Butylated hydroxytoluene (BHT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), polyunsaturated linoleic acid, and beta-carotene (β-Carotene) were provided from Sigma-Aldrich (Sigma-Aldrich, Deisenhofen, Germany). Low-melting agarose and Ficoll-Paque (density 1.077 g/ml) were attained from Thermo Fisher Scientific (Rockford, IL, US) and GE Healthcare (Madison, WI, USA), respectively. Luria-Bertani, Mueller-Hinton, and Sabouraud dextrose Media were purchased from Merck Company (Darmstadt, Germany). Also, other pure analytical grade compounds (>99% purity) were attained from the Iranian commercial resources.
β carotene
Manufactured by Merck Group
Sourced in United States, Germany, Italy, France, Spain, United Kingdom, Switzerland, Canada, Poland, Japan, Chile, New Zealand, Portugal, China, India, Mexico, Australia, Macao
β-carotene is a carotenoid compound commonly used in laboratory research and product development. It functions as a provitamin, which means it can be converted into vitamin A in the body. β-carotene is a natural colorant and antioxidant with potential applications in various industries.
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Lab products found in correlation
Linoleic acid, by Merck Group (102 mentions)
Gallic acid, by Merck Group (85 mentions)
Lutein, by Merck Group (76 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (67 mentions)
Quercetin, by Merck Group (64 mentions)
Ascorbic acid, by Merck Group (61 mentions)
Lycopene, by Merck Group (52 mentions)
Methanol, by Merck Group (46 mentions)
Folin ciocalteu reagent, by Merck Group (44 mentions)
Chlorogenic acid, by Merck Group (38 mentions)
Zeaxanthin, by Merck Group (36 mentions)
Rutin, by Merck Group (34 mentions)
Caffeic acid, by Merck Group (33 mentions)
Chloroform, by Merck Group (31 mentions)
α carotene, by Merck Group (29 mentions)
Acetonitrile, by Merck Group (29 mentions)
α tocopherol, by Merck Group (27 mentions)
Tween 40, by Merck Group (27 mentions)
Tween 20, by Merck Group (27 mentions)
Catechin, by Merck Group (27 mentions)
436 protocols using β carotene
1
Antioxidant and Free Radical Scavenging Assays
2
Assay for Antioxidant Activity of β-Carotene
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The method used in this study was developed by Miller (1971) [28 (link)]. A solution of 2 mg/mL of β-carotene (Sigma-Aldrich; Madrid, Spain) in chloroform, ACS reagent grade (Merck; Darmstadt, Germany) was prepared. To an amount of 1 mL of the β-carotene solution, 200 mg of Tween® 40 and 20 mg of linoleic acid (Sigma-Aldrich; Madrid, Spain) were added. The chloroform was evaporated in a rotary evaporator at 40 °C. The emulsion was completed by adding 50 mL of ultra-pure water obtained from a Milli-Q™ system and vigorously shaken.
In 200 µL of each sample was added 5 mL of the β-carotene and linoleic acid emulsion. The solutions were kept at 50 °C for 2 h and then their absorbance was read in an Evolution 300 UV–Vis spectrophotometer (Thermo Scientific™, England) at 470 nm, against water. The absorbance of the control assays (with 200 µL of ethanol, 5 mL of the β-carotene and linoleic acid emulsion) was read before and after submitting the sample to 50 °C for 2 h. The Antioxidant Activity Coefficient (AAC) was calculated by Equation (2).
in which, AS is the absorbance of the samples after 2 h of reaction, the AC0 is the absorbance of the control assay at the initial time (t = 0 min), and the AC2 is the absorbance of the control assay after 2 h.
In 200 µL of each sample was added 5 mL of the β-carotene and linoleic acid emulsion. The solutions were kept at 50 °C for 2 h and then their absorbance was read in an Evolution 300 UV–Vis spectrophotometer (Thermo Scientific™, England) at 470 nm, against water. The absorbance of the control assays (with 200 µL of ethanol, 5 mL of the β-carotene and linoleic acid emulsion) was read before and after submitting the sample to 50 °C for 2 h. The Antioxidant Activity Coefficient (AAC) was calculated by Equation (2).
in which, AS is the absorbance of the samples after 2 h of reaction, the AC0 is the absorbance of the control assay at the initial time (t = 0 min), and the AC2 is the absorbance of the control assay after 2 h.
