*4.2. Unconventional Extraction Techniques*

The supercritical fluid extraction method is an unconventional extraction technique that can be used for krill oil extraction and is known to be solvent-free [81]. It is considered environmentally friendly as it uses supercritical carbon dioxide, which is safe, non-toxic and chemically inert. Nevertheless, carbon dioxide is not an ideal molecule for the extraction of all the lipid content from krill as it fails to extract PLs [82]. For this reason, the addition of 5– 20% of ethanol to supercritical carbon dioxide extraction has been tested and demonstrated to improve PL solubility and thus lipid recovery [83]. The use of this technique presents some issues, including its limited processing capacity, the high cost of the equipment and the risk of de-solvation by the liquid state of ethanol at 25 ◦C.

Subcritical fluid extraction with compressed butane and propane, performed at low temperature and pressure, is commonly used for oil extraction [84]. In a study conducted by Xie et al., the use of subcritical butane (30 ◦C, 0.3 to 0.8 MPa) gave results that are comparable to those of hexane in terms of yield and krill oil quality, although it was faster and the extraction process required less solvent [73]. In addition, in a study by Sun et al., subcritical butane extraction was proven to give the extract that was richest in astaxanthin and tocopherol, and the lowest in oxidation degree, compared to the solvent extraction technique [85]. However, even in this case, the high cost of equipment and limited processing capacity are the main issues for a successful scale-up process.

Enzyme-assisted extraction is a novel pre-treatment technique than can increase lipid yield, as it can release the bound molecules using specific enzymes (e.g., amylase, protease, glucanase, cellulase and pectinase) [86]. These enzymes can selectively enhance the extractability of lipid compounds, improving the breakdown of cell walls and destroying lipid bodies. The high quality of the extracted oil and the mild process conditions also make this technique attractive for the extraction of krill oil [75]. Bruheim et al., have obtained satisfactory results with enzyme-assisted extraction for krill oil. After an initial krill-disintegration process, the small particles were treated with water and heat and the hydrolytic enzymes were then added to hydrolyze the material and improve lipid extraction. Finally, the enzymes were deactivated and, after removal of the solid material, the PL-protein complex was separated and dried, and the krill oil was extracted [75]. Finally, in a study by Lee et al., a combination of an enzyme-assisted extraction process and an ultrahigh-pressure (10 to 300 MPa) reactor made krill easily liquefiable and ensured full contact with the proteases [87].

The high costs and longer hydrolysis times are still challenging for the use of this technique in large-scale industrial applications.
