus of the present study. Anthocyanins are water-soluble pigments localized in the vacuole of the plant cell and protected by a membrane known as tonoplast. [PERSON] and others [10] have demonstrated that PEF treatment on onion tissues at a low field strength (0.07 kV/cm) can cause initial membrane breakdown at the cell membrane, while an electric field strength beyond 0.2 kV/cm could also lead to tonoplast membrane breakdown. Therefore, a possible explanation for the loss of purplish-red pigments in the onion cells could be that the PEF conditions under investigation were able to permeabilized the tonoplast of the onion cell. As a result, leaching of anthocyanins from the vacuole to the intra- or extra-cellular spaces would take place while inducing massive mixing of the cell contents at the vacuole, the cytoplasm, and the cellular spaces that completely reduced anthocyanins to their colorless form following PEF.