*4.1. Conventional Extraction Techniques*

Solvent extraction is the most conventional method to obtain krill oil. However, different solvents must be used to extract all the lipids from krill because of the diversity of the polarities of the lipid compounds in the biomass. As reported in a study by Xie et al., ethanol and isopropanol can extract most of the phospholipids, although the obtained krill oil is lacking in other lipid components [21]. Hexane is the most common solvent for vegetable oil production due to its low cost, high extraction efficiency and because it is easily eliminated and leaves few traces of residues in food sources. Nevertheless, data on the use of hexane for krill oil production has demonstrated that this solvent only has moderate PL-extracting ability [28,73].

Acetone has proven to be effective for the extraction of minor molecules from krill, but not for PLs. In this regard, a combination of nonpolar and polar solvents is a good compromise for the extraction of both the PLs and other molecules from krill [74]. Two-step acetone and ethanol extraction techniques are the most common methods used for krill [71], as other methods, including the Folch method (which is popular for lipid extraction from animal tissues), are not commercially feasible because of the toxicity of the solvents used (e.g., chloroform and methanol) [75]. Similar results have been obtained using a single-step extraction method with a mixture of acetone and ethanol as the solvents (1:1, v/v) [76]. Moreover, lipid efficiency can be improved by combining the extrusion pre-treatment of krill meal with the solvent extraction technique [77].

Environmental concerns, which have a real impact even in krill oil production, are one of the main limitations with the use of solvents. In addition, this technique is timeconsuming and labor intensive because of the multiple extraction and evaporation steps. However, it remains the most economical and easily scalable.

Krill oil can also be extracted using non-solvent techniques including mechanical pressing, which is commonly used for oilseed extraction, such as in sesame oil and sunflower oil [78]. However, this method is inefficient compared to solvent extraction, for two reasons in particular: the low lipid content present in fresh krill (0.5–3.6%) and the difficulty of the mechanical processing used for krill [77].

Non-solvent sequential procedures, such as cooking, decanting, pressing and centrifuging, are able to successfully separate krill oil from the mixture (Figure 2). In fact, the release of PLs from the lipid fraction of fresh or defrosted krill—after conventional grinding into a slurry which involves mechanical disruption procedures and centrifugation—does not facilitate the complete separation of the oil fraction due to the amphipathic nature of PLs, which act as emulsifying agents in the formed slurry [79]. Katevas et al., have developed a method that can extract both the PL fraction and the neutral lipid-enriched krill oil. In this method, the first cooking step is conducted at 90◦ C without grinding and agitation, thus avoiding the emulsification process. The same authors concluded that fresh krill is a better option than defrosted krill as it was able to avoid or reduce the emulsification process, which may occur in defrosted krill due to the formation of ice crystals (after freezing) and the consequent disruption of krill tissue [70].

Although the non-solvent extraction technique avoids the use of solvents, making it safer and more eco-friendly than solvent-extraction methods, it presents many drawbacks. First, it is poorly scalable because of the high investment in equipment and the high costs of energy required. Secondly, the cooking procedure at high temperatures may induce lipid oxidation in the products. Last but not least, this technique has only been observed to give a total lipid yield of 2.2% and does not extract all of the available oil from krill. For these reasons, mechanical separation can be used to initially obtain part of the krill oil, but also to produce krill meal, which can be subsequently treated with other techniques including supercritical fluid extraction and solvent extraction [80].

**Figure 2.** An overview of non-solvent extraction technique (Adapted from Katevas et al. [70].