3
Carotenoid Profiling by TLC
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Pigment extract was analyzed by thin-layer chromatography (TLC) on silica gel plates using the following mixture of solvents as mobile phase: acetone:petroleum ether (20:80, v/v), methanol:chloroform (7:93, v/v) for bacterioruberin and monoanhydrobacterioruberin, and petroleum ether:diethyl ether (99:1 v/v) for β-carotene and lycopene. β-carotene (Sigma Aldrich®, St. Louis, MO, USA) was used as standard at a concentration of 1 mg/mL. The carotenoid content was analyzed based on retention factor (Rf) values.
4
HPLC Analysis of n-Hexane Extract and Fractions
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The HPLC analysis of n-hexane extract and its fractions (F1 and F2) was performed using an Agilent 1260 infinity series HPLC-DAD system (Agilent Technologies, Waldbronn, Germany) equipped with a binary gradient Agilent 1260 prep pump (G1361A) and an autosampler Agilent 1260 prep ALS (G2260A). Agilent diode array detector 1260 DAD VL (G1315D) was employed for the detection of carotenoids. The separation was performed using an Agilent normal phase (NP) silica column (ZORBAX RX-Sil, 5μm, 4.6 X 150 mm). The following solvents (A) n-hexane and (B) acetone were used at a flow rate of 1 mL/min using a gradient between solvents A and B following the method of Prum et al. [24 (link)] with some modifications as follows: B was run at 0 to 30% for 5 min, 30 to 50% for 15 min, 50 to 100% for 3 min, and maintaining 100% of B until the end of the separation at 30 min. The peaks were integrated at 450 nm. β-carotene (Sigma-Aldrich) was used as a standard to identify the isolated β-carotene.
5
Antioxidant and Anti-inflammatory Assays
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The solvents used in this study were obtained from VWR International s.r.l. (Milan, Italy). Gallic acid, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, ellagic acid, quercetin, catechin, rutin, ascorbic acid, propyl gallate, butylated hydroxytoluene (BHT), β-carotene, linoleic acid, pancreatic lipase, Tween 20, sodium potassium tartrate, sodium chloride, sodium carbonate, Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), o-dianisidine (DIAN) color reagent, peroxidase-glucose oxidase (PGO), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, (ABTS) solution, sodium acetate, β-carotene, linoleic acid, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Dulbecco’s Modified Eagle Medium (DMEM), dimethyl sulfoxide (DMSO), and Fetal Bovine Serum (FBS) were purchased from Sigma-Aldrich s.r.l. (Milan, Italy). l -Glutamine and penicillin/streptomycin were purchased from Gibco, Life Technologies (Waltham, MA, USA).
6
Comprehensive Phytochemical Analysis of Plant Samples
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The total carotenoid content was determined using the method reported by Pasqualone et al. [54 (link)] and a calibration curve was prepared by different concentrations of β-carotene (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) in order to express the carotenoid content in the sample as mg/kg of β-carotene on dry matter.
The total anthocyanin content was determined using the method reported by [54 (link)] and was expressed as mg/kg of cyanidin 3-O-glucoside. A calibration curve was prepared by using a different concentration of the cyanidin 3-O-glucoside standard (Phytoplan, Heidelberg, Germany).
The content of total phenolic compounds (TPC) and the antioxidant activity (AA) were assessed following the method proposed by Pasqualone et al. [55 (link)]. In order to express the total phenolic compounds content as mg/g of ferulic acid on dry matter, a calibration curve with ferulic acid at different concentrations was prepared. The antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay and expressed as μmol 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent/g on dry matter.
All determinations were carried out in triplicate.
The total anthocyanin content was determined using the method reported by [54 (link)] and was expressed as mg/kg of cyanidin 3-O-glucoside. A calibration curve was prepared by using a different concentration of the cyanidin 3-O-glucoside standard (Phytoplan, Heidelberg, Germany).
The content of total phenolic compounds (TPC) and the antioxidant activity (AA) were assessed following the method proposed by Pasqualone et al. [55 (link)]. In order to express the total phenolic compounds content as mg/g of ferulic acid on dry matter, a calibration curve with ferulic acid at different concentrations was prepared. The antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay and expressed as μmol 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent/g on dry matter.
All determinations were carried out in triplicate.
