Uronic acid determination

The total uronic acid was quantified according to Filisetti-Cozzi and Carpita (1991). Five mg of each de-starched cell wall were weighed and 2 mL of concentrated sulfuric acid were added. The reactions were incubated for 10 min on ice under stirring (1,250 rpm), followed by addition of 1 mL of deionized water. This procedure was repeated once. The incubated mixtures were diluted to 10 mL and centrifuged at 4,000 g for 10 min at room temperature. Forty μL of 4 M sulfamic acid/potassium sulfamate solution (pH 1.6) and 2.4 mL of 75 mM sodium borate in sulfuric acid was added to aliquots of supernatant (400 μL). The homogenized solutions were incubated at a 100°C for 20 min, then cooled on ice for 10 min. Eighty microliter of m-hydroxybiphenyl in 0.5% NaOH were added and vortexed for color development. The samples were read at 525 nm in Spectrophotometer Genesys 10S UV-VIS ThermoScientific®. A standard curve using D-galacturonic acid was performed in the concentration range of 5–40 μL/400 μL.