Simulated In Vitro Gastrointestinal Digestion

All samples were digested using a static in vitro digestion system consisting of a simulated oral phase, gastric phase, and intestinal phase, with modifications.^24,25 The compositions (%, w/w) of the simulated salivary fluid (SSF), simulated gastric fluid (SGF, pH 3.0 ± 0.05), and simulated intestinal fluid (SIF, pH 7.0 ± 0.05) were as reported.²⁴

For the oral phase, for 5 mL samples (fucoxanthin content of 100 μg), 3.5 mL of SSF stock solution (37 °C), 25 μL of CaCl₂ (0.3 M), and 1.475 mL of Milli-Q water (Veolia water, Veolia Water Solutions and Technologies Netherlands B.V.) were added and mixed. For the experiment of oxidation prevention, 0.02 and 0.04% BHT were added.

Subsequently, to start the gastric phase, 5 mL of oral bolus was mixed with 3.75 mL of SGF (37 °C). Then, 0.8 mL of porcine pepsin stock solutions of 25 000 units/mL (2000 units/mL in final chyme) in SGF (37 °C) was added, followed by 2.5 μL of CaCl₂ (0.3 M). Last, about 0.1 mL of HCl (1 M) was added to reach pH 3. Finally, 0.349 mL of Milli-Q water was added to obtain the final volume (10 mL). Then, the chyme was shaken at 37 °C for 2 h without light. All reactors were filled with nitrogen when closed before starting the digestion. In the experiments at different gastric pH, pH was adjusted to 4 and 5 before starting the gastric phase with HCl.

At the end of the gastric digestion step, about 0.075 mL of NaOH (1 M) was added to reach pH 7.0, inhibiting the gastric enzyme activity. Thereafter, to 10 mL of gastric chyme, 5.5 mL of SIF (37 °C), 2.5 mL of porcine pancreatin (trypsin activity of 800 units/mL and lipase activity of around 280 units/mg) in SIF (37 °C), 1.25 mL of fresh bile stock solutions (10 mM in the final intestinal chyme), 20 μL of CaCl₂ (0.3 M), and 0.655 mL of Milli-Q water were added. Finally, the chyme was incubated in a shaking water bath for 2 h at 37 °C, and the digestion was stopped by cooling immediately in an ice bath. To study the kinetics of fucoxanthin micellarization, samples were collected at 0, 10, 20, 30, 60, and 120 min, respectively. For the inhibition of lipase activity, 10 μg/mL orlistat was added to pancreatin dissolved in SSF and the mixture was incubated at 37 °C for 30 min before undertaking the simulated pancreatic digestion.²⁶ In this experiment, samples were only collected after 120 min. Also, for the experiment with BHT, samples were collected at the end of intestinal digestion. All intestinal digests were centrifuged for 10 min at 20000g (4 °C), and the supernatants were collected and filtered through a 0.22 μm membrane filter (Phenomenex, Netherlands). Fucoxanthin in the filtered supernatants was supposed to have been incorporated into the micellar phase. All tubes used here were filled with nitrogen before starting the digestion procedure, and all procedures were conducted under red light.