## 2 Results and Discussion
### Microbial Analysis  
The microbial stability of PEFJ during refrigerated storage (4 \({}^{\circ}\)C) is shown in Figure 6. At T0, immediately after the process, our results show the great potentiality of the PEF process in achieving microbial inactivation, more evidently in the counts of mesophilic viable bacteria on plate count agar (PCA) solid plates and spoilage microorganisms on orange serum agar (OSA) solid plates, as well as in the count of yeasts and molds on sabouraud dextrose agar (SAB) solid plates. Indeed, the initial microbial load of freshly squeezed blood orange juice was 3.18 \(\pm\) 0.06 Log CFU/mL in PCA, 2.85 \(\pm\) 0.09 Log CFU/mL in SAB, and 3.28 \(\pm\) 0.01 Log CFU/mL in OSA, before treatment (UJ). The PEF process was able to significantly inactivate microbial growth, as at T0 there were no culturable organisms present in the juice for PCA and OSA plates, while yeasts and molds reached a microbial load equal to 1.70 \(\pm\) 0.09 Log CFU/mL. This kind of microbial inactivation induced by the PEF process is similar to levels reported in previous findings by [25; 37], who reported microbial counts below 1 Log CFU/mL in PEF-treated orange juice processed at 40 kV/cm for 97 us and 35 kV/cm for 1000 us, respectively. Moreover, the lower effect of PEF on the inactivation of yeasts and molds with respect to bacteria was also previously reported in another study [37]. PEF technology has been proposed as a promising food disinfection method based on its ability to induce irreversible cell membrane electro-permeabilization, which leads to the death of the bacteria [12]. It has been demonstrated, based on the use of advanced SEM, that after PEF exposure of vegetative bacteria, alterations in the cell shape and the presence of cell debris occur, affecting cell wall integrity and inducing plasma membrane permeabilization [12]. Indeed, the authors explored the influence of the electric field strength on the inactivation of vegetative bacteria by applying 1000 pulses of 5 us from 2 to 7.5 kV/cm. They found that between 2 and 6 kV/cm, the inactivation is due to reversible permeabilization by electroporation, and the inactivation induces the irreversible permeabilization of the cell wall only for higher electric fields. Based on this, it was presumable that in our PEF process, applied at an energy density equal to 180 kJ/Kg, electroporation may be reversible. Thus, in order to unequivocally determine the commercial refrigerated shelf-life of our PEF-treated blood orange juice, we carried out samplings at 5-day intervals during refrigerated storage at 4 \({}^{\circ}\)C. This temperature was chosen to mimic refrigerated storage during retail and household storage. As expected, as storage continued, microbial counts began to increase. The microbial value of 6 Log CFU/mL was \begin{table}
\begin{tabular}{c c c} \hline \hline  & **Total Polyphenols Content** & **ORAC Units** \\  & **(mg GAE */L)** & **(\(\mu\)mol TE/100 mL)** \\ \hline PEFJ-0 days & 867.77 \(\pm\) 0.63 AB & 3398.87 \(\pm\) 31.71 \\ PEFJ-5 days & 862.28 \(\pm\) 0.85 B & 3974.88 \(\pm\) 140.97 \\ PEFJ-10 days & 872.99 \(\pm\) 1.12 A & 3772.46 \(\pm\) 54.57 \\ PEFJ-15 days & 828.30 \(\pm\) 0.27 D & 3503.03 \(\pm\) 309.02 \\ PEFJ-20 days & 836.16 \(\pm\) 0.80 C & 3735.00 \(\pm\) 23.06 \\ \hline \hline \end{tabular} \({}^{\star}\)GAE: Gallic acid equivalents.  
\end{table} 
Table 3: Total polyphenol content (A) and ORAC units (B) in PEFJ (PEF-treated juice) during further refrigerated storage at 4 \({}^{\circ}\)C. Data are expressed as means of three analytical replicates \(\pm\) standard deviation. Means in the same column followed by different letters are significantly different: \(p\leq\) 0.01—capital letter; \(n=3\). considered unacceptable for juice quality [45], and it was almost reached after 20 days of refrigerated storage. As a matter of fact, microbial load of PEFJ at 20 days was 5.93 \(\pm\) 0.10 Log CFU/mL in PCA, 6.07 \(\pm\) 0.02 Log CFU/mL in SAB, and 6.04 \(\pm\) 0.01 Log CFU/mL in OSA.