Macroalgae as Protein Sources—A Review on Protein Bioactivity, Extraction, Purification and Characterization

The increased demand for protein sources combined with a decrease in the available land and water resources have led to a growing interest in macroalgae as alternative protein sources. This review focuses on strategies for macroalgae protein extraction, enrichment and characterization. To date, the protein extraction methods applied to algae include enzymatic hydrolysis, physical processes and chemical extraction. Novel methods, such as pulsed electric field, microwave-assisted, pressurized liquid and supercritical fluid extractions, and the application of smart solvents are discussed. An overview of the use of membranes and other processes to generate high-value protein concentrates from algae extracts is also presented, as well as some examples of the methods used for their characterization. The potential bioactivities from macroalgae-derived proteins and peptides, including novel glycoproteins and lectins, are briefly reviewed.


Introduction
A "Biorefinery" is defined as a "sustainable and synergetic processing of biomass into marketable food and feed ingredients, products (chemicals and materials) and energy" by the International Energy Agency (IEA) [1]. Different strategies can be integrated to maximize the extraction of valuable components, with subsequent use of the remaining fractions as raw materials for further processing in a cascading approach, while minimizing waste streams. This concept applied to macroalgae adds value to these bioresources and contributes to sustainable development. It is therefore crucial to choose the best available technologies for an efficient sequential separation of valuable seaweed fractions, in particular of seaweed proteins. Seaweed proteins/peptides due to their availability, amino acid composition and bioactive properties constitute a good option to fulfill the increased demand for protein sources. This review records the available technologies for extraction and enrichment of macroalgal proteins, as well as the methods for their characterization.

Macroalgae Background
The term algae refers to a large diversity of unrelated phylogenetic entities, ranging from picoplanktonic cells to macroalgal kelps [2]. In contrast to terrestrial plants that share a common ancestor, algal diversity includes several distantly related groups of mainly photoautotrophic organisms from aquatic habitats. The high diversity of macroalgae at high taxonomic levels and related long evolutionary history displayed by complex phylogeographies are reflected in the observed richness and diversity of algal components. Algal chemistry is largely linked to evolution, but phenotypic modifications can also be a

Proteins in Seaweed
Proteins from macroalgae of marine origin have been the focus of several studies due to their potential bioactivity. The protein content varies according to phylum. Brown algae generally have a low protein content (3 to 15% dw), which contrasts with the protein content of green (9 to 26% dw) and red algae (20 to 47% dw) [15][16][17]. A recent review lists the protein content of macroalgae species from different geographical regions [18]. In the latter phylum, it is worthy to highlight the species belonging to the genus Porphyra, where the protein content can reach 47% dw [19]. These concentrations are comparable to those found in high-protein vegetables, namely (% dw), leafy greens and legumes (mint-30.9; cilantro-22.2; spinach-26.5; cauliflower-29.9; soybean-35 to 40; chickpea-20 to 25) and major cereals (wheat-8 to 15; barley-8 to 15; rice-7 to 9; corn-9 to 12) [20].
The protein content of macroalgae varies within seasonal cycles. One example is the protein content of the red seaweed Palmaria palmata collected at the French Atlantic coast, which showed fluctuations between 9 and 25% dw in protein content. The highest values occurred during winter and spring [15]. Seaweed may contain non-proteinic nitrogen, from nitrates, pigments or nucleic acids, which results in an overestimation of their protein content (usually estimated by the Kjeldahl method, with a general Nitrogen-to-Protein conversion factor of 6.25). Specific Seaweed-Nitrogen-to-Protein (SNP) conversion factors for brown, red and green seaweeds have been reported [21]. Detailed essential amino acids (EAA) and non-EAA profiles, total protein content and protein/non-protein nitrogen amounts of four macroalgae species obtained from an integrated multi-trophic aquaculture system in Ria de Aveiro (from ALGAplus Ltd., Ílhavo, Portugal), have been reported [22].

Phycobiliproteins
Protein-pigment complexes such as the phycobiliproteins (PBPs) are one of the most important groups of marine proteins. In red seaweed, these complexes are the main light-harvesting pigments and account for up to 50% of the total protein content [23]. Phycobiliproteins are the only water-soluble algal pigments. They are a family of fluorescent proteins covalently linked to tetrapyrrole groups, known as bilins. They constitute a structure attached to the cytoplasmic surface of thylakoid membranes named phycobilisomes (unlike carotenoids and chlorophylls that are located in the lipid bilayer) [23]. PBPs are grouped into four classes: phycoerythrin (PE), phycocyanin (PC), phycoerythrocyanins (PEC) and allophycocyanin (APC) [14]. The most common phycobiliprotein in many red seaweeds is known as R-phycoerythrin (R-PE). Isolation of PE has been reported from many species, for instance, Gelidium pusillum [24], Grateloupia turuturu [25,26] and Rhodymenia pseudopalmata [27].

