## 2 PPO Effect on Fresh-Cut Produce and Its Mechanism  
As is known, browning of fresh fruit and vegetables reduces quality and is often the limiting factor regarding their shelf-life [8]. Enzymatic browning requires the presence of four different components: oxygen, an oxidizing enzyme, cooper and a suitable substrate such as phenols that are found mostly in vacuoles and apoplast/cell wall compartments [10]. Therefore, browning should be avoided by removing one of these components. The most important oxidizing enzyme is PPO, but there are others such as lipooxidase, which catalyzes peroxidation reactions, phenol peroxidase (EC 1.11.1.7 or POD), which contribute to enzymatic browning oxidizing hydrogen donors in the presence of hydrogen peroxide or phenylalanine ammonia lyase (PAL), that transforms the amino acid L-phenylalanine to trans-cinnamic acid [11; 12]. Thus, phenol compounds synthesized by PAL might be oxidized by PPO. The latter catalyzes two reactions: a) hydroxylation of monophcnols to o-diphenylnols (monophenolase or cresolase activity) and b) oxidation of o-diphenylnols to o-quinones (diphenylnolase or catecholase activity). The hydroxylation reaction is kinetically slower than the oxidation reaction. As a result, quinones are obtained as products. Due to their subsequent reactions (which are non-enzymatic), melanin accumulation takes place, and fruits and vegetables show brown, black or red pigments (depending on the polyphenic substrate) related to browning [11; 12] (see Figure 3).  
Figure 2: References related to: “PPO” and “PPO + fresh-cut” [7].  
In detail, PPO is an enzyme that contains two cooper atoms in its active center (see Figure 4). Depending on its enzymatic form transition (met, oxi or deoxi), PPO might have catecholase or mono-phenolase activity.  
On the other hand, an additional discoloration effect was found for some fruit tissues, such as in the case of strawberries, probably due to deterioration of anticonaines pigments [13]. During minimal processing, tissue integrity and the compartmentalization of cells are damaged, and all browning components are put in contact (that is, phenols and enzymes such as PPO or POD and oxygen). Thus, another way to retain browning is keeping compartmentalization in vegetable and fruit tissues. In addition, some authors have pointed out the possibility of synergistic effects between PPO and other enzymes such as POD [14].  
Due to the fact that PPO mainly affects browning, it should be taken into consideration how color parameter evolution is during browning. Indeed, many authors have pointed out a close relationship between PPO activity and browning, concluding that PPO activity could be considered as a good browning indicator [15; 16; 17; 18; 19]. On the other hand, some authors have not found relevant correlation between these aspects, explaining that PPO  
Figure 4: Detailed mechanism for polyphenol oxidase activity.  
Figure 3: Main degradation steps of phenolic compounds.  
activity differences could affect browning to a lesser extent, with browning evolution being negligible during storage for human eye [20; 21]. In addition, other authors have not found any correlation between phenolic compound content and browning [21]. Thus, taking into account the CIELAB color space, an increase in browning is related to L* decrease and a* increase, and subsequently the hue angle (h*) usually shows a diminution as well [15; 22; 23]. Consequently, this color decay might imply a reject by consumers when purchasing minimally processed products, reducing their shelf-life considerably.