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.