Bioactive peptides and carbohydrates from seaweed for food applications: Natural occurrence, isolation, purification, and identification

Abstract Macroalgae or seaweed are relative un-explored and promising sources of novel molecules for the food industry including peptides and carbohydrates for their use as functional foods and nutraceuticals. Several algae-derived bioactive compounds have shown a wide range of biological activities both in vitro and in vivo, i.e. antihypertensive and antioxidant, that are strongly associated with the chemical structure of the peptides or carbohydrates. Multiple improvements in the purification and analytical tools to characterize these compounds have been reported in recent years, aiming to gain further insight into the complexity of different molecular structures of bioactive peptides and carbohydrates. This paper discusses the variable composition of algae and the opportunities of the use of this biomass to obtain novel functional bioactive peptides and carbohydrates for functional food applications. The main biological activities of the discovered bioactive peptides and carbohydrates together with the analytical procedures used to purify and characterize multiple compounds are also discussed.


Introduction
Traditional food ingredients have gained increased scientific attention for their potential to generate functional foods and nutraceuticals. Functional foods are defined as foods that may impart a health benefit to the consumer that goes above and beyond basic human nutrition [1]. Several successful examples in the market include the use of fortified products with omega-3 polyunsaturated fatty acids of algal origin [1] and beverages containing bioactive peptides such as Evolus® or Calpis® [2].
Algae are a rich source of multiple valuable macro-and micro-nutrients, including proteins, carbohydrates, phenols, vitamins, and minerals [1,3]. These compounds were traditionally exploited by the food and animal feed industries due to their nutritional value, i.e. as a source of proteins rich in essential amino acids [4] or the use of non-digestible carbohydrates from seaweed as a source of dietary fibre [5]. This rich and variable composition of algae is being explored for its potential to obtain functional ingredients including bioactive peptides and carbohydrates [1,[5][6][7][8]. Bioactive peptides are sequences of 2 to 30 amino acids in length that display hormone-like beneficial properties when released from their parent protein [9,10]. Biological effects described to date include antihypertensive, antioxidant, antithrombotic, antimicrobial, and immunomodulatory properties [1,5]. Currently there are a number of products in the market containing bioactive peptides, including a peptide soup containing the peptides LKPNM obtained from dried bonito and a sardine-derived product rich in the di-peptide VY [1]. Moreover, algal carbohydrates such as alginates or carrageenan are industrially commercialized as thickening and gelling agents with multiple applications in the food, textile, biotechnological, and biomedical industries [11]. These marine carbohydrates have recently come under the spotlight as functional food ingredients [5]. Seaweed carbohydrates such as alginates, carrageenan, and fucoidan showed a wide range of biological activities including antiinflammatory, anticoagulant, antioxidant, antiproliferative, and immunostimulatory activities both "in vitro" and/or "in vivo" [5]. Furthermore, algal polysaccharides such as laminarin are non-digested in the upper gastrointestinal tract and are considered as dietary fibre [5]. The consumption of dietary fibre has a positive influence on human health with beneficial impacts including a reduced risk of suffering from colon cancer and constipation, but also reduced hypercholesterolemia, obesity, and diabetes [12].
The biological effects displayed by these marine bioactive peptides and carbohydrates depend on the chemical structure of these molecules [1,13]. These differences could be attributed to biological factors affecting the seaweed biomass (i.e. season and location of collection), but extraction and purification [5,14]. The increased interest in developing functional bioactive peptides and polysaccharides represents a challenge for analytical scientist trying to identify the relationship between the chemical structure of these compounds and their biological activities. Multiple analytical approaches have been used to date to purify and analyse the chemical structure of bioactive peptides and carbohydrates [5,15]. This paper discusses the variable composition of algae and the opportunities of the use macroalgae to obtain novel functional bioactive peptides and carbohydrates for functional food applications. The main biological activities of the discovered bioactive peptides and carbohydrates together with the analytical procedures used to purify and characterize multiple compounds are also discussed.

Chemistry and biochemistry of macroalgae
Macroalgae or seaweed comprise a heterogeneous group of approximately 10,000 species [16], being only few of them used for food applications, mainly as food additives or flavouring materials, especially in Asian countries. Indeed, seaweed is served in approximately 21% of meals in Japan [17] and some eastern varieties are increasingly consumed in western countries since oriental food became more popular.
