Potential Use of Marine Seaweeds as Prebiotics: A Review

Human gut microbiota plays an important role in several metabolic processes and human diseases. Various dietary factors, including complex carbohydrates, such as polysaccharides, provide abundant nutrients and substrates for microbial metabolism in the gut, affecting the members and their functionality. Nowadays, the main sources of complex carbohydrates destined for human consumption are terrestrial plants. However, fresh water is an increasingly scarce commodity and world agricultural productivity is in a persistent decline, thus demanding the exploration of other sources of complex carbohydrates. As an interesting option, marine seaweeds show rapid growth and do not require arable land, fresh water or fertilizers. The present review offers an objective perspective of the current knowledge surrounding the impacts of seaweeds and their derived polysaccharides on the human microbiome and the profound need for more in-depth investigations into this topic. Animal experiments and in vitro colonic-simulating trials investigating the effects of seaweed ingestion on human gut microbiota are discussed.


Introduction
Marine seaweeds have been consumed whole by East Asian populations for centuries, if not millennia, appearing in traditional recipe books in many countries [1]. Additionally, their human consumption in Western countries has been increasing in the latest decades because of their association with improved human health. Some benefits of their consumption include a lower incidence of cancers, decreased blood pressure and blood sugar, and antiviral, anti-inflammatory, immunomodulatory or neuroprotective activities [1,2]. A mechanistic link proposed to explain the prevention of such diseases by seaweed consumption implicates the presence of diverse health-promoting bioactive compounds in seaweeds, including sulphated polysaccharides, polyphenols, pigments (chlorophylls, fucoxanthins, phycobilins), carotenoids, omega-3 fatty acids or mycosporine-like amino acids [1,3,4]. In some cases, these compounds are not produced by terrestrial plants or are not consumed in adequate quantities as part of a typical Western diet.
Nowadays, with a continuously expanding world population, fresh water is an increasingly scarce commodity, and the world's desertification processes continue. About 45% of the world's land surface is considered drylands, while 12 million hectares of land are degraded yearly through lack of water and related processes [5]. According to the Food and Agriculture Organization of the United Nations [6], agricultural productivity is persistently declining at over 1% per year. It is thus reasonable to expect that in the next decades there will be a need for increasing algae production to replace or supplement microbiota in animal models and showed the ability to increase the population of beneficial microbiota and maintain the homeostasis of the GM [15,22].
With respect to the antioxidant activity of seaweeds, this protective affects depends mainly on phlorotannins, secondary metabolites that exert their great antioxidant activity via the scavenging of reactive oxygen species [19]. Secondary metabolites of seaweeds are also responsible for the anti-cancer activity, whose includes different mechanisms such as repairing the intestinal barrier by intensifying the expression of the tight junction proteins via increasing the phosphorylation of MAPK and ERKT/2 genes [18], and activating the caspase cascades. Other potential mechanisms are reducing the expression of cyclin-dependent kinases and matrix metalloprotease family [17] and inducing decreased levels of pro-apoptotic metabolic signals [19].

Polysaccharides from Marine Seaweeds
Indigestible dietary polysaccharides attract attention as functional food ingredients with health benefits [7]. Most carbohydrates entering the colon are fermented in the proximal colon, which is considered a saccharolytic environment. As digesta moves through the distal colon, carbohydrate availability decreases, and proteins and amino acids become the main metabolic energy sources for bacteria in this region. The main end-products of saccharolytic fermentation are short chain fatty acids (SCFA), which contribute towards the host's daily energy requirements. On the contrary, the end-products of proteolytic fermentation include metabolites such as phenolic compounds, and nitrogenous ones like amines and ammonia, some of which are carcinogens. This means that the GM exerts a key contribution to the human energy balance and nutrition, by extending the host metabolic capacity to indigestible polysaccharides. In addition, intestinal microorganisms contribute to develop and maintain the host immune system, defending the host from colonization by opportunistic pathogens [2]. The effects of polysaccharides on the GM are generally evaluated by the contents of SCFAs, the composition and the abundance of beneficial intestinal bacteria [20].
Polysaccharides in today's human diet originate primarily from terrestrial plant cell walls, while other sources, such as seaweeds, are less represented [8]. Studies indicate that polysaccharides and oligosaccharides derived from seaweeds can modulate intestinal metabolism, including fermentation, inhibit pathogen adhesion and evasion, and potentially treat inflammatory bowel disease [8,23]. Some seaweed polysaccharides also demonstrate anticoagulant [24], antitumor [25], anti-inflammatory [26], antiviral, antihyperlipidemic [27] or antioxidant activity [28]. Other research has focused on their use as prebiotics to aid in limiting the occurrence of non-transmissible chronic diseases common in Western countries, such as obesity, diabetes, cardiovascular diseases or some types of cancer [2]. Nevertheless, many seaweed fibres are high-molecular-weight polymers that need to be transformed into oligosaccharides to increase their fermentability by the GM [2].
Depending on the taxonomic classification of algae, polysaccharides can vary greatly in their composition [8]. Seaweeds feature an integrated network of biopolymers in their cell walls, mainly formed by polysaccharides associated with other compounds, such as proteins, proteoglycans, polyphenols and some mineral elements, like calcium and potassium [8]. It is because of this complexity that for most seaweed polysaccharides, the exact structures, constituents and chemistry are not fully known [29]. Depending on the algal taxa, both structural and storage polysaccharides may vary. Structural polysaccharides are the most abundant, and their composition can be influenced by the seaweed species [29], as well as environmental factors, such as salinity, water temperature and sunlight intensity [30]. Some of the structural polysaccharides are carboxylated or sulphated, which can affect their fermentability [2].
