Some (candidate) prebiotics occur naturally in seaweeds and marine microalgae, and some of their PS (native or somehow modified, such as LMW-PS) were already recognized and accepted as dietary prebiotics: GOS, AGAROS, XOS, neoagaro-oligosaccharides (NAOS), alginate-derived oligosaccharides (ALGOS), arabinoxylans, galactans, β-glucans, although the fulfillment of the criteria still has to be proved for some of them. However, these algal PS are not degraded by enzymes in the upper part of the GI tract. Therefore, they can be used as dietary prebiotics (fibers), as they also enhance the growth of lactic acid bacteria (LAB) [ 29 ].

Hemicelluloses may include most of the algal PS, which are usually branched polymers embedded in algal cell walls, present within the cell or even produced and released into the culture medium. These are heteropolymers that can be easily hydrolyzed by hemicellulases or by acid or basic diluted solutions. In addition to the PS that are mostly considered as soluble fibers, seaweeds also contain cellulose, which is a linear non-branched polymer made up by only anhydrous glucose residues linked together by β-(1,4) bonds. Cellulose and lignin are insoluble fibers, resistant to microbial and human enzymes [ 21 ].

In what concerns PS from microalgae, there is not much information on these complex polymers. Except for a β-glucan inand a homogalactan in, most of the other PS are heteropolymers of several different monosaccharides. The glycosidic bonds were described for only a few PS, including those fromandHowever, the structures for the repeating mono-, di- and oligosaccharides were already described for the PS ofand 45 ].

Carrageenans are widely used in foods, for example, as gelling agents in plant-derived gelatines. Polysaccharides from green seaweeds may also consist of (gluco)mannans () and a rare (1,3)-β-mannan in, while ulvan is the main PS present in green macroalgae (). Rhamnans (), galactans () and other, more complex PS may appear as well [ 45 ].

The main carbohydrates of red seaweeds are floridean starch (as reserve/storage) and S-galactans (carrageenans and agarans), as is the case ofand, andand, respectively. Usual linkages and principal monomers are (1,3)-α--galactose, and (1,4)-β-3,6-anhydrous galactose or (1,4)-β--galactose (alternating). As it was mentioned earlier, AGAROS can be obtained by acid-hydrolyzing α--anhydrous galactose bonds [ 37 ]. These oligosaccharides were already showed to provide several health benefits [ 21 ]. Some genera may present xylomannans (and), and xylogalactans () as well.

Alginates are the principal carbohydrates inand(20%–29% DW), which may also present fucoidans in lower percentages (10%–11% DW) [ 153 155 ].andalso contain laminaran, a β-glucan, with (1,3)- and (1,6)-β-glucose linkages, with some other sugar residues linked laterally. Galactofucans may appear in some brown macroalgae () as well [ 45 ].

An extensive review on the PS from marine algae was recently published [ 45 ]. The authors focused on the overall structures of the PS from the various big groups of seaweeds (phaeophytes, rhodophytes and chlorophytes) and the most-studied genera of marine microalgae, including some cyanobacteria. Additionally, the monosaccharide compositions and the linkage types were described, as well as some of the di- and oligosaccharides that were already referred to as being part of the PS of some microalgae. For example, brown seaweeds contain mostly fucoidans, soluble homo- or heteropolymers, with-fucose as the main sugar residue; fucoidans are irregularly branched sulphated HMW-PS, whose monomers are usually linked by (1,3)- and (1,4)-α (alternating) bonds.

3.2. Prebiotic Benefits of Algal Biomass and Fibers, Oligo- and Polysaccharides

158,159, The health benefits and biological activities of polysaccharides produced by both marine macro and microalgae were recently reviewed [ 45 46 ]. Additionally, the results of several clinical trials showing the health benefits of marine microalgae biomass intake and microalgal-derived products were also reviewed and indicated in another recent work [ 44 ]. Scarce information exists regarding the effects on microbiota, though. Courtois [ 26 ] came forward with an explanation for these long-chain HMW-PS to present such prebiotic/beneficial effects at the cell level, most of the time preventing or decreasing reactive oxygen species (ROS) production. This researcher proposed that such HMW-polymers must first be hydrolyzed through digestion or fermented by the gut microorganisms before being assimilated as smaller oligosaccharides and entering the cells, where the oligomers are active [ 46 157 ]. After being ingested, algal PS can resist hydrolysis in the upper part of the GI tract, until they reach the large intestine [ 64 160 ].

