# Extraction of Bioactive Compounds from _Ulva lactuca_
## 2 Materials and Methods
### Methods  
#### 2.3.1 Biochemical Characterization  
Lipid Content  
Lipid extraction was conducted based on the Folch method [24]. Particularly, 0.5 g of seaweed were extracted with 15 mL of chloroform/methanol (1:2) mixture, in an ultrasonic bath (Elma TRANSONIC DIGITAL) for 4 min and left overnight at room temperature in the dark. Then, the sample was centrifuged (HERMLE Z 206 A) at 3000 rpm for 10 min. The supernatant was transferred into a separating funnel where the chloroform phase was collected in a pre-weighed flask. The chloroform phase was then concentrated in a rotary evaporator (Hei-VAP Advantage HL G3, Heidolph, Germany) at 45 \({}^{\circ}\)C and 150 mbar to recover the lipids. Total lipids were gravimetrically determined as wt% of the biomass.  
For the determination of the protein content, the extraction procedure described by [PERSON] et al., 2019 [25] was followed. _U. lactuca_ biomass was stirred overnight at 4 \({}^{\circ}\)C with distilled water 5% _w/v._ The supernatant was separated using a centrifuge at 5000 rpm for 15 min. Mercaptethanol (0.5% _v/v_) was added to the pellet, pH was adjusted to 12.0 with the addition of 1 M NaOH solution and left for 2 h under stirring at room temperature. The supernatant was separated again via centrifugation. The supernatants from both centrifugation cycles were mixed and the pH was adjusted to 7.0 using 1 M HCl solution. Solid ammonium sulfate was added to the supernatant up to 85% saturation. The solution was kept for 1 h at room temperature and then centrifuged in order to separate the precipitate. The sediment was then washed several times with deionized water, oven-dried (at 100 \({}^{\circ}\)C overnight) and measured in order to calculate the protein content gravimetrically as wt% of the biomass.  
The polysaccharide content was determined based on the method of [PERSON] et al., 2006 [26]. According to this method, 100 mL of distilled water was added to five grams of previously defatted _U. lactuca_ biomass and the extraction was carried out at 80-90 \({}^{\circ}\)C under continuous stirring for 2 h. The supernatant was collected after centrifugation at 3000 rpm for 10 min in a pre-weighed flask. The supernatant was then concentrated using a rotary evaporator at 50 \({}^{\circ}\)C and 74 mbar and precipitated with 4 volumes of ethanol. Centrifugation and concentration were repeated. The extracted polysaccharide was washed with distilled water, freeze-dried, and weighed.  
_U. lactuca's_ polysaccharide content was gravimetrically determined as wt% of the biomass.  
Determination of Mineral Ash  
Ash content was determined gravimetrically. Specifically, 2.5 g of _U. lactuca_ biomass were weighed in a crucible and combusted for 3 h at 550 \({}^{\circ}\)C using a muffle furnace (Thermolyne 47900). Water and other volatile materials are vaporized, and organic substances are burned in the presence of oxygen to CO\({}_{2}\), H\({}_{2}\)O, and N\({}_{2}\). After the combustion completion, the crucible was placed in a desiccator and weighed soon after cooling.  
The mineral ash content of _U. lactuca_ macroalgae was gravimetrically determined as wt% of the biomass.