Algae can be divided into three main groups or phyla: brown (Phaeophyceae), red (Rhodophyceae), and green (Chlorophyceae). Moisture content of fresh marine algae is very high and can account for up to 94% of the biomass [18]. In addition, algae have a highly variable composition, with large differences in their final content in minerals (including calcium, phosphorus, and potassium), vitamins, proteins, lipids, and fibre [8]. Seaweed composition, i.e. protein and carbohydrate contents, depends not only on the species, but also on the time of collection and habitat, and on external conditions such as temperature, light intensity, and nutrient concentration in water [16]. As mentioned previously, the protein content of seaweed differs depending on different factors. In addition, comparison of the protein content among algae is difficult because of methodological differences, especially during protein extraction, and the large number of species identified to date [19]. Although brown seaweed usually contains a low protein content when compared to that of green or red seaweed, Lourenço, Barbarino, De-Paula, Pereira and Marquez [19] reported a relatively high protein content ranging between 10 and 15% for the species Chnoospora minima, Dictyota menstrualis, Padina gymnospora, and Sargassum vulgare. Common brown seaweed includes Laminaria digitata, Ascophyllum nodosum, Fucus vesiculosus, and Himanthalia elongata. The average protein content of Laminaria digitata was recently calculated as 6.8%, with highest and lowest protein levels in the first and third quarter of the year, respectively [20]. This value was similar to that obtained by Peinado, Girón, Koutsidis and Ames [21], who calculated the protein content of Laminaria digitata as 5.8%. Comparable results were observed in this study for other brown algae species such as Laminaria hyperborea, Saccharina latissima, and Alaria esculenta with average protein levels ranging between 6.8 and 11.0% of the total dry weight (DW) [20] and Ascophyllum nodosum, Pelvetia canaliculata, Fucus vesiculosus, and Fucus spiralis which presented an average protein content of 5.2, 7.3, 5.8, and 5.9%, respectively [21].
Although previous studies highlighted that in some green seaweed, such as those species belonging to the genus Ulva, the protein content can range between 10 and 26% of the total DW [22], a more recent study reported that the protein content of Ulva lactuca, harvested between May and June, was below 8.6% [23]. Maehre, Malde, Eilertsen and Elvevoll [23] obtained similar results from other green algae such as Cladophora rupestris and Enteromorpha intestinalis, which were also harvested between May and June in Scotland, and had a total protein content of 3.4 and 11.3%, respectively. In addition, Lourenço, Barbarino, De-Paula, Pereira and Marquez [19] calculated the protein content of the green algae Caulerpa fastigiata, Caulerpa racemosa, Codium decorticatum, Codium spongiosum, Codium taylorii, and Ulva fasciata, collected in Brazil in autumn, and results suggested that the protein content varied between 11 and 20%, depending on the species.
Numerous seaweed-derived proteins have been identified, and these can be used for the generation of biologically active peptides with health-promoting properties. However, among the different classes of proteins identified, it is important to emphasize lectins and phycobiliproteins. Lectins are glycoproteins with carbohydrate-binding properties which allows them to agglutinate microbes, yeasts, tumour cells, and erythrocytes [25]. Lectins are very interesting for a diversity of applications in immunological and histochemical studies [26] as well as in agricultural and medical applications due to their antimicrobial, antitumor, and antiviral activities [27]. In addition, phycobiliproteins are brilliantly coloured and highly fluorescent components which are currently used in numerous fluorescence-based techniques such as immunofluorescence, fluorescence-activated cell sorting, and fluorimetric microplate assays [28].

Amino acid composition
The amino acid composition of seaweed proteins has been repeatedly studied and compared to that of other foods. Most seaweed contain all the essential amino acids, and aspartic and glutamic acids were suggested to constitute a large part of their amino acid fraction [22]. Indeed, these two residues represent 22 and 44% of the total amino acids in certain brown seaweed [29]. Brown seaweed have been suggested as rich sources of threonine, valine, leucine, lysine, glycine, and alanine previously, and amino acids such as cysteine, methionine, histidine, tryptophan, and tyrosine were recorded at lower levels [18]. Lourenço, Barbarino, De-Paula, Pereira and Marquez [19] studied the amino acid profile of four brown seaweed species namely Chnoospora minima, Dictyota menstrualis, Padina gymnospora, and Sargassum vulgare. All samples were collected in Brazil in June and September, and showed a high content of aspartic and glutamic acid, and a relatively high (over 8%) content of leucine.
The green seaweed Ulva pertusa contains high levels of proline [30], and the amino acid profile of the green seaweed Ulva lactuca revealed that this specie contained all the essential amino acids, in levels which were comparable to the dietary recommendations proposed by the Food and Agriculture Organisation of the United Nations (FAO) and the World Health Organisation (WHO) [31]. Lourenço, Barbarino, De-Paula, Pereira and Marquez [19] reported high concentrations of aspartic and glutamic acids in six species of green algae, namely Caulerpa fastigiata, Caulerpa racemosa, Codium decorticatum, Codium spongiosum, Codium taylorii, and Ulva fasciata. The authors also recorded a relatively T. Lafarga, et al.
