Carotenoid was extracted as described in our previous study (20 (link)). High-performance liquid chromatography (HPLC) was performed for carotenoid quantification in samples. Samples in a volume of 10 μL were loaded on an infinity Lab Proshell 120 EC-C18 column (4.6 × 150, ODS 4 μm). Two solvents A (96% methanol) and B (100% methyl-terc-butyl ether) were used as mobile phase in the following gradient to analyze carotenoid: min/solvent A%/solvent B% was (0/99/1; 8/60/40; 13/46/54; 15/0/100; 18/0/100; 21/99/1; 25/99/1) at a flow rate of 1 ml/min. Column thermostat temperature was set as 35°C and detection wavelength was set as 450 nm, using a diode-array detector (Agilent Technologies, Santa Clara, CA, United States). The following standards were used to identify the carotenoids in transformants: β-carotene, canthaxanthin, astaxanthin, echinenone (Sigma–Aldrich). The total carotenoids were quantified by a spectrophotometer at 450 nm and measured using an extinction coefficient of 2,500 (A1% = 2,500) as previously described (21 ).
Canthaxanthin
Manufactured by Merck Group
Sourced in United States
Canthaxanthin is a synthetic carotenoid compound used as a food colorant and a nutritional supplement. It is a red-orange pigment that can be used to color various food products. Canthaxanthin is approved for use as a food additive in many countries.
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Lab products found in correlation
β carotene, by Merck Group (11 mentions)
Astaxanthin, by Merck Group (10 mentions)
Zeaxanthin, by Merck Group (6 mentions)
Lutein, by Merck Group (5 mentions)
N hexane, by Merck Group (3 mentions)
Lycopene, by Merck Group (3 mentions)
Fucoxanthin, by Merck Group (3 mentions)
Echinenone, by Merck Group (2 mentions)
Acetone, by Merck Group (2 mentions)
Butylated hydroxytoluene bht, by Merck Group (2 mentions)
Methanol of lc ms grade, by Merck Group (2 mentions)
β cryptoxanthin, by Extrasynthese (2 mentions)
E β apo 8 carotenal, by Merck Group (2 mentions)
α carotene, by Merck Group (2 mentions)
Zeaxanthin, by Extrasynthese (2 mentions)
13 z β carotene, by Biosynth (2 mentions)
9 z β carotene, by Biosynth (2 mentions)
Lutein, by Cayman Chemical (2 mentions)
Chlorophyll a, by Merck Group (2 mentions)
Diode array detector, by Agilent Technologies (1 mentions)
16 protocols using canthaxanthin
1
Carotenoid Quantification by HPLC
2
Carotenoid Extraction and Characterization
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Four carotenoid standards (β-carotene ≥ 95%; canthaxanthin ≥ 95%; zeaxanthin ≥ 95%; and astaxanthin ≥ 97%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The salts were purchased from J.T. Baker and Sigma-Aldrich. Glucanex® and hemicellulase were obtained from Sigma-Aldrich and FoodPro® CBL from Chen-Ding Enterprises Co., Ltd. (Taipei, Taiwan). Acetone and n-hexane (LC grade) were from MERCK (Darmstadt, Germany). For chromatography analysis, LC-grade methanol (MeOH) was obtained from MERCK, and LC-grade methyl tert-butyl ether (MtBE) from Duksan Pure Chemicals (Ansan, South Korea). Both hydrochloric acid and ammonia solutions for pH value adjustment were bought from MERCK (Darmstadt, Germany). The tested salts for chelation were purchased from Alfa Aesar (Ward Hill, MA, USA), J.T. Baker, MERCK, and Sigma-Aldrich. The water was double distilled and deionized (≥18 MΩ·cm resistivity at 25 °C). All standard solutions were prepared using LC-grade Acetone.
