## 2 Protein Extraction  
### 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 carrageenanans 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  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Species** & **Peptide** & **Bioactive Activity** & **Ref.** \\ \hline _Bryopsis_ spp. & Kahalalide F & Antitumoral activity & [57] \\ \hline _Galaxaura filamentous_ & Galaxamide & Anti-proliferative activity against human epithelial cancer cell lines & [60] \\ \hline \multirow{3}{*}{_Palmaria palmata_} & Asn-Ile-Gly-Gln & Anti-inflammatory activity & [61] \\ \cline{2-4}  & Val-Tyr-Arg-Thr; Leu-Asp-Tyr; Leu-Arg-Tyr; Phe-Glu-Gln-Trp-Ala-Ser & [62] \\ \hline \multirow{3}{*}{_Porphyna vezoensis_} & Ile-Tyr; Met-Lys-Tyr; Ala-Lys-Tyr-Ser-Tyr; Ley-Arg-Tyr & ACE-I inhibitory activity & [63] \\ \cline{2-4}  & Ala-Lys-Tyr-Ser-Tyr & & [64] \\ \hline \multirow{3}{*}{_Undaria pinnatifida_} & Val-Tyr; Ile-Tyr; Phe-Tyr; Ile-Trp & & [65] \\ \cline{2-4}  & Val-Tyr; Ile-Tyr; Ala-Trp; Phe-Tyr; Val-Trp; Ile-Trp; Leu-Trp & Anti-hypertensive & \\ \hline \hline \end{tabular}
\end{table}
Table 2: The bioactivities of several macroalgal peptides.  
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.