Algal Research 48 (2020) 101909 high content of the residues leucine and alanine for all the studied species. In addition, leucine, threonine, isoleucine, valine, and methionine are well represented in the essential amino acid fraction of Palmaria palmata, with similar leucine and valine concentrations to those generally reported for ovalbumin, and similar isoleucine and threonine contents to those recorded for leguminous proteins [22]. The amino acid profile of the red seaweed Hypnea charoides and Hypnea japonica revealed that both species contained all the essential amino acids (excluding tryptophan), which accounted for approximately 42-48% of the total amino acid content [31]. Red seaweed proteins also contain high quantities of glutamic and aspartic acid. However, previous studies suggested that the quantities of glutamic and aspartic acid in red seaweed were significantly lower than those observed in brown and green seaweed [32]. Lourenço, Barbarino, De-Paula, Pereira and Marquez [19] reported a concentration of aspartic and glutamic acid ranging between 10 and 15% in numerous red algae species including Acanthophora spicifera, Aglaothamnion uruguayense, Cryptonemia seminervis, Gracilariopsis tenuifrons, Laurencia flagellifera, and Porphyria acanthophora. Relatively high concentrations (over 7%) of leucine, valine, and glycine were recorded for the red seaweed Porphyria tenra [30], and concentrations of approximately 10% of alanine, threonine, proline, and histidine were reported for Acanthophora delillii [33]. Similar results were obtained for the red algae Hypnea muciformis, Sebdania polydactlyla, and Scinia indica, which had an approximate proline concentration of 10% [33].
Overall, glutamic and aspartic acid were the most abundant amino acids in most species. Methionine content was reported to be low in most of the species, and the mean values for individual amino acids were, in the majority of the reviewed studies, similar in brown, green, and red algae.

Carbohydrate contents in macroalgae
Seaweed also contain large amounts of polysaccharides with important functions for the macroalgal cells including structural and energy storage [5]. The total polysaccharide concentrations in macroalgae ranged from 4 to 76% of DW with the highest contents described in Ascophyllum, Porphyra and Palmaria; although other green species such as Ulva showed contents of up to 65% on a DW basis [18]. Seaweed polysaccharides include relevant bioactive compounds such as alginate, carrageenan, fucoidan, and laminarin. Furthermore other phycocolloids from macroalgae, i.e. agar, are commonly used in the food and animal feed industries as stabilisers, thickeners, and emulsifiers [13,18].
Carrageenans (CRGs) are sulphated linear galactans, consisting of alternating β-1,4and α-1,3-linked D-galactosyl residues (D-and Gunits). Carrageenans are characterized depending on the disaccharide repeating unit and the degree of sulphation of the molecule [37]. The three most industrially exploited types, namely Kappa, Iota, and Lambda are distinguished by the presence of one, two, and three estersulphate groups per repeating disaccharide unit respectively. However, the sulphate contents of the commercial carrageenans can vary depending on the seaweed specie or batch [38]. Chondrus crispus and Kappaphycus sp. contain up to 71 and 88% of carrageenan, respectively [18].
Fucoidans are cell wall polysaccharides present in brown macroalgae which play a crucial role in the protection of macroalgae against environmental challenges [5]. Fucoidans are fucose containing sulphated polysaccharides formed of a backbone of (1 → 3)-linked α-L-fucopyranosyl or alternating (1 → 3)-and (1 → 4)-linked α-L-fucopyranosyl residues with variable degrees of sulphation [5]. These polysaccharides are a chemically diverse group with molecular weights ranging from 43 to 1600 kDa [39]. The content and composition of fucoidan varies depending on the season and seaweed specie [5]. Fucus vesiculosus had fucoidan levels ranging from 16 to 20%, while Undaria pinnatifida contained approximately 1.5% [18]. Furthermore, recent studies also showed differences in the fucoidan content depending on the stage of growth of the seaweed. Cultured Laminaria japonica showed the highest contents of fucoidan in October, when the seaweed blades were matured rather than at younger stages of development [40].
Laminarins are a group of energy storage polysaccharides composed by 1,3-linked β-D-glucose monosaccharides with variable branching at β-(1,6) [5]. The laminarin structure may vary in the degree of branching and degree of polymerization depending on the seaweed specie, season, and other parameters such as extraction and purification procedures [5]. Laminarin is present in high yields in Laminaria sp. (up to 32%, depending on the season) and Saccharina sp., but these molecules have been also described in Ascophyllum, Fucus, and Undaria sp. [18]. Seasonal studies on Laminaria sp. showed higher accumulation of storage carbohydrates during summer and autumn that will be used during the winter as an energy source for new tissue growth [21].

Bioactive peptides derived from edible seaweed
As mentioned previously, algae are known to contain high concentrations of high quality polysaccharides, minerals, and vitamins, as well as bioactive compounds including proteins, lipids, and numerous polyphenols [18]. Bioactive peptides have been generated from a wide variety of natural sources including meat [10,41,42], milk [43,44], cereals [45,46], and fish [47,48]. Although there has been a growing demand to isolate bioactive peptides from marine algae [9,49], the number of biologically active peptides generated from seaweed is still limited. A number of functional foods containing seaweed-derived peptides are currently commercialized, mainly in Japan. The Japanese Ministry of Health and Welfare established a policy for approving some selected functional food products as Foods for Specified Health Uses (FOSHU) whose health claims are legally permitted [50]. Seaweedderived peptide-containing products with FOSHU approved antihypertensive claims include Wakame peptide jelly (Riken Vitamin Co., Ltd., Tokyo, Japan) and Nori peptide S (Shirako Co., Ltd., Tokyo, Japan) [51].