i.e. , their molecules may be broken apart into mono- and/or oligomers. Some of the best-characterized enzymes produced by such microorganisms—bifidobacteria and lactobacilli—are xylases and (glycosyl) hydrolases (α- and β-galactosidases, α-glucosidase, fucosidase) [158,159,161, in vitro to the effects of LMW-PS prebiotics from algal origin, such as those from Gelidium , and laminaran and alginate/ALGOS, with the formation of SCFAs [165,166,167, in vitro studies, some positive effects on the number of lactobacteria were observed in pigs fed fucoidans [169,170, However, HMW-PS may be hydrolyzed by the enzymes of some colonic microorganisms,, their molecules may be broken apart into mono- and/or oligomers. Some of the best-characterized enzymes produced by such microorganisms—bifidobacteria and lactobacilli—are xylases and (glycosyl) hydrolases (α- and β-galactosidases, α-glucosidase, fucosidase) [ 29 162 ]. Some species of bifidobacteria can also hydrolyze arabinans, arabinogalactans, arabinoxylans from plant or algal origin, as, possessing the arabinofuranohydrolases, they are able to ferment arabinofuranosyl-monomers [ 29 ]. Those LMW-saccharides are then fermented by other groups of bacteria into SCFAs, which can be monitored by GC-MS, for example [ 163 ]. In addition to intestinal bacterial enzymes, laminaran may also be degraded by laminarases and laminarinases [ 64 ]. Moreover, before being used, fucoidans may be hydrolyzed into oligomers by fucoidanases, which are enzymes produced by some bacteria and mollusks from marine environments, but not by colonic microbiota [ 164 165 ]. Furthermore, there are proofs of the microbiota changes when subjectedto the effects of LMW-PS prebiotics from algal origin, such as those from, and laminaran and alginate/ALGOS, with the formation of SCFAs [ 61 168 ]. Nevertheless, and despite the results obtained fromstudies, some positive effects on the number of lactobacteria were observed in pigs fed fucoidans [ 166 171 ] ( Table 4 ).

Table 4. Prebiotic effect of algal biomass, their extracts and oligo- and polysaccharides.

Table 4. Prebiotic effect of algal biomass, their extracts and oligo- and polysaccharides. Oligo-/PS Algal Genus Effects In Vitro/in Vivo (Animal Model) References alginate - ↑ Bifidobacterium rats [61] ↑ Lactobacillus NAOS (native and hydrolysates, DP 4–12) - ↑ Bifidobacterium mice/rats ( in vitro ) [158] ↑ Lactobacillus ↓ Bacteroides and enterococci ↓ pH in medium ↓ putrefactive microorganisms laminaran - ↑ Bifidobacterium rats ( in vitro ) [165] ↓ putrefactive compounds laminaran + fucoidan - ↑ lactobacilli weanling pigs [172] ↓ diarrhoea extracts Undaria/Porphyra ↓ enzymes responsible for the transformation of pro- into carcinogens rats [173] biomass Ascophyllum ↑ Lactobacillus / Escherichia coli weanling pigs [174] biomass/extracts Laminaria ↑ SCFAs weanling pigs [175] ↓ ammonia in the colon fucoidan - ↑ lactobacteria pigs [166,171,176] ↑ fatty acids alginate - ↑ beneficial bacteria of microbiota humans [60] ALGOS and native or LMW-PSs Gelidium ● positive effects on the microbiota and on the production of SCFAs

↓ putrefactive compounds rats [61,165,167,168] ↓ putrefactive microorganisms FUCOS - ↑ beneficial bacteria - [158,168] AGAROS - ↓ pro-inflammatory cytokines - [177,178] ● act against glycosidase extracts Gelidium ↑ bifidobacteria; best with Gelidium -extract in vitro [179] Gracilaria Ascophyllum ↑ total SCFAs, and acetic and propionic acids; best with Gelidium -extract biomass Chondrus ↑ beneficial bacteria rats [127] ● improvement of microbiota ↑ SCFAs ● improvement in the histo-morphology of the colon ↑ holding-water capacity of stool ● enhancement of immune system:

