*3.2. Antioxidant Activity (DPPH and ABTS Method)*

Figures 3 and 4 report the results of antioxidant activity, obtained with DPPH and ABTS antiradical activity methods, of differently obtained carrot and apple juices, respectively.

**Figure 3.** Antioxidant activity (DPPH and ABTS method) of carrot juices obtained from pretreated mash. Different letters indicate significant differences (*p* < 0.05) between samples.

**Figure 4.** Antioxidant activity (DPPH and ABTS method) of apple juices obtained from pretreated mash. Different letters indicate significant differences (*p* < 0.05) between samples.

For carrot juice (Figure 3), a significantly higher antioxidant activity was obtained for carrot juices preheated to 80 ◦C with or without additional PEF or OH treatment with the ABTS method. Higher retention of bioactive compounds with DPPH method was observed for carrot mash pretreated at 80 ◦C coupled with PEF treatment. However, detected with DPPH method, the application of OH treatment reaching 40 and 60 ◦C reduced the antioxidant activity, in comparison with that of juice from the untreated control carrot mash. With ABTS method, no significant difference was found (Figure 3).

Significantly higher antioxidant activity, detected with both DPPH and ABTS methods, was obtained for apple juices preheated to 80 ◦C with or without additional PEF or OH treatment (Figure 4). Instead, OH reaching 40 ◦C and 60 ◦C reduced the antioxidant activity, with both used method, compared with that of the apple control juices, which might be due to the activation of degradative enzymes, such as peroxidase and polyphenoloxidase that lead to bioactive compounds' oxidative degradation.

Fruits and vegetables are good sources of natural antioxidants, containing carotenoids, vitamins, phenolic compounds, flavonoids, dietary glutathione and endogenous metabolites. However, the majority of the antioxidant activity of fruits and vegetables is derived from phenolic compounds (hydroxycinnamic acids, flavan-3-ols, anthocyanidins, flavonols and dihydrochalcones) rather than vitamin C and E, or β-carotene, due to their stronger activity against peroxil [27]. The peroxidase and polyphenoloxidase enzymes lead to the degradation of phenolic compounds and a subsequent loss of nutritional and sensorial values such as browning and off-flavour [28]. Moreover, heat treatment may influence the binding properties of bioactive compounds causing their higher release, but at the same time increase enzymatic or nonenzymatic degradation processes that can cause subsequent negative effects on quality of processed products [15].

In fact, higher temperature leads to the inactivation of the oxidative enzymes, thus reducing degradation effects and resulting in higher antioxidant activity in the juice. Moreover, additional effects other than those from thermal treatment need to be taken into account, since the electropermeabilization, induced by PEF and OH treatment, may contribute to the increased release of antioxidant compounds.

The detected difference between the two different methods used could be due to the fact that DPPH method is more sensitive to detect flavanones, while ABTS for the radical scavengers such as vitamin C [29].

For carrot and apple juice, PEF treatment without preheating did also not affect the extractability of bioactive compounds, which is in accordance with Shilling et al. [15], who reported no significant differences on total antioxidant activity between control and PEF-treated apple mash for different electric fields (1, 3 and 5 kV/cm).

#### *3.3. Enzyme Activity*

#### 3.3.1. POD Activity

Process pretreatment for the juice production is an important operation in order to improve the quality of the vegetable and fruit raw materials as well as to avoid the activation of degradative enzymes such as POD and PPO that consequently provoke pigments and nutrients loss [30].

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Peroxidase (POD) activity for differently obtained carrot juice is shown in Figure 5.

**Figure 5.** Peroxidase (POD) activity in carrot juice obtained from pretreated mash. Different letters indicate significant differences (*p* < 0.05) between samples.

This study revealed that the effect of only PEF treatment at 20 ◦C and 40 ◦C preheating could not reduce the activity of POD in both carrot (Figure 5) and apple juice [26], while for the samples preheated at 60◦C, the reduction of POD activity was observed.

The highest POD inactivation could be reached by preheating to 80 ◦C with and without additional PEF or OH treatment. All pretreatment conditions with temperature at or up to 60 ◦C and 40–80 ◦C by OH treatment led to a decrease in POD activity in the carrot juice, while for apple juices, a greater reduction of the activity (from 50% to 90%) was achieved by 60 and 80 ◦C preheating temperatures with and without additional PEF or OH application. Enzyme inactivation in the juice, after higher PEF treatment intensities, for microbial inactivation and preservation purposes, is mainly related to secondary effects such as local temperature distributions, electrochemical reactions or formation of free radicals, instead of primary effects of electric field. For the treatment of mash, the PEF treatment intensity can be considered 10-fold lower and having no direct effect on fruit and vegetable mash ingredients.

POD activity decreased with increasing temperature, and almost no POD activity was detected in juice extracted when the treatment temperature reached 80 ◦C, especially in apple juice samples. High temperature leads to an increase in the internal energy of the enzymes, thus consequently causes the break of bonds that determine the three-dimensional structure of enzymes [18].

