Dx 500 hpaec pad system

Hydrolyzed monosaccharides were generated by trifluoroacetic acid and H₂SO₄ hydrolysis (Shiga et al., 2009 (link)). The supernatants obtained were analyzed for neutral sugars and uronic acids by HPAEC-PAD according to methods of Shiga et al. (2009) (link). Briefly, 1 mg of polysaccharides obtained by extractions (WSF and OSF) was hydrolyzed with 1 mL of 2 M TFA at 120°C for 60 min in a screw-capped conical vial and centrifuged (2000 × g, 5 min, 25°C). Supernatants were transferred to new vials, dried under an N₂ stream, and separated for analysis. The same procedure was applied for ASF and IF, but the precipitates that resulted from TFA hydrolysis (the cellulose-rich residues) were dried under an N₂ stream and rehydrolyzed with 0.9 mL of 2 M H₂SO₄ at 120°C for 90 min. After hydrolysis, supernatants were neutralized with 0.1 mL of 50% NaOH (w/w) and analyzed in a DX 500 HPAEC-PAD system (Dionex, Sunnyvale, CA, USA). Neutral sugars (L-arabinose, D-galactose, D-glucose, D-fucose, D-mannose, L-rhamnose, and D-xylose) and uronic acids (D-glucuronic and D-galacturonic acid) were used as standards (Sigma; St. Louis, MO, USA).

The low molecular weight peaks from WSF at 1 and 5 DAH were separated by ultrafiltration using Millipore Amicon Ultra-4 centrifugal filter units (MWCO 30 kDa). The oligosaccharide profiles were analyzed using a DX 500 HPAEC-PAD system (Dionex, Sunnyvalle, CA, USA) as described by Jonathan et al. (2012) (link). Briefly, samples were diluted in 500 μL of water, injected (25 μL) and their profiles were analyzed in a CarboPac PA-1 column (2 mm × 250 mm) (Dionex). Oligomers derived from neutral sugars were eluted (0.3 mL/min) with a linear gradient of 0.02–0.05 M NaOH for 3 min and 0.05–0.075 M NaOH for 10 min, followed by isocratic elution of 0.1 M NaOH for 2 min. Oligomers derived from uronic acids were then eluted with a gradient of 0–1 M NaOAc in 0.1 M NaOH for 50 min. Finally, the column was washed with 1 M NaOAc in 0.1 M NaOH for 7 min followed by 0.1 M NaOH for 3 min. Equilibration was done by eluting 0.02 M NaOH for 20 min. Standards sugars (L-arabinose, D-galactose, D-glucose, D-fucose, D-mannose, L-rhamnose, D-xylose, D-galacturonic acid, and digalacturonic acid) and oligosaccharides (maltotriose, maltopentaose, maltohexaose, and trigalacturonic acid) were used as external standards (Sigma).