Carotenoid column

Contents of lutein and β-carotene were evaluated using HPLC with a diode array detector (at 440 nm), on a YMC Carotenoid column (3 μm particle size, 150 x 4.0 mm; YMC, Japan). Carotenoids were extracted using 80% acetone (1 g of sample grounded with liquid nitrogen 10 ml^-1 of solvent), centrifuged (5 min, 349 x g), and filtrated through a 0.45-μm nylon membrane syringe filter (VWR International, USA). The HPLC 10A system (Shimadzu, Japan) equipped with a diode array (SPD-M 10A VP) detector was used for analysis. Peaks were detected at 440 nm. The mobile phase consisted of A (80% methanol, 20% water) and B (100% ethylacetate). Gradient: 0 min; 20% B, 2.5 min; 22.5% B, 20–22.5 min; 50% B, 24–26 min; 80% B, 31–34 min; 100% B, 42–47 min; and 20% B, flow rate 1 ml min^-1. The sensitivity of all chromatographic methods was established using a method validation procedure outlined by Edelenbos et al. [24 (link)].

When grown on R2A medium, strain DY-R2A-6^T exhibited carotenoid accumulation, resulting in the production of a pink-yellow pigment. To extract the total carotenoids, fresh cells (100 mg) were mixed with 1 mL of methanol (HPLC grade) and sonicated for 10 min. The methanol extract was then centrifuged at 8,000 rpm for 10 min and the supernatant was collected and passed through a 0.2-μm membrane filter. The UV–Vis absorption spectrum (200–800 nm) of the total carotenoid extracts was recorded using a microplate spectrophotometer (Multiskan SkyHigh, Thermo Fisher Scientific). The obtained data were compared with those of standards and published values.
To further identify the pigment, the methanol extract was analyzed using an HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a YMC carotenoid column (250 × 4.6 mm.D, S-5 μm, YMC, Japan). The separation column was maintained at a temperature of 35°C, with solvent A, consisting of methanol: MTBE: water (85, 10:5, v/v), and solvent B, consisting of 100% MTBE, as the mobile phase. The flow rate was set to 1 mL/min, the wavelength was set at 450 nm, and a 20-μL sample was injected and analyzed at EZmass, Inc. (Korea).¹²

β-carotene was extracted using 80% acetone (1 g of sample ground with liquid N and 10 ml^-1 of solvent) and then centrifuged (5 min, 4000 rpm min^-1) and filtered through a 0.45-µm nylon membrane syringe filter (VWR International, United States). The contents of β-carotene were evaluated using a Shimadzu HPLC (Japan) instrument equipped with a diode array detector (SPD-M 10A VP) on a YMC Carotenoid column (3 µm particle size, 150 × 4.0 mm) (YMC, Japan). The mobile phase consisted of A (80% methanol, 20% water) and B (100% ethyl acetate). The gradient was as follows: 0 min; 20% B, 2.5 min; 22.5% B, 20–22.5 min; 50% B, 24-26 min; 80% B, 31–34 min; 100% B, 42–47 min; and 20% B, flow rate 1 ml min^-1 (Edelenbos et al., 2001 (link)). The peak was detected at 440 nm and identified using an external calibration method.

All solvents were purchased from Sigma-Aldrich (St Louis, MO). Isolated lutein and zeaxanthin were obtained as described previously [14 (link)]. Astaxanthin and -carotene were the kind gift of Prof. Hideki Hashimoto; the latter was further purified by thin-layer chromatography, using hexane as mobile phase. The galloxanthin fraction from HPLC of chicken retinas (see below) was further purified using a YMC Carotenoid column (5 μm particle size, 4.6 x 250 mm) with an isocratic delivery of acetonitrile. For retinal samples, the eyes were isolated from the head of euthanized chickens. After incision, the anterior segment was cut off with scissors. The iris, lens, ciliary body and anterior sclera were dissected out, and most of the vitreous removed. The central retina was trepanned with a 10 mm circular punch, separated from the underlying retinal pigment epithelium and overlying vitreous, and placed on glass microscope slides for use in confocal Raman measurements. The slides were viewed under an epi-fluorescence microscope using visible and UV illumination (Leica DM5000, Germany).

Xanthophylls (neoxanthin, violaxanthin, lutein, and zeaxanthin), carotenes (carotenes α and β), and chlorophylls (chlorophylls a and b) were extracted using 80% acetone (1 g of sample ground with liquid N₂ and 10 ml of solvent), centrifuged (5 min, 4,000 rpm), and filtered through a 0.45-μm nylon membrane syringe filter (VWR International, United States). The contents of carotenes, xanthophylls, and chlorophylls were evaluated using a Shimadzu HPLC (Japan) instrument equipped with a diode array detector (SPD-M 10A VP) on a YMC Carotenoid column (3 μm particle size, 150 × 4.0 mm) (YMC, Japan). The mobile phase consisted of A (80% methanol, 20% water) and B (100% ethyl acetate). The gradient was as follows: 0 min; 20% B, 2.5 min; 22.5% B, 20–22.5 min; 50% B, 24–26 min; 80% B, 31–34 min; 100% B, 42–47 min; and 20% B, flow rate 1 ml min^–1 (Edelenbos et al., 2001 (link)). The diode array detector was employed at 440 nm, and the absorption spectra of xanthophylls, carotenes, and chlorophylls were identified using an external standard calibration method.

A single colony of the candidate strains to be fermented were inoculated into 3 mL SC-Ura medium at 30 °C, 220 rpm for 24 h, and then the preculture was transferred into a 14 mL shaking tube containing 5 mL fresh SC-Ura with an initial OD₆₀₀ = 0.1. The batch cultivation was conducted at 30 or 20 °C, 220 rpm for 5 days.
After fermentation, 1 mL of cell culture was collected by centrifugation at 8000× g rpm for 5 min and washed with deionized water. The cells were resuspended with 0.5 mL acetone and petroleum ether (9:1) mixture and broken with glass beads (1 g, 0.50–0.75 mm) by vortex for 10 min. The mixture was centrifuged at 12,000× g rpm for 5 min and the supernatant was transferred to a new tube. The extraction was repeated by adding 0.5 mL acetone and petroleum ether (9:1) mixture again and sonicated until the pellets were colorless.
Lycopene was analyzed using reversed-phase high performance liquid chromatography using isocratic elution and UV detection at 472 nm (Agilent 1260 Infinity II). A YMC Carotenoid column (250 mm × 4.6 mm, S-5 µm, Catalog number: CT99S05-2546WT) was used. Mobile phases A (methanol/MTBE/water = 81/15/4, v/v) and B (methanol/MTBE/water = 6/90/4, v/v) were eluted as 5%, 100%, and 100%, at 0, 7 and 15 min (MTBE: methyl tert-butyl ether). The flow rate was 1 mL/min column temperature was 30 °C and the injection volume 30 µL.

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)].