*2.1. Polysaccharides*

Brown macroalgae are known to produce different types of polysaccharides and/or fibres which, despite their variability, represent major components that can reach up to 70% of their dried weight (DW) [19]. In fact, previous reported data set the polysaccharide contents of relevant species, namely *L. japonica*, *F. vesiculosus*, *A. nodosum*, *Saccharina longicruis*, *U. pinnatifida* and *Sargassum vulgare* at 37.5%, 65.7%, 69.6%, 57.8%, 35.2% and 67.8% DW, respectively [20–23]. Amongst them, alginates, fucoidans and laminarins are the most representative ones.

Alginic acids or alginates, i.e., the salts of alginic acid, are the main polysaccharides in brown seaweeds [24], reaching up to 16.9% DW in *S. vulgare*, 20% DW in *S. longicruris*, 24% DW in *A. nodosum*, 32% DW in *Sargassum carpophyllum*, 40% DW in *Laminaria hyperborean* [25], 41% in *Sargassum siliquosum* and even to 59% DW in *F. vesiculosus* [26]. Within the cell wall, these polysaccharides are known to be partially responsible for the seaweed's flexibility [3] and therefore, expectedly, brown seaweeds grown under turbulent conditions usually have superior alginate contents than those of calm waters. In terms of structure, alginic acids or their corresponding extracted salts consist of α-l-guluronic acid (G) and <sup>β</sup>-d-mannuronic acid (M) (1→4)-linked residues arranged either in heteropolymeric (MG) and/or homopolymeric (M or G) blocks (Figure 1A–C). Regardless, the variations caused by diverse factors (e.g., algae species, seasonability, parts of the algae) are expected [16]. Noteworthy, alginates are considered one of the most important food colloids, with many applications in several industries such as foods, paper, pharmaceutical or cosmetics [27]. In fact, G-blocks in the presence of ions, such as Ca2<sup>+</sup> form is the so-called egg-box, thus granting stiffness to the overall structure and conferring gel-forming properties to these polysaccharides [28]. Therefore, they are usually used as thickeners, gels, emulsifiers and stabilizers in order to improve quality parameters, especially in food grade products [29]. In addition to their wide applications, more recently, dietary alginates are being associated with positive health benefits in the gastrointestinal tract and appetite regulation, as well as antihypertensive and anti-diabetic effects [30]. Alginates are also considered great prebiotics as they were demonstrated to significantly promote the growth of several bacteria, including *Bifidobacterium bifidum*, *Bifidobacterium longum* and *Lactobacilli*, alongside with the increase of acetic acid, propionic acid and several short chain fatty acid metabolites, while decreasing deleterious metabolites, including faecal sulphide, phenol, *p*-cresol, indole, ammonia and skatole [31].

Fucoidans i.e., metabolites belonging to the fucans family, also have a structural role in brown algae, mostly preventing dehydration [3]. Their reported content in *Phaeophytae* is variable, ranging from approximately 6–8% DW in *L. japonica*, 3.2–16% DW in *U. pinnatifida*, and 3.4–25.7% DW in *F. vesiculosus* [10,32]. These polysaccharides are mainly composed of fucose and sulphate, although the presence of other types of monosaccharides (glucose, galactose, mannose, xylose and uronic acids), acetyl groups and proteins also occur [33]. Despite being molecules with high structural diversity, the representative backbone of fucoidans consists of (1→3)- and (1→4)-linked <sup>α</sup>-l-fucopyranose residues, and these polysaccharides are commonly divided in two types, the first being characterized

by long chains of (1→3)-linked <sup>α</sup>-l-fucopyranose residues (mainly present in *L. saccharina*, *L. digitata*, *C. okamuranus*, and *Chorda filum*) and the second consisting of alternating (1→3)- and (1→4)-linked α-l-fucopyranose residues (characteristic from *A. nodosum* and *Fucus* spp.) (Figure 1D,E) [24,34].

