Alberto Zavarise

β-Glucosidases (βGLs) play important roles in plant secondary metabolism by hydrolyzing glycosylated compounds involved in defense, signaling, and the formation of bioactive and flavor-related metabolites. In this study, a β-glucosidase from Fragaria × ananassa (Fra-βGL; FxaC_10g48920.t1) was identified and biochemically characterized in comparison with a commercial β-glucosidase from Prunus dulcis (Pru-βGL). The Fra-βGL cDNA was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified for biochemical characterization. Substrate screening demonstrated that Fra-βGL hydrolyzed the 7-O-glucosides of quercetin, kaempferol, and luteolin, whereas no detectable activity was observed toward the tested 3-O-glucosides, anthocyanin glucosides, arbutin, or phloridzin under the assay conditions. Kinetic analysis revealed higher catalytic efficiencies of Fra-βGL than Pru-βGL toward all four substrates examined. Fra-βGL showed its highest turnover rate and catalytic efficiency toward the synthetic substrate p-nitrophenyl-β-D-glucopyranoside (pNP-Glc), with a K_cat of 60.33 s⁻¹ and a K_cat/K_m of 10.18 mM⁻¹·s⁻¹. Among the naturally occurring flavonoid glucosides, Fra-βGL exhibited the highest catalytic efficiency toward quercetin 7-O-glucoside (4.14 mM⁻¹·s⁻¹), approximately fivefold higher than that of Pru-βGL (0.824 mM⁻¹·s⁻¹). Thermal stability analysis showed progressive loss of activity with increasing temperature, with Pru-βGL retaining greater residual activity than Fra-βGL at elevated temperatures. Molecular docking suggested structural features that may contribute to the observed preference of Fra-βGL for flavonoid 7-O-glucosides over the corresponding 3-O-glucosides. Collectively, these findings demonstrate the substrate selectivity and distinct catalytic properties of Fra-βGL and identify Fra-βGL as a candidate for further investigation in the context of flavonoid glycoside metabolism in strawberry.

Pulsed electric field (PEF) and high-pressure processing (HPP) are non-thermal treatments, developed to ensure preservation of food products whilst maintaining taste and valuable nutrients. In this study, we investigated their potential for the inactivation of 3 commercial exogenous pectinases (polygalacturonase, pectin transeliminase, pectin esterase) commonly used in juice processing for clarification of juices. The inactivation of these enzymes after processing is mandatory by European law. Clear apple juice was treated with both non-thermal processing methods, as well as with thermal pasteurization as the standard method. For HPP, 3 pressures (250, 450, and 600 MPa) and different holding times (from 2 to 12 min) were tested. For PEF, 3 electric field intensities (10, 13, and 15 kV/cm) and different specific energy values (from 121 to 417 kJ/kg). Standard thermal pasteurization resulted in a complete inactivation of all tested pectinases. HPP treatment only showed marginal effects on polygalacturonase and pectin transeliminase at the highest pressure and holding times, which are beyond levels used in industrial settings. For PEF, dependence upon high electric field strength and specific energy values was evident; however, here too, the effect was only moderate at the levels attainable within the scope of this study. Assuming a continued linear relationship, usable results could be achieved in an industrial setting, albeit under more extreme conditions.

The browning of fruit juices and nectars is a common issue in the beverage industry and is a particular problem in strawberry nectars, where it significantly reduces the shelf-life. Polyphenol oxidases (PPOs), which are multicopper enzymes responsible for the oxidation of a wide plethora of polyphenols in plants, have been widely assumed to be involved in the enzymatic browning of strawberry nectar. To investigate the possible involvement of PPOs, the substrate specificity of four recombinant PPOs and their gene expression pattern in 10 cultivars of Fragaria × ananassa at five ripening stages were determined. This allowed us to obtain adequate amounts of enzymes to study them independently and without interfering matrix effects. All four PPOs possess monophenolase activity, which was particularly high for PPO4. PPO3 did not show sufficient stability for the kinetic studies. The other three showed a high preference for the flavan 3-ol catechin with a 2-fold higher catalytic efficiency compared to dopamine for PPO1 and PPO2. At a neutral pH, they also showed activity with cyanidin but not with pelargonidin, which is the prevalent anthocyanidin type in strawberry. The enzymes showed a high affinity but only low turnover rates for the dihydrochalcone phloretin, resulting in an inhibitory effect that was strong enough to extend the shelf-life of the strawberry nectar by one week if phloretin was added in high concentrations (600 µM). PPO1 and PPO2 were prevalently expressed in all fruit stages. The gene expression of the four PPOs did not correlate with the color stability of the nectars of the 10 varieties and also showed a random expression pattern during fruit development. The limited activity in acidic conditions and the low substrate specificity for pelargonidin does not point to a crucial role for PPOs in the browning of strawberry nectar, but the high catalytic efficiency with catechin as a substrate could contribute to anthocyanin degradation via mechanisms such as copolymerization.