*2.3. Functional Properties Determination*

### 2.3.1. Proximate Composition of Fresh Pasta

Protein (total nitrogen × 5.7), ash, and lipids content were determined using the AOAC method 979.09, 923.03, and 945.38 F, respectively [39]. Total dietary fiber content was determined by enzymatic gravimetric as described by the AOAC Official Method 991.43. Moisture content was determined by a moisture analyzer (Mod. MAC 110/NP, Radwag Wagi Elektroniczne, Radom, Poland) at 120 ◦C. The carbohydrate content was determined as difference. The determinations were carried out in triplicate.

### 2.3.2. In Vitro Starch Hydrolysis

In vitro gastrointestinal digestion of fresh pasta and starch hydrolysis was determined according to Liljeberg et al. [40], simulating the in vivo digestion of starch. Briefly, aliquots of fresh pasta samples, cooked until the optimal cooking time and containing 1 g of starch (determined in cooked fresh pasta), were subjected to an enzymatic process (pancreatic amylase and pepsin-HCl), and the released glucose content was measured with D-fructose/D-glucose Assay Kit (Megazyme, Wicklow, Ireland). Simulated digests were dialyzed (cut-off of the membrane: 12,400 Da) for 180 min. Aliquots of dialysate, containing free glucose, and partially hydrolyzed starch were sampled every 30 min and further treated with amyloglucosidase. Then, free glucose was determined using the above-mentioned enzyme-based kit and finally converted into hydrolyzed (digested) starch in pasta. Control white wheat bread was used as the control to estimate the hydrolysis

index (HI = 100). The predicted glycemic index (pGI) was calculated using the equation pGI = 0.549 × HI + 39.71 [41]. Each sample was analyzed in triplicate.