3
Microalgal Oleoresin Characterization
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The study materials consisted of oleoresin samples of natural carotenoid complex (~10%wt) extracted from the microalga H. pluvialis (Flotow) strain Steptoe (Nevada, USA) with the help of the SC-CO2 method. The oil obtained from H. pluvialis was used in the preparation of the oleoresin. Chilean Company Atacama Bio Natural Products Inc. (Iquique, Chile) kindly donated oleoresin samples to us. General details of the H. pluvialis production are offered on the company’s website (https://www.atacamabionatural.com/web/index.php/about-us/our-process ). All chemicals used in this study were of analytical grade, except for those used for HPLC and GC analyses (high-performance liquid chromatography and gas chromatographic, respectively), which were of chromatographic grade (methanol, acetonitrile, ethyl acetate, water, and n-hexane). The carotenoids astaxanthin, lutein, canthaxanthin, and β-carotene, chemicals for the analysis of total phenols, cholesterol esterase, and reagents for determining antioxidant capacity were procured from Sigma-Aldrich, Santiago, Chile at ≥98% purity. For fatty acid identification and quantification, a standard fatty acid methyl ester (FAME) mix, C4–C24, by Supelco Analytical (Bellefonte, PA, USA) was used, and tripentadecanoin >99% (Nu-Check Pre, Inc., Elysian, MN, USA) was used as the internal standard.
4
Quantification of Leaf Pigments
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Leaf areas of interest were harvested, snap‐frozen in liquid nitrogen and lyophilized until they were completely dry. Approximately 4 mg of this freeze‐dried tissue (corresponding to samples from different leaves pooled together) was mixed with 375 µL of methanol and 25 µL of a 10 % (w/v) solution of canthaxanthin (Sigma) in chloroform. Extraction and separation of chlorophylls and carotenoids were then performed as described (Emiliani et al., 2018 ). Eluting compounds were monitored using a photodiode array detector. Peak areas of chlorophylls at 650 nm and carotenoids at 470 nm (lycopene, lutein, β‐carotene, violaxanthin, neoxanthin, canthaxanthin) or 280 nm (phytoene) were determined using the Agilent ChemStation software. Quantification was performed by comparison with commercial standards (Sigma).
5
Identification and Quantification of Carotenoids by TLC
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The extracted carotenoids from the previous section were saponified and separated on a precoated TLC Aluminiumoxid 60 F254 neutral plate (Merck). For saponification, the dry pigment was recovered by evaporating the extraction solvent (see Section 4.9 ), and dissolved it in 500 μL of a methanol/chloroform mixture (2: 1 v/v). A 50 μL portion of alkaline methanol (112.2 g KOH per L methanol) was added. This mixture was incubated at 40 °C for 30 min. The liquid phase was recovered by centrifugation (10,000× g, 10 min) and loaded on the TLC plate. Solutions of authentic astaxanthin, canthaxanthin, and β-carotene (Sigma) were used as standards. The mobile phase was 75:25 by volume mixture of petroleum ether and acetone. Chromatograms were developed at room temperature in a glass chromatographic chamber saturated with the vapor of the mobile phase. The developed plate was dried in the air in the dark.
6
Carotenoid Quantification in Plasma
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The analyzed samples were −80°C stored K3-EDTA fasting plasma that had been drawn at baseline. Handling of all samples and standards was always done under cool conditions and avoiding exposure to light. Carotenoid standards: astaxanthin, canthaxanthin, E-β-apo-8′-carotenal, α-carotene, β-carotene, fucoxanthin, and lycopene were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lutein was purchased from Cayman Chemical (Ann Arbor, MI, USA), zeaxanthin and β-cryptoxanthin were provided by Extrasynthese (Genay, Lyon, France). 13-Z-β-carotene and 9-Z-β-carotene were purchased from Carbosynth (Newbury, Berkshire, UK).
Methanol of LC-MS grade, n-hexane, ethanol, and methyl tert-butyl ether (MTBE) of HPLC grade, blank human plasma, and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich. Ammonium acetate (AMAC) and acetic acid of HPLC grade were purchased from Panreac Quimica SLU (Barcelona, Spain). Ultrapure water (Milli-Q) was generated by a Millipore system (Bedford, MA, USA).
Methanol of LC-MS grade, n-hexane, ethanol, and methyl tert-butyl ether (MTBE) of HPLC grade, blank human plasma, and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich. Ammonium acetate (AMAC) and acetic acid of HPLC grade were purchased from Panreac Quimica SLU (Barcelona, Spain). Ultrapure water (Milli-Q) was generated by a Millipore system (Bedford, MA, USA).
