*2.3. Extraction of Glucans*

The employed extraction procedure was a conventional method involving two stages to sequentially remove starch and proteins from the powder, namely an aqueous alkali extraction at pH 10 and 55 ◦C and acidic precipitation, respectively [12,18–20].

Preliminary treatments of this method involve sequential extractions with three solvents in order of increasing polarity, to remove lipids and soluble material such as vitamins, polyphenols, monosaccharides, disaccharides, and others [21]. The flour (3 g) was first extracted with acetone (3 × 30 mL each), then with methanol (3 × 30 mL each) and finally with 70% aqueous ethanol (3 × 30 mL each). All the extractions were performed at room temperature (t = 2 h). For starch precipitation, residue solid was added to a solution of 20%, *w*/*v* sodium carbonate in distilled water (pH 10, 30 mL), and the obtained suspension was warmed at 55 ◦C in a water bath with stirring for 30 min and then was centrifuged (Model J2–21, Beckman Instrument Co., Mississauga, ON, Canada) at 5000× *g* for 30 min. The solid was removed, and the pH of the supernatant was added to HCl 2 M until a pH of 4.5 was reached (isoelectric point of proteins), and centrifuged at 5000× *g* for 30 min to separate proteins [22,23], which were discarded. An equal volume of absolute ethanol was added to the supernatant in order to precipitate glucans. After 12 h at 4 ◦C, the solution was centrifuged at 5000× *g* for 30 min. The precipitate was resuspended in ethanol, filtered, rinsed with ethanol and freeze-dried. The extractions were performed in triplicate on three samples of husk powder from each agroclimate locality and the results were expressed as mean ± standard deviation. The crude glucans were measured and characterized by FT-IR, SEM and DSC.