Green seaweeds contain mostly sulphated structural polysaccharides, like ulvans (the most abundant, representing 8-29% of dry weight) and sulphated galactans, xylans and mannans. These polymers are composed mainly of rhamnose, xylose, glucose, glucuronic acid and sulphates, with smaller amounts of mannose, arabinose and galactose [31,32]. These polysaccharides are not fully fermented by the human GM [33,34]. Conversely, the main carbohydrate in storage is starch.
Through a long time of co-evolution between GM and host, the intestinal microbes have evolved diverse strategies for degrading polysaccharides from terrestrial plants [38]. However, because the consumption of seaweeds polysaccharides was not common over human evolution, the human GM did not acquire the same efficacy to degrade seaweed polysaccharides. Thus, although humans possess the enzymes necessaries to degrade some algal polysaccharides, such as starches, they are unable to digest the most complex polysaccharides [2,38]. An elegant work carried out by Hehemann et al. [39], showed that specific genes coding for enzymes with potential capacity to degrade seaweed polysaccharides, as porphyranases or agarases, can be transferred from a member of marine Bacteroidetes, Zobellia galactanivorans to the GM bacterium Bacteroides plebeius in Japanese individuals. As a consequence, GM of those subjects acquires the ability to degrade porphyran and agarose, as compared to the GM from North American individuals, who are incapable of degrading it [39].
De Jesus Raposo et al. [40] suggested that most seaweed polysaccharides can be regarded as dietary fibre, as they are resistant to digestion by enzymes present in the human gastrointestinal tract, reaching the distal gut. In this allocation, polysaccharides are fermented and become food for the commensal bacteria, stimulating their growth. For this reason, great efforts have been placed on developing efficient methods for seaweed polysaccharides extraction, purification and structural characteristics elucidation in order to improve their bioavailability, especially for insoluble fibre [2].
It was previously shown that variations in the chemical structure of a prebiotic can impact its selective fermentation by bacteria [41]. For this reason, there were published large works regarding the investigation of the potential prebiotic effect of single polysaccharides, often contained in seaweeds, but employing pure standards [42][43][44]. However, it should be considered that seaweeds contain other components than also can affect GM. Consequently, trials employing whole seaweeds are required to investigate their real effect in human GM. Furthermore, although several works investigated the effects of seaweeds in livestock gut microbiota, they are more oriented to study the effects of seaweeds in animal production and welfare. An elegant review was recently published where they can be checked [32].

Other Bioactive Compounds from Marine Seaweeds
In addition to polysaccharides, seaweeds also contain other bioactive compounds, called secondary metabolites much of them with antioxidant activity [45]. Among these, polyoletides (such as phlorotannins), isoprenoids (such as terpenes, carotenoids and steroids), alkaloids and shkimates (such as flavonoids) are the main groups of secondary metabolites found in algae [46]. Compared with other macroalgae, red seaweeds are richer sources of these secondary metabolites [47]. The human health benefits afforded by these bioactive compounds include anti-inflammatory, antioxidant, anticoagulant, antiviral, antimicrobial, antidiabetic, antitumor, antihypertensive, antiallergic and immunomodulatory activities [45][46][47][48].
Exceptionally, phlorotannins or polyphenols are recognized as structural classes of polyketides, found primarily in brown algae. These compounds can also reach the large intestine where GM can convert them into beneficial bioactive metabolites. Phlorotannins are highly hydrophilic components formed by polymerization of monomeric units of phloroglucinol (1,3,5-trihydroxybenzene). There are six main groups: fucols, floretoles, fucofloretols, fuhalols, isofuhalols and eckols, and all display strong antioxidant properties and act against oxidative stress [46]. Certain polyphenols are used as prophylactics against problems such as cardiovascular diseases, cancers, arthritis and autoimmune disorders [47]. In addition, some phlorotannins have been shown to decrease blood glucose levels after carbohydrate-rich meals. This action is achieved by interfering with the enzymes amylase and sucrase that intervene in the digestion and assimilation of these carbohydrates. In addition to their effects on the metabolic functions of the host, phlorotannins also appear to have some antibacterial activities [47], which may explain the low production of AGCC derived from seaweeds in which they are an important part [7]. However, like other phenolic compounds, bacterial growth inhibition occurs selectively in microbial populations, including some pathogens, and its antibacterial effect is minor in commensal bacteria [7]. Because of this selective inhibition of bacterial pathogens, large whole seaweeds and seaweeds ethanolic extracts has been used to extend the shelf life of fresh fishery foods, such as Fucus spiralis [48,49], Bifurcaria bifurcata [50], Cytoseira compressa [51] or Gracilaria verrucosa [52] Another of its potentially therapeutic functions is that extracts with high phlorotannins content have demonstrated a potent inhibitory action on the growth of cancerous cell lines [53,54].
Bromophenols present in marine algae have attracted much attention in the field of antimicrobial agents [55]. Previous studies indicate that marine bromophenols possess promising antibacterial [56,57] and antiviral activities [58]. In addition, symphyocladin G, a new bromophenol adduct derived from the red seaweed Symphyocladia latiuscula, is found to have antifungal activity against Candida albicans [59]. Several bromophenols isolated from the red alga Odonthalia corymbifera, are promising candidates for antifungal agents in crop protection [56]. These properties are not exclusive to red algae because compounds, such as bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, isolated from brown algae Leathesia nana, showed cytotoxic activity against some cancer cells [54], and exhibited antibacterial activity against several strains of Gram-positive and Gram-negative bacteria [56].
