2.5. Quantification of Glycolipids and Phospholipids

Glycolipids were quantified by measuring the sugar content using the orcinol method [44]. For that, 100 µg of each dried lipid extract in 2 mL of orcinol solution (0.2% in 70% H₂SO₄) were incubated at 80 °C for 20 min. After cooling to room temperature, the absorbance was read at 505 nm (Multiskan GO, Thermo Scientific, Hudson, NH, USA). For determination of the sugar content in each lipid extract, a calibration curve was obtained by performing the same reaction with glucose standards (up to 40 μg) prepared from an aqueous glucose solution (5 mg mL⁻¹). The content of glycolipids was estimated by multiplying the sugar content by 2.8 [45].

Phospholipids quantification was achieved by measuring the phosphorous amount using the molybdovanadate method [46], as done in our previous work [44]. The lipid hydrolysis was performed by adding 70% perchloric acid (125 μL) to each dried lipid extract (50 µg in a glass tube washed with 5% nitric acid), followed by incubation on a heating block at 180 °C for 1 h. After cooling to room temperature, Milli-Q water (825 μL), ammonium molybdate (2.5 g 100 mL⁻¹ in Milli-Q water; 125 μL), and ascorbic acid (0.1 g mL⁻¹ in Milli-Q water; 125 μL) were added to each sample, vortexing after each addition. Samples were placed in a water bath at 100 °C for 10 min and then allowed to cool in a cold-water bath. Phosphate standards containing up to 2 μg of phosphorus (P) were prepared from a sodium dihydrogen phosphate dihydrate solution (100 μg mL⁻¹ of P), using the same procedure as samples, excluding the heating on the block heater. After reading the absorbance at 797 nm on the microplate UV-Vis spectrophotometer, the content of phospholipids was estimated by multiplying the amount of P by 25 [47].