Lycopene

Intracellular carotenoids were extracted from cellular pellets according to the previous method [4 (link)]. Briefly, 10–50 μL bacterial culture was collected and centrifuged. Cell pellets were washed with PBS and were resuspended in 20 μl of water, followed by addition of 180 μl of acetone. The high performance liquid chromatography (HPLC) method was modified as previously described. Briefly, the analysis employed an Agilent 1260 Infinity LC System equipped with a ZORBAX, Eclipse Plus C18, 4.6 × 250 mm, 5 μm column and diode array detector (DAD). Isocratic condition (50% methanol, 48% ethyl acetate, and 2% water) was maintained at 1.5 mL/min for 5 min. The carotenoids were detected at wavelength of 450 nm. Standard curves were generated using commercial standards of ε-carotene (CaroteNature, Switzerland) and lycopene (Santa Cruz Biotechnology, Dallas, TX).

Carotenoids were extracted as described above and analyzed using high-performance liquid chromatography (1260 Infinity II, Agilent, CA, USA) equipped with a C30 column (YMC Carotenoid column, 250 mm, 5 μm pore size). Mobile phase A consisted of 15:81:4 Methyl tert-Butyl Ether (MTBE):methanol:water by volume, and mobile phase B consisted of 81:15:4MTBE: methanol:water by volume. Using a flow rate of 1.0 mL/min at 20 °C, a linear elution gradient from 100% A to 100% B over 15 min was followed by 12 min of 100% B before returning to mobile phase A over 3 min. HPLC standards (astaxanthin, lycopene, β-carotene, zeaxanthin, and canthaxanthin) were purchased from Santa Cruz Biotechnology for identification of carotenoid retention times. zeaxanthin was used to identify isozeaxanthin as this compound cannot be purchased, and these isomers are known to co-elute using C18 chromatography [20 (link)].