Fucoidans: Downstream Processes and Recent Applications

Fucoidans are multifunctional marine macromolecules that are subjected to numerous and various downstream processes during their production. These processes were considered the most important abiotic factors affecting fucoidan chemical skeletons, quality, physicochemical properties, biological properties and industrial applications. Since a universal protocol for fucoidans production has not been established yet, all the currently used processes were presented and justified. The current article complements our previous articles in the fucoidans field, provides an updated overview regarding the different downstream processes, including pre-treatment, extraction, purification and enzymatic modification processes, and shows the recent non-traditional applications of fucoidans in relation to their characters.


Introduction
Polysaccharides, nucleic acids, and peptides are considered the main three types of bioactive polymeric macromolecules [1]. Among these, polysaccharides serve various roles in living cells including structural functions, where cellulose and chitin represent the major components of the different cell wall matrices [2,3], energy storage (e.g., starch and glycogen) [4,5], and hydration and signaling functions (e.g., mucilage and alginic acid) [6,7].
Particularly, marine homo-and heteropolysaccharides are derived from marine organisms, which represent a large part of global biodiversity [8]. Among these are the algal polysaccharides, such as fucoidan and alginate in brown seaweeds, carrageenan in red seaweeds and ulvan in green seaweeds. These were reported to have interesting nutraceutical, biomedical, pharmaceutical and cosmeceutical applications, including dietary fibers; anti-inflammatory, anti-tumor, anti-oxidant, hepatoprotective and anti-coagulant properties; and drug carrier functionality. Therefore, they have been extensively investigated during the last few decades [9][10][11][12][13], especially after the emergence of glycobiology and glycomics [14][15][16][17].
Polysaccharides such as dietary fibers of brown algae are abundant and diverse (e.g., alginates, cellulose, fucoidans and laminarins) constituting the major components (up to 75%) of the dried thallus weight (% DW) [18][19][20]. Previous work investigated their abundance in different species, reporting Fucus, Ascophyllum, Saccharina, and Sargassum to contain 65.7, 69.6, 57.8 and 67.8 % DW, respectively [21,22]. Specifically, fucoidans are found in the cell walls and extracellular matrices of brown algae in addition to more than 265 genera and 2040 species of marine invertebrates (e.g., sea cucumbers), where they perform vital structural functions [23][24][25][26]. Fucoidans are assumed to act as cross-linkers between the major threads of cellulose and hemicellulose, promoting cellular integrity and maintaining cellular hydration (especially during drought seasons) [27]. They also act in other In 2014, the annual production of cultivated seaweeds reached 27.3 million tons [68], representing 27% of the total marine aquaculture production, while the global market of marine biotechnology (blue biotechnology) for industrial applications has been expected to achieve US $4.8 billion in 2020 and grow to US $6.4 billion by 2025 [69].
Species of brown macroalgae (Phaeophyceae) are distributed among the orders Fucales and Laminariales, which are the major commercial sources of the algal sulfated polysaccharides, in addition to Chordariales, Dictyotales, Dictyosiphonales, Ectocarpales, and Scytosiphonales. Moreover, phylogenetic analysis showed that Fucales are one of the largest and most diversified orders within Phaeophyceae, having eight families (41 genera and 485 species), named Ascoseiraceae, Cystoseiraceae, Durvillaeaceae, Fucaceae, Hormosiraceae, Himanthaliaceae, Sargassaceae, and Seirococcaceae [70]. Figure 1 illustrates the distribution of several examples of well-known brown algae species which are considered potential sources of sulfated polysaccharides dominating tropical to temperate marine forests and intertidal regions. The data were based on Wahl, et al. [71]. Furthermore, like terrestrial plant tissue culture (PTC), several biotechnological attempts were performed to cultivate and/or regenerate thallus from different species of brown seaweeds using seaweeds tissue culture [72]. They include micropropagation, callus induction and protoplast isolation [69,[73][74][75]. They are very promising techniques as it may not only help to overcome the previously mentioned fucoidans production heterogeneity challenges [53] but also provide a sustainable supply [76]. However, compared to PTC, STC is still not well-enough established to be used for production of hydrocolloids and fucoidans [77] or cultivation in closed, well-controlled bioreactors, as in case of the red algae organism Agardhiella subulata [78].