↑ Ig-A and G biomass Spirulina ↑ L. casei , L. acidophilus , S. thermophillus and other beneficial bacteria, such as Bifidobacterium in vitro [87,180,181] ↓ harmful bacteria: P. vulgaris , B. subtilis , B. pumulis biomass Isochrysis ↑ lactic acid bacteria rats [182]

S -galactofucan was already detected in human blood after the intake of Undaria dried biomass or extracted and purified S -galactofucan [ Laminaria spp. or their extracts showed prebiotic effects on pigs, with an increase of SCFAs in the colon, and in the number of bifidobacteria and lactobacteria detected in the cecum and large intestine, respectively [ in vivo results were obtained with rats, through the determination of putrefactive compounds in their feces [158, Additionally and despite the lack of proper enzymes to digest fucoidans in the GI tract, this type of PS was already detected in human blood and urine after oral administration [ 183 ]. Therefore, it is highly probable that humans can use and transform fucoidans [ 29 ]. Furthermore, the LMW-fucoidan derivative-galactofucan was already detected in human blood after the intake ofdried biomass or extracted and purified-galactofucan [ 184 ]. In addition, fucoidan fromspp. or their extracts showed prebiotic effects on pigs, with an increase of SCFAs in the colon, and in the number of bifidobacteria and lactobacteria detected in the cecum and large intestine, respectively [ 175 176 ]. Nevertheless, most of theresults were obtained with rats, through the determination of putrefactive compounds in their feces [ 61 165 ].

in vitro , with better effects than those of fructooligosacharides (FOS) or lactose, a group of studied prebiotics. Moreover, ALGOS inhibit the growth of putrefactive microorganisms [158,168, Fucoidan (FUCOS) and ALGOS have also proven to increase the number of beneficial bacteria, with better effects than those of fructooligosacharides (FOS) or lactose, a group of studied prebiotics. Moreover, ALGOS inhibit the growth of putrefactive microorganisms [ 61 185 ].

et al. [ In vitro studies conducted by these researchers showed that these NAOS hydrolysates and native NAOS could be metabolized by bifidobacteria and lactobacilli, but the NAOS-B with DP 8–12 were best fermented. A decrease in the pH of the culture medium may be an indication that those beneficial bacteria had better fermented those oligosaccharides. However, SCFAs were not determined. Additional evidence of the prebiotic properties of these oligosaccharides was shown by a significant increase in the numbers of the various species of Bifidobacterium and Lactobacillus , but not enterococci, which decreased. A positive change in the numbers of beneficial bacteria was also observed in vivo , in the feces and cecal contents of rats and mice, and the growth of bacteroides was inhibited together with other putrefactive microorganisms [ et al. [ in vitro studies with different algal-derived oligosaccharides, following an increase of the number of beneficial bacteria (mostly bifidobacteria and lactobacilli), is probably due to the production of SCFAs. However, these end-products of the bacterial fermentation of the oligosaccharides were not determined, the same happening with several animal models [ As it happens with fucoidans, NAOS and AGAROS, before being ingested, should be first obtained by hydrolysis by β-agarases and α-agarases, respectively; α-agarases hydrolyze α-(1,3) bonds, while β-agarases break β-(1,4) linkages [ 158 186 ]. However, these two hydrolases are produced by non-colonic bacteria [ 158 ]. NAOS hydrolysates with different DP (DP 4–12) were tested in rats by Hu 158 ].studies conducted by these researchers showed that these NAOS hydrolysates and native NAOS could be metabolized by bifidobacteria and lactobacilli, but the NAOS-B with DP 8–12 were best fermented. A decrease in the pH of the culture medium may be an indication that those beneficial bacteria had better fermented those oligosaccharides. However, SCFAs were not determined. Additional evidence of the prebiotic properties of these oligosaccharides was shown by a significant increase in the numbers of the various species ofand, but not enterococci, which decreased. A positive change in the numbers of beneficial bacteria was also observed, in the feces and cecal contents of rats and mice, and the growth of bacteroides was inhibited together with other putrefactive microorganisms [ 158 ]. Hu 158 ] verified that the prebiotic effectiveness of NAOS, especially NAOS-B (DP 8–12), was higher than that of other known oligosaccharides (FOS and GOS) as well. AGAROS had already been proven to be able to inhibit the release of pro-inflammatory cytokines and to act against the enzyme glycosidase [ 177 178 ]. The decrease in the pH instudies with different algal-derived oligosaccharides, following an increase of the number of beneficial bacteria (mostly bifidobacteria and lactobacilli), is probably due to the production of SCFAs. However, these end-products of the bacterial fermentation of the oligosaccharides were not determined, the same happening with several animal models [ 61 158 ] ( Table 4 ).