With respect to terpenes, there are about 200 different diterpenoids, of which some have important cytotoxic and antiviral, antimicrobial and antiparasitic activities (such as against Leishmania) [60,61]. These compounds are found in red and brown algae.
Flavonoids and their glycosides are present in green, brown and red algae. These compounds possess antioxidant properties and have demonstrated action against arteriosclerosis and cancer [45]. Within this group, fucoxanthin, β-carotene and violaxanthin stand out. Besides its strong anticancer activity, fucoxanthin has promise in preventing obesity [62]. The correlation between a carotenoid-rich diet and a low risk of cardiovascular and ophthalmological diseases has been supported by recent research with different types of carotenoids in cellular systems and human intervention studies [63]. Specifically, flavonoids from Enteromorpha prolifera influenced the GM balance in diabetic mice, increasing the presence of Alistipes, Lachnospiraceae and Odoribacter genera [63]. Alistipes spp. is one of the most abundant bacterial genera in the mouse intestine and is capable of fermenting glucose and lactic acid to produce propionic, acetic and succinic acid, which modulate the release of intestinal hormones, thereby influencing the release of insulin and appetite. It is perhaps for this reason that E. prolifera is traditionally used in China as a natural herb to treat diseases associated with inflammation [63]. It has recently been observed that a polysaccharide of E. prolifera could be used as a novel agent to treat obesity and hyperlipidaemia [62].
Other important secondary metabolites contained in seaweeds and responsible of important beneficial effect in human health are peptides, such as lectins [48]. Lectins primarily show antiviral, antibacterial, and antifungal activities. Specially one type of lectin, griffithsin, showed important antiviral activity and is nowadays considered a promise antiviral agent, with great potential concerning the prevention of sexually transmitted infections [48], including HIV [64]. Other important peptides are renin inhibitor tridecapeptide [65] and dipeptide [66], which demonstrated hypotensive effect dipeptide. Phycoerythrin [43] and kahalalide F [67] are other important peptidic compounds isolated from seaweeds than showed antitumor effect.
Besides the so-called secondary metabolites, seaweeds contain other minor nutrients of immense importance for human health. Phycobiliproteins, responsible for the characteristic bright pink appearance of red algae, are classified into phycoerythrin (red) and phycocyanin (blue). These pigments are used commercially in food, nutraceuticals, and for their therapeutic properties, mainly antimicrobial, antioxidant, anti-inflammatory, neuroprotective, hepatoprotective, immuno-modulatory and anticancer effects [67][68][69][70][71][72]. Such compounds may improve the efficacy of standard anticancer drugs, decrease their side effects, and act as photosensitisers for the treatment of tumour cells [30].

Effects of Seaweed Polysaccharides on Human Health
Compounds with prebiotic activity, such as oligosaccharides, lactulose, fructo-oligosaccharides (FOS), inulin, galacto-oligosaccharides and arabinoxylano-saccharides are used as functional ingredients in the food industry [10]. While most of the above compounds are now derived from terrestrial plants, some studies have shown that polysaccharides and oligosaccharides derived from marine algae can also modulate intestinal metabolism, including fermentation, inhibit adhesion and invasion of pathogens, and treat inflammatory bowel disease [23,73]. Furthermore, these compounds have demonstrated anticoagulant, antioxidant, immunomodulatory, antitumor and antiviral activities [10].
Being much less degradable by enzymes from the human upper gastrointestinal tract than their terrestrial plant counterparts, polysaccharides from marine algae reach a greater proportion in the descending colon. For this reason, some authors [42,74] have found that polysaccharides from marine algae, such as alginate, agarose oligosaccharides and κ-carrageenan oligosaccharides, have a higher prebiotic activity than FOS in vitro. Specifically, sulphated polysaccharides from marine algae show anticoagulant, antiviral, antitumor, anti-inflammatory, antibacterial, immunological, antioxidant and many other biological and physiological activities [8,75]. Sulphated polysaccharides include fucoidans (l-fucose and sulphated ester groups) from brown seaweeds, agars and carrageenans (sulphated galactans) from red seaweeds, and ulvans (sulphated glucuronoxylorhamnan) and other sulphated glycans from green seaweeds [8].
The consumption of these sulphated polysaccharides can block the adhesion of leukocytes to the epithelium of blood vessels, preventing the migration of these cells to the site of inflammation [76]. These polysaccharides often stimulate the growth and activity of beneficial bacteria by acting as substrates for fermentation in the large intestine, leading to the production of SCFA, with multiple functions that help maintain health [8]. As previously mentioned, seaweed polysaccharides differ in their properties and compositions from one type of algae to another [46], so their effects on the human GM will also differ.
The GM, especially in its most distal parts, harbors many bacteria, archaea, protozoa and viruses, which along with their genetic material, is collectively referred to as the gut microbiome (GMB) [77]. This GM is composed of up to 12 different bacterial phyla of which more than 90% belong to the Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes [78], while the remaining phyla are much less constant and numerous [79]. The most frequent bacterial species in the colon, which is where the highest bacterial concentration exists [78], belong mainly to the families Bacteroidaceae, Prevotellaceae, Rikenellaceae, Lachnospiraceae and Ruminococcaceae [77]. The GM presents a diverse set of functions important to human health, such as the extraction of energy from a broad spectrum of nutrients, the production of vitamins, the promotion of immune homeostasis and the prevention of colonization of the intestine by pathogens [80]. One of the most important functions of the GM is in the prevention of chronic low-grade inflammation [81]. Host genetics define the chemistry and physics of the GM, including the availability of nutrients and the threshold of activity required to induce an immune response. Consequently, intestinal microbial communities are composed of species that have evolved to occupy specific ecological niches in the gut, including the ability to metabolize specific molecules available from the host or to evade host defenses [77].
