Astaxanthin, DHA and EPA Content and Bioaccessibility
The content of astaxanthin, DHA, and EPA was individually determined following salivary, gastric, and intestinal digestion, employing the methods described previously. After in vitro digestion, a portion of the digesta was subjected to centrifugation at 8000 rpm/min at 25°C for 30 min. The resulting clear middle layer, formed after centrifugation, was assumed to contain the micellar fraction that solubilized astaxanthin, DHA, and EPA. This micellar phase was extracted using the wet oil extraction method and prepared for HPLC analysis.
The actual content of astaxanthin, DHA, and EPA was calculated based on their respective standard curves. Subsequently, the bioaccessibility was determined. The effective bioaccessibility (%) of astaxanthin, DHA, and EPA was calculated using the following formula: Astaxanthin/DHA/EPA bioaccessibility (%) = 100 × Cmicelle / Cinitial, where Cmicelle represents the concentration of astaxanthin, DHA, and EPA in the micellar fraction, while Cinitial represents their initial concentrations in the samples.