Carbopac pa1 biolc

Individual sugar concentrations were determined by HPLC (Thermo Scientific 5000+ HPLC‐PAD system; Thermo Fisher Scientific Inc Waltham, Massachusetts, USA) using a multistep gradient. A flow rate of 0.3 ml min⁻¹ was used on a CarboPac PA1 column (Guard column: Dionex CarboPac PA1 BioLC 2 × 50 mm and main column: Dionex CarboPac PA1 BioLC 2 × 250 mm). The column was equilibrated before injection with a pre flow of 18 mM sodium hydroxide (NaOH). During a total running time of 50 min, the following solutions were used: A, water; B, 100 mM NaOH; C, 100 mM NaOH with 1 M sodium acetate. During the first 20 min, 18% of B was applied, followed by a 10 min linear gradient to 40% C and 0% B, and 100% C for 5 min. To rinse the acetate, 100% B was used for 5 min, and 10 min of 18% B was used to rinse the column. The quantification was performed based on external standard calibration. Reference sugars (Sigma‐Aldrich, Zwijndrecht, Netherlands) were used in a concentration range from 2.5 to 200 μM. The data obtained are the results of two independent biological replicates and for each replicate three technical replicates were assayed.