Determination of uronic acid content

The content of uronic acids was determined with the biphenol method [59] with the prior hydrolysis of polysaccharides with sulfuric acid [60]. Lyophilized samples (around 10 mg) were dissolved in 0.1 ml of concentrated sulfuric acid in a cooling bath with shaking for 5 min. Then, 0.1 ml of sulfuric acid, 0.05 ml of water, 0.05 ml of water and 0.7 ml of water were added with shaking between each portion. After the centrifugation (10 min, 2000 x g, RT), 0.1 ml of the supernatant was taken, and 10 μl of 4 M aminosulfonic acid of pH 1.6 was added and 600 μl of 75 mM sodium tetraborate in concentrated sulfuric acid was added. After careful mixing, the samples were incubated for 20 min at 100 °C, cooled down and 20 μl of m-hydroxybiphenyl (0.15 %) in 0.5 % NaOH was added, and they were left at room temperature for 10 min. The content of uronic acids was measured spectrophotometrically at 525 nm. As a standard for the calibration curve, glucuronic acid was used.