Antioxidant peptides
A free radical can be defined as a chemical substance capable of independent existence with one or more unpaired orbital electrons, and can be produced either from normal cell metabolism in situ of from external sources such as radiation or pollution [52]. When free radicals are produces in excess, and cannot gradually be destroyed, their accumulation in the body generates a phenomenon called oxidative stress [52]. The regulation of oxidative stress is an important factor in both, tumour development and responses to anticancer therapies [53]. Antioxidants can counteract oxidative stress, and these are either produced by the human body in situ or incorporate through diet.
Seaweed are a rich source of antioxidants. For example, the known antioxidant peptides carnosine and glutathione, which are generally present in animal muscle, were identified in seaweed species previously [54]. In addition, as mentioned previously, seaweed can contain relatively high protein content, and seaweed-derived proteins can be used for the generation of antioxidant hydrolysates and peptides. For example, Heo, Park, Lee and Jeon [55] generated a large amount of antioxidant hydrolysates of proteins isolated from Ecklonia cava, Ishige okamurae, Sargassum fullvelum, Sargassum horneri, Sargassum coreanum, Sargassum thunbergii, and Scytosipon lomentaria using the commercial enzymes Alcalase, Flavourzyme, Neutrase, Protamex, and Kojizyme. In this study, antioxidant activity was assessed using four different antioxidant scavenging assays and results demonstrated that the antioxidant potential of the Alcalase hydrolysates of Sargassum horneri were dose-dependent and thermally stable. In a more recent study, Cian, Martínez-Augustin and Drago [56] obtained different enzymatic hydrolysates from co-products of Porphyra columbina using alcalase, trypsin, and combinations of both. In addition, Harnedy, O'Keeffe, and FitzGerald (2017) generated an enzymatic hydrolysate of Palmaria palmata using the food-grade enzyme Corolase PP. The generated hydrolysate was sequentially fractionated using solid phase extraction and semi-preparative reversed-phase high performance chromatography (RP-HPLC) and the peptide SDITRPGNM showed the highest oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) activity with values of 152.43 ± 2.73 and 21.23 ± 0.90 nmol TE/μmol peptide, respectively. Bioavailability of peptides with antioxidant properties has been also evaluated. Indeed, Cian, Garzón, Ancona, Guerrero and Drago [57] observed that antioxidant activity of peptides derived from Pyropia columbina increased after a simulated gastrointestinal digestion.

Bioactive peptides and their role in diseases related to metabolic syndrome
The term metabolic syndrome describes a combination of medical disorders which increase the risk of developing cardiovascular disease, and these include obesity, diabetes, hypertension, lipid disorders, and alterations in the thrombotic potential [58]. Some of these disorders can be prevented or controlled by the inhibition of a number of enzymes and regulation systems by the use of chemically synthesised drugs or the ingestion of nutraceuticals or functional foods.
Venous thromboembolism (VTE) is a common and underestimated condition and one of the main causes of death and disability in highincome countries [59]. Blood coagulation is a complex mechanism with numerous coagulation factors and enzymes involved, and because of this, thrombus formation can be inhibited using different mechanisms [60]. The role of anticoagulants such as aspirin in the primary prevention of VTE has been evaluated in various clinical studies that associated this drug with a reduction of up to 50% in the risk to suffer VTE [61]. However, treatment with aspirin was also associated with an increased risk of bleeding. For this reason, investigations into the identification of novel antithrombotic compounds from different sources, including seaweed, are on-going. Antithrombotic peptides have been previously generated from milk proteins [60]. Up to the best of our knowledge, the peptide NMEKGSSSVVSSRMKQ, recently reported by Indumathi and Mehta [62], was the first antithrombotic peptide generated from seaweed. This peptide was generated by hydrolysis of Porphyra yezoensis proteins using pepsin. In vitro anticoagulation results showed a dose-dependent prolongation of activated partial thromboplastin time, a medical test that characterises blood coagulation, and the authors suggested that the peptide interacted with the clotting factors involved in the intrinsic pathway of coagulation. In addition, Indumathi and Mehta [62] recently isolated the anticoagulant peptide NMEKGSSSVVSSRM(+15.99)KQ from a Nori hydrolysate. Anticoagulation was measured by microtiter plate reader and was found to be non-cytotoxic and to have a similar order of activity as that of heparin.
