## 4 Discussion
Both temperature and pH had significant effects on UV absorbance. Temperature followed a quadratic curve with a minimum absorbance at the middle of the range and, at the end, experience maximum near-RT and near-boiling. The compounds that were hypothesized to precipitate with the increased extraction of other molecules (observed in the yield) are possibly responsible for the UV absorbance (likely, the proteins). In the 50% (_v/v_) ethanol, even though the yield increased at mid temperature and decreased at higher ones, UV-absorbance was minimal at mid temperature increased with higher temperature. Chemical characterization of the extracts using chromatographic and spectroscopic techniques could assist in describing these mass tranfer nuances. The effect of pH was seemingly impacted by temperature, with neutral pH being better for UV absorbance recovery with hot extraction, following a quadratic curve, while room temperature extracts had no- to linear-effect with increased ethanol. The solubility of UV-absorbing proteins and MAAs might be impacted by pH since these molecules contain a certain balance of carboxylic and amine groups, which varies their state of protonation with pH [45]. Considering that yield is lower under alkaline conditions, it is likely that an increase in UV-absorbance of alkaline extracts at room temperature is, however, caused by a lower co-extraction of non-absorbing molecules, rather than a direct effect on the extraction of UV-absorbing molecules. Considering extraction yield and UV-absorbance together (total UV-absorbance, see Figure 8), it is clear that room temperature and acidic solvents, especially those containing ethanol, are the best extraction method (concerning pH and temperature) for UV-absorbing molecules recovery from _G. turuturu_. It is important to observe how significant the impact of pH adjustment is. In this study, when working at room temperature, the addition of HCl to the solvent allowed increments in UV-absorbing molecules a total recovery of 33% to 62% for 50% ethanol and 0% (_v/v_) ethanol, respectively. This represents a significant increase in profitability and therefore in the sustainability of industrially exploring this species of seaweed with a simple modification of the extraction protocol. Finally, the antioxidant activity of the second Box-Behnken was addressed (Figure 9). Ethanol inclusion in the solvent of extraction, as expected, let to an increase in ORAC at room temperature. However, upon manipulation of temperature, pH, or both, ethanol led to a decrease in ORAC at low inclusion levels that only resumed the increasing trend with inclusions near 50% (_v/v_). It might be that pH and temperature manipulation leads to higher recovery of polysaccharides of different chemical natures, and that this recovery is different with high-, low-, and absent-ethanol. Regardless, at any given ethanol inclusion level, the effects of temperature and pH followed similar trends: an increase in pH led to a linear increase of ORAC; temperature, yield, and UV-absorbance had a minimum at the middle of the range, meaning either cold or hot extractions were privileged to medium-heat ones, possibly due to the phenomena of precipitation. The total ORAC analysis (Figure 10) revealed skewed patterns of effects and interactions between temperature and pH. However, for all ethanol percentages, ORAC-reactive antioxidants were extracted most at near-boiling temperatures, with little to no effect of pH, suggesting that higher temperature (under pressure) hydroethanolic extraction of _G. turuturu_ may lead to even higher recoveries than those obtained in this study. Probably of a different chemical nature, with 50% (_v/v_) ethanol, cooler acidic extractions also resulted in the accumulation of substantial amounts of ORAC-active compounds.