## 4 Discussion
Typically, for SLEs, temperature increases were expected to result when yield increased and more so when using water as a solvent. Solubility of organic substances tends to increase with temperature, and mass transfer phenomena become facilitated by kinetic energy possessed at higher temperatures [42; 43]. The adjustment of pH is not something for which a trend is typically assigned,as it highly depends on the solvent and matrix being extracted; if pH changes results in higher or lower solubilities depends entirely on each molecule's characteristics [7]. For seaweed, however, because the matrix has a high content of polysaccharides and proteins, both macromolecules for which pH drastically alters their solubilities, pH adjustment is expected to result in very different extracts. The yield of the extracts produced for the second optimization (Figure 6) revealed some unexpected effects. In the water extract, heating led to a decrease in yield that only reverted to an increase in the middle of the range (approximatley 60 \({}^{\circ}\)C), reaching a maximum yield at 95 \({}^{\circ}\)C, but had comparable values at room temperature. In the 25% (_v/v_) ethanol, the yield was not affected by temperature at a neutral pH. In the 50% (_v/v_) ethanol, the yield did increase with temperature, but in the middle of the range, it decreased to values comparable to room temperature. None of these three phenomena are in accordance with the SLE theory, but have been observed for seaweed extracts before; conditions of higher solvent availability/solubility sometimes result in lower yields [7]. In this case, it might be that, for water extracts, the compounds extracted at room temperature, plus the extra compounds extracted at 60 \({}^{\circ}\)C, end up interacting (for example, protein-polysaccharides adsorption), precipitating, and being removed along with the extracted biomass particles. The same phenomena might occur in the 50% (_v/v_) ethanol for the high-end temperatures tested. By modifying the pH, it is less odd that events like these occur or cease to occur. Side chains of proteins and polysaccharides become either totally protonated (in acid pH) or totally deprotonated (in alkaline pH) to a point where adsorption phenomena and solvation phenomena change significantly [42; 43]. In _G. turuturu_, pH adjustment did not significantly impact the water-based extraction at room temperature, because whatever was being extracted had low molecular weight carbohydrates for which protonation was less relevant in terms of solubility than higher-molecular weight ones. At a near-boiling temperature, however, a linear increase of yield with pH suggested that alkalinity promoted extraction, which was expected since alkaline hydrolysis was one of the methods to dissolve cellulosic biomass and solvate their carbohydrates [44]. With ethanol in the solvent, this trend was reversed and we observed an almost-linear increase of yield and decreased pH. These events may be related to an increased ease of mass transfer due to a certain degree of acid and alkaline-hydrolysis of the cellulose cell walls. If that is the case, then in ethanol-containing solvents it has been demonstrated that acidic pH increased the solubility of _G. turuturu_ metabolites, perhaps by disrupting the hydrogen bonds between proteins and such other solubules. UV-absorbance of the extracts in the second Box-Behnken (Figure 7) followed a similar trend across the different ethanol percentages, which makes sense considering that in the initial optimization it was in the vicinity of 40 mg:mL\({}^{-1}\) with 60 min of extraction, and the ethanol percentage had a low effect (pattern was similar). This reinforced that ethanol percentage, using this LSR and time, did not change extract composition in terms of UV-absorbing molecules (the proteins, the MAAs, and some polyphenols). However, like the first RSM, higher ethanol did promote higher absolute UV-absorbance values, possibly by decreasing the co-extraction of non-absorbing molecules.