Downstream Processes
Fucoidans are anionic polymers occurring in highly complicated matrices in cell walls and intercellular spaces along with other carbohydrate polymers (e.g., alginate, cellulose and laminarin), polyphenols and proteins [79]. Additionally, due to the sulfate ester groups, fucoidans are water soluble polysaccharide polymers [80] exhibiting high affinity to other cell wall components, especially polyphenols [81]. Therefore, various and complicated downstream processes are required to remove such extraneous substances before and after precipitation with ethanol or cationic surfactants to obtain high-purity fucoidans [82,83]. The processes always include pre-treatment, extraction and purification stages as shown in Figure 2.

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.
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 great 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:H 2 O: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) CaCl 2 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). 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]).
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].
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 Na 2 CO 3 or (NH 4 ) 2 CO 3, 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][126][127][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].

Separation Physical Methods
Filtration, dialysis and centrifugation, either for the algal biomass or precipitates, are also among the downstream processes after pre-treatment and extraction steps [131][132][133]. Cross-flow filtration and dialysis against water are usually performed using different molecular weight cut-off (MWCO) membranes for isolation of fucoidans from smaller compounds depending on the high molecular weight of fucoidans [134] and also for fractionation purposes, where low molecular weight fucoidans (LMWF) can be separated from high molecular weight analogues (HMWF) [49].
In addition, filtration, concentration, and fractionation are simultaneously performed using centrifugal concentrators (Vivaspin ® ) equipped with membranes with certain MWCO, like in protein purification. However, in some cases, especially in the presence of bulk masses or high concentrations of salts and small contaminants, the use of centrifugal concentrators becomes practically and economically unsuitable for fucoidans purification. In such cases, bulky contaminants result in membrane clogging leading to its deterioration and increasing the production cost.

Purification
Despite the previously mentioned purification steps, residuals of co-extracted contaminants are still present, and resulting fucoidans are still crude-type. [27]. Therefore, further selective purification steps are needed to obtain a high-quality product for reproducible and accurate biological investigations. Some researches adopted simple, non-chromatographic steps, such as bleaching of the crude fucoidans (NaClO 2 in dilute HCl) followed by precipitation with cetyltrimethylammonium bromide [135] or by cold overnight incubation in aqueous buffered solution of calcium acetate (20 mM, pH = 6.5 -7.5) followed by dialysis [136]. In addition, membrane filtration was reported to produce fucoidans fractions of different molecular weight [137].
However, other chromatographic purification techniques were discussed in our previous publications [41,53,98,102]. Almost all the chromatographic techniques are based on the permanent negatively charged sulfate ester groups distributed on the polymer backbone which allow selective fucoidans capture. However, carboxylated (e.g., alginate) and phosphorylated (e.g., nucleic acids) compounds might interfere [138,139]. Therefore, the pH value of the applied solvents is critical during chromatographic purification. One option for this uses anionic exchange resins (e.g., diethylaminoethyl cellulose or DEAE-cellulose), which was performed at pH 7.2 using 0.1 M sodium phosphate buffer [140]. An alternative is cationic dyes (e.g., toluidine blue-or perylene diimide derivative), modified resins or chitosan functioning in buffered solutions [27,102]. Both anion exchange and dye affinity chromatography involve the use of highly concentrated NaCl elution solvents. As a result, a subsequent purification step using chromatographic gel permeation [141] or dialysis [140] is required to remove salts, increasing the production costs. Other methods based on the use of biological macromolecules, such as lectins and anti-thrombin III, were also reported [53].
Novel innovative purification techniques were recently developed, such as selective solid phase extraction for purifying fucoidans and other complex seaweeds polymers by molecularly imprinted polymers (MIP) [142,143] or MIP modified by deep eutectic solvents [142,143]. Abdella et al., developed a green and time-saving purification protocol using genipin cross-linked toluidine blue immobilized-chitosan beads employing fucoidans affinity to cationic thiazine dyes [102].