Seaweed-derived bioactive peptides identified to date also include enzymatic inhibitors of the enzymes renin (EC 3.4.23.15) and angiotensin-I-converting enzyme (ACEeI; EC 3.4.15.1). Inhibition of renin, ACEeI, and other enzymes involved in the renin-angiotensin-aldosterone system (RAAS) plays a key role in the treatment of hypertension [63]. These enzymes can be inhibited by the use of chemically synthesised drugs or the ingestion of functional foods. There are a number of bioactive peptide-containing functional foods which are commercially available worldwide. For example, the Japanese company Senmi Ekisu Co. Ltd. obtained generally recognized as safe (GRAS) status for its hydrolysed sardine muscle protein from the Food and Drug Administration (FDA) of the United States (US). This product, labelled as Valtyron®, is rich in the di-peptide VY with known ACE-I inhibitory and antihypertensive benefits [64]. In addition, the European Food Safety Authority (EFSA) issued a positive opinion regarding the safety of hydrolysed sardine muscle protein, for use as a novel food ingredient in 2010 [7]. Bioactive algae-derived hydrolysates and peptides with in vitro ACE-I inhibitory activity include enzymatic hydrolysates of Spirulina platensis (cyanobacteria) and Polysiphonia urceolata, which inhibited the activity of ACE-I by half at concentrations ranging between 0.1 and 1.0, depending on the enzymatic treatment [49], and the tetrapeptides AIYK, YKYY, KFYG, and YNKL, which were identified from a peptic digest of Undaria pinnatifida and showed ACE-I IC 50 values of 213, 64, 90, and 21 μM, respectively [65]. In addition, Qu, Ma, Pan, Luo, Wang and He [66] generated several enzymatic hydrolysates of Porphyra yezoensis using commercially available enzymes and suggested papain, protamex, alcalase, and neutrase as the most efficient enzymes, from those studied, for the generation of ACE-I inhibitory peptides from Porphyra yezoensis. The ACE-I inhibitory activity of the alcalase hydrolysates, generated following these methods, was stable after storage at temperatures ranging between 4 and 37°C. As mentioned previously, inhibition of enzymes involved in the RAAS, such as renin, also plays a key role in the treatment of hypertension. Indeed, renin regulates the initial, rate-limiting step of the RAAS and inhibition of renin has long been considered the logical therapeutic target for blocking the system [67]. Only few renin inhibitory peptides have been reported to date, and these were mainly generated from pea [68] and blood [15,69] proteins. However, Fitzgerald, Aluko, Hossain, Rai and Hayes [7] recently reported the renin inhibitory peptide IRLIIVLMPILMA, which showed an in vitro renin IC 50 value of 3.34 mM, and was identified from a papain hydrolysate of Palmaria palmata. Up to the best of our knowledge, this is the only seaweed-derived renin inhibitory peptide identified to date. The peptide IRLIIVLMPILMA and the papain hydrolysate from where it was identified not only inhibited renin in vitro, but also showed in vivo antihypertensive activities after oral administration in spontaneously hypertensive rats (SHRs) at a dosage of 50 mg/kg body weight [7].
Moreover, seaweed-derived peptides have been also shown to be efficient inhibiting the enzymes dipeptidyl peptidase-IV (DPP-IV; EC 3.4.14.5) and platelet-activating factor acetylhydrolase (PAF-AH; EC 3.1.1.47). DPP-IV degrades and inactivates glucagon-like peptide-1 (GLP-1) and gastric-inhibitory peptide (GIP), two incretin hormones which contribute to the enhancement of glucose-induced insulin secretion [70]. In addition, PAF-AH is thought to be involved in the generation of lysophosphatidylcholine (LPC) and oxidized non-esterified fatty acids (NEFA), pro-inflammatory mediators which lead to the recruitment and activation of leukocytes and induce apoptosis and impaired removal of dead cells [71]. Inflammation has been also linked to obesity, hypertension, and other disorders related to metabolic syndrome previously [72]. Seaweed-derived DPP-IV inhibitory peptides identified to date include the peptides ILAP, LLAP, and MAGVDHI, recently reported by Harnedy, O'Keeffe and FitzGerald [73]. The peptides ILAP, LLAP, and MAGVDHI were generated from an aqueous protein extract of Palmaria palmata using Corolase PP and showed DPP-IV IC 50 values of 43.4, 53.7, and 159.4 μM, respectively. In addition, Fitzgerald, Gallagher, O'Connor, Prieto, Mora-Soler, Grealy and Hayes [74] identified the PAF-AH inhibitory tetra-peptide NIGK, which was generated from Palmaria palmata using papain and showed a PAF-AH IC 50 value of 2.3 mM.

Seaweed-derived bioactive carbohydrates
Macroalgal carbohydrates display a wide range of biological properties including antiinflammatory, immunostimulatory, antioxidant, anticoagulant, antiviral, antiproliferative, and antitumour properties [5,13]. These biological activities could be attributed to the high content of sulphated polysaccharides of seaweed (i.e. carrageenans, and fucoidan), which cannot be found in terrestrial plants [13]. Furthermore, seaweed polysaccharides such as alginates, fucoidan, and laminarin, are non-digested in the upper digestive track of the animals, being macroalgae considered as a rich source of dietary fibre [5,13]. Despite these beneficial properties, seaweed carbohydrates are being currently exploited industrially for their physicochemical properties, i.e. the ability of alginates and carrageenans to form gels, allowing their use as thickening, gelling, and protein-suspending agents [13,38]. Alginates from brown macroalgae showed a wide potential for its application in the biomedical and bioengineering fields, due to its gelling capacity, biocompatibility, biodegradability and lack of toxicity [75]. Nutritionally, alginates are dietary fibres and contribute to the gut's health due to its water binding properties and thus its effects in faecal bulking, decreasing the colonic transit times which could be beneficial in preventing colonic cancer [76]. Furthermore, the Food and Drug Administration (FDA) named alginic acid and its salts as generally regarded as safe (GRAS) ingredients for oral administration [75].
