2.2. In Vitro Gastrointestinal Digestion Procedure

In vitro digestion protocol (Figure 1) was performed according to the standardized method reported by Minekus et al. [15]. Of note, only gastric and intestinal phases were included. Preparation and composition of Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) electrolyte stock solutions strictly followed the procedure of Minekus et al. [15] and identical dilutions were achieved during in vitro digestion experiments. Adaptations regarding sample collection and handling options were, however, operated. All digestion experiments were performed in triplicate (n = 3).

Flow diagram of the simulated in vitro digestion procedure. AEVM: Anthocyanin enriched extract from Vaccinium myrtillus, SGF: Simulated Gastric Fluid, SIF: Simulated Intestinal Fluid.

Regarding gastric step, 5 mL of AEVM solution (10 mg/mL in distilled water) was mixed with 3 mL of SGF electrolyte stock solution and 1 mL of pepsin solution made up in SGF stock solution (20,000 U/mL). A final volume of 10 mL was obtained after addition of calcium chloride (0.075 mM in final gastric mixture), water, and pH adjustment to 3.0 with 1 M HCl. A two hours incubation at 37 °C was performed with constant shaking at 50 rpm in an orbital shaking incubator (NB-205 L, N-Biotek, Bucheon-si, South Korea). The obtained gastric mixture was divided in two equal parts, 5 mL was employed for intestinal digestion, and 5 mL was reserved for chemical and biological evaluations.

For intestinal phase, 5 mL of gastric mixture was mingled with 3 mL of SIF stock solution and 1 mL of pancreatin solution made up in SIF stock solution (1000 U/mL). A final volume of 10 mL was obtained after addition of calcium chloride (0.3 mM in final intestinal mixture), water, and pH adjustment to 7.0 using 0.1 M NaOH. Similarly to gastric phase, a 2 h incubation was performed using a shaking incubator (37 °C, 50 rpm).

Gastric samples were first diluted in distilled water (1/2) to normalize AEVM concentration among different samples. Gastric and intestinal samples were immediately deproteinized by adding 4 volumes of ethanol and were centrifugated at 4300 rpm for 15 min (Centrifuge 5804 R, Eppendorf, Montesson, France). Supernatants were then divided in aliquots of 1 mL, which were stored at −80 °C until further analysis. Undigested sample of AEVM was also prepared (2.5 mg/mL in distilled water) to serve as reference. It was submitted to equivalent deproteinization, centrifugation and conservation than digestive samples.