## 5 Conclusions
All authors have read and agreed to the published version of the manuscript. This research was funded by Fundo Azul--Direccao-Geral de Politica do Mar, Ministerio do Mar, Portugal, grant number FA_05_2017_033. Funding received by national funds from FCT--Fundacao para a Ciencia e a Tecnologia, I.P., in the scope of the project UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit Institute for Bioengineering and Biosciences--iBB and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy--i4 HB is acknowledged. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Aim/Method** & **Main Advantages** & **Main Drawbacks** & **Ease of Scale-Up **\({}^{**}\)** \\ \hline _Cell wall disruption_ & & & \\ \hline Osmotic shock & Low energy; selectivity & Cost of chemicals; diluted product & +++ \\ Mechanical shearing & No chemicals; high yield & Energy cost; heat damage \({}^{*}\) & +++ \\ Enzyme treatment (EAE) & Low energy; selectivity & Cost of enzymes; product & ++ \\  & & contaminated with enzyme & ++ \\ Ultrasonication (UAE) & No chemicals; high yield & Energy cost; heat damage \({}^{*}\); & ++ \\  & & equipment cost & ++ \\ Pulsed electric field (PEFE) & No chemicals; high yield & Energy cost; heat damage \({}^{*}\); & + \\  & & equipment cost & + \\ Microwave (MAE) & No chemicals; high yield & Energy cost; heat damage \({}^{*}\); & + \\  & & equipment cost & \\ \hline _Protein extraction_ & & & \\ \hline Aqueous solubilization (water, & Mild conditions; & Limited recovery yield; & +++ \\ alkali, buffer) & bioactivity preserved & diluted product & +++ \\  & Mild conditions; bioactivity & Cost of chemicals; improvements & \\ Aqueous biphasic systems & preserved; achievable high & depend on cost and & ++ \\  & recovery yield and selectivity; & environmental impact & \\ Pressurized liquid extraction & & Energy cost; heat damage \({}^{*}\); & \\ (PLE)/Accelerated solvent & Higher recovery yield & equipment cost; possible product & ++ \\  & extraction (ASE) & contamination by co-solvents & \\ Enzymatic protein & Higher recovery yield; tailored & Cost of enzymes; loss of protein & \\ hydrolysis (EAE) & protein modification; possible & bioactivity/function & ++ \\  & gain of peptide bioactivities & & \\ \hline _Protein enrichment_ & & & \\ \hline Precipitation & High recovery yield; & & \\  & mild conditions & Cost of chemicals; low selectivity & +++ \\ Membrane separation (MF, UF, & High recovery yield; no chemicals; & Cost of membranes; & +++ \\ NF, RO) & mild conditions & low selectivity & +++ \\ Adsorption/Chromatography & High selectivity; high & Cost of adsorber media, & \\  & recovery yield & instrumentation and & ++ \\  & & buffer chemicals & \\ Supercritical fluid & High selectivity for low & Energy cost; equipment cost; & \\ extraction (SFE) & polarity impurities & possible product contamination & + \\  & by co-solvents & \\ \hline \hline \end{tabular} \({}^{*}\) Need of thermal control. \({}^{**}\) From + (minimum) to ++++ (maximum).  
\end{table} 
Table 8: Comparison of the relevant aspects of protein extraction methods. **Acknowledgments:** Work developed within the scope of the Smart Valorization of Macroalgae project with the support of IBERAGAR S.A., SPAROS Ltd and IPMA. **Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.