Nutrients can interact directly with the GM to promote or inhibit its growth. In this sense, the ability of the GM to extract energy from specific components of the diet offers a direct competitive advantage to specific members of the GM, allowing them to proliferate at the expense of other members [81]. Thus, diet affects not only the composition and absolute abundance of intestinal bacteria but also their growth kinetics [82]. In this context, the most influential nutrients are indigestible carbohydrates, which can be of both terrestrial and marine algae origin [81].
The human genome encodes a limited number of hydrolases capable of hydrolyzing the glycosidic bonds of polysaccharides in dietary fibre (collectively referred to as CAZymes). Consequently, many polysaccharides, such as resistant starch, inulin, lignin, pectin, cellulose and FOS, reach the large intestine undigested. In contrast, the GMB codes tens of thousands of CAZymes. In the presence of bacteria harboring key enzymes involved in carbohydrate metabolism, these complex polysaccharides can thus be degraded and metabolized in vivo [83]. The bacteria able to degrade these complex polysaccharides are called primary degraders and include members of the genera Bacteroides, Bifidobacterium and Ruminococcus, Roseburia, Facealibacterium, Anaerostides or Coprococcus. A relative abundance of these genera in our GM infers that during a food shortage, these bacteria can alternate between energy sources by using sensors and regulatory mechanisms that control gene expression [21,81]. Hydrolases act on polysaccharides to generate oligosaccharides and monosaccharides. Secondary fermentation of these compounds by the GM produces SCFA, specifically acetic, propionic, butyric, lactic and succinic acids, which initiate a complex metabolic network [81].
The GM of hunter-gatherer, rural and agricultural populations are usually more bacterially diverse than in modernized urban societies [84] and so require a greater functional repertoire to maximize their energy intake from dietary fibres. Conversely, the consumption of a diet composed mainly of products of animal origin causes an enrichment in the GM of genera of bile-tolerant bacteria, such as Alistipes, Bilophila and Bacteroides, and the almost total exhaustion of bacteria that metabolize polysaccharides, such as Roseburia, Eubacterium rectale subgroup and Ruminococcus bromii [81].
Clinical studies investigating prebiotic effects have some disadvantages with respect to ethical constraints, as well as limited sampling possibilities from the colon and limited measurements of in situ SCFA production. These concerns are commonly avoided by applying an in vivo approach [41], that are the most common in the investigation of seaweed effects on human GM, as is described below. Contrariwise, in vitro studies show important limitations because only represent the first step of a long process, and the results observed in vitro can be magnified, diminished, or totally different in a more complex and integrated system [48]. An additional limitation is that, due to the short fermentation time in in vitro studies, they fails to capture the complete picture of cross-feeding interactions between gut microbes, and which may not fully correlate with the long-term effects of seaweed compounds on GM [41].

Polysaccharides from Brown Seaweeds
Although the prebiotic and immuno-modulation properties of brown algae have been studied both in animal models and in vitro, humans intervention studies are also needed to assess whether there is a direct association between these uses of algae and the human GM, but are currently restricted due to ethical concerns [78]. The most relevant results obtained from examining the impacts of brown seaweeds on the GM can be found in Table 1. In this table it were included results about the prebiotic effect of brown seaweed species from genus Ecklonia [7,85], Sargassum [86][87][88], Laminaria [82,[89][90][91][92], Ascophyllum [93][94][95], Fucus [23,63], Undaria [90], Saccorhiza [96] or Porphyra [97]. As can be seem in Table 1, in most cases, the administration of whole brown seaweed or brown seaweed-extracted polysaccharides resulted in an increase of SCFA production, stimulating of beneficial bacteria grown such as Lactobacillus [7,82,85,86,95], Bifidobacterium [7,82,85,87,92] or Faecalibacterium [7,58,87]. In some cases, the brown seaweed or brown seaweed-extracted polysaccharides also inhibited the growth of potentially pathogen bacteria [73,86]. In some cases, it were reported other beneficial effects not strictly related with action on GM, such as reducing serum inflammatory markers [23], reducing serum levels of lipopolysaccharide-binding protein [44], increasing CAZymes [44], reducing activity of fecal bile salt hydrolase activity [96], or reduced the expression or diabetes-related genes [15].
Laminar storage polysaccharides, typical of brown seaweeds, are low-molecular-weight, linear polysaccharides composed of glucose units with a low degree of branching [79]. Besides affecting mucin composition and SCFA concentration, laminins can affect the adherence, translocation and proliferation of bacteria in the gut [98,99]. At the same time, laminins stimulate the proportion of Bifidobacterium, which generates a prebiotic potential. In other research, laminarin has been shown to promote an immune response [98], and could be useful for inhibiting the production of putrefactive substances from undigested proteins [100]. In vitro batch fermentation of laminarin for 24 h promoted an increase in Bifidobacterium and Bacteroides, and propionate and butyrate production [42]. Contradicting results by other researchers indicated that laminarin was not selectively fermented by Lactobacillus and Bifidobacterium, but could modify the composition, secretion and metabolism of the jejunal, ileal, caecal and colonic mucosa to protect against bacterial translocation [32]. In addition, laminarin increased the presence of Clostridium spp. and Parabacteroides distasonis in rats [101].