Enzymatic Modification of Native Fucoidans
Owing to their high molecular weight, therapeutic applications of native fucoidans face many challenges including structure elucidation, solubility, manufacturing, and handling [63,116], in addition to safety as a food supplement [175]. Structure elucidation and quantitation of native fucoidans is highly complicated and requires advanced or hyphenated spectroscopic techniques such asHPLC-MS/MS as it applied in Sea Cucumbers fucoidans [176,177]. Also, these techniques must be applied after a step of enzymatic or acid hydrolysis to transform the fucoidans polymers to oligomers. According to their molecular weight, fucoidans are classified into three classes: LMWF (<10 kDa), medium molecular weight fucoidan (MMWF) (10-10000 kDa), and HMWF (>10000 kDa) [31]. LMWF demonstrated better bioavailability and bioactivities than HMWF [178,179]. As a consequence, several articles reported physical, chemical and enzymatic modification of the native HMWF to get LMWF of higher biological activity [62]. Specifically, enzymatic modification of macroalgal polysaccharides, including fucoidans by either fucoidanases or sulfatases, is characterized by regioselectivity and stereospecificity. This new trend is considered crucial and highly promising for current and future applications of polysaccharides [180].
Nevertheless, our publications in 2009 particularly reviewed the specific enzymatic degradation of fucoidans induced by fucoidanases (EC 3.2.1.44) and α-L -fucosidases (EC 3.2.1.51), mainly those isolated from marine bacteria [35]. Cumashi, et al. studied the chemical structures of different fucoidans isolated from a number of brown algal species [181]. Their proposed models, which were highly appreciated and recommended by many researchers [60], showed the backbone of fucoidans to be mainly an alternating α-(1-4) and α-(1-3) linked L -fucopyranoside. Regarding the sulfation pattern, C-2 is usually substituted with sulfate ester groups in addition to alternating C-3 or C-4 in L-fucopyranose residue, according to the glycosidic linkages. In addition, branched chain polymers were also found as in F. serratus. Other minor sugar units (e.g., mannose, galactose, glucose and xylose) occur as well in fucoidans structure; however, their distribution pattern and positions are still unknown [60,181]. Now, the mechanism of enzymatic degradation can be described in relation to fucoidans chemical structures.
Despite the increasing number of publications investigating fucoidanase activity of different marine species cell extracts, few of these enzymes have been isolated and characterized. Moreover, genome sequences encoding few fucoidanases have been published, including Ffa2 and FFA1 from Formosa algae KMM 3553 T [182,183], FcnA from Mariniflexili fucanivorans SW5T [184]. Therefore, specificity of fucoidanases, type of cleaved glycoside bond, structure-activity relationship studies and enzyme stability are still poorly described. It was only observed that identified microbial fucoidanses act only on fucoidans isolated from their respective symbionts [185]. In fact, fucoidanases have not actually been fully utilized yet as a powerful tool either for the structural studies of fucoidans or production of defined and well-characterized bioactive fragments of extracted fucoidans, as shown in Table 2.

Conclusion and Future Prospective
As multifunctional molecules, fucoidans have received special interest based on their proven efficacy in different fields. The current article reviewed many aspects related to fucoidans' production, mainly from brown algae. Biogenic source and downstream processes were shown as major factors determining their application, which is affected by molecular weight and quality grade of fucoidans. Therefore, the alteration of fucoidans' native structure was recommended, especially as performed by fucoidanases. Their production in nanoform or in combination with other polymers can improve or modify their potential uses, allowing their expanded potential as therapeutic agents, e.g., in anti-cancer applications [202].
Production of high-quality purified fucoidans is urgently required to clarify the relationships between chemical structure and the various bioactivities attributed to fucoidans, eliminating any interference from contaminants. However, it was observed in some cases that crude extracts and presence of co-extracted contaminants, especially polyphenolic phlorotannins, have advantageous cosmeceutical effects due to their powerful anti-oxidant activity [203,204].
Novel techniques, either in cultivation or downstream processes, have been established, increasing the global production yields and reducing ecological and economic problems. A new advance toward achieving such goals was established by optimization of water extraction via measurement of kinetic parameters [205]. In addition to this, it is expected that most future trends in marine biotechnology research will focus on the cell wall and extracellular matrix components of brown algae, including fucoidans' biosynthetic genes and production regulators [23,53,63,[206][207][208]. Such trials may enable the scientific community to produce more bioactive molecules of fucoidans with defined characteristics, including degree of polymerization, sulfate content and pattern, in reproducible manners.