in vitro study was carried out by Ramnani et al. [ Gelidium , Gracilaria and Ascophyllum , and observed that LMW-PS effectively induced changes in the microbiota. However, the effectiveness was greater with Gelidium extract, with a significant increase in the number of bifidobacteria. Furthermore, a shift up in SCFAs was observed with a significant increase in acetic and propionic acids after fermentation of the oligo- and polysaccharides from those seaweeds. The highest production of total SCFAs, and acetic and propionic acids, was also noticed after the fermentation of Gelidium -extract [ A somewhat differentstudy was carried out by Ramnani 179 ]. They subjected human feces (with respective microbiota) to native and LMW derivatives from alginate and agar, along with extracts fromand, and observed that LMW-PS effectively induced changes in the microbiota. However, the effectiveness was greater withextract, with a significant increase in the number of bifidobacteria. Furthermore, a shift up in SCFAs was observed with a significant increase in acetic and propionic acids after fermentation of the oligo- and polysaccharides from those seaweeds. The highest production of total SCFAs, and acetic and propionic acids, was also noticed after the fermentation of-extract [ 179 ].

et al. [ in vitro experiments. However, while the alginate induced an augmentation in the acetic acid levels, laminaran caused an increase in propionic, butyric and lactic acids. They also noticed that the release of putrefactive compounds decreased. During a concomitant experiment with rats, Kuda et al. noticed that only laminaran and LMW-alginate caused a positive effect on the organic acids in the cecal contents. Furthermore, as it happened with an in vitro experiment with human feces, putrefactive compounds decreased up to 60% in the cecum of the rats fed diets supplemented with laminaran and alginate from seaweeds. This might be due to the ability of these PS to inhibit the growth of putrefactive microorganisms. It is important to note that putrefactive compounds are related to the appearance of colon cancer and, thus, can be looked at as biomarkers for this disease [ in vitro or in vivo , which stimulated the growth of bifidobacteria as well. These researchers also found that a β-glucan from the microalga Euglena gracilis caused an increase in the stool bulk, but could not be fermented by human microflora [ A similar study was carried out by Kuda 165 ], in order to verify the effectiveness of laminaran and alginate (LMW- and HMW-PS). They observed that both laminaran and alginate caused an increase in the production of total organic acids in the human feces inexperiments. However, while the alginate induced an augmentation in the acetic acid levels, laminaran caused an increase in propionic, butyric and lactic acids. They also noticed that the release of putrefactive compounds decreased. During a concomitant experiment with rats, Kudanoticed that only laminaran and LMW-alginate caused a positive effect on the organic acids in the cecal contents. Furthermore, as it happened with anexperiment with human feces, putrefactive compounds decreased up to 60% in the cecum of the rats fed diets supplemented with laminaran and alginate from seaweeds. This might be due to the ability of these PS to inhibit the growth of putrefactive microorganisms. It is important to note that putrefactive compounds are related to the appearance of colon cancer and, thus, can be looked at as biomarkers for this disease [ 165 ]. Similar results were obtained by using a laminaran-oligosaccharide (DP 22) eitheror, which stimulated the growth of bifidobacteria as well. These researchers also found that a β-glucan from the microalgacaused an increase in the stool bulk, but could not be fermented by human microflora [ 187 ] ( Table 4 ).