An in vitro study conducted with the species Sargassum thunbergii revealed a dramatic increase in the population of beneficial bacteria (from 17% to 28%), while a group of harmful Firmicutes decreased from 75% to 64% after 48 h of fermentation [87]. No noticeable changes were found in Proteobacteria or Actinobacteria. At the genus level, an increase in Lactobacillus, Bifidobacterium, Roseburia, Parasutterella and Fusicatenibacter appeared after incubation for 24 h, followed by an increase in Faecalibacterium and Coprococcus at 48 h of incubation [87]. Bifidobacterium, Coprococcus and Parasutterella have been negatively correlated with non-alcoholic steatohepatitis, hepatocellular carcinoma and diabetes [102], while Ruminococcus, Roseburia and Faecalibacterium are producers of butyric acid and are facilitate the degradation of polysaccharides and fibres [103]. Fusicatenibacter was positively associated with increased serum leptin in obese rats [104], which reduces their appetite. All these findings highlight the prebiotic potential of S. thunbergii by its modulation of the composition and abundance of beneficial GM.
An in vitro study using S. wightii in MRS broth evaluated their antioxidant activity and prebiotic score comparing L. plantarum and Salmonella Typhimurium relative growths. The study showed that the prebiotic activity score was positive, promoting selectively the growth of L. plantarum with respect to the pathogen S. Typhimurium. Specifically, a prebiotic effect by 1.42-fold more growth stimulation of L. plantarum than S. Typhymurium [86].
In other work Chen et al. [58] showed an increase in fucoidan from A. nodosum in an in vitro assay simulating the human digestive tract was due to an increase in Bacteroidetes, Firmicutes and SCFA. At the genus level, the genera Bacteroides, Phascolarctobacterium, Oscillospira and Faecalibacterium increased, while the levels of Fusobacterium, Megamonas, Parabacteroides, Clostridium and Dorea decreased relative to the samples to which the algae A. nodosum had not been added [46]. demonstrated the in vitro prebiotic activity of a mixture of fucoidans and alginates obtained from A. nodosum, leading to an increase in the growth rate of L. delbrueckii and L. casei to levels similar to those observed after administration of inulin, a standard commercial prebiotic [46]. Other authors [93] conducted a study in rats, which were administered polysaccharides extracted from A. nodosum, and they were seen an increase in both acetate, propionate and butyrate SCFAs.
According to Zaporozhets et al. [76], fucoidans obtained from F. evanescens stimulate the colonic growth of beneficial Bifidobacterium species, such as B. longum B379M and B. bifidum 791B. Lean et al. [23] administered F. vesiculosus-derived fucoidan extracts to mice and, interestingly, found a reduction in markers associated with inflammatory bowel diseases.
When Wister rats were fed with feed enriched with alginates or laminarins, An et al. [101] found a notable decrease in the number of metabolites resulting from putrefaction, such as indole, H 2 S and phenol. This result was subsequently confirmed in both in vitro and rat models by Nakata et al. [100], who also found a decrease in ammonium levels with alginate. At the phyla level, alginate increased the levels of Actinobacteria, while laminarins increased the levels of Proteobacteria. At the genus level, Bacteroides was markedly more abundant in the group fed with alginate, and B. capillosus was the most frequent species. In rats fed with laminarin-enriched feed, Parabacteroides, Lachnospiraceae and Parasutterella bacterium were detected in greater abundance than in control rats. Nguyen et al. [44] studied laminarin supplementation in a mice high-fat diet. They could see a decrease in Firmicutes and an increase in the Bacteroidetes phylum, especially the genus Bacteroides.
Ramnani et al. [94] performed in vitro fermentation with A. nodosum-derived alginates, which increased Bifidobacterium and SCFAs. An increase in the proportion of Bacteroidetes to Firmicutes was observed as well in fermentations added with sulphated polysaccharides extracted from A. nodosum versus controls. Increased levels of Bacteroidetes and decreased levels of Firmicutes have been associated with a reduced risk of obesity in humans [79]. Increase in total SCFA in laminarin-added culture medium than in glucose-added. Laminarins supplementation increased the colon luminal content of mucin, while decreased luminal mucin in jejunum, ileum and caecum in rats [89] In vivo trial using 28 female Sprague-Dawley rats Supplementation with 10% of dried L. japonica for 4 weeks. Control rats were fed with basal diet

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Reduction in Firmicutes to Bacteroidetes ratio and decrease of pathogenic bacteria such as Clostridium, Escherichia and Enterobacter Increase in total SCFA, and butyric acid. Lower production of acetic acid propionic acids [90] In vivo trial using six female BALB/C mice per group Mice received normal diet, high-fat diet or high-fat diet added with laminarins at 1% w/w in a high-fat diet ad libitum for 4 weeks. After finishing, highly-fat diet was provided for an additional 2 weeks Evidence that probiotic bacteria in the gastrointestinal tract utilize dietary alginate was reviewed by Shang et al. [38]. Among the studies, Kuda et al. [73] found that supplementation with sodium alginate and laminarin of brown algae inhibited the adhesion and invasion of pathogens, such as S. Typhimurium, Listeria monocytogenes or Vibrio parahaemolyticus. Other authors reported an increase in Lactobacillus and Ruminococcus in the intestine of mice fed fucoidans from A. nodosum, besides a reduction in the opportunistic Peptococcus bacteria [95].
