Aacc method 76

Starch content was evaluated by the enzymatic assay Total Starch AOAC Method 996.1 1 and AACC Method 76.13 (Megazyme ®, Ireland). Briefly, 50 mg of extract were suspended in 200 μl of ethanol 80% v/v and 1 ml of 2 M KOH. Samples were magnetically stirred for 20 min at 4°C. Then, 4 ml of sodium acetate pH=3.8 were added, followed by the addition of 50 μl of α-amylase (8300 U/mL) and 50 μl of amyloglucosidase (AMG, 3300 U/ml). Samples were incubated for 30 min with intermittent mixing on a vortex mixer, then centrifuged for 10 min at 3000 rpm to recover the supernatant. In order to evaluate total starch content, a reaction mixture was prepared as follows in a quartz cell: 1 ml H₂O, 25 μl of sample, 50 μl of a buffer solution pH = 7.6, 50 μl NADP+/ATP. The solution was incubated for 3 min at room temperature and then the absorbance was read at 340 nm against the blank. Then, 10 μl of a solution containing hexokinase (HK) and glucose-6-phosphate-dehydrogenase (G6PDH) was added. After an incubation of 5 min at room temperature, the absorbance was read against the blank again at 340 nm. Data are expressed as g of starch per 100 g of extract.

The enzyme assay was performed using commercial enzymes adopted from the total assay procedure method (AOAC Method 996.11/ AACC Method 76.13) from Megazyme (Megazyme Inc., Bray, Ireland). Thermostable α-amylase (3,000 U/mL) was diluted in MOPS buffer (Sigma-Aldrich, St. Louis, MO) at pH 7.0 followed by an incubation in C₂H₃NaO₂ buffer (200 mM) at pH 4.5 and amyloglucosidase concentrate (3,300 U/mL). Total starch content was determined by the enzymatic hydrolysis of 100 mg of fine powder from each sampled stem core. The samples were hydrolysed using 3 mL of thermostable α-amylase to extract maltodextrin from starch at 100 °C over 6 min (vortex at 2, 4 and 6 min intervals). The slurry samples were incubated at 50 °C in a water bath with 4 mL of C₂H₃NaO₂ buffer, followed by 0.1 mL of amyloglucosidase for 30 min to hydrolyse maltodextrin to glucose. The amount of glucose present was measured using an UV-VIS spectrophotometer at 510 nm (Perkin Elmer Lamda 35 UV/VIS Spectrophotometer, Perkin Elmer, MA, USA).