Alginates were also proved to act as prebiotics in weaning piglets, by increasing the number of enterococci and improving bacterial diversity in the intestine [ 188 ]. Furthermore, an alginate-derived LMW-polymannuronate improved cecal microflora and lactic and acetic acids in the cecum of broiler chickens [ 189 ]. Despite the low number of individuals used in the trial, Terada and colleagues [ 60 ] already studied the prebiotic effects of alginate in humans two decades ago. They observed that alginate stimulated the growth of beneficial bacteria in the colon and inhibited harmful microorganisms, with the consequent (significant) decrease in putrefactive compounds in the feces. Alginate-fed individuals also presented higher levels of total SCFAs, and acetic and propionic acids.

Chondrus crispus also possesses prebiotic properties. These researchers fed rats a diet supplemented with C. crispus and verified that the animals’ microbiota was improved. The beneficial bacteria increased, as did the levels of the acetic, propionic and butyric SCFAs. An improvement of the histomorphology of the colon and an increase in the water-holding capacity of the feces were observed as well, as favorable effects provided by the biomass of the red seaweed. The immune status was also enhanced, as the levels of immunoglobulins A and G increased ( In another study, Liu and co-workers [ 127 ] showed that the biomass from the microalgaealso possesses prebiotic properties. These researchers fed rats a diet supplemented withand verified that the animals’ microbiota was improved. The beneficial bacteria increased, as did the levels of the acetic, propionic and butyric SCFAs. An improvement of the histomorphology of the colon and an increase in the water-holding capacity of the feces were observed as well, as favorable effects provided by the biomass of the red seaweed. The immune status was also enhanced, as the levels of immunoglobulins A and G increased ( Table 4 ).

Arthrospira platensis can promote the growth of beneficial bacteria, such as Lactobacillus casei , Streptococcus thermophilus , and L. acidophilus in special [ Proteus vulgaris , Bacillus subtilis and B. pumulis , for example) were suppressed in an in vitro study [ Spirulina promoted the growth of L. acidophilus and Bifidobacteria as well [ Isochrysis galbana is another marine microalga with high contents of both soluble and insoluble fibers, and it is promising as a prebiotic since the numbers of LAB increased in the feces of rats treated with I. galbana [ In addition to seaweeds, some microalgae are also known to have prebiotic properties. For example, the biomass ofcan promote the growth of beneficial bacteria, such as, andin special [ 87 180 ]. Furthermore, harmful pathogenic bacteria (and, for example) were suppressed in anstudy [ 87 ]. When added to yogurt, the biomass frompromoted the growth ofandas well [ 181 ]. However, none of the effects on the production of SCFAs were determined in any of these studies.is another marine microalga with high contents of both soluble and insoluble fibers, and it is promising as a prebiotic since the numbers of LAB increased in the feces of rats treated with 182 ]. Nevertheless, some more studies are necessary in order to confirm the effectiveness of this microalga on the colon histomorphology and production of organic acids ( Table 4 ).

There is also evidence that some algal PS are able to regulate the numbers of altered microbiota in mice [ 29 166 ]. Even so, these studies on the “selectivity of growth and/or activation of one species or a certain group of colon microorganisms” are scarce. Most of the research performed only evaluated the health benefits of the microflora in general.

For some of the benefits to be effective at the cell level, poly/oligosaccharides must present in specific glycan sequences that will match the respective receptors in the cells. This is related to the flexibility and rotation/torsion capacity of the molecules around their anomeric link. This flexibility is influenced by the substituents and the solvents, however [ 26 190 ].

191,192, The prebiotic properties provided by seaweeds and marine microalgae should not be restricted to their PS and lignin, but should rather be extended to monosaccharides, enzymes, polyunsaturated fatty acids (PUFAs), peptides, polyphenols, and alcohols, as it was demonstrated for similar compounds from other origins [ 35 193 ].

In the near future, the possibility of using PS from marine algae or oligosaccharides resultant thereof, through several degrading techniques, to modulate the microbiome, and, consequently, to prevent diseases is foreseen. These techniques may include new enzymes from bacteria and mollusks from marine origin.