3.9. In Vitro Multi-Step Digestion Model

The in vitro digestion degree of lipids was assessed using a modified method described by Versantvoort et al. [41] and Chang et al. [18] (Table 7). All simulated digestive juices were prepared and used on the day of the experiment, and their compositions are presented in Table 7. The completely melted lipid (100 mg) and saliva juice (1.2 mL) were mixed in an Erlenmeyer flask in a shaking water bath for 5 min at 37 °C and 80 rpm, and gastric juice (2.4 mL) was added and allowed to react for 2 h. NaHCO₃ solution (0.4 mL), bile juice (1.2 mL), and duodenal juice (2.4 mL) were added, and the mixture was hydrolyzed for 30, 60, and 120 min. After the reaction, a lipase inhibitor (100 μL, 0.2 g of 4-bromophenylboronic acid/mL in methanol) was added to stop digestion [42]. For the extraction of hydrolyzed lipids, n-hexane was added, mixed, and centrifuged at 1763× g for 5 min, and the supernatant was collected (repeated three times). Next, 1 N HCl (0.5 mL) was mixed with the remaining lower part for 1 min, then n-hexane (10 mL) was mixed and centrifuged, and the supernatant was collected (repeated three times). The all n-hexane extraction was combined, and then the solvent was removed by using N₂. Next, 10 mL of ethanol: n-hexane (1:1, v/v) and 1 mL of 1% phenolphthalein in ethanol were added and titrated with 0.05 N KOH solution. The in vitro digestion degree of each lipid was expressed as the released FFA (%) using Equation (2) [43]:

The composition of synthetic juices for the in vitro multi-step digestion model ⁽¹⁾.

⁽¹⁾ Versantvoort et al. [41]; Chang et al. [18]. ⁽²⁾ Mean ± SD.