Moreover, with the increasing of temperature, the enzyme activity decreased and required a particular temperature–time combination for complete inactivation. Inadequate temperature led to a decrease in the enzyme activity time rather than complete inactivation, which may cause browning effect. In fact, Bhat et al. [18] reported similar results for OH-treated bottle gourd juice, where the temperature of 60 and 70 ◦C seemed to be not enough for complete enzyme inactivation, which instead was observed at 80 ◦C for 4 min.

Icier et al. [31] showed that OH treatment could be used for POD inactivation on pea puree at the range of 30–50 V/cm combined with the water blanching. Elez-Martínez et al. [32] reported a completely POD deactivation in orange juice after the application of PEF treatment at 35 kV/cm for 1500 μs.

Moreover, the variations of colour for carrot and apple juice pretreated with 80 ◦C with both PEF and OH applications could be explained by the decrease of enzyme activity, in fact, a correlation between colour and POD activity was found to be 0.8902 and 0.5166, respectively, for carrot and apple juice.

#### 3.3.2. PPO Activity

Polyphenoloxidase (PPO) activity of differently obtained apple juice samples is shown in Figure 6.

**Figure 6.** Polyphenoloxidase (PPO) activity of apple juice obtained from pretreated mash. Different letters indicate significant differences (*p* < 0.05) between samples.

PPO is an oxidoreductase enzyme which catalyses the oxidation of phenolic compounds in o-quinones, which are subsequently polymerized into brown pigments [33]. Heating treatment seems to be the most effective applied treatment for the stabilization of food products against microbial and enzyme activity. Nevertheless, thermal treatment has been shown to cause negative effects on quality and related nutritional compounds [9]. The mechanism of enzyme inactivation is not completely clear. Current results show empirical proof of protein modification by electrical fields [34] that may provoke a deformation or structural change of a protein, due to the interaction between the external electric field and the functional groups of the protein that allow its unfolding [32].

PPO activity was significantly decreased by PEF application at room temperature compared with that of the untreated control sample. In addition, a greater inactivation was achieved when the treatment temperature reached 80 ◦C, as well as with OH treatment and just preheating. Moreover, PPO inactivation was even more effective when a combination of temperatures and PEF or OH applications were used.

Similar results were reported by Turk et al. [35]. PPO activity was reduced in apple cider mash pretreated with PEF at 1 kV/cm for 100 μs. The loss of PPO activity could be explained also by the inhibition of the enzyme with oxidised phenolic compounds, in particular procyanidins [36].

Previous work reported similar results for PPO deactivation; the residual PPO activity was 35% after 14 min with OH treatment at 70 ◦C, by applying 35 V/cm in grape juice [30].

Liang et al. [37] found a significant decrease (33%) in PPO activity in freshly squeezed apple juice when preheated at 50 ◦C and treated with PEF at 27 kV/cm for 58.7 μs.

Saxena et al. [20] found a reduction of PPO activity up to 97.8 % by applying 32 V/cm with OH treatment at 90 ◦C for 5 min in sugarcane juice. Moreover, a greater increase in residual PPO activity was visible at 90 ◦C by increasing the holding times of OH treatment (5, 10, 15 and 20 min). The increase of the enzyme activity with the holding time at constant temperature was attributed to the pulsating OH treatment that may cause biochemical reactions by changing the molecular spacing and may result in a better interaction between substrate and enzyme [38]. A recent review on the impact of electric fields on enzymes is provided by [39].

#### **4. Conclusions**

Obtained results emphasize the role of thermal treatment for the inactivation of enzymes, reflected by improved colour values for juices exposed to 80 ◦C, independent of the PEF or OH application. The inactivation of POD and PPO was more pronounced when a temperature of 80 ◦C was achieved for both carrot and apple mash (around 90%).

However, a better retention of plant secondary metabolites from carrot and apple mashes could be achieved by additional PEF or OH application. PEF treatment was found to improve the release of such compounds, whereas OH contributed to a very fast volumetric heating that reduced the overall thermal load that the sample was exposed to. Based on the results, a combination of thermal and electric field pretreatments is required for the controlled release, inactivation and retention of ingredients. Thermal effects contributing to the colour, bioactive compounds retention and enzyme inactivation were found to be still important when applying nonthermal cell disintegration techniques such as PEF. However, both electrotechnologies, PEF and OH were found to positively contribute to improved juice quality by enhanced ingredient release and retention.

**Author Contributions:** C.M., T.F. and H.J. conceived and designed the experiments; C.M. and K.R. performed the experiments; C.M., K.R. and U.T. analysed the data; H.J. contributed reagents/ materials/ analysis tools; S.R. as Supervisor; C.M. contributed Writing-Original Draft Preparation; C.M., T.F., U.T., S.R., M.D.R. and H.J. contributed Writing-Review and Editing.

**Funding:** This work was partly supported by the Austrian Research Promotion Agency (FFG Project No. 859077).

**Acknowledgments:** Part of the equipment used in this study was financed by EQ-BOKU VIBT GmbH and belongs to the Center for Preservation and Aseptic Processing. Cinzia Mannozzi acknowledges the Marco Polo Programme (University of Bologna) for the travel grant.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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