Other macroalgal polysaccharides such as laminarin and fucoidan are also considered as dietary fibres [5]. Laminarin modulates the intestinal metabolism by affecting the biochemical and microbiology of the human gut microflora [77]. In vivo studies showed a down-regulation of pro-and antiinflammatory cytokines in post-weaning pigs supplemented with laminarin [78]. Laminarin and fucoidan supplementation to sows during the periods of pregnancy and lactation showed an increase on immunoglobulin concentrations in the colostrum and a decrease of E. coli in suckling [79] and weaned piglets [80].
Further biological properties described for fucoidan with potential biomedical applications include the antioxidant and antiinflammatory activities, anticancer, anticoagulant and antithrombotic activities related to the structure and the degree of sulphation of the polysaccharide [81,82]. Fucoidan from Fucus evanescens showed anticoagulant activities similar to other drugs such as heparin in vitro and in vivo [83].
Other sulphated polysaccharides such as carrageenans have also gathered scientific attention due to its biological properties including anti-inflammatory and immunomodulatory, but also antitumor, antihyperlipidemic, antiviral and anticoagulant properties using different in vitro and in vivo models [38]. Carrageenan gels from Chondrus crispus had antiviral properties against HIV virus and other sexual transmitted diseases such as herpes simplex virus [38]. As in the case of fucoidan, the main explanation for the anticoagulant activities of carrageenan appeared to be related with the degree of sulphation of the molecules with λ-carrageenan showing the highest antithrombotic activities followed by κ-carrageenan [18].

Purification of bioactive peptides and carbohydrates
For the determination of the sequence of a peptide and to perform individual activity assays, an effective purification of the target specie is necessary. Purification of peptides and obtaining them in sufficient quantities is a complex and expensive process. Ultrafiltration membrane systems are fast and economic devices that are used to separate peptides with the desired MW by choosing the correct molecular weight cut-off (MWCO). Several studies enriched enzymatic hydrolysates and peptides derived from not only seaweed but other food sources using MWCO membranes. For example, Bondu, Bonnet, Gaubert, Deslandes, Turgeon and Beaulieu [84] utilized 1 and 10 kDa MWCO membranes for the purification of proteins and peptides derived from several macroalgae varieties including Ulva lactuca, Solieria chordalis, Palmaria palmata, and Saccharina longicruris. A similar strategy was followed by Beaulieu, Sirois and Tamigneaux [85] to purify peptides from Palmaria palmata. However, membranes have been reported to be poorly reproducible and could remove peptides below the desired MW [86]. Interactions between the peptide and the membrane can occur. The most hydrophobic peptides can adsorb on the membrane and be depleted from the sample [87]. Therefore, the separation and purification of not only peptides but also polysaccharides from seaweed for later identification is commonly achieved using one or multiple chromatographic techniques which have the advantage that can be used for qualitative and quantitative analysis as well as for preparative purposes.
Liquid chromatography (LC) has also been used extensively for the purification of bioactive peptides include mainly RP-HPLC, using columns packed with silica particles modified with C-18 chains [9,54,73,74,88]. Several lengths (50-400 mm), inner diameters (1.4-2.6 mm), and particle sizes (1.7-5.0 μm) were utilized in order to achieve a suitable resolution between the target compounds. In addition, in relation to mobile phases used, mixtures of formic acid in water and formic acid in acetonitrile are the most commonly used (Table 1). UPLC applies the principles of separation of HPLC. UPLC uses low particle size columns (less than 2 μm in diameter) and UPLC instrumentation (solvent and sample manager units) to operate at higher pressures, increasing the resolution, sensitivity, and speed of the analysis [89]. UPLC has been used recently not only for the purification of bioactive compounds but also to characterize the amino acid composition of 21 seaweed species from Norway [90]. LC is not regularly used for the purification of polar compounds such as carbohydrates. However, this separation technique could be used to analyse the monosaccharide composition of carbohydrates. Recently Liu, Wang, Song, He, Ren, Cong and Wu [91] analysed the monosaccharide composition of fucoidan extracts using a C-18 column (ZORBAX Eclipse XDB, 5 μm, 4.6 mm × 250 mm) following the acidic hydrolysis and derivatization of the compounds using 1-phenyl-3-methyl-5-pyrazolone (PMP) for its detection with UV at 250 nm. UPLC systems were also used to purify ad characterize multiple carbohydrates in products of plant [92,93] and animal origin [94]. Recently, Adrien, Dufour, Baudouin, Maugard and Bridiau [96] used UPLC coupled with MS to analyse the monosaccharide composition of seaweed extracts from Himanthalia elongata, Laminaria digitata, Ascophyllum nodosum, Fucus vesiculosus, Ulva lactuca, and Chondrus crispus with anticoagulant properties.