Glycoproteins and Lectins
Glycoproteins (GPs) are carbohydrate-binding proteins. Glycans can be conjugated to peptide chains by N-glycosyl linkages and/or O-glycosyl linkages. Protein glycosylation can happen co-or post-translation. GPs are located in the cell wall and on cell surfaces or are secreted [14]. A few seaweed glycoproteins have been isolated by hot-or cold-water extraction. GP-rich fractions obtained from Ulva spp. showed a higher content in proteins than in neutral sugars [28]. Extracts of Ulva lactuca with high contents of both carbohydrates and protein suggested the presence of GPs [29]. Yoshiie et al. (2012) [30] have performed the structural analysis of the N-glycans expressed in several seaweed species. The study revealed that only high-mannose glycans were present in the 15 macroalgae addressed (red, brown and green), while the complex type of N-glycans containing β1-2 xylosyl and α1-3 fucosyl residues, which is encountered in terrestrial plants, was not found. The same authors discovered that the M9 structure is predominant in the N-glycans of the GPs of Zoostera marina and Sargassum fulvellum. Therefore, they claim the interest of both species as good sources of N-glycans used, e.g., as chaperone-like oligosaccharides in protein assembly [31].
Phycolectins are a group of carbohydrate-binding proteins in macroalgae. Lectins interact with specific glycan structures that constitute the soluble and membrane-bound glycoconjugates [14]. Seaweeds are particularly good sources of novel lectins. A few examples are griffithsin, a mannose-specific lectin isolated from the red algae Griffithsia spp. [32]; SfL1 and SfL2 from Solieria filiformis [33]; and HRL40 from Halimeda renschii [34]. Although macroalgae phycolectins have been characterized, very little is currently known about their structural and biochemical properties [14].
Arabinogalactan proteins, which belong to the hydroxyproline-rich glycoproteins, have been reported in the cell wall of few species of seaweeds, namely, in the green seaweeds Codium vermilara [35] and C. fragile [36].

Enzymes
Seaweeds are rich sources of enzymes. Alkaline phosphatase (Zn-containing metalloproteinase that catalyzes the non-specific hydrolysis of phosphate monoesters) is widely encountered in seaweeds, namely, in Ulva pertusa [37]. Alternative oxidases (proteins involved in the electron flow through the electron transport chain and in the regulation of the mitochondrial retrograde signaling pathway) have also been described, namely, from Caulerpa cylindracea [38]. The fibrinolytic enzyme (trypsin-like serine protease) has also been isolated from green algae, such as Codium fragile and Codium latum [39,40]. Rubisco, a protein that catalyzes carbon dioxide fixation and oxygenation, has been obtained from Kappaphycus alvarezii [41].

Peptides and Amino Acids
Although the structure and biological properties of algal proteins are still relatively poorly documented, the amino acid composition of several species of macroalgae is known [42]. Most species contain all the EAA for humans, which may represent about 50% of the total amino acids. Besides, their proteins are also rich in aspartic and glutamic acid residues (22.7 g/100 g protein in several red algae) [23]. The levels of some amino acid residues, such as threonine, lysine, tryptophan, cysteine, methionine and histidine, are higher than those found in terrestrial plants [18]. Seaweed amino acid analyses have demonstrated profiles similar to those of ovalbumin (52.4% EAA) and leguminous plants (41.62% EAA) [15,43]. At this point, it is worth mentioning that the interest in marine proteins may not be directly linked to the proteins themselves, but rather to the bioactivity potential of the peptides that can be derived from them [44,45]. Bioactive peptides usually contain 3-40 amino acid residues, and their activities stem from both their amino acid composition and sequence. They are generated from parent proteins either through digestion processes in the gastro-intestinal tract or produced through fermentation or other processes involving enzymatic hydrolysis [46,47].
The free amino acid fraction of macroalgae is composed primarily of alanine, taurine, omithine, citrulline and hydroxyproline. Laminine, kanoid amino acids and mycosporinelike amino acids have also been found in marine macroalgae [48]. The successful production of bioactive peptides that originated from hydrolyzed proteins of Palmaria palmata, Solieria chordalis, Ulva lactuca and Saccharina longicruris has been reported [49].

