2.7. In vitro lipolysis study

In vitro lipolysis was carried out according to our previously published method (Yu et al., 2012). In short, a lipolysis buffer was prepared with Tris maleate, sodium chloride, calcium chloride dihydrate, sodium taurodeoxycholate, and phosphatidylcholine in concentrations of 50, 150, 5, 20, and 5 mM, respectively. Pancreatin was freshly prepared for each study by mixing 1 g of pancreatin powder with 5 mL lipolysis buffer and centrifuging at 2000 rpm for 10 min; the supernatant was collected and stored on ice. To begin the lipolysis study, a sample containing 250 mg oil phase was mixed with 9 mL of fed state buffer in the glass reaction vessel, which was put in an oil bath with temperature maintained at 37 ± 1 °C. The mixture was stirred at 200 rpm for 10 min and its pH was monitored by a pH meter. Before 1 mL of ice-chilled pancreatin was added to initiate the digestion, the pH of the mixture was adjusted to 7.50 ± 0.02 by adding 0.2 M sodium hydroxide solution. This temperature and pH were maintained during the lipolysis study to neutralize the free fatty acids (FFA) released from the lipid digestion. The volume of sodium hydroxide added at each time point was recorded for later analysis. The percentage of FFA released was calculated using eq. (1):

V_NaOH is the volume of titrant in liters, m_NaOH is the molarity of sodium hydroxide, Mw_lipid is the apparent molecular weight of MCT oil obtained from eq. (2), and W_lipid is the weight of oil in the digestion system in grams. Molecular weight of the triglycerides was estimated using the saponification value (SV) of the MCT oil (97% caprylic triglycerides), i.e. 384.

Immediately upon completion of the lipolysis study, the resulting lipolysis solutions were subjected to ultracentrifugation (Type 60 Ti rotor, Beckman Coulter) for 40 min at 40,000 rpm. After ultracentrifugation, the digestion medium was separated into an opaque sediment phase, an aqueous phase containing formulated PMFs micelles in the middle, and an oil phase at the top. The micelle phase was collected using a syringe and the volume was recorded. For HPLC analysis, 200 μL of lipolysis supernatant sample (0.22 μm filtered) was mixed with 400 μL of DMSO. The concentration of PMFs in the micelles was analyzed using HPLC. The bioaccessibility (%) of PMFs was calculated using eq. (3):