Microalgae represent diverse branch of microorganism that can produce a wide range of unique functional ingredients that can be used in food, cosmetics, pharmaceuticals, and energy. Among them, Haematococcus pluvialis is known for accumulating the highest levels of a potent natural antioxidant, astaxanthin, which has demonstrated positive health effects. Therefore, the aim of numerous studies has been to develop novel and efficient extraction techniques to produce high-quality (purity and antioxidant activity) extracts, while complying with the Green Chemistry Principles. Supercritical CO2 (scCO2) emerges as an alternative to organic solvents because of its high selectivity and bioactivity-preserving qualities. Nevertheless, astaxanthin is a large molecule with low solubility in scCO2 that usually requires long extractions at high pressures. Ethanol has been used as co-solvent to increase astaxanthin solubility in scCO2. In this work, a Box-Behnken experimental design was used to study the effects of operating pressure (20-35 MPa), temperature (40-70 °C), and ethanol content in scCO2 (0-13%, w/w) on the yield, astaxanthin content, and antioxidant activity of H. pluvialis extract. Results showed that ethanol content in CO2 has a more significant effect on all responses than pressure and temperature. These results lead us to investigate the effect of a further increase in ethanol content, up to the region of gas-expanded liquids. We studied the effects of temperature (30-60 °C) and ethanol content (50-70%, w/w) at a fixed pressure (7 MPa) on the same response variables using CO2-expanded ethanol (CXE). Results showed that temperature and ethanol content had a significant influence on astaxanthin yield and antioxidant activity. Also, the overall responses of CXE surpassed scCO2 extractions to match conventional extraction with acetone, maintaining high quality extracts, thus validating the use of this new type of green technology for extraction of high-value compounds. © 2014 Elsevier B.V.

The authors want to thank Spanish Projects AGL2011-29857-C03-01 and CONSOLIDER INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Ministerio de Economía y Competitividad) and the European project MIRACLES (KBBE.2013.3.2-02: The CO2 algae biorefinery). Fabián A. Reyes would like to thank CONICYT-Chile for supporting his fellowship in CIAL-CSIC, Spain.

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