2.3. In Vitro Gastrointestinal Digestion

The in vitro gastrointestinal digestions consisted of enzymatic digestion, including three sequential steps: oral, gastric, and intestinal. For the simulated digestion, the standardized static COST INFOGEST-based SGD protocol was followed [22].

Stock solutions, simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared in the same molarity as reported in Table 1. All solutions were prepared daily and pre-warmed at 37 °C before use. Additionally, salivary α-amylase (oral phase) was prepared in SSF to a final concentration of 75 U/mL, pepsin (gastric phase) in SGF to a final concentration of 2000 U/mL, as well as pancreatin (small intestine phase) in SIF to a final concentration of 100 U/mL (based on trypsin activity). Bile salts (10 mM) were prepared in SIF.

Composition of simulated digestive fluids used for the in vitro digestion.

* Ca²⁺ concentration in the final digestion mixture. ** Sufficient pancreatin to provide 100 U/mL of trypsin (TAME Units).

Equal amounts of five dry-cured loins of the same batch were minced using a Premax manual mincer. Dry-cured loin samples (5 g) were sequentially incubated for 2 min with 5 mL SSF (pH 7.0), 120 min with 10 mL SGF (pH 3.0), and 120 min with 20 mL SIF (pH 7.0).

In the oral phase, 5 g of dry-cured loin were transferred to a 50 mL beaker and mixed thoroughly with 4 mL SSF solution, 0.5 mL of salivary α-amylase solution, 25 μL of 0.3 mol/L CaCl2 and 475 μL of distilled water. Then, the obtained mixture was incubated in a water bath for 2 min at 37 °C. Gastric digestion was continued by immediate addition of 8 mL of SGF, 5 μL of 0.3 mol/L CaCl₂, and 1.03 mL of distilled water to the oral bolus, and pH was adjusted to 3.0 with 0.47 mL volume of 6 M HCl. Next, 0.5 mL of porcine pepsin was added and continuously kept under shaking (120 rpm) at 37 °C for 2 h. Then, intestinal digestion was followed by the addition of 8.5 mL of SIF, 40 μL of 0.3 M CaCl₂ and 2.5 mL of bile salts (25 mg/mL) to the mixture. After adjusting the pH to 7 with 2.7 mL of 1 M NaOH, 5 mL of a pancreatin solution and 1.26 mL distilled water were added and kept under agitation (120 rpm) at 37 °C for 2 h. Blank samples containing only simulated fluids were prepared. Enzymatic incubations were performed in quintuplicate.