*3.1. Pre-Treatment*

After harvesting algal biomass from beaches, the biomass should be washed thoroughly with tap water to remove sands and epiphytes, then dried and milled to increase the area-to-mass ratio. Several pre-treatment steps are performed before the extraction step to release fucoidans from intercalating components, ease the following extraction process, improve the extraction yield, and decrease the possible interferences from co-extracted components in purification and biological investigations.

Previous experiments tried to remove pigments (e.g., chlorophyll, flavins and carotenoids) and lipids in specific bleaching and defatting steps with acetone, toluene, charcoal or 80%–85% (*v*/*v*) ethanol [34,84,85]. Since fucoidans are negatively charged molecules, they remained unaffected by incubation with organic solvents (e.g., acetone, toluene or hexane:isopropanol (3:2) mixture) during pre-treatment of the dried algal biomass. Such extracts were further treated to obtain carotenoids, represented by fucoxanthin in brown algae [86], lipids and fatty acid metabolites (especially essential polyunsaturated fatty acids (PUFA) and fucosterol), adding to nutraceutical applications of brown algae [87,88]. In contrast, activated carbon materials, such as charcoal, adsorb the target fucoidans molecules, adversely affecting the final production yield [79].

Other studies tried to exclude the tightly non-covalently bound polyphenolic compounds represented by phloroglucinol-type phlorotannins [89], which contribute to the light to dark brown color of the crude fucoidans extract (along with fucoxanthin) [41,81]. They reported comparatively high phlorotannins content, reaching approximately one fifth of the brown algae dry weight [25]. Phlorotannins perform major structural and physiological functions, like tannins found in plants, including defense against biotic and abiotic stresses [90,91]. Despite of the grea<sup>t</sup> pharmacological importance of phlorotannins [92,93], their presence in high-quality fucoidans is not acceptable because of the possibility of interference with the anti-oxidant [25,52,94] and anti-tumor activities of fucoidans [95]. Therefore, the natural phenolics content of fucoidans should be determined before the measurement of their biological activities [96]. Therefore, nearly all pre-extraction protocols for fucoidans involved strategies to remove such contaminants, e.g., incubation with EtOH:H2O:HCHO (16:3:1) (*v*/*v*/*v*) at pH 2. Under such conditions, formaldehyde enhances the crosslinking and polymerization of such polyphenolic contaminants and the high volume of ethanol results in protein denaturation [41,60,97,98]. However, the toxicity of formaldehyde limits its utilization in pre-treatment protocols [51].

Furthermore, pre-treatment steps are performed to remove other carbohydrates such as alginate, the major hydrocolloids in brown algae [99]. This is commonly removed by formation of water-insoluble calcium complex either before [60] or during the extraction procedure using 1%–4% (*w*/*v*) CaCl2 followed by a filtration or centrifugation step to remove the formed precipitate [58,98,100,101]. These previously mentioned procedures were optimized using successive incubation, centrifugation or filtration, washing and drying for the main extraction step of the dried, milled algal biomass, as described in Figure 3. The application of such an optimized protocol resulted in a dried, pre-treated powder representing 71% (*w*/*w*) of the starting material [98]. Despite these results, downstream processing of fucoidans, except with enzymatic modification, starts with a small scale (e.g., 5–10 g of the dried algal biomass) to optimize parameters like dried biomass to solvent ratio, temperatures, pH, and incubation time, based on preliminary quality and yield of crude fucoidans measured by infra-red spectroscopy (IR), simple sugar tests and elemental analysis. After this, transfer to large scale production could be accomplished using larger biomass quantities (e.g., 500–1000 g).

**Figure 3.** Overview of optimized pre-treatment steps of the dried algae biomass before fucoidans extraction. All steps were performed at 25 ◦C overnight and the ratio between dried algal biomass to solvent was 1:10, except for the acetone step, which was 1:20 (modified after [98,102]).

Due to several complicated pre-treatment steps, general protocols always employ a single incubation step using the ternary mixture composed of CH3OH:CH3Cl:H2O (4:2:1) (v/*v*/*v*) [103], binary mixture of CH2Cl2:EtOH (94.2:5.8, *v*/*v*) [104], or aqueous ethanol (e.g., 95% *v*/*v*) [105,106] to remove pigments [107], polyphenols [51,103] and lipids [108]. Nevertheless, pre-treatment steps may be insufficient for complete removal or prevention of some residual co-extraction.

Notably, all these procedures were carried out at room temperature in organic solvents and high volumes of ethanol, in which fucoidans are insoluble. Theoretically, the native structural backbone should not be affected. However, similar polymeric carbohydrates such as laminarin may still be present, contaminating the extract after these steps.

Recently, in order to decrease pollution of organic toxic solvents, compressional-puffing pre-treatment was applied for *Sargassum hemiphyllum* and *S. glaucescens* fucoidans. The pre-treatment method was based on mechanical pressure at higher temperatures that loosen the cell wall matrix before the step of extraction. Such methods succeeded in increasing the production yield, but they affected the molecular features of the fucoidans, including molecular weight [109,110].