7
Quantifying Beta-Carotene in Flour Samples
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Extraction of ß-carotene content was done as described in Sadler et al. (1990) , with minor modifications. One gram of a sample flour was mixed with one gram CaCl2.2H2O and 50 mL extraction solvent (50% hexane, 25% acetone, and 25% ethanol, containing 0.1% BHT) by shaking for 30 min at room temperature. After adding 15 mL of distilled water, the solution was frequently mixed by shaking for a further 15 min. The organic phase, containing the β-carotene was separated from the water phase, using a separation funnel, and filtered using Whatman filter paper No.1. The extraction procedure was conducted under subdued light to avoid degradation ofcarotenoids. The stock β-carotene (Sigma Aldrich from USA) standard solution was made by dissolving accurately weighed 0.01 g β-carotene in the solvent (50% hexane, 25% acetone, and 25% ethanol) used to extract samples and made the volume to one hundred milliliter using the same solvent. From stock solution, series of standard solutions (0.1, 0.2, 0.4, 0.6, 0.8 and 1 μg/mL, R2 = 0.994) were used to construct calibration line from which β -carotene was estimated and expressed in mg/g. The absorbance of the sample extract and β-carotene standard solutions was measured at 450 nm wavelength using UV-Vis spectrophotometer.
8
Engineered Strain Beta-Carotene Quantification
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All engineered strains were cultured in the YPD medium for the 144 h of shake-flask fermentation. The cells were harvested every 24 h for the measurement of cell growth, dry cell weight (DCW), and β-carotene. One milliliter culture broth was used for the measurement of DCW. β-carotene was extracted as described previously with minor modifications (Gao et al., 2017 (link)). In brief, 1 mL cells were harvested by centrifuging at 12,000 × g for 5 min. The obtained cells were re-suspended in 0.5 mL dimethyl sulfoxide, and then incubated for 15 min at 55°C followed by 45°C for 15 min after an equal volume of acetone was added. The samples were then centrifuged at 12,000 × g for 5 min. Supernatants containing β-carotene were filtered through a 0.45 μm filter. The β-carotene analysis was performed by high-performance liquid chromatography (HPLC, Agilent Technologies 1260 Infinity Series System, CA, United States) with the UV signal at 450 nm and a C18 column (4.6 mm × 250 mm). The mobile phase consisted of 50% acetonitrile, 30% methanol, and 20% isopropanol (v/v/v), and the flow rate was 1 mL/min at 30°C. The standard β-carotene was purchased from Sigma-Aldrich (Darmstadt, Germany).
9
Carotenoid Extraction and Analysis Protocol
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After storage, samples were centrifuged (10 min at 4 °C, 16,000 × g) and 100 µL was transferred to another light-protected Eppendorf tube and used for sample preparation. Acetone was removed using a speedvac and carotenoids were resuspended in 100 µL acetonitrile:methanol;70:30 (v/v) by vortexing. The solution was next transferred to MS vials with glass inserts. Similar method was used for the standard solutions (1 or 2 mg/mL) of all reported compounds (astaxanthin (Sigma Aldrich), canthaxanthin (Supelco), β-carotene (Sigma Aldrich) and lycopene (Supelco)), which were also stored at −20 °C and mixed, evaporated and resuspended in acetonitrile:methanol;70:30 (v/v) at a final concentration of 3 µM for each compound. This standard was diluted in acetonitrile:methanol;70:30 (v/v) to prepare the other standards for a calibration curve based on 0, 0.1, 0.25, 0.5, 1 and 3 µM concentrations.
10
Carotenoid Extraction and Analysis in Orange Varieties
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Carotenoids were extracted and analyzed in the pulps of mature fruits of the four varieties (December and January for Navel oranges, and March and April for Valencia oranges) as described by Rodrigo et al. [26 (link)], using a Waters a liquid chromatography system (HPLC) equipped with a 600E pump, a photodiode array detector (DAD), model 2998 and Empower3 software (Waters, Barcelona, Spain). A C30 carotenoid column (250 × 4.6 mm, 5 μm) was coupled to a C30 guard column (20 × 4.0 mm, 5 μm) (YMC, Teknokroma, Spain). The carotenoids were identified by absorbance spectra and retention time; peaks were integrated at their individual maximal wavelength and their contents were calculated using the appropriate calibration curves of lycopene (Extrasynthese) for lycopene, neurosporene and δ-carotene, lutein (Sigma), β-carotene (Sigma), β-cryptoxanthin (Extrasynthese), zeaxanthin (Extrasynthese), anteraxanthin (CaroteNature) for anteraxanthin and mutatoxanthin and violaxanthin (CaroteNature) for violaxanthin isomers and luteoxanthin. Phytoene, phytofluene and ζ-carotene were previously purified by thin-layer chromatography from carotenoid extracts of Pinalate orange fruits [26 (link)]. The spectroscopic characteristics of all carotenoids detected in the pulps of Navel, Kirkwood, Valencia and Ruby oranges are shown in Table S1 .
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