*3.4. In Vitro Digestion Process*

The in vitro digestion was conducted in a glass bioreactor equipped with 4 inlets, allowing the introduction of the pH electrode, programming of the active acidity, dosage of biochemical agents, and appropriate media as well as collection of analytical samples. Samples for the in vitro digestion process were prepared by taking 23 g of products and dissolving them in demineralized water to a volume of 230 mL. During the process, the total polyphenol content, antioxidation potential, and *β*-glucuronidase activity were determined.

The bioreactor was thermally stable and the reactions were carried out at 37 ◦C. The conditions of the process in the bioreactor were designed in such a way as to comprise the following stages of the model: the 'stomach', the 'small intestine', and the 'large intestine' (Figure 3). The parameters of the digestion process were selected on the basis of our previous investigations [27,30]. *Stomach stage*: the digestion process was started by simulating digestion in the stomach. In this process, 60,000 U pepsin (Sigma-Aldrich, St. Louis, MO, USA) suspended in 2 mL 0.1 M HCl was added to the sample. The pH was then adjusted to 2.0 and the digestion continued for 4 h. *Small intestine stage*: after 4 h of digestion, the pH was adjusted to 6.0 by the addition of 1 M NaHCO3, followed by the addition of 10 mL of a pancreatic-intestinal extract composed of 0.02 g of pancreatic extract (Sigma-Aldrich, St. Louis, MO, USA) and 0.12 g of bile salt (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 10 mL 0.1 M NaHCO3. The pH was then adjusted to 7.4 with 1 M NaHCO3, and human intestinal microflora prepared according to the method described by Knarreborg et al. [85] was added at a total count of about 10<sup>6</sup> cfu/mL. Intestinal digestion was carried out for 2 h. *Large intestine stage*: after the digestion process in the small intestine, the pH was adjusted to 8.0 by adding 2 M NaHCO<sup>3</sup> and the fermentation was continued for another 18 h. A nitrogen stream was passed through the bioreactor to ensure anaerobic conditions

**Figure 3.** Gastrointestinal tract model.