Applications of Macroalgal Proteins and their Derivatives
Marine macroalgae are rich sources of structurally diverse bioactive compounds with valuable pharmaceutical and biomedical properties. They can also be used as functional health-promoting ingredients in food and feed [50,51]. Besides the benefits, attention must be given to the potential deleterious effects of food and feed derived from contaminated seaweed and to the legislation regulating these applications [51]. The functional properties of proteins are mainly associated with their ability to form and/or stabilize gels and films, foams, emulsions and sols [52]. Macroalgae or macroalgal extracts have shown effects on the immune status and intestinal health of several monogastric farm animal species, including pigs [53], broiler chicken [54] and fish [55]. These bioactive properties could be related to the presence of specific proteins and/or peptides.

Bioactive Proteins
Among all the macroalgal proteins, lectins and phycobiliproteins have received particular attention due to their reported biological activities [42]. The main biological activity associated with lectins is their hemagglutinating activity against erythrocytes [42], whereas phycobiliproteins are used in biomolecule labeling for fluorescent immunoassays, immunohistochemistry assays and fluorescence microscopy, and as natural colorants for food and cosmetic applications [56]. Different phycobiliproteins have exhibited antioxidant, antiinflammatory, neuroprotective, hypocholesterolemic, hepatoprotective, antiviral, antitumor, liver-protecting, atherosclerosis treatment, serum-lipid-reducing and lipase-inhibition activities [56]. The bioactivities of several macroalgal proteins are shown in Table 1. Eucheuma serra and Galaxaura marginata Lectins Antibacterial activity against the fish pathogen Vibrio vulnificus. [57] Griffithsia spp. Novel lectin Strong anti-HIV activity. [42] Hypnea cervicornis and Solieria robusta Lectins Anti-inflammatory and mitogenic activities in mice spleen lymphocytes; growth inhibition of mice leukemia cells L1210 and mice FM3A tumor cells. [42] Hypnea cervicornis Mucin-binding agglutinin Antinociceptive and anti-inflammatory activity. [42]
An extensive list of bioactivities of the peptides derived from specified seaweeds, including methodologies for their enzymatic production conditions, respective bioactivity evaluation assays and citations, can be found in [18]. Table 2. The bioactivities of several macroalgal peptides.

Species
Peptide Bioactive Activity Ref.

Challenges and Impact of Cell Structures
The successful extraction of macroalgal proteins highly depends on their accessibility since most of them are produced intracellularly. Therefore, the complex nature of algal cell walls is the main challenge for the use of seaweed as protein sources. Algal cells possess many intracellular enzymes and proteins [66] and their cell wall is composed of a highly integrated network of biopolymers, mainly polysaccharides, which interact with water and metal cations, amongst other molecules [67]. The cell wall can be divided into three main domains: the fibrillar wall, the amorphous matrix and the glycoprotein domain [23]. The fibrillar polysaccharides and the glycoproteins form a reticulated network that is embedded in the gel-like amorphous matrix. The fibrous part is the most chemically inert and mechanically resistant cell wall component, with cellulose being the most significant compound amongst others such as xylan and hemicellulose. Very little is known about the glycoprotein domain, which is constituted by glycoproteins that contain cellulose-binding domains. The gel-like matrix is made of carboxylic and/or sulfated polysaccharides, like sulfated galactans such as carrageenans and agarans, and usually extends into the intercellular spaces between adjacent cells [23]. Other biopolymers, such as proteins and polymeric phenolics, can also participate in cell wall formation.
The presence of polysaccharide-bound cell wall mucilage, including anionic or neutral polysaccharides, and polyphenols reduce the protein extractability and requires additional steps for fractionation and purification. Polysaccharides induce strong electrostatic interactions [68], whereas polyphenols may form reversible hydrogen bonds with proteins or undergo oxidation. Oxidized phenolic compounds can react with amino acids and form insoluble complexes [69].
The morphology of different seaweed species has also been suggested to be an important factor in protein extraction, with tougher thallus forms reported to require increased processing. The raw biomass from seaweed after harvesting must be preserved by drying or freezing or used fresh as soon as possible to avoid protein degradation [14]. The increased protein extractability from oven-dried biomass was suggested to have been due to the decomposition of the phenolic compounds, as well as increased disruption of the anionic or neutral polysaccharides found within the cell wall of the seaweed [46].
The combination of extraction methods and purification techniques is necessary to improve the protein yield. The greater the scale, the bigger the challenge because methods with low time, cost and energy consumption (i.e., environmentally friendly) are required.

