2.5.2. Reducing Sugars Concentration

The concentration of total reducing sugars was determined according to the Somogyi Nelson method [17–19], using glucose as a standard for calibration. Two hundred microliters of solids-free, properly diluted samples were mixed with 200 µL of the Somogyi reagent and incubated for 40 min at 100 ◦C. After cooling to room temperature, 200 µL of the Nelson reagent were added and incubated for 15 min. Then, 400 µL of acetone and 1 mL of water were added, and the absorbance was measured at 610 nm in a UV-vis spectrophotometer (Agilent Technologies Inc., Santa Clara, CA, USA).

2.5.3. High Performance Liquid Chromatography (HPLC) Analysis of Monosaccharides Composition

The composition of low molecular weight sugars (xylose and fructose) was determined via ion-exchange chromatography on an Agilent 1100 Series HPLC system (Agilent, Santa Clara, CA, USA) with a Diaspher-110-Amin column 5 µm 4 × 250 mm; the eluent was acetonitrile–water at 75:25 (*v*/*v*), the flow rate was 1 mL/min and the sample's volume was 10–100 µL. To prepare samples acetonitrile (0.8 mL) was added to 0.2 mL of hydrolysate, followed by centrifugation for 5 min at

9000× *g*. Solutions of xylose and fructose (Megazyme, Victoria, Australia) at the concentration of 1 g/L were used as standards.
