**1. 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 [9–13], especially after the emergence of glycobiology and glycomics [14–17].

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) [18–20]. 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 [23–26]. 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 reproductive, immune and cell-to-cell communicative roles [23]. As recommended by the International

Union of Pure and Applied Chemistry (IUPAC), fucoidans is a general term used to describe sulfated L-fucose-based polymers including sulfated fucans cited by the Swedish scholar Kylin, as well as other fucose-rich sulfated heteropolysaccharides [23,28]. Their chemical structures, in terms of monomeric composition and branching, are quite simple in marine invertebrates compared to their analogues in brown algae [13,29].

Hundreds of articles have thoroughly discussed and reviewed the biological, pharmacological and pharmaceutical applications of fucoidans [30–33], including nanomedicine, [34] which has made it a hot topic in the last few decades [35–37]. All these studies tried to investigate fucoidans molecular mechanisms in relation to their chemical structure and physicochemical properties. Therefore, different hypotheses were suggested for each activity, such as anti-tumor [31,38–40], anti-coagulant [41,42], anti-viral [43,44] and anti-inflammatory activity[45,46]. These investigations revealed that various factors are relevant, such as molecular weight, sulfation pattern, sulfate content and monomeric composition [47–49]. For example, different fractions were produced with different physicochemical properties in our previous experiments; sulfation pattern and sulfate content were highly related to anti-viral and cytotoxic activities against HSV-1 and Caco-2 cell lines, respectively, while molecular weight and sugar composition were potential factors in anti-coagulation activity [41,50]. In addition, degree of purity was reported as an influential factor [32], where co-extracted contaminants (e.g., phlorotannins or polyphenols) could lead to significant interference in anti-oxidant activity and, consequently, cosmetic applications [51,52].

Therefore, several key production challenges regarding fucoidans were discussed in our last review article in order to obtain a product that follows the universal good manufactured practice (GMP) guidelines. The article discussed sources of heterogeneity in extracted fucoidans, including the different biotic (e.g., biogenic, geographical and seasonal factors) and abiotic (e.g., downstream processes) factors affecting the fucoidans physicochemical and chemical properties [53]. Others patented production techniques that have assisted in the marketing of several commercial fucoidans by well-known companies (e.g., Sigma-Aldrich®, Algues and Mer and Marinova®) derived from *Fucus vesiculosus* and other brown algae species [54–56].

Furthermore, the improvement of fucoidans activity was investigated, targeting several points. Among these was the modification of the chemical structure of the native fucoidans scaffolding, including depolymerization [57,58] and over-sulfation [59]. These modifications could be attempted chemically [60], enzymatically [35,61] or physically [62]. Predetermined synthesis of oligomers [63,64] and low molecular weight polymers with defined monomeric units [65] is also involved. Additionally, fractionation of fucoidans is a common approach during extraction and purifications steps by applying different extraction and purification conditions (e.g., pH, time, molarity of NaCl) [49,55].

The current article aimed at complementing our previously published article discussing the reasons for heterogeneity of fucoidans [53]. It reviewed and evaluated the different downstream processes used in production as the most important abiotic factors affecting the fucoidans quality and structural features; it then addressed recent uncommon applications and prospective bioproduction of fucoidans. In addition, the updated status of enzymatic structural modifications of fucoidans, especially by fucoidanases, were presented.

#### **2. Global Market and Cultivation of Brown Algae**

Marine hydrocolloids (e.g., agar, carrageenan and alginate) are of particular industrial interest, with worldwide annual production of approx. 100,000 tons and a value above US \$1.1 billion [66]. Based on FAO periodical reports (FAO, 2014, 2016), among the top seven most-cultivated seaweeds, three taxa are mainly used for hydrocolloids production; these include Rhodophyta (e.g., *Eucheuma* sp. and *Kappaphycus alvarezii*) for carrageenan production and *Gracilaria* sp. for agar production [67]. These data encouraged the global marine market to escalate the production yield by finding alternative, eco-friendly seaweed cultivation techniques, such as sea farming or aquaculture and biotechnology [53]. 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].

**Figure 1.** Global distribution of the major brown seaweeds' species. They dominate tropical to temperate marine forests and intertidal regions.

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 [69,73–75]. 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].
