2.11.4. Supercritical Carbon Dioxide (Sc-CO2) Extraction with Ethanol

The extraction of starfish meal was conducted using supercritical fluid extraction (SFE) with an MV-10 ASFE System (Waters, Milford, MA, USA). The extraction was conducted with previously optimized extraction conditions [18]. Briefly, 13 g of dried starfish meal (<1 mm) was packed in 25 mL extraction vessels connected to a Sc-CO₂ inlet and extract outlet line and then stored in the oven. CO₂ was pumped into the extraction vessel to attain the desired pressure using a high-pressure pump after passing through a cooling heat exchanger. The flow rate of the CO₂ was 5 mL/min and two different flow rates were set for the co-solvent (EtOH) of 1 mL/min and 3 mL/min. The extraction temperature and pressure were 45 °C and 275 bar, respectively. These parameters were selected based on work reported for the extraction of phospholipids from salmon by-product [18]. The extraction was conducted in one cycle for both static and dynamic extractions. The dynamic extraction stage was conducted under different conditions for a total of 90 min. For the first 70 min of dynamic extraction and 10 min of static extraction, the extraction was conducted in the presence of the co-solvent, and for the last 10 min, the flow of the co-solvent was stopped while keeping the same flow rate of CO₂ to remove all of the remaining solvents from the extraction vessel. After the extraction was completed, the system was depressurized, and the extract was collected. The remaining solvent in the extract was removed using a stream of nitrogen. Finally, the extract was stored in a freezer at −20 °C until it was required for further analysis.