Enzymatic Analysis of Sugars in Tomato

To determine the levels of glucose, fructose, and sucrose, an enzymatic method was used as described previously [40 (link)]. An amount of 25 mg of lyophilized tomato fruits was mixed with 500 µL 80% (v/v) aqueous ethanol and incubated in a thermomixer at 1000 rpm at 80 °C for 1 h, followed by cooling on ice and centrifugation at 14,000× g at 4 °C for 5 min. The supernatant was collected and transferred to a new tube for evaporation to dryness (60–78 °C). The residue was dissolved in 250 µL water and subsequently used for sugar analysis in three technical replicates. Reactions were conducted in a 96-well plate with reaction mixtures each containing 100 µL diluted supernatant (7%) and 0.1 U glucose-6-phosphate dehydrogenase (Merck, Darmstadt, Germany) diluted in 100 µL buffer (100 mM Imidazole/HCl, pH 6.92, 10 mM MgCl₂, 2 mM ATP, 4 mM NAD). The reaction was performed in a microplate reader (Sunrise) and recorded at 340 nm wavelength for 3 min. Subsequently, 0.1 U hexokinase (Merck) was added to determine glucose level. The contents of fructose and sucrose were determined in the same way using 0.2 U phosphoglucose isomerase (Merck) and 1 U invertase (Merck), respectively. Levels were calculated using glucose, fructose, and sucrose as standards [23 (link)].