Nevertheless, the direct application of one LC method does not allow the detection of the investigated compounds, especially if they are present in the mixture in low quantities. Therefore, multidimensional approaches are generally used. Ion exchange chromatography (IEC) is one of the classic methods among multidimensional approaches. IEC achieves the separation of peptides and carbohydrates based on the adsorption of the charged analytes on to immobilized ion exchange groups of an opposite charge in the stationary phase of the column and its elution by changing the concentration or pH of the mobile phase [5]. Carbohydrates from seaweed are normally separated using IEC containing positively charged resins as stationary phase with affinity for negatively charged analytes (anion-exchange chromatography (AEC)). AEC is one of the most commonly used purification techniques used to separate alginates and sulphated polysaccharides such as fucoidan [5] and carrageenan [38]. The sulphate ester groups linked to the backbone of these seaweed polysaccharides exhibit high anionic charges, which facilitate the use of AEC following different experimental conditions described in detail in Table 2. The elution of the adsorbed carbohydrates from different resins was performed mainly by stepwise [97] or linear gradient [98][99][100] of NaCl or NaOH to achieved multiple polysaccharide fractions. In addition, IEC purification of fucoidan extracts followed by ESI-MS analysis was used to determine the monosaccharide composition of the different fucoidan fractions obtained from Sargassum fusiforme [97], while IEC followed by ESI-MS/MS was used to characterize fucoidan carbohydrates with anticancer activity from Padina boryana [101]. ESI-MS/MS was used to characterize the monosaccharide units of immunomodulatory and anti-HIV fucoidan fractions from Sargassum mcclurei, Sargassum polycystum and Turbinara ornate [102] and from Sargassum crassifolium and Padina australis [103], respectively. SEC is also a common stem in multidimensional fractionation of bioactive peptides. Today, SEC is the most widely accepted and used analytical method for the measurement of MW distributions of biopolymers [104]. Briefly, SEC is a liquid chromatographic technique in which a sample solution is introduced into a column filled with porous packing, often silica-based, carried through the column by a solvent, and separated by differential pore permeation [104]. Unlike IEC, polymers do not bind to the inert material of the columns and thus the composition of the buffer has no effect on the separation of the compounds, enabling to use the type of solvent that better suits the preservation of the compounds of interest for further use or purification and chemical characterization [5]. SEC can provide a rapid estimate of the MW of a peptide, obtained under denaturing conditions, when all polypeptides have the same random coil structure [105]. Small peptides with just a few amino acid residues in length do not usually adopt stable structures in solution. However, in rare occasions small peptides were shown to form stable secondary structures. For example, the fragment Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) of the Alzheimer β-amyloid peptide was demonstrated to undergo from a random coil to a β-sheet structure previously [106]. Peptide bonds absorb UV light with a maximum around 187 nm. However, because of interference from buffer components, wavelengths ranging between 210 and 220 nm are the most frequently used [105]. SEC has been used for the determination of the MW distributions of multiple seaweed carbohydrates as seen in Table 2. The separation of different MW alginates and fucoidans was achieved by the use of a single column or various columns connected successively, being the elution of the different compounds monitored by refractive index detector (RI) [97,100,101,107] or evaporative light-scattering detector (ELSD) [108]. Both IR and ELSD are commonly used to detect compounds that cannot be efficiently detected with UV spectra. Recent innovations in the purification of carbohydrates include the separation of polysaccharides such as dextran, fucoidan, alginic acid, and laminarin using ionic liquid-modified silica rather than the conventional SEC silica [109].

Sequence validation
The identification of the sequence responsible of the observed bioactivities has been largely neglected or was performed to identify the most probable sequence. Edman degradation was the first approach used for peptide identification [87]. However, due to their accuracy and sensitivity, chromatographic techniques, especially LC-MS/MS methods have become more popular over the last decade and are considered as a reference for bioactive compound analysis (Table 1). Other technologies such as nuclear magnetic resonance (NMR) have been also studied. MS is an analytical technique that can provide useful data on the structure and concentration of the analytes of interest in a sample after their conversion to ions [103]. In MS the analytes are ionised using high-energy electrons, causing a molecular fragmentation of the analytes that pass through a mass-to-charge analyser and are detected as a function of mass-to-time or mass-to-charge ratio (m/z) [110]. In the last decade, MS has become increasingly important for analysis of peptides and carbohydrates due to its high sensitivity, accuracy, and fast processing times compared to NMR and other detectors [38]. MS techniques commonly used to analyse macroalgal peptides and carbohydrates include electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) which differ on the strategy followed to ionize the analytes. ESI uses electrical energy to transfer ions from an initial solution into a gas stage prior to its detection with MS. This ionization into gas requires the dispersion of charged droplets, followed by the evaporation of the solvent and ejection of ions from the highly charged droplets [111]. ESI-MS is a sensitive, robust and reliable method capable to detect femtomole quantities of multiple compounds, including non-volatile and thermolabile analytes [111]. ESI-MS coupled Table 2 Experimental conditions used for the purification of macroalgal carbohydrates. with LC for molecular fractionation prior to MS analysis (LC/ESI-MS) or to MS in tandem (LC/ESI-MS/MS) are powerful methods recently used to analyse peptides and carbohydrates in a complex biological samples, including seaweed extracts [97,101]. The conversion of the analytes to a gaseous phase performed during ESI could be difficult to achieve when analysing complex proteins and carbohydrates due to their high polarity and mass [106]. In MALDI, the analytes are normally mixed with a matrix to form a solid-state mixture that will be later irradiated with UV laser pulses (337-355 nm), generating ions analysed based on its mass-to-charge ratio by MS. Choosing the appropriate matrix for an analyte is crucial for successful MALDI analysis [112,113]. MALDI continues to be a major technique for the analysis of carbohydrates [112]. MALDI-TOF mass spectra was used to analyse the structure of antiinflammatory κ/β-carrageenans from red algae Tichocarpus crinitus [114]. Furthermore, MALDI was recently used to analyse fucoidan fractions with multiple biological properties from seaweed, including Saccharina cichorioides [115], Saccharina gurjanovae [116], Sargassum muticum [117] and Alaria angusta [118].

