2.2.4. Total Polyphenol Content and Ferulic Acid Determinations

Simulated In Vitro Oral-Gastric-Duodenal Digestion Process of BSG Extract

The simulated in vitro oral-gastric-duodenal digestion of BSG extract was performed following the protocol by Minekus et al. with some modifications [27]. The used reagents are reported below: potassium chloride (KCl), dihydrogen potassium phosphate (KH2PO4), sodium carbonate (NaHCO3), magnesium chloride (MgCl2), ammonium carbonate (NH4)CO3, calcium chloride (CaCl2), sodium chloride (NaCl), hydrochloric acid (HCl), and sodium hydroxide (NaOH). All were provided by Carlo Erba (Milan, Italy). Pancreatin from porcine pancreas (extract of pig bile), α-amylase from *Bacillus licheniformis*, pepsin from porcine gastric mucosa and porcine bile extract, formic acid solution (1 M), water, methanol, and acetonitrile LC–MS grade were sourced from Sigma-Aldrich, Merck KGaA (Milan, Italy).

In brief, 5 g of the BSG extract were dissolved in 3.5 mL of previously prepared simulated salivary fluid (SSF). Then, 0.5 mL (1500 U/mL) of fresh α-amylase solution were added to both samples. In the end, water was added to reach a final volume of 10 mL and the sample was incubated for 2 min at 37 ◦C. The bolus obtained in the previous phase was mixed with 7.5 mL of simulated gastric fluid (SGF) and 1.6 mL (25,000 U/mL) of fresh pepsin; the pH was then adjusted to 2.00 ± 0.02 using 1 M HCl. The sample was brought up to 20 mL volume and the mixture was incubated at 37 ◦C for 2 h in a shaking water bath. Subsequently, gastric chyme was incubated with 5 mL of fresh pancreatin (800 U/mL) and 2.5 mL of fresh bile mixture (160 mM) to reach a final volume of 32.5 mL. The sample was finally made up to a 40 mL final volume; the pH was adjusted to 7.00 ± 0.02 using 1 M NaOH and incubated at 37 ◦C for 2 h. At the end of the digestion process, the oralgastric-duodenal digested sample was freeze dried and maintained at 4 ◦C pending total polyphenol content determination and RP-HUPLC-MS analysis.
