Enzymatic Browning in Sugar Beet Leaves (<i>Beta vulgaris</i> L.): Influence of Caffeic Acid Derivatives, Oxidative Coupling, and Coupled Oxidation

Sugar beet (<i>Beta vulgaris</i> L.) leaves of 8 month (8<sub>m</sub>) plants showed more enzymatic browning than those of 3 month (3<sub>m</sub>). Total phenolic content increased from 4.6 to 9.4 mg/g FW in 3<sub>m</sub> and 8<sub>m</sub>, respectively, quantitated by reverse-phase-ultrahigh-performance liquid chromatography–ultraviolet-mass spectrometry (RP-UHPLC–UV-MS). The PPO activity was 6.7 times higher in extracts from 8<sub>m</sub> than from 3<sub>m</sub> leaves. Substrate content increased from 0.53 to 2.45 mg/g FW in 3<sub>m</sub> and 8<sub>m</sub>, respectively, of which caffeic acid glycosyl esters were most important, increasing 10-fold with age. Caffeic acid glycosides and vitexin derivatives were no substrates. In 3<sub>m</sub> and 8<sub>m,</sub> nonsubstrate-to-substrate ratios were 8:1 and 3:1, respectively. A model system showed browning at 3:1 ratio due to formation of products with extensive conjugated systems through oxidative coupling and coupled oxidation. The 8:1 ratio did not turn brown as oxidative coupling occurred without much coupled oxidation. We postulate that differences in nonsubstrate-to-substrate ratio and therewith extent of coupled oxidation explain browning.