In vitro fermentation experiments conducted by Charoensiddhi et al. [85] demonstrated the growth-promoting effect of E. radiata extracts on beneficial bacteria, such as Bifidobacterium, Lactobacillus and Clostridium coccoides, and SCFAs production was stimulated as well. Later, the same authors [7] found increased levels of beneficial bacteria, such as Bifidobacterium, Lactobacillus and C. coccoides associated with the phlorotannin-enriched fermentation of E. radiata. Higher numbers of Lactobacillus, Faecalibacterium prausnitzii, C. coccoides, Firmicutes and E. coli were observed for phlorotannin-supplemented fermentation compared with inulin fermentation [7]. In contrast, the number of Enterococcus in both fermentations decreased approximately ten-fold relative to the initial counts.
Other authors tested the effects of supplementation of two brown algae (U. pinnatifida and L. japonica) on the GM and body status of laboratory rats [90]. In both instances, the animals' body weight was reduced, which was thought to be mediated by the influence of the seaweed on the composition of the intestinal microbial communities associated with obesity, reducing the proportion of Firmicutes with respect to Bacteroidetes, and the populations of pathogenic bacteria, such as Clostridium, Escherichia and Enterobacter [90]. Similarly, L. japonica increased beneficial bacteria and SCFA, and decreased the pH level [82], while β-glucans extracted from L. digitata increased Bifidobacterium and propionic and butyric acids in vitro, in addition to lowering pH [92].
β-Glucans obtained from another Laminaria species (L. digitata) were able in an in vitro test [92] to increase Bifidobacterium and propionic and butyric acids, in addition to lowering pH. A study by Strain et al. [91] in vitro investigated the effect of a polysaccharide-rich raw extract obtained from L. digitata. A significant alteration of the relative abundance of several families, including Lachnospiraceae and genera such as Streptococcus, Ruminococcus and Parabacteroides of human faecal bacterial populations was seen. Concentrations of acetic acid, propionic acid, butyric acid and total SCFA were significantly higher.
Finally, Huebbe et al. [96] conducted a study on mice that were administered polysaccharides from S. polyschides with a high-fat diet. A metabolic improvement was seen including normalization of blood glucose, reduction of plasma leptin, reduction of fecal bile salt hydrolase activity and secondary bile acids in these mice.

Polysaccharides from Red Seaweeds
The most relevant results obtained from the investigation of red seaweeds effect on GM can be found in Table 2 As can be seem in Table 2, as was described previously for brown seaweeds, administration of red seaweeds or seaweed-extracted polysaccharides resulted in an increase of SCFA production, stimulating of beneficial bacteria grown such as Lactobacillus [86] or Bifidobacterium [57,94,107], whereas inhibited the growth of potentially pathogen bacteria [57,86]. It was also reported red seaweeds activity on the prevention of naproxen-induced gastrointestinal damage [106].
Agarose stands out among the polysaccharides isolated from red algae that cannot be digested by human intestinal enzymes. When seaweed is consumed, whether as an edible food or food additive, agarose reaches the most distal portions of the gastrointestinal tract, where it is fermented and metabolized by the GM [108,109]. As described by Ramnani et al. [94], low-molecular-weight agarose exerted a prebiotic effect in vitro by promoting the growth of Bifidobacterium and increasing SCFA concentrations in the medium. In vivo trial using male Wistar rats (six per group) Rats were pretreated with 0.5% carboxymethylcelloluse (controls) or 0.5% w/v of of sulphated polysaccharides from Gracilaria birdiae, twice daily for 2 days. After 1 h, naproxen (80 mg/kg) was administered twice a day for 2 days Molar mass distribution was found to be within 2.6 × 10 6 and 3.8 × 10 5 g/mol, while the soluble carbohydrate, protein, and sulfate contents were 85.5%, 2.5%, and 8.4%, respectively

No relevant variation was observed in GM populations
Prevention of naproxen-induced gastrointestinal damage determined by macro-and microscopic findings [106] In  Bajury et al. [107] conducted an in vitro colon model in which they evaluated the prebiotic capacity of K. alvarezii. This study showed an increase in SCFA (particularly acetate and propionate) and Bifidobacterium. In the other hand, decrease in C. coccoides and E. rectale. These results suggested that K. alvarezii might have the potential as a prebiotic ingredient. A study published by Zhang et al. [110] focused on the beneficial effect of low-melting-point agarose (in the form of neoagaro-oligosaccharides) on the GM during the relief of intense exercise-induced fatigue in mice. Results showed the abundance of Bacteroidetes and Proteobacteria increased and decreased, respectively, during the attenuation of fatigue and its associated gastrointestinal problems. Ladirat et al. [111] found that mice fed 2.5% (w/v) neoagaro-oligosaccharides for seven consecutive days achieved a much more pronounced increase in the population of Lactobacillus spp. and Bifidobacterium spp. in their GM relative to those fed 5% (w/v) FOS for 14 consecutive days. Likewise, it was demonstrated that agaro-oligosaccharides could be used as a prebiotic to encourage the growth of beneficial strains of bacteria, such as B. adolescentis ATCC 15703 and B. infantis ATCC 15697. Low-molecular-weight agar has demonstrated a bifidogenic effect, along with an increase in SCFA acetate and propionate concentrations, after 24 h of in vitro fermentation with human faeces inoculant [94].
