*3.2. Extraction*

As previously mentioned, fucoidans are principally anionic water-soluble macromolecules. Therefore, they can be extracted from the pre-treated biomass using a simple hot- or cold-water incubation. Afterwards, the extracted fucoidans can be precipitated by high volumes of solvents with a low dielectric constant (e.g., >70% (*v*/*v*), > 2.5 volume ethanol [111,112], <2 volume acetone [113]) or cationic surfactants (e.g., hexadecyltrimethylammonium bromide (Cetavlon®) 10% (*v*/*v*)) [55] via an affinity complex formation at low temperatures (4 ◦C) to remove the undesired salts from the sulfated polysaccharides [52]. This specific precipitation reaction between fucoidans and Cetavlon® is applied in screening tests of microorganisms for putative fucoidanase activity [114].

Ale et al. published comprehensive articles discussing the history of extraction, including the different classical extraction methods of fucoidans, and reported that extraction procedures significantly affect the polymers monomeric composition, even for the same organism [60,115]. Beyond simple hot water extraction [58,116], attempts were made to increase the selectivity and extraction yields, including extraction in acidic [117], alkaline [118], and buffered [41,119] aqueous solutions. However, a neutralization step is required, using Na2CO3 or (NH4)2CO3, directly after extraction to guard against the non-specific acidic hydrolysis of the polymer [101,115]. Such drastic pH changes affect the chemical and physicochemical properties of fucoidans during the extraction step.

Currently, besides the previously discussed classical extraction methods based on thermal energy, extraction protocols based on vibrational energy have been developed. These protocols are based on microwave-assisted (MAE) [120,121] or ultrasound-assisted (UAE) [94,122] extraction steps to elicit cell wall degradation which improves the polymer release into aqueous solvent. These protocols were optimized either using an approach that modified one factor at a time or a multiple factorial design, setting the polymers production yield, monomeric composition and biological activities as the measured responses.

Recently, combined sulfated polysaccharides extraction protocols were optimized from different brown algae species using hydrothermal-assisted extraction (HAE) followed by sequential ultrasound and thermal technologies [123]. Similarly, subcritical water extraction was applied to increase the production yield of fucoidans from *Nizamuddinia zanardinii* [124]; such mild conditions may be advantageous to preserve the native chemical backbone and physicochemical characters of fucoidans.

Recently, as a trial to reduce such undesirable effects, enzyme-aided or assisted extraction (EAE) protocols are being developed using enzymes instead of harsh chemicals and high extraction temperatures during extraction. These include cellulase, papain, laminarinase, alginate lyase, and protease, which are present in products of Novozymes [79,125–128]. In addition, other cost-effective and time-saving techniques are reported, like those for terrestrial plant polysaccharides, such as extraction under vacuum to lower the boiling point of water and hence avoid possible heat-induced fucoidans degradation [129]. Alternatively, 0.5% (*w*/*v*) ethylenediaminetetraacetic acid (EDTA) was applied at 70 ◦C for simultaneous extraction of *Laminaria japonica* fucoidans and removal of pigments [130].
