Carotenoid Extraction and Analysis

Carotenoids were extracted using methods specific to the material being examined. For the pericarp tissues, the extraction and saponification were conducted as described by Kim et al. (2016) (link). For the E. coli transformants, cell pellets were obtained using centrifugation, from which the carotenoids were extracted with 600 μl HPLC-grade acetone (Honeywell, Charlotte, USA), as described by López-Emparán et al. (2014) (link).
For the extracts derived from both the pericarps and E. coli cell pellets, the carotenoids were analysed using ultra-performance liquid chromatography (UPLC) on an Acquity UPLC-H-Class system (Waters). The compounds were separated using an Acquity UPLC HSS T3 column (2.1×100, 1.8 μm) at 35 °C. The mobile phase was a binary solvent system consisting of phase A (acetonitrile/methanol/methylene chloride, 65/25/10, v/v/v) and phase B (distilled water). The gradients were programmed according to the method described by Kim et al. (2016) (link). For the qualitative and quantitative analyses, 11 carotenoid standards were purchased from Sigma-Aldrich, namely antheraxanthin, capsanthin, capsorubin, lutein, neoxanthin, violaxanthin, zeaxanthin, α-carotene, α-cryptoxanthin, β-carotene, and β-cryptoxanthin.