2.3.3. Prebiotic Activity Assay

To evaluate the prebiotic activity of AR inulin-enriched fresh pasta, samples were previously subjected to in vitro gastrointestinal digestion according to the method used by Kamiloglu and Capanoglu [42], with slight modifications. The in vitro gastrointestinal digestion was performed, comprising of a pepsin-HCl digestion for 3 h at 37 ◦C (to simulate gastric digestion) and pancreatin digestion with pancreatin and bile salts for 3 h at 37 ◦C (to simulate small intestinal digestion). As reported by Caponio et al. [43], 10 mL of each fresh pasta extract was added to α-amylase (56 mg/mL) (Sigma-Aldrich Chemistry, St. Louis, MO, USA) and to 10 mL of pepsin solution composed of NaCl 125 mM/L + KCl 7 mM/L + NaHCO3 45 mM/L + pepsin (Sigma-Aldrich Chemistry, St. Louis, MO, USA) at 3 g/L. Then, the pH was adjusted to 2 using HCl and incubated at 37 ◦C for 180 min in a water bath under shaking. After incubation, an aliquot of the gastric-digested extract was added in equal volume to an intestinal solution. The intestinal solution was simulated by dissolving 0.1 g/100 mL of pancreatin (Sigma-Aldrich Chemistry, St. Louis, MO, USA) and 0.15 g/100 mL bile salts (Oxoid™, Hampshire, UK). The pH was adjusted to 8 using NaOH and incubated at 37 ◦C for 180 min in a water bath under shaking. After incubation, an aliquot of intestinal-digested extract was ultra-filtrated with 3000 Da membrane (Vivaspin 20, Sartorius, Goettingen, Germany) to eliminate free carbohydrates. The retentate fractions were diluted in water and then filtered using 0.45 μm Whatman filter paper and further analyzed for prebiotics activities, as follows.

Twenty-two probiotic strains of probiotics and one strain of *Escherichia coli* (*E. coli*) available in the culture collection of the Department of Plant, Soil, Food Science of University of Bari, Italy were used to carry out the experiments in fecal batches. The fecal medium (FM) was constituted as previously described [44] without the addition of glucose. This was labelled as FM (absence of carbohydrates), FMPC (FM + pasta not containing inulin), FMP5 (FM + pasta with 5% of inulin), FMP10 (FM + pasta with 10% of inulin), FMP15 (FM + pasta with 15% of inulin). For those fecal samples containing pasta, this was added in a ratio of 1:5 (*w/v*) in media after cooking and digestion was simulated as previously described. Viable probiotics and *E. coli* were inoculated in fecal media at a cell density of 7 UFC/mL (log10), measured through OD at 620 nm. Inoculated batches were incubated in anaerobic conditions for 36 h at 37 ◦C, under slight stirring (150 rpm). After the incubation, plate counts for lactic acid bacteria and *E. coli* were respectively made in De Man, Rogosa, and Sharpe agar (MRS) and Violet Red Bile Glucose agar (VRBGA). Both agar media were purchased from Oxoid Ltd. (Basingstoke, Hampshire, England, UK). Probiotic growth was also profiled in terms of ΔpH, as the difference between final (36 h) and initial (pH 7.0 ± 0.02) values of pH.
