Extraction and Purification of Carotenoid

The yellow pigments of strain KCCP11226 were extracted according to our previous study [9]. In brief, 100 ml of cultured cells were harvested by centrifugation at 10,000 ×g for 10 min and extracted in 5 ml methanol overnight. Next, 5ml of hexane and 2.5ml of distilled water were added to the methanol extract. After centrifugation at 8,000 ×g for 10 min, the organic phase containing carotenoids was transferred to a 15 ml tube. The organic phase was evaporated and then dissolved in 1 ml of petroleum ether. The pigmentation levels of crude carotenoids were measured at an absorption wavelength of 470 nm (A₄₇₀) using a spectrophotometer (Shimadzu, Japan).

For purification of 4,4′-diaponeurosporene, the isolated carotenoids were salted out with 5 N NaCl solution and re-extracted with an equal volume of ethyl acetate (EtOAc). The upper phase was loaded onto an anhydrous sodium sulfate column (BioBasic, Canada) for dehydration and then evaporated. The dried carotenoids were dissolved in EtOAc, filtered, and purified by loading on a silica gel column [18]. The total amount of purified carotenoids was quantified using the previously reported extinction coefficient of 4,4′-diaponeurosporene [19, 20].