Conventional Extraction Methods
As mentioned above (Section 2.1), the extraction and utilization of algal proteins evidently depends on the disruption of the cell wall. To ensure extraction of intracellular proteins, additional stress factors are often applied, which improve the extraction efficiency.
So far, algal proteins have not been fully described, and hence they are simply divided into four main classes based on their solubility, namely, albumins, which are soluble in water; globulins, which are soluble in salt solutions; glutelins, which are soluble in dilute acids or bases; and prolamins, which are soluble in 70% alcohol in water [66]. Sequential extraction steps are often carried out to ensure the extraction of different types of proteins, unless a specific type is being targeted.
Algal proteins are conventionally extracted by means of aqueous, acid, and alkaline solution-based methods, followed by centrifugation for extract clarification and fractionation and enrichment techniques, such as ultrafiltration, precipitation and/or chromatography [70]. Aiming at the preservation of protein structure and affinity interactions ability, mild extraction conditions at defined pH ranges may be required [33,[71][72][73].
Physical cell disruption methods, such as osmotic shock, freeze/thawing, shearing or grinding, can enhance the extraction of protein from some seaweeds. The conventional disruption pre-treatments and protein extraction methods are presented in Table 3.

Enzyme-Assisted Extraction
Enzyme-Assisted Extraction (EAE) is often the preferable method to extract proteins and/or their hydrolysates from seaweed [46]. Polysaccharidases can be applied as a cell disruption treatment prior to protein extraction to increase the protein yield. Several polysaccharidases (κ-carrageenase, β-agarase, xylanase and cellulase) have been used in protein extractions from red seaweed species, namely, Chondrus crispus, Gracilaria verrucosa and Palmaria palmata [74]. The use of cocktails that contain multiple hydrolytic activities (cellulase, hemicellulase and β-glucosidase) for carrageenan, agar, alginate and cellulose is a promising option to increase the extraction yields [75]. The selective degradation of structural proteins, such as glycoproteins of the cell wall, might be possible by using subtilases or serine proteases [76]. Different digestion enzymes have also been used to release bioactive peptides from parent proteins, with chymotrypsin, trypsin and pepsin being the most used [14]. Combining EAE with other processes (e.g., enzymatic hydrolysis combined with alkaline extraction) is usually the go-to approach (Table 3) [77].
Once again, the enzyme choice is highly correlated with the desired end product (e.g., intact proteins, specific proteins or bioactive peptides). Regarding feasibility, every case needs to be looked at individually. If a high enzyme:substrate ratio is required, an enzymatic treatment might not be viable at the industrial scale, particularly due to the cost of the enzymes.

Ultrasound-Assisted Extraction
Ultrasound-Assisted Extraction (UAE) has been shown to be an attractive technology when used on cellular matrices. It acts by creating compression and decompression through sound waves at frequencies higher than 20 kHz. UAE can be applied to numerous food sources, particularly in the modification of plant micronutrients to improve the bioavailability, simultaneous extraction and encapsulation, quenching radical sonochemistry to avoid degradation of the bioactive compounds, as well as to increase the bioactivity of the phenolics and carotenoids by targeted hydroxylation [78]. The bioavailability of seaweed proteins can also be improved by the degradative effect of sonochemistry, which is not produced by the ultrasound waves directly but rather by acoustic cavitation. This happens when the static pressure falls below the vapor pressure of the liquid and the formation and growth of vapor bubbles occurs. Such bubbles, under the subsequent pressure peak, violently collapse, leading to peeling, erosion, particle breakdown and degradation of the solid-liquid surfaces. Solvent penetration into the cells is facilitated, as is the release of the intracellular compounds to the bulk solvent [79].
The application of ultrasound can be divided into two different categories, namely, low intensity-high frequency (100 kHz-1 MHz) and high intensity-low frequency (between 20 and 100 kHz) ultrasound, the latter being the type that is typically used for the disruption of cell walls and membranes [80]. Higher extraction yields are usually achieved at shorter processing times and lower temperatures, making this method suitable for the extraction of thermolabile compounds [81]. Under these conditions, solvent consumption is also lower, facilitating the downstream processing of the target compounds [82].
An ultrasound pre-treatment reportedly increased protein extraction from Ascophyllum nodosum by 540% and 27% when followed by acid or alkaline treatment, respectively, when compared with extraction performed with no pre-treatment. It also resulted in a reduced processing time (from 60 to 10 min). Extraction of R-phycoerythrin has been deemed effective by combining EAE and UAE in Grateloupia turuturu [26], due to a synergistic effect. In Gelidium pusillum, UAE followed by maceration and extraction in phosphate buffers allowed the recovery of 77% and 93% of R-PE and R-phycocyanin (R-PC), respectively [83]. Some examples are presented in Table 3.

