## 3 Extractions Processes
### Ulvan Extraction  
Ulvans differ in their molecular weight, sugar composition, and sulfation ratio; these factors influence the biological properties. Therefore, increasingly sensitive, and reliable analytical methods, the mechanisms by which ulvan interferes with the different biological systems, and the knowledge of the smallest structure required for bioactivity have induced companies to produce well-defined ulvans (second generation). The extraction method, chemical characterization and quantity need to be considered for industrial production. However, much effort is currently required to explore the structural diversity of the ulvan broad family for an industrial perspective in relation to its biological properties. Since ulvan is a complex polymer associated to proteins and with others cell-wall components of _Ulva_[38], the ulvan extraction procedure is a critical point.  
Macceration or Soxhlet extraction with solvents (ethanol or methanol) are important steps for defatting the macroalgae material to remove amino acids, peptides, lipids, and pigments. They are used as conditions to obtain high pure molecular weight and better extraction yields from 2.8 to 13.8% [84]. Alternatively, algae powder can be incubated 24 h with acetone to remove lipids and pigments [85]. However, [PERSON] et al. [63] considered that such pre-treatments have no effect on either extraction efficiency or the quality of ulvan extracted and for this reason are considered unnecessary.  
To enhance extraction efficiency and to provide better ulvan yield, _Ulva_ biomass is dried in air at 70 \({}^{\circ}\)C [68] and finely milled to obtain high recovery and pure product [63]. The maximum ulvan extraction efficiency depends also on the active growing period of the macroalgae [44]. _Ulva_ is usually obtained with hot water in the autoclave for 1-2 h at 90 or 110 \({}^{\circ}\)C (1.97 atm) or heated (1, 2 or 3 h) [63; 67]. The extraction is improved by the presence of divalent cation chelating agents, such as ammonium oxalate or sodium oxalate [68]. The extract can be treated with enzymes such as protease (proteinase K) for proteolytic digestion to remove contaminants [86]. Other methods include multilevel extraction procedures, using water followed by the addition of NaCO\({}_{3}\) and NaOH or acid solutions and ammonium oxalate, but the stability may be decreased at low pH [72]. The extract is separated from the biomass by filtration and/or centrifugation. Regardless of the extraction method, precipitation of ulvan is achieved by adding 2-2.5 volumes of ethanol (70 or 96%, cold or not) and is the most common method used in the laboratory to recover the polymer, while ethanol-soluble compounds, such as metabolites, remain in solution. Although ethanol precipitation is a suitable method, it is limited by the low solubility of salts and high solubility of low molecular weight ulvan in ethanol-water mixtures. Later, the ulvan aqueous extract is filtered, concentrated in a rotary evaporator, and air-dried or even freeze-dried [67]. As a final step, the precipitation methods are followed by efficient desalting and removal of small molecules contaminants by dialysis membrane, ultrafiltration, or even chromatography [63].