7
HPLC Analysis of Carotenoid Production
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Carotenoid production was analyzed by HPLC analysis. First, carotenoids were extracted from the cell fraction of the cultivation broth using a methanol:acetone (7:3) mixture. Extraction was performed at 60 °C and 600 rpm for 15 min. After centrifugation at 14,000 rpm and 10 min the supernatant was used for high performance liquid chromatography (HPLC). The Agilent 1200 series system (Agilent Technologies, Waldbronn, Germany) was used with a reversed phase column system. Carotenoids were detected with a diode array detector (DAD) through recording of the UV/visible (Vis) spectrum. For quantification the extracted wavelength chromatogram at λmax 470 nm was used. Calibration curves were generated with standard substances: β-carotene, canthaxanthin and astaxanthin (Sigma-Aldrich). The column system consisted of a precolumn (LiChrospher 100 RP18 EC-5, 40 × 4 mm) and a main column (LiChrospher 100 RP18 EC-5, 125 × 4 mm). Methanol (A) and methanol:water (9:1) (B) were used as the mobile phase. A gradient at a flow rate of 1.5 mL/min was used as the following; 0 min B: 0%, 10 min B: 100%, 32.5 min B: 100%.
8
Carotenoid Analysis in Fasting Plasma
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K3-EDTA fasting plasma samples from the baseline blood extractions were analyzed. These samples were drawn in the first visit of the study, just after being randomly assigned to an intervention group and stored at −80 °C until use. All samples and standards were always handled avoiding exposure to light and under cool conditions. Carotenoid standards: astaxanthin, canthaxanthin, E-β-apo-8′-carotenal, α-carotene, β-carotene, fucoxanthin, and lycopene were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lutein was provided by Cayman Chemical (Ann Arbor, MI, USA), zeaxanthin and β-cryptoxanthin were purchased from Extrasynthese (Genay, Lyon, France). 13-Z-β-carotene and 9-Z-β-carotene were purchased from Carbosynth (Newbury, Berkshire, UK). Standards were stored in powder form at −20 °C and protected from light.
Methanol of LC-MS grade, n-hexane, ethanol and methyl tert-butyl ether (MTBE) of HPLC grade, blank human plasma and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich. Ammonium acetate (AMAC) and acetic acid of HPLC grade were purchased from Panreac Quimica SLU (Barcelona, Spain). Ultrapure water (Milli-Q) was generated by a Millipore system (Bedford, MA, USA).
Methanol of LC-MS grade, n-hexane, ethanol and methyl tert-butyl ether (MTBE) of HPLC grade, blank human plasma and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich. Ammonium acetate (AMAC) and acetic acid of HPLC grade were purchased from Panreac Quimica SLU (Barcelona, Spain). Ultrapure water (Milli-Q) was generated by a Millipore system (Bedford, MA, USA).
9
HPLC Analysis of Carotenoids in Samples
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Extracts were analyzed by HPLC with an automatic injection pump (Agilent Technology HPLC 1260) and diode array detector (model G4212B) in 5 μm columns (ZORBAX SB-C8, 4.6 cm x150 mm). The mobile phase was activated by a quaternary pump (model VL 1260). Solvents used were: solvent A, methanol, 1 N ammonium acetate (70:30 v/v); and solvent B, methanol at a flow rate of 1 mL min−1 for 20 min at room temperature. The gradient system was according the following procedure (minutes; %solvent A, % solvent B): (0; 75, 25), (1; 50, 50), (15; 0, 100), (18; 0, 100), and (18.5; 75, 25) [57 ]. astaxanthin and other carotenoids were identified at 440 nm and compared against known standards for retention time and absorption spectrum. Known standards were astaxanthin, zeaxanthin, β-carotene, canthaxanthin, and chlorophyll-a (Sigma-Aldrich).
10
Carotenoids and Tocopherols Quantification
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Lutein, zeaxanthin, β-cryptoxanthin, canthaxanthin, astaxanthin, apocarotenal, physalien, β-carotene, lycopene and tocopherols (α, β, γ and δ) were from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Acetonitrile, methanol, ethanol, dichloromethane, acetone, n-hexane and ascorbic acid were of analytical-grade purity and purchased from Merck (Darmstadt, Germany). Water was prepared using a Millipore Simplicity 185 S.A., 67120, water purification system (Molshem, France). The standard FAME Mixture of 37 components was purchased from Food Industry FAME Mix, RESTEK (Bellefonte, PA, USA) (lot 24676).
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