Development of Engineered Stationary Phases for the Separation of Carotenoid Isomers

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of b-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan realtime PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, z-carotene desaturase, carotene cis-trans-isomerase, b-lycopene cyclase, and b-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that b-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, a/b-lycopene cyclase, b-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or z-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the b-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-transisomerism. Possible implications are discussed.

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“…cis-Isomers of b-carotene were analyzed and identified with the help of published data (Sander et al, 1994) using a gradient system consisting of…”

Section: Carotenoid Extraction and Analysismentioning

confidence: 99%

Why Is Golden Rice Golden (Yellow) Instead of Red?

et al. 2005

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“…To quantify carotenoids in the crude extract, carotenoid esters have been hydrolyzed using a non-base-dependent hydrolysis reaction [20] to minimize side reactions [21,22]. Methods for the separation of carotenoid isomers have been well-established using C 30 reverse phase chromatography [23,24]. Use of this stationary phase in combination with diode array (DAD) and mass spectrometry (MS) detection allows for an efficient means of monitoring the possible formation of the less desirable isomers that may arise as a result of an inferior extraction process.…”

Section: Introductionmentioning

confidence: 99%

Pressurized fluid extraction of carotenoids from Haematococcus pluvialis and Dunaliella salina and kavalactones from Piper methysticum

Denery

Dragull

Tang

et al. 2004

Analytica Chimica Acta

147

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Pressurized fluid extraction (PFE) was examined as an alternative technology for the extraction of carotenoids in the green algae Haematococcus pluvialis and Dunaliella salina and kavalactones in Piper methysticum. The extraction process was optimized by varying the key extraction factors of solvent, sample-solvent ratio, temperature, and time. The selectivity and efficiency of extraction parameters were determined with high performance liquid chromatography (LC) and LC-mass spectrometry (LC-MS). Results showed that PFE utilization of conventional solvents under controlled temperature and pressure in an oxygen and light-free environment could result in the use of less solvent in a shorter period of time. PFE showed higher or equal extraction efficiencies as compared with traditional solvent extractions while maintaining the integrity of chemical components. PFE showed high potential for extraction of natural products and nutraceuticals, particularly labile and light sensitive chemicals.

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“…The same HPLC systems, software, column, and solvents were used. The gradient used 30 was different from egg yolk astaxanthin quantification. Briefly, 100% solvent A changed to 100% solvent B linearly over 90 min.…”

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confidence: 99%

Supplementation of Laying-Hen Feed with Palm Tocos and Algae Astaxanthin for Egg Yolk Nutrient Enrichment

Walker

Wang²,

Xin³

et al. 2012

J. Agric. Food Chem.

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Adding supplements to hen feed can increase egg nutritional value. Astaxanthin, tocotrienols, and tocopherols are potent antioxidants that provide health benefits to humans. We hypothesized that the addition of these nutrients to hen feed would result in an increased nutrient content in egg yolk with minimum changes in functional properties. Laying hens (Hy-Line W-36 breed) were fed four diets with different supplementation levels of palm toco concentrate and algae biomass containing astaxanthin for 8 weeks. Egg yolks were analyzed for physical, chemical, and functional properties. The feed with the highest nutrient concentration was also studied for stability of these antioxidants using the Arrhenius approach. No significant differences were observed in functional properties except for emulsification capacity and sensory characteristics among eggs from different diet treatments. Changes in egg yolk color reached the maximum values at day 8. Incorporation of tocopherols and tocotrienols increased until day 8, astaxanthin incorporation increased until day 10, and all decreased thereafter. Feed nutrients resulted in a dose-response relationship of these compounds in the egg yolk. The transfer efficiency ranged from 0 to 9.9% for tocotrienols and tocopherols and from 7.6 to 14.9% for astaxanthin at their peak values. Results of the Arrhenius accelerated stability study showed significant differences in the shelf life of various nutrients, and these results can be used to properly formulate and store the feed materials. Agricultural and Food Chemistry, 60, no. 8 (2012): 1989-1999 ABSTRACT: Adding supplements to hen feed can increase egg nutritional value. Astaxanthin, tocotrienols, and tocopherols are potent antioxidants that provide health benefits to humans. We hypothesized that the addition of these nutrients to hen feed would result in an increased nutrient content in egg yolk with minimum changes in functional properties. Laying hens (Hy-Line W-36 breed) were fed four diets with different supplementation levels of palm toco concentrate and algae biomass containing astaxanthin for 8 weeks. Egg yolks were analyzed for physical, chemical, and functional properties. The feed with the highest nutrient concentration was also studied for stability of these antioxidants using the Arrhenius approach. No significant differences were observed in functional properties except for emulsification capacity and sensory characteristics among eggs from different diet treatments. Changes in egg yolk color reached the maximum values at day 8. Incorporation of tocopherols and tocotrienols increased until day 8, astaxanthin incorporation increased until day 10, and all decreased thereafter. Feed nutrients resulted in a dose−response relationship of these compounds in the egg yolk. The transfer efficiency ranged from 0 to 9.9% for tocotrienols and tocopherols and from 7.6 to 14.9% for astaxanthin at their peak values. Results of the Arrhenius accelerated stability study showed significant differences in the shelf life of vari...

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Development of Engineered Stationary Phases for the Separation of Carotenoid Isomers

Cited by 345 publications

References 55 publications

Why Is Golden Rice Golden (Yellow) Instead of Red?

Why Is Golden Rice Golden (Yellow) Instead of Red?

Pressurized fluid extraction of carotenoids from Haematococcus pluvialis and Dunaliella salina and kavalactones from Piper methysticum

Supplementation of Laying-Hen Feed with Palm Tocos and Algae Astaxanthin for Egg Yolk Nutrient Enrichment

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