*2.4. Analytical Procedures*

The moisture contents of HS and AS were assayed according to the T-264-cm-97 standard method [29]. The T-249-cm-85 method [30] was employed for measuring the contents of structural carbohydrates and Klason lignin. This method consisted of a two step quantitative acid hydrolysis performed with 72% and 4% H2SO4, respectively. The method led to an insoluble lignin residue (Klason lignin) and to a liquid phase. Samples from the liquid phase were filtered through 0.45 µm cellulose acetate membranes and analyzed by High Performance Liquid Chromatography (HPLC) using an Agilent 1200 series instrument (Agilent Technologies, Santa Clara, CA, USA), fitted with a refraction index detector (RID). Samples were assayed for monosaccharides (glucose, xylose, and arabinose), organic acids (acetic acid), and furans (furfural and hydroxymethylfurfural) using a 300 × 7.8 Aminex HPX-87H column (BioRad Life Science Group, Hercules, CA, USA) kept at 50 ◦C and eluted with 0.003 N H2SO<sup>4</sup> at a 0.6 mL·min−<sup>1</sup> flow rate. The ash content was assayed according to the T-211-om-02 method [31].

Samples of AL were filtered through 0.45 µm cellulose acetate membranes and assayed by HPLC as described above. Aliquots of AL were subjected to quantitative posthydrolysis (4% of H2SO<sup>4</sup> at 121 ◦C for 20 min). The increases in the concentrations of monosaccharides and acetic acid caused by posthydrolysis provided the measure of oligomers concentration and their degree of substitution with acetyl groups. The content of total nonvolatile compounds (NVC) was measured by oven-drying samples at 105 ◦C until constant weight. All the analytical determinations were performed in triplicate.

Uronic acids were determined spectrophotometrically at 520 nm, using galacturonic acid as a standard [32].

High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAED–PAD) was used to assess the types of oligomers present in the reaction media. The analyses were performed using an ICS300 instrument (Dionex, Sunnyvale, CA, USA) equipped with a 250 × 2 mm CarboPac PA-1 column in combination with a 25 × 2 mm CarboPac PA guard column [33]. Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry (MALDI TOF–MS) analyses were employed to allow a detailed structural characterization of the oligomers contained in AL. Assays were performed using an Autoflex III smartbeam instrument (Bruker Daltonics, Bremen, Germany) operating in linear positive ion mode. Spectra were acquired and treated using the Flex Control 3.0 and Flex-Analysis 3.0 software (Bruker Daltonics), respectively [34]. Commercial XOS (degree of polymerization, DP 2-6) from Megazyme (Wicklow, Ireland) were used as standards for HPAED–PAD and MALDI TOF–MS analyses.

The total phenolic content (TPC) was determined using the Folin–Ciocalteu assay [35], and the results are expressed as Gallic Acid Equivalents (GAE). The determination of antioxidant activities was carried out using the 2,2-diphenyl-1-picrylhydrazyl assay ((DPPH) [36], Trolox-Equivalent Antioxidant Capacity assay (TEAC) [37], and Ferric Reducing Ability of Plasma assay (FRAP) [38] methods. Identification of phenolic compounds was carried out by High Performance Liquid Chromatography with Diode Array Detection (HPLC–DAD) [39].
