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.

Among fruits, the apple is unique for producing large amounts of the dihydrochalcone phloridzin, which, together with phloretin, its aglycone, is valuable to the pharmaceutical and food industries for its antidiabetic, antioxidant, and anticarcinogenic properties, as well as its use as a sweetener. We analysed the phloridzin concentration, total phenolic content, and antioxidant activity in the peel, flesh, seeds, juice, and pomace of 13 international and local apple varieties. In the unprocessed fruit, the seeds had the highest phloridzin content, while the highest total phenolic contents were mostly found in the peel. In processed samples, phloridzin and the total phenolic compounds especially were higher mostly in juice than in pomace. Moreover, the total phenolic content was much higher than the phloridzin content. Juice showed the highest antioxidant activity, followed by the peel and flesh. Across all samples, antioxidant activity did not directly correlate with phloridzin concentrations, suggesting that the antioxidant activity ascribed to phloridzin may need re-evaluation. In the Ferric Reducing Antioxidant Power (FRAP) assay, phloridzin only showed antioxidant activity at high concentrations when compared to its aglycone, phloretin. Considering the large amounts of apple juice produced by the juice industry, residual pomace is a promising source of phloridzin. For technical use, processing this phloridzin to phloretin would be advantageous.

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.