Torularhodin

For cell disruption and carotenoid extraction [65 (link)], 400 mg dry cells was disrupted by grinding them with SiO₂, and carotenoids were extracted with 10 mL of acetone at room temperature. The extract solution was centrifuged at 4500 rpm for 5 min, and the supernatant was collected, while the precipitate was ground and subjected to a second extraction. The supernatants from the two extractions were mixed together, and the OD was measured at the 445 nm (β-carotene) wavelength to determine the total carotenoid content [66 ]. All procedures were carried out in triplicate.
The carotenoid composition was determined using high-performance liquid chromatography (HPLC) [67 (link)]. The instrument and column used were the Agilent 1260 Infinity II and ZORBAX Eclipse XDB-C18 (4.6 mm × 250 mm × 5 µm; Agilent). Torulene, torularhodin, γ-carotene, and β-carotene pigments (CaroteNature, Münsingen, Switzerland) were used as standards for the HPLC quantitative analysis.

Total lipid content was quantified gravimetrically following extraction with Folch reagent (2:1 chloroform/methanol) as described previously [19 (link)]. Carotenoids were extracted with acetone and quantified by high performance liquid chromatography (HPLC) as described previously [20 (link)]. Carotenoid standards β-carotene, torulene, and torularhodin were obtained from carotenature, GmbH (Ostermundigen, Switzerland).

Twenty μL of sample was injected into the column (SETREX IEX H+ 300 × 8 mm column, Polymer IEX H form, 8 μm) and the sugars content was analyzed using HPLC (SYKAM chromatograph, Sykam GmbH, Eresing, Germany combined with a RI detector set at 35°C and with thermostatic control at 35°C in order to avoid fluctuations in detector responses). Samples were eluted isocratically using 5 mM H₂SO₄ as the mobile phase, at the flow rate 1 mL/min. The residual glucose was identified using the recognised standard; under these conditions glucose eluted at 5.2 min.
Twenty μL of carotenoid extract was injected into the column (BIONACOM Velocity CRT column; 250 × 4.6 mm, 5 μm) and the carotenoid composition was analyzed using HPLC (SYKAM S 1125) equipped with a diode array detector (DAD). The applied solvents were acetone (solvent A) and water (solvent B). The solvent flow rate was 1 mL/min with a gradient of 80% A, 20% B at 0 min, 100% A at 12 min, 100% A at 18min, 80% A and 20% B at 19 min, and maintained for 30 min. The carotenoid pigments were identified using the recognised standards: β-carotene (Sigma Aldrich), γ-carotene (CaroteNature, Münsingen, Switzerland), and torulene (CaroteNature), torularhodin (CaroteNature)).