In vitro Simulated Digestion
The in vitro digestion model was structured in three stages: mouth, stomach, and intestinal tract, each involving specific simulated digestive fluids. The compositions of these fluids, namely simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF), were prepared in accordance with a previous study by El-Messery et al. (2020). The digestion experiments were conducted within a shaking water bath at 37°C.
In the simulated salivary stage, approximately 10 mL of samples were blended with an equal volume of SSF and gently stirred at 37°C and 120 rpm for 10 min.
Following salivary digestion, the simulated gastric stage involved the addition of 20 mL of SGF containing 3.2 mg/mL pepsin, 7 mL/L hydrochloric acid, and 2 g/L sodium chloride to the digestive fluid. The pH was adjusted to 2.5 using 0.1M HCl. The digestive fluid was then stirred at 37°C and 120 rpm for 2 h.
In the simulated intestinal stage, after gastric digestion, the digest was promptly adjusted to pH 7 using a 0.1 M NaOH solution. Subsequently, 2.5 mL of pancreatic lipase (4.8 mg/mL), 4 mL of bile salt solution (5 mg/mL), 1 mL of CaCl2 solution (750 mM), and 0.5 mL of glucosamine were added to the sample. The pH of the reaction system was monitored and maintained by titrating 0.1 M sodium hydroxide solution into the reaction vessel over a period of 2 h at 37°C.
Throughout the digestion process, the cumulative volume of added NaOH at different time points (20 min, 40 min, 60 min, 80 min, 100 min, and 120 min) was recorded, and the pH was maintained at 7.0. The corresponding release rate of free fatty acids (FFA) was calculated using the following equation:
\(\text{FFA}\left(\%\right)=\frac{VNaOH\times mNaOH\times Mlipid}{\text{Wlipid}}\times 100\%\)
where VNaOH represents the volume (L) of sodium hydroxide required to neutralize the FFAs produced, mNaOH is the molarity of the sodium hydroxide solution (0.1 M), Wlipid signifies the total weight of lipid initially present in the reaction vessel, and Mlipiddenotes the molecular weight of KO.