###### Abstract  
Polyphenol oxidase (PPO) is the key enzyme responsible for enzymatic browning. To extend the shelf life of shredded lettuce, knowledge about biochemical PPO properties is required. The characterization of the enzyme from shredded, cold-stored lettuce was performed using pyrotectend and the endogenous substrate (ES) (lettuce phenolics). The optimum pH and temperature for PPO activity were 5 and 50 \({}^{\circ}\)C, respectively. Natural infusions used as the PPO inhibitors (IC\({}_{50}\)) were ranked as follows: lavage (0.09%), majoram (0.13%), orange peel (0.14%), oregano (0.15%), basil (0.22%), lemon peel (0.24%), parsley leaves (0.58%), and wheat bran (1.06%). Among well-recognized PPO inhibitors, kojic acid (0.00043%), ascorbic acid (0.00053%), and L-cysteine (0.00085%) were the most effective. Among the metal ions, MgCl\({}_{2}\), FeCl\({}_{2}\), and CaCl\({}_{2}\) at 0.5 mM inhibited the PPO activity most effectively (by 28%, 27%, and 21%, respectively). The substrate used (pyrocatechol/ES) significantly influenced the enzyme inhibition. Using pyrocatechol, the lovage extract acted in a mixed mode (K\({}_{\text{mi}}\) = 27.8 mM, V\({}_{\text{maci}}\) = 2.03 MU), while the ES acted according to the non-competitive mode (K\({}_{\text{mi}}\)= 0.57 mg GAE/mL, V\({}_{\text{max}}\) = 0.0046 U). The study confirms that natural extracts are more effective than L-cysteine when the ES is used. A pre-storage treatment with an infusion may be potentially used to improve the quality of shredded lettuce. polyphenol oxidase; iceberg lettuce; storage; enzyme inhibition; natural plant extracts; endogenous substrate; shelf life +
Footnote †: journal: Applied sciences  
are used to extend the shelf life of low-processed food. Among the physical methods, the most noteworthy are blanching, pulsed electric field, high hydrostatic pressure, cold plasma technology, and radiation technologies. Unfortunately, despite the numerous advantages of these methods, they have certain limitations; namely, they may negatively affect the texture of the final product and cause significant losses of vitamins. Due to the above limitations, they can be applied to a limited group of products, such as liquid products (e.g., juices and smoothes); however, they are not recommended for preserving low-processed food. Chemical methods limiting enzymatic browning include treatments with reducing agents, chelating agents, and acidulants. Even though these compounds preserve the natural properties of low-processed food and are relatively inexpensive, in recent years, more and more producers have consciously stopped using them. Furthermore, a significant drawback of these agents is the lack of biodegradability (e.g., sulfites) [6; 7]. Acidulants, such as ascorbic acid and citric acid, decrease the pH below the optimal value for enzyme activity. Moreover, our previous studies have shown that the acidification of low-processed vegetables could stimulate the growth of molds and yeasts [8]. Finally, enzyme inhibitors, such as kojic acid or EDTA, are successfully applied, but, recently, they have consequently been eliminated by consumers more aware of their effect on health and the natural environment. In light of that, new technologies or tools, especially those of natural origin, are wanted. The quality of low-processed products can be preserved or enhanced by the application of natural plant extracts [9; 10]. This approach also has additional advantages; instead of inhibiting the enzymatic browning process, it may improve the pro-health properties, maintain a high microbiological purity, or simultaneously fortify with desirable components (e.g., phenolics and microelements). To use this new technology consciously, it is important to approach the subject from several key perspectives. In the beginning, a detailed characterization of enzymes involved in the generation of undesirable changes is required. Of course, special attention should be placed here on the inhibitory profile and substrate specificity.