**Said Toro-Uribe 1, Jhair Godoy-Chivatá 1, Arley René Villamizar-Jaimes 2, María de Jesús Perea-Flores <sup>3</sup> and Luis J. López-Giraldo 1,\***


Received: 1 March 2020; Accepted: 28 April 2020; Published: 27 May 2020

**Abstract:** A full factorial design (ascorbic acid/l-cysteine inhibitors, temperature, and time as factors) study was conducted to enhance inhibition of polyphenol oxidase (PPO) activity without decreasing cocoa polyphenol concentrations. The data obtained were modelled through a new equation, represented by Γ, which correlates both high polyphenol content with reduced specific PPO activity. At optimized values (70 mM inhibitory solution at 96 ◦C for 6.4 min, Γ = 11.6), 93.3% PPO inhibition and total polyphenol of 94.9 mg GAE/g were obtained. In addition, microscopy images confirmed the cell morphological changes measured as the fractal dimension and explained the possible cell lysis and denaturation as a result of heat treatment and chemical inhibitors. Results also showed that PPO enzyme was most suitable (higher *v*max/*Km* ratio) for catechol, with a reduction in its affinity of 13.7-fold after the inhibition heat treatment. Overall, this work proposed a suitable and food-safe procedure for obtaining enriched polyphenol extract with low enzyme activity.

**Keywords:** polyphenol oxidase; cocoa polyphenols; heat treatment; enzyme inactivation