Deionized water and NaOH
Aqueous treatment 10 g of algal powder; 200 mL deionized water; 4 • C; stirred overnight.

Novel Methods
The design of a biorefinery usually follows a fractionation approach, so as to maximize the number, quality and quantity of the extractable products and thus enhance the value of the bioresource. This is why novel extraction methods are being sought; e.g., in seaweed, which might yield a more complete valorization of the macroalgae by obtaining pure extracts in the first separation steps and, concomitantly, not causing harm to the subsequently extracted components.

Pulsed Electric Field Extraction
Pulsed Electric Field Extraction (PEFE) is used as a cell disruption method, particularly in microalgae. It involves the application of short, high-voltage electric current pulses to perforate a cell wall or membrane. Depending on the intensity, amplitude, duration, number and repetition frequency of the external electric pulses, reversible or irreversible pores (electroporation) are formed in the membranes. Irreversible pore formation is of particular importance for extraction of bioactive compounds from natural matrices [14]. Treatments with an electric field strength from 0.7 to 3 kV/cm, a specific energy of 1-20 kJ/kg, a couple of hundred of pulses and a total time duration lower than 1 s are usually used for natural products extraction [80]. When applied to U. lactuca, a higher protein content was observed, namely, 59 µg mL −1 against the 23 µg mL −1 obtained in the control [84]. When 50 pulses of 50 kV, applied at a 70.3 mm electrode gap, were used in the extraction of proteins from Ulva spp., a sevenfold increase in total protein content was obtained when compared to the use of osmotic shock [86]. The applied conditions can possibly limit the scale up of the method.

Microwave-Assisted Extraction
Microwave-Assisted Extraction (MAE) is another procedure used to enhance protein extraction from biological matrices. When microwave energy is transferred to the solution or suspension, the vibration/oscillation of the polar molecules occurs, causing inter-and intra-molecular friction. This effect combined with the movement and collision of a large number of charged particles leads to the heating of the matrices. Intracellular heating ultimately leads to pressurization effects that induce the breakdown of cell walls and membranes, in addition to electroporation effects [80]. Since MAE has been mostly used for analytical purposes (sample preparation), the results found in the literature focus on the reduction of extraction times rather than on its effects on parameters such as protein functionality [80].
This method can be carried out in open vessels (atmospheric pressure) or closed ones, under controlled pressure and temperature. In closed vessels, the solvent can be heated above its normal boiling point by manipulating the pressure, accelerating the mass transfer of the compounds from the natural matrix to the bulk solvent [87]. The higher the dielectric constant of the solvent and/or algal matrix, the greater the energy absorbed by the molecules and the faster the extraction temperature is reached [88,89], which makes polar solvents, such as water, the preferred choice.

Pressurized Liquid Extraction
Pressurized Liquid Extraction (PLE), or Accelerated Solvent Extraction (ASE), combines temperatures that range from 50 to 200 • C and pressure ranging from 35 to 200 bar. These parameters are set for values lower than the solvent critical temperature and pressure, keeping it in its liquid state [20]. Both temperature and pressure increase the mass transfer rate. High pressures cause the solvent to reach temperatures higher than its normal boiling point and these higher temperatures enhance the solubility of the extractable components and reduce the viscosity and surface tension of the solvent [80]. Water is the most widely used solvent but other solvents, such as propane and dimethyl ether (DME), can also be used. Since DME is partially miscible with water, it allows the simultaneous extraction of non-polar target metabolites and the removal of water from wet matrices [90][91][92].