Over the last years, extensive biological activities (e.g., antitumor, antioxidant, anticoagulant, antithrombotic, immunoregulatory, antiviral, anti-inflammatory among others) have been demonstrated with promising preclinical results, as recently reviewed [35]. As an example of in vivo studies, the effectiveness of a *F. vesiculosus* fucoidan injection towards oxidative stress in hyperoxaluric rats was demonstrated by Veena et al. [36] to be mediated by the stimulation of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Moreover, Huang et al. [37] reported that the ingestion of fucoidans isolated from *L. japonica* reduced the serum levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol in hyperlipidaemic rats, while increasing the enzymatic activity of lipoprotein lipase, hepatic lipase and lecithin cholesterol acyltransferase. In addition, their relevance in obesity and/or diabetes was also highlighted, in particular, by Xan et al. [38], who reported *F. vesiculosus* fucoidans' ability to inhibit α-glucosidase in vitro and to decrease the fasting blood glucose and glycosylated haemoglobin levels of db/db mice, as well as by Kim et al. [39], when administrating *U. pinnatifida* fucoidans to the same animal model. Although there is limited evidence to implicate a role of fucoidans in the gut microbiota, some works reported that fucoidans from different brown algae species greatly contributed for the increase in the growth of *Bifidobacterium*, *Lactobacillus* and *Ruminococcaceae,* either in mice or human faecal samples [31].

**Figure 1.** The structure of representative polysaccharides found in brown algae: (**A**–**C**) alginic acids; (**D**–**E**) fucoidans from *A. nodusum*/*F. vesiculosus* and *S. latissima*, respectively; (**F**–**G**) laminarins M and G chains.

Laminarins, also named laminarans or leucosins, on the other hand, belong to the glucan family and serve as reserve metabolites in brown algae [40]. These are commonly found in the fronds of *Laminaria* and *Saccharina* macroalgae and, to a lesser extent, in *Ascophyllum*, *Fucus* and *Undaria* species [41]. In general, they are relatively small polysaccharides composed of β-(1→3)-linked glucose monomers, containing large amounts of sugars and a low fraction of uronic acids (Figure 1F,G) [42]. Depending on the type of sugar at the reducing end, they are classified in two distinct types, specifically the M chains, which have a terminal 1-O-substituted d-mannitol, and the G chains, possessing a terminal glucose [16]. The content of these polysaccharides is also season-dependent, since seaweeds show no production or very less amounts in the winter and maximum production during summer and autumn [23]. As previously stated, *Laminariales* are known to produce high amounts of laminarins, with contents reaching up to 35% DW, particularly in *L. saccharina* and *L. digitata* [40]. Other reported values of laminarins content comprise those of *A. esculenta*, *U. pinnatifida*, *A. nodosum* and *F. serratus* (11.1%, 3%, 4.5% and up to 19% DW, respectively) [22–24,40].

The bioactivities of laminarins are scarcely exploited, but still they are considered as fibres and therefore can be partially or totally fermented by the endogenous intestinal microflora. This was demonstrated by Devillé et al. [43], when comparing the results from in vitro digestibility tests, where no hydrolysis of this fibre occurred, to those of in vivo tests, for which no traces of laminarin were detected in the faeces of fed Winstar rats after ingestion.

It should be noted that oligosaccharides from brown macroalgae polysaccharides may also exhibit interesting bioactivities, which can differ from those of the original polysaccharides. In this topic, alginate oligosaccharides have been claimed to possess radical scavenging activities with the great potential for application in the food industry [44], and even promising effects on neuro-inflammation, promoting microglial phagocytosis. This could be of great relevance for their application as a nutraceutical agent for neurodegenerative diseases, such as Alzheimer's disease [45]. In turn, in vivo experiments on renovascular hypertensive rats revealed that fucoidan oligosaccharides exhibited anti-hypertensive effects comparable to those of captopril, i.e., an approved drug used for the treatment of hypertension [46].
