2.8.3. Gastrointestinal Digestion Experimental Procedure

Lipid digestion kinetics were monitored for a total of 90 min (30 min gastric step followed by 60 min intestinal step). A Metrohm 902 Titrando pH-stat titration apparatus equipped with a Dosino 800 dosing apparatus (Herisau, Switzerland) was used for the studies. The experimental procedure was initiated with the addition of an amount of formulation corresponding to 3 mg of FEN into a temperature-controlled reaction vessel containing 10 mL of freshly prepared FaSSGF (37 ◦C, pH 1.6). Gastric lipolysis was carried out for 30 min and was initiated by the addition of 100 µL *Candida* lipase (600 tributyrin units lipase activity). Following gastric lipolysis, 18 mL of FaSSIF media was added (pH 6.5) to the reaction vessel, and the pH was automatically adjusted by the pH-stat apparatus to maintain pH at 6.5 ± 0.01. Following a pH-equilibration time of 3–5 min, intestinal lipolysis was initiated by the addition of 2 mL freshly prepared pancreatin extract, bringing the total volume to 30 mL. Fatty acids (FA) produced as a result of digestion were instantly titrated with 0.6 M NaOH to maintain a constant pH of 6.50 ± 0.01. The cumulative volume of NaOH was converted to the number of moles of NaOH consumed, which in turn corresponded to the number of moles of FA released. Data obtained from NaOH titration were used to compare the rate and extent of lipid digestion for the different formulations. It is important to note that NaOH titration during the gastric step does not reflect extent of lipolysis since the FA produced are protonated at pH 1.6 and could therefore not be recorded during gastric lipolysis [37].