Another type of polysaccharide with prebiotic function found in red algae are the group of carrageenans, which are derived from D-galactose, and approved as food additives [79]. In rats fed 2.5% C. crispus, of which carrageenan is a major polysaccharide, B. brevis, as well as SCFA, increased considerably, while pathogens Clostridium septicum and Streptococcus pneumonia noticeably decreased compared with the basal diet [57]. Elevation of plasma immunoglobulin levels was also found in rats fed with C. crispus, resulting in improved host immunity. Consistent with the prebiotic activity of carrageenan, carrageenans isolated from red algae G. filicina and E-spinosum promoted the growth of Bifidobacterium [10].
Research led by Di et al. [105] found that the polysaccharides of Gracilaria rubra increased the relative abundances of Bacteroides, Prevotella and Phascolarctobacterium in vitro compared with the control group. Bacteroides spp. assists the host with degrading polysaccharides and contains codifying genes of glucosidase enzymes [39]. Prevotella is another beneficial genus with the potential to participate in the metabolism and utilization of plant polysaccharides. The genus Phascolarctobacterium is associated with the production of SCFA [110].
Many other bacterial genera, such as Legionella, Sutterella, Blautia, Holdemania, Shewanella and Agarivorans, were decreased as a consequence of intake of C. crispus supplements in rats [57]. Decreases in the presence of Streptococcus were also observed. In conclusion, carrageenans from C. crispus could act as a fermentable substrate for probiotic bacteria present in the gastrointestinal tract, thereby promoting the growth of probiotic groups, while inhibiting certain groups of pathogenic bacteria [57]. Another study in chickens described an overall impact of administering whole red algae (Sarcodiotheca gaudichaudii and C. crispus) on the intestinal mucosa, increasing the height and surface of the villi in these animals [112]. Moreover, the abundance of beneficial bacteria, such as B. longum and Streptococcus salivarius increased, while some harmful bacteria species, such as C. perfringens, decreased [112] Rodrigues et al. [88] used extracts from the red algae O. pinnatifida and S. muticum in an in vitro fermentation system, which increased the production of acetate and propionate, and the population of Bifidobacterium. In work published by Silva et al. [106], in which extracts of sulphated polysaccharides from G. birdiae were administered to laboratory rats, gastrointestinal damage induced by naproxen was prevented, although it did not produce notable variations in the GM of these rats.
An in vitro study using A. spicifera in MRS broth evaluated their antioxidant activity and prebiotic score with L. plantarum and S. Typhimurium. The study showed that the prebiotic activity score was positive, promoting the growth of L. plantarum and suppressing the growth of the pathogen S. Typhimurium. Specifically, a prebiotic effect by 0.84-fold more growth stimulation of L. plantarum than S. Typhymurium [86].

Polysaccharides from Green Seaweeds
Unlike brown and red algae, the current evidence for the fermentation capacity of green algae and their polysaccharides is scarce, partly because their fermentation requires a specific activity of α-l-rhamnosidase in the gastrointestinal tract, which is infrequent [113]. The most relevant results obtained from the investigation of green seaweeds effect on GM can be found in Table 3. In this table, results about the prebiotic effect of green seaweed species from genus Enteromorpha [38,82,86,[113][114][115] and Ulva [115] were included. Administration of green seaweeds or seaweed-extracted polysaccharides also resulted in an increase of SCFA production, stimulating of beneficial bacteria grown such as Lactobacillus [38,86,115], Bifidobacterium [38], or Akkermansia [38] whereas inhibited the growth of potentially pathogen bacteria [81,114]. Other beneficial actions were reported such as decrease lipopolysaccharide-binding protein in female mice [38], diminished histopathological lesions of inflammatory infiltrations in distal colon [114], or modulating diabetes-related genes expression in diabetic mice [22].
Ulvans are one of the most frequent polysaccharides in green algae. This polysaccharide is a water-soluble sulphated heteropolysaccharide [79]. Ulvans contains sulphate and uronic acids, and so produce undigestible ionic colloids, has ion-exchange capacity and can bind to bile acids, consequently increasing the excretion of bile acids with cholesterol-lowering or antihyperlipidemic effects [2,79]. Antioxidant and immunomodulatory properties are other beneficial actions elicited by ulvans [116,117].
Ulvans has also been studied for its possible prebiotic potential. Kong et al. [82] performed an in vitro assay using Enteromorpha with a high content of ulvans, but there were no noticeable variations in the populations of Enterococcus, Lactobacillus and Bifidobacterium compared with controls. In contrast, in a recent in vitro faecal fermentation analysis, ulvans stimulated the growth of Bifidobacterium and Lactobacillus populations and promoted the production of SCFA, such as lactic and acetic acids [42]. In a previous study by Ren et al. [114], both whole Enteromorpha and polysaccharides extracted from Enteromorpha improved inflammation associated with loperamide-induced constipation in mice. In those mice, the GM showed an increase in Firmicutes and Actinobacteria compared with the control mice, whereas the relative amounts of Bacteroidetes and Proteobacteria decreased.
In work by Shang et al. [38], an extract of E. clathrata was administered to mice, resulting in marked decreased concentrations of genera, such as Enterobacter, Staphylococcus and Streptococcus. Surprisingly, such supplementation also dramatically reduced the population of A. muciniphila in the intestine. These observations indicate a possible unfavorable effect of these polysaccharides on the GM. Contrarily, these polysaccharides were reported to increase the abundance of A. muciniphila, Bacteroides, Alloprevotella, Ruminococcaceae and Blautia in the intestinal tract of mice, and decrease the abundance of Peptococcus, Rikenellaceae and Alistipes [96,109].