Supercritical Fluid Extraction
Supercritical Fluid Extraction (SFE) is an alternative extraction technique that produces extracts with very low contents of polar impurities [93]. It is a green technology, since a concentration step can most often be skipped after the extraction process [94]. A fluid becomes supercritical if subjected to temperature and pressure conditions above its critical point values (P c ; T c ). Experimental studies using SFE are usually limited to the region of P c < P ≤ 6P c and T c < T ≤ 1.4T c [95]. Under supercritical conditions, some of the properties of the fluid become indistinguishable from those of its gaseous state, with a density similar to that of a liquid, but with diffusivity and viscosity matching those of a gas. This makes supercritical fluids capable of a faster and deeper penetration into the solid particles of the matrix to be processed [93]. The fluid must be chosen carefully, especially when dealing with thermolabile compounds. Carbon dioxide, with T C and P C values of 31.1 • C and 73.9 bar, is by far the most used supercritical solvent [96]. In addition to being ideal for the extraction of thermolabile compounds, it has low viscosity, low surface tension, high diffusivity, is non-toxic, non-flammable, widely available and chemically inert under the conditions relevant for the extraction process. The fact that it is gaseous at normal pressure and temperature eliminates the need for a solvent evaporation step after extraction [96]. The greatest limitation of supercritical CO 2 is that it is not suitable for extraction of polar compounds [97].
The addition of an organic modifier, such as ethanol or methanol, can greatly improve the extraction efficiency with supercritical CO 2 [96]. Other possible supercritical solvents, such as water, methanol, ethanol, acetone, chloroform, ethyl acetate and toluene, are usually avoided when extracting bioactive compounds, since their T c value is above 200 • C [80].
The SFE technology has been used for processing seaweeds and other plant-based materials mainly to extract non-polar and small-size molecular components, such as pigments (chlorophylls, carotenoids) and some lipids (e.g., sterols) that are valuable as food and pharmaceutical ingredients. Proteins, polysaccharides, long-chain fatty acids and phlorotannins have also been recovered from seaweeds under PLE using subcritical solvents, and/or subcritical water and organic modifiers (SWE) [98]. Therefore, SFE may be a first processing step in seaweed biorefinery strategy [85] wherein the protein extraction step, from the first-extracted biomass, could be done either by PLE, SWE or conventional methods, after optimizing the operating conditions to increase the selectivity, as recommended for biorefineries of other plant-based resources [98].

Switchable or Smart Solvents
Switchable solvents are a new class of smart extraction solvents that can switch from a non-ionic form to an ionic liquid through contact with CO 2 gas bubbles. Exposure to N 2 allows them to return to their non-ionic form [14]. Ionic liquids (ILs) are composed of large asymmetric organic cations (imidazolium, pyrrolidinium, pyridinium, ammonium or phosphonium) and different inorganic or organic anions, such as BF 4− , PF 6− , Cl − and Br − [80]. They are very versatile, since their polarity, hydrophobicity, viscosity and other properties highly depend on the cationic or anionic constituents selected, hence being referred to as "designer solvents". They have low melting points (below 100 • C) and their non-flammable and non-volatile nature makes them an attractive choice for the development of safer processes [80].
The basis of the extraction mechanism with ILs lies in their interaction with macroalgal cell walls. As mentioned, cellulose is a core component of algae cell walls, although its proportion varies between species. The use of a solvent capable of disrupting the intermolecular H-bond interactions leads to cellulose dissolution [99]. Indeed, several ILs have been found to dissolve large amounts of cellulose [100]. These interactions are relevant because they ultimately lead to the complete or partial disruption of cell walls [101].

Protein Enrichment Methods
Using seaweed as a protein resource requires the processing of biomass to deliver a concentrated form of high-quality protein. The protein concentration from plant materials has traditionally been achieved directly by extracting and isolating the protein, or indirectly by extracting the non-protein components. In contrast with other plant materials, isolating and concentrating proteins from seaweed is a relatively unexplored domain and most protocols tend to focus solely on the initial extraction methods [102]. Purification of the extracted proteins represents a challenge, especially for novel proteins because of their unknown physicochemical properties. The selection of the method also depends on the final application of the product and the scale of production. The extraction method used also influences the purification method chosen. For example, reducing agents, such as N-acetyl-L-cysteine, which are used to improve cell wall-associated protein extraction, might have to be avoided, depending on the applications of the extracted proteins.
Single or combined enrichment methods can be employed. These methods include precipitation, membrane technologies and chromatography [14]. A combination of membrane technologies could be used to isolate seaweed proteins using the same principles used to obtain protein isolates in the dairy industry. Microfiltration (MF) could be used to remove the cell wall components, ultrafiltration (UF) could be used to isolate proteins with a molecular weight between 1 and 200 kDa, nanofiltration (NF) could be used to remove monovalent salts and reverse osmosis (RO) to reduce the final volume [103]. Ultrafiltration has been applied after supercritical CO 2 and ultrasonic-assisted extraction to isolate polysaccharides from Sargassum pallidum [25] and after hot water extraction in Ulva fasciata [104]. Most protocols reported in the literature often describe methods for the purification and isolation of specific proteins, namely, R-PE. Some examples are presented in Table 4.  [108] AEX-anion-exchange chromatography; EBA-expanded bed adsorption; MWCO-molecular weight cut-off; PES-polyethersulfone; RC-regenerated cellulose; SEC-size-exclusion chromatography.