Compounds of interest
NMR spectroscopy is one of the most powerful analytical tools available to date to determine the structure of bioactive compounds [38,119,120]. NMR is based on the principle of nuclear spin or the fact that when a molecule is placed in a magnetic field, this momentum will be aligned either in the same or opposite direction to the field, resulting in 2 states separated by an energy gap and thus, in resonance frequency [119]. This difference in resonance frequency will depend on the chemical environment of the nucleus in a molecule and on the magnetic field strength in an effect known as chemical shift [119]. Most elements have at least one naturally occurring NMR active isotope with variable frequency of occurrence i.e. the natural abundance of 1 H is almost 100% being easier to detect by NMR compared to 13 C and 15 N isotopes that appeared naturally in 1.1 and 0.4% respectively [119]. For characterization of carbohydrates, the resonances of 1 H NMR in the anomeric region (4.4-5.5 ppm) and the 13 C NMR spectra (95-110 ppm) provide useful information on the number of monosaccharide units of the carbohydrates [119]. 1 H NMR resonances bellow 1 ppm indicate the presence of CH 3 -groups and above 2 ppm reveal N-acetyl and/or Oacetyl groups [119]. NMR have been used extensively to characterize the structure of seaweed carbohydrates. Carrageenan can be qualitatively and quantitatively characterized by NMR techniques, allowing the determination of molar rations and the presence monosaccharides present in the extracts. Both 1 H and 13 C NMR spectroscopy are applicable for these purposes, but 1 H NMR has the advantage of a relatively high sensitivity and lower time of analysis [38,119]. 1 H NMR was also used to identify and characterize the structural units of alginates (MM, GG, MG or GM blocks) from multiple seaweed species [34,100]. 1 H NMR and 13 C NMR spectra together with 2D NMR was also used to characterize a fucoidan fraction with antiangiogenic activity from macroalgae Sargassum fusiforme [97]. 13 C NMR analysis was also performed to characterize fucoidan fractions with hepatoprotective activities from Kjellmaniella crassifolia [91]. NMR spectra using 1 H and 13 C among other techniques were also used by Youssouf, Lallemand, Giraud, Soulé, Bhaw-Luximon, Meilhac, D'Hellencourt, Jhurry and Couprie [121] alginates extracted from Sargassum binderi and Turbinaria ornata and carrageenans from the red macroalgae Kappaphycus alvarezii and Euchema denticulatum. NMR was recently used for the characterization of a glycoprotein with 36.24% carbohydrate, composed of rhamnose, galactose, glucose, and mannose with a mole ratio of 38:30:26:6 [122]. NMR spectra proved that the above mentioned glycoprotein, isolated from the green seaweed Codium decorticum, contained protein and carbohydrate portions with (1 → 4)-linked β-galactose residues and β-linked glucose residues. Senthilkumar and Jayanthi [122] demonstrated the anticancer properties of this compound by different cell lines.

Conclusions
Seaweed are relative un-explored and promising of novel molecules for its use as functional foods and nutraceuticals including bioactive peptides and carbohydrates. Several seaweed derived bioactive compounds have shown a wide range of biological activities both in vitro and in vivo. Among the biological activities reported by bioactive peptides and polysaccharides the most promising include antihypertensive, antioxidant, anticoagulant, antitumour, anti-inflammatory, and immunostimulatory activities. These biological activities have increased the scientific interest in developing functional ingredients and to study the relationship between the chemical structure of the compounds and the biological activities. Multiple analytical approaches have been used to date to purify and analyse the chemical structure of bioactive peptides and carbohydrates. Recent improvements in the purification and characterization methodologies gradually increased the use of novel chromatographic techniques i.e. ultra-performance liquid chromatography (UPLC) and analytical techniques such as NMR spectroscopy ( 1 H and 13 C NMR) for qualitative and quantitative analysis and mass spectrometry (ESI and MALDI) to gain further insight into the complexity of different molecular structures of bioactive peptides and carbohydrates.