*3.9. In Vitro Glycemic Index (GI)*

The in vitro GI of the biscuits was determined by evaluation of the in vitro starch digestibility according to the method described by Reis and Abu-Ghannam [55] with slight modifications, using the digestion procedure described in Section 3.7. Aliquots of 1 mL of the hydrolyzed solution were taken at 10, 20, 30, 60, 90, 120, and 180 min intervals, respectively. They were mixed with 4 mL of absolute ethanol to deactivate the enzymes. The glucose content of the hydrolysates was determined using the GOPOD method. The values were expressed as mg glucose/g sample. Glucose content was plotted as a function of time and the areas under the hydrolysis curves (AUC) were calculated. The hydrolysis index (HI) for each sample was calculated as the ratio between the AUC of the sample and the AUC for the reference food (white bread). The HI values were normalized for the total carbohydrate available

in each sample and reference and expressed as percentages. The GI was predicted according to the equation described by Goñi, Garcia-Alonso, and Saura-Calixto [56]:

$$\text{GI} \, (\%) \, = \, \text{39.71} + \, 0.549 \times \text{HI} \, \tag{6}$$
