Gopod kit

The GI was determined in the brans, first, measuring the content of available starch using the total starch assay kit of Megazyme (Bray, Ireland). Then, the in vitro starch hydrolysis rate was determined as described by Gularte and Rosell [51 (link)]. Glucose analysis was performed using a GOPOD kit (Megazyme, Bray, Ireland). Hydrolysis index (HI) and glycemic index (GI) values were calculated as proposed by Granfeldt et al. [52 (link)].

The product profiles of the various GTFB preparations were investigated by incubating enzyme in sodium acetate buffer (25 mM, pH 4.7, 1 mM CaCl₂) at 37 °C for 24 h, with maltose, maltotriose and maltoheptaose separately as substrates [2 (link)]. After 24 h or 72 h reactions, all tubes were boiled for 5 min followed by 15,000 × g centrifugation for 5 min. The supernatants were kept for high-pH anion-exchange chromatography (HPAEC) analysis as described below.
GTFB hydrolysis rates were assessed by measuring glucose release. GTFB enzyme solutions (50 μl) with different protein concentrations were added to a final 500 μl reaction system with 10 mM maltoheptaose. At a time interval of 5 min, 50 μl samples were taken and the reaction terminated by addition of 25 μl 0.4 M NaOH, followed by neutralization with 25 μl 0.4 M HCl. The GOPOD kit (Megazyme) was used to detect glucose, as a measure for hydrolysis activity [6 (link)]. One unit of hydrolysis activity was defined as the amount of enzyme that produces 1 μmol glucose per min. Transferase activity was measured by determining maltose generation when GTFB enzyme was incubated with amylose (0.25 %, w/v) as donor and glucose (10 mM) as acceptor substrate. One unit of transferase activity was defined as the amount of enzyme that produces 1 μmol maltose per min.

Determination of GI was performed by a two-step’s procedure. Firstly, the content of total starch was quantified using the total starch assay kit of Megazyme, Bray, Ireland (K-TSTA 08/16). Then, the in vitro starch hydrolysis rate was determined according to Gularte and Rosell [33 (link)], with slight modifications. Samples containing 50 mg of available starch were dissolved in tris-maleate buffer (0.1 M, pH = 6, 2 mL) and combined with 2 mL of an enzymatic solution containing porcine pancreatic α-amylase (460 U mL⁻¹) and amyloglucosidase (6.6 U mL⁻¹). Aliquots were taken during the incubation period (150 min) and immersed in boiling water for 5 min to inactivate enzymatic activity present in the aliquots. After cooling the sample in ice, 150 µL of absolute ethanol was added and the sample was centrifuged (4 °C, 10,000× g for 5 min). The pellet was washed with 150 µL ethanol:water (1:1, v/v) and the supernatants were pooled together and stored at 4 °C for the subsequent colorimetric analysis of reducing sugars using the GOPOD kit (Megazyme, Bray, Ireland). Expected GI values were calculated from hydrolysis index (HI) values, as proposed by Granfeldt [34 ].

The GI in WB was determined by, first, measuring the content of available starch using the total starch assay kit of Megazyme (K-TSTA 08/16). Then, the in vitro starch hydrolysis rate was determined as described by Gularte and Rosell [29 (link)]. Glucose analysis was performed using a GOPOD kit (Megazyme, Bray, Ireland). Hydrolysis index (HI) and glycemic index (GI) values were calculated as proposed by Granfeldt [30 (link)].

For the evaluation of glycaemic index (GI) in non-extruded and extruded samples, first, the content of available starch was measured using the total starch assay kit of Megazyme (K-TSTA). Afterwards, the in vitro starch hydrolysis rate was measured as described by Gularte and Rosell [47 ], with some modifications. Samples with 50 mg of available starch were dissolved in tris-maleate buffer (0.1 M, pH = 6, 2 mL) and then 2 mL enzymatic solution containing porcine pancreatic α-amylase (460 U mL⁻¹) and amyloglucosidase (6.6 U mL⁻¹) was added. Aliquots were incubated for 150 min, and then were kept in boiling water for 5 min to stop the enzymatic reaction and cooled in ice. Then, a volume of 150 µL of absolute ethanol was added and the sample was centrifuged (4 °C, 10,000× g for 5 min). The pellet was washed with 150 µL ethanol:water (1:1, v:v) and the supernatants were pooled together and stored at 4 °C for the following colorimetric analysis of reducing sugars using the GOPOD kit (Megazyme, Bray, Ireland). GI values were expressed from hydrolysis index (HI) values, as proposed by Granfeldt [48 ].

Before determination of glycemic index (GI), available starch of the samples was first evaluated using a total starch assay kit of Megazyme (K-TSTA 08/16). Afterwards, the in vitro starch hydrolysis rate was determined as described by Gularte and Rosell [102 (link)], with slight modifications. The samples containing 50 mg of available starch were dissolved in 2 mL Tris-maleate buffer (0.1 M, pH = 6) and then, 2 mL of enzymatic solution containing porcine pancreatic amylase (460 U/mL) and amyloglucosidase (6.6 U mL⁻¹) were added. Aliquots were taken during the incubation period (150 min) and immediately placed in boiling water for 5 min to stop the enzymatic reaction, and then cooled on ice. Then, a volume of 150 µL of absolute ethanol was added and the sample was centrifuged (4 °C, 10.000× g for 5 min). The pellet was washed with 150 µL of ethanol:water (1:1, v/v) and the supernatants were pooled and stored at 4 °C for the subsequent colorimetric analysis of glucose, using a GOPOD kit (Megazyme, Bray, Ireland). Hydrolysis index (HI) and glycemic index (GI) values were calculated following formulas proposed by Grunfeld [103 ].