*2.4. α- and β-Glucan Content*

β-glucan content of husks was determined in triplicate using β-glucan assay kit from Megazyme Ltd., Bray, Co. Wicklow, Ireland [17]. The dry sample (100 mg) was weighed into a 25 mL flask and 12 M ice-cold sulphuric acid solution (2 mL) was added. The solution was vortexed and left to incubate for 2 h in an ice-water bath, and then it was diluted with distilled water (12 mL) and left for 2 h in a boiling-water bath (T = 100 ◦C). After cooling the temperature, 10 M KOH solution (6 mL) and 200 mM sodium acetate buffer (pH 5) were added. After centrifugation (1500× *g*, t = 10 min), an aliquot (0.1 mL) of the supernatant was mixed with 0.1 mL of a mixture of *exo*-1,3-β-glucanase (20 U/mL) plus β-glucosidase (4 U/mL) at 40 ◦C for 60 min. Finally, the content of glucose in the solutions was determined by incubating the solution with glucose-oxidase/peroxidase (GOPOD, 3.0 mL) at 40 ◦C for 20 min.

For obtaining total glucan content, the solution was analysed by the spectrophotometer at λ = 510 nm (Model UV-vis, JASCO, V-550) against the blank and the D-glucose standard solution (1 mg/mL), incubated with GOPOD reagent. The blank was prepared by adding

0.2 mL of 200 mM sodium acetate buffer at pH 5.0 to 3 mL of GOPOD. For obtaining α-glucan content, the husk flour (0.1 g) was added with 2 M KOH (2 mL), 1.2 M sodium acetate buffer (pH 3.8, 8 mL) and incubated with 0.2 mL of amyloglucosidase plus invertase for 30 min at 40 ◦C. After centrifugation (1500× *g*, t = 10 min), the supernatant (0.1 mL) was mixed with sodium acetate buffer (200 mM, pH 5.0, 0.1 mL) and GOPOD (3 mL). The difference between total and α-glucan contents gave the β-glucan content.