Protein Characterization Methods
Characterization and/or identification of isolated proteins is usually carried out by direct comparison with standard molecules and/or data collected from the available literature. This approach is successful until unknown compounds are brought into consideration for which standards are not available [14]. Exploratory methods thus have to be used.

SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to identify the molecular weight of dominant protein subunits bands (Table 5). This method has been used to identify the protein profiles of different seaweeds, such as Ulva spp. [28], Gracilaria changii [109], Caulerpa lentillifera, Caulerpa racemose and Kappaphycus spp., amongst others [110].

Chromatography
As mentioned in Section 3, chromatographic methods are widely used for separation and purification of seaweed proteins and they can be used also for their characterization. High-Performance Liquid Chromatography (HPLC) is often used coupled with other chromatographic methods, such as Size-Exclusion Chromatography (HPSEC) [106]. Amino acid profiles after protein digestion can be analyzed with gaseous or liquid chromatography (GC or LC) with a derivatization step, which includes treatment with ortho-phtalaldehyde (OPA) or fluorenylmethoxy chloroformate (FMOC) [115,116]. However, assessment of the amino acid profile without derivatization by using anion exchange chromatography (HPAEC-PAD) has been reported [117]. Examples of chromatographic methods used for seaweed protein isolation and characterization are shown in Table 6. HPSEC and SDS-PAGE; 4-20% Precast Mini-Protean ® linear gel; two serially connected columns-1st with 5 µm particle size, 150 Å pore size, 2nd with 5 µm particle size and 300 Å pore size. [118]

Spectrometry
Fourier Transform Infrared (FTIR) spectroscopy is used to obtain information about the structural composition of proteins, especially when it comes to their secondary structural conformation [27]. Mass spectrometry (MS) can be an accurate protein identification tool and electrospray ionization (ESI) and matrix-assisted laser desorption ionizations/time-of-flight (MALDI-TOF) can also be important tools [27]. Examples of their application to seaweed proteins are shown in Table 7.
The lyophilized protein concentrate was pelletized with potassium bromide (1/100 ratio w/w) and the spectral analysis was carried out using FTIR. [119] Macrocystis pyrifera and Chondracanthus chamissoi Reverse phase high performance liquid chromatography (RP-HPLC) with inductively coupled plasma mass spectrometry (ICP-MS). [120]

Conclusions
Given the current interest in sustainable protein sources, macroalgae have been identified as promising alternatives due to their protein quality and bioactivities. Seaweed proteins and seaweed-derived proteins are still poorly explored, and the development of appropriate and feasible extraction and purification processes is of growing interest in order to incorporate them in pharmaceutical, nutraceutical, cosmeceutical or food and feed applications. The major identified challenges lie on improving the protein yield and on the disruption of the polysaccharide-rich cell wall. Cell disruption techniques play indeed an essential role in the successful extraction and enrichment of algal protein at larger scales. Additionally, the protein contents and quality highly depend on parameters such as the species, harvesting season, location and growing conditions. Conventional protein extraction methods, such as enzymatic hydrolysis and various physical processes, are laborious, time-consuming and costly, and may require the use of solvents. Novel methods, such as PEF, MAE, PLE and SFE, have been predominantly used in the extraction of compounds other than proteins, and are still poorly explored in algae. Table 8 briefly compares relevant aspects of these conventional and novel extraction methods. To achieve progress in this protein extraction area, future work addressing the applicability of these novel methods is necessary. The same applies to membrane technologies, which also show great promise to pre-purify/concentrate or even isolate (in membrane chromatography) compounds, but are still fully underexploited regarding the protein extraction from macroalgae.