Hpaec pad ics 5000 system

Monosaccharides were identified as reduced and acetylated derivatives (alditol acetates) by gas chromatography coupled to mass spectrometry (GC-MS) as described previously (34 (link)). The absolute configuration of the monosaccharides was determined by GC-MS analysis of acetylated (R)-2-butylglycoside derivatives as described previously (34 (link)). The products were analyzed by GC-MS and identified with standards prepared from monosaccharides with known configurations with (R)- and (S)-2-butanol. GC-MS was performed with a Trace GC ULTRA system (Thermo Scientific) equipped with an NMTR-5MS capillary column (30 m by 0.25 mm) by using a temperature gradient of 170°C (3 min) to 250°C at 5°C/min with a DSQ II MS detector.
The amount of CWPS extracted by HF from cell walls was determined by quantification of monosaccharides after acid hydrolysis with 4 M trifluoroacetic acid (TFA) for 3 h at 110°C. Monosaccharides were separated on a CarboPac PA20 column (Dionex) by HPAEC-PAD (ICS 5000 system; Thermo Fisher Scientific). Quantification was performed by comparison with standard amounts of each monosaccharide.

Starch was enzymatically determined as described previously (Cordenunsi and Lajolo, 1995 (link)). Briefly, frozen and grinded banana pulp was soaked in 0.5 M NaOH. After neutralization with acetic acid, the starch was precipitated with 80% ethanol, separated by centrifugation, and hydrolyzed with amylase (Megazyme, Wicklow, Ireland) and amyloglucosidase (Megazyme). The resulting glucose was quantified enzymatically using the glucose oxidase/peroxidase system. Soluble sugars were analyzed by high-performance anion exchange chromatography coupled with a pulse amperometric detector (HPAEC-PAD) (Shiga et al., 2011 (link)). Briefly, frozen and grinded banana pulp was extracted three times with 80% ethanol at 80°C. The ethanol was evaporated using a vacuum concentrator (Savant CS2 10A, Thermo, Waltham, MA, United States). Then, the residue was reconstituted in water, filtered, and analyzed by HPAEC-PAD ICS5000 system (Thermo) equipped with a CarboPac PA1 column (4 × 250 mm, Thermo) using 18 mM NaOH as the mobile phase. Analyses were performed in triplicate and results were expressed as g/100 g of fruit.