An in vitro faecal fermentation of xylans derived from Palmaria palmata reported that xylose was fermented after 6 h, and the SCFA content increased simultaneously [32]. This study did not determine the bacterial composition. Nonetheless, Xylans and xylo-oligosaccharides extracted from terrestrial plants, such as wheat husks and corn, are considered potential prebiotics due to evidence of bifidogenesis, improved plasma lipid profile and positive modulation of immune function markers in healthy adults [118]. Not provided [115] In vitro fermentation system using fresh fecal samples from three healthy donors 0.8 g of fucoidans obtained from Enteromorpha prolifera for 48 h. Blank samples contained no polysaccharide

Not provided
Decrease in Enterobacter spp. in E. prolifera fucoidans-added samples Not significant changes [82] In vivo trial using 24 Kunming female mice Loperamide at a dosage of 9.6 mg/kg/twice a day via oral gavage for 2 weeks was provided to mice to induce slow-transit constipation in mice. Afterwards, E. prolifera and polysaccharides extracted from E. prolifera added in fed at a 1:5 w/v ratio was administered for 7 days Not provided E. prolifera increased bacterial diversity, Bacteroidales, Firmicutes, Actinobacteria, and decreased Bacteroidetes and Proteobacteria. Extracts from E. prolifera increased Prevotellaceae, Firmicutes, Actinobacteria, and decreased Bacteroidetes and Proteobacteria Both E. prolifera and E. prolifera extracts diminished histopathological lesions of inflammatory infiltrations in distal colon. Both E. prolifera and E. prolifera extracts reduced serum levels of nitric oxide (inhibitory neurotransmitter) and showed laxative effects [114] In vivo trial using 24 Kunming male mice Treated mice were fed with high sucrose/high fat diet for 5 weeks. Next type-2 diabetes was induced by intraperitoneal administration of streptozotocin at 45 mg/kg for 3 days.
Diabetic mice were administered with 150 mg/kg E. prolifera extracts or its flavonoid-rich fractions less than 3 kDa, respectively, for 4 weeks Not provided E. prolifera extracts increased the proportion of Alistipes, Lachnospiraceae and Odoribacter, while both extracts reduced the proportion of Ruminiclostridium and Akkermansia in GM of diabetic mice Flavonoids from E. prolifera reduced blood glucose in mice, reduced mRNA expressions of JNK1/2 gene and increased the expression of PI3K, IRS1 and AKT genes in diabetic mice [22] Gal: galactose; GlcA: glucuronic acid; Glc: glucose; GM: gut microbiota; kDa: kilodaltons; Man: mannose; MRS: man Rogosa and Sharpe; MTCC: Microbial Type Culture Collection and Gene Bank; NCIM: National Centre of Integrative Medicine; Rha: rhamnose; SCFA: Short chain fatty acids; Xyl: Xylose. E. clathrata is an edible green seaweed possessing polysaccharides with numerous bioactivities, including anticoagulant, immunomodulatory, antioxidant, anticancer and anti-obesity effects [38]. It was reported that the polysaccharides of E. clathrata exerted diverse prebiotic effects on A. muciniphila, Bifidobacterium and Lactobacillus in male and female mice [38]. The results were most evident in the male mice because of a sex-specific effect on the GM, as sex hormones play a key role in determining the composition of intestinal microorganisms [109]. In other work from the same authors, male mice were supplemented with polysaccharides of E. clathrata in the diet, which increased the abundance of Bacteroides, Prevotella, Alloprevotella, Eubacterium and Peptococcus, and decreased the proportion of the cancer-related Helicobacter [109]. In the female counterparts, the abundance of Odoribacter, Clostridium IV, Oscillibacter and Alistipes spp. increased, and the proportions of beta-proteobacteria decreased [38].
An in vitro study using E. compressa in MRS broth evaluated their antioxidant activity and prebiotic score with L. plantarum and S. Typhimurium. The study showed that the prebiotic activity score was positive, promoting the growth of L. plantarum and suppressing the growth of the pathogen S. Typhimurium. This seaweed exhibited the highest score of prebiotic activity (1.44-fold), stimulating the growth of L. plantarum than S. typhimurium [86].
The natural products of marine macroalgae have shown notable antidiabetic potential by interfering with carbohydrate metabolism. For example, E. prolifera contains many bioactive compounds, such as sulphated polysaccharides, which could improve glucose metabolism, in addition to displaying anti-inflammatory, antiviral and anticoagulant functions [22].

Conclusions
Although substantial evidence of the prebiotic effect of seaweed and seaweed extracts has been published in recent years, these studies have been performed using in vitro digestion systems simulating the human colon, or in animal models. Animals, such as mice or rats, differ widely from humans in the GM composition, immune function, diets, metabolism and other key aspects, so extrapolating the results obtained from animal models to humans may not be valid. In vitro systems replicate more similarly the human intestinal microbiota, but are less-dynamic systems than the real human colonic environment. Additionally, other factors should be considered, such as the possible effect of other secondary compounds contained in seaweeds on the GM composition, or the potential to transfer genes from marine bacteria to human GM bacteria coding for enzymes that could degrade seaweed polysaccharides. Thus, not all people will respond equally after seaweed ingestion. The decrease in terrestrial agriculture and disposable water is likely to increase the consumption of algae by humans in the near future. Meanwhile, there is a profound need for more in-depth investigations into the potential prebiotic effects of marine seaweeds and their derived polysaccharides on the human GM.