Dataset Information

Isolation and functional characterization of two dioxygenases putatively involved in bixin biosynthesis in annatto (Bixa orellana L.).

ABSTRACT: Carotenoid cleavage dioxygenases (CCDs) are enzymes that have been implicated in the biosynthesis of a wide diversity of secondary metabolites with important economic value, including bixin. Bixin is the second most used pigment in the world's food industry worldwide, and its main source is the aril of achiote (Bixa orellana L.) seeds. A recent transcriptome analysis of B. orellana identified a new set of eight CCD members (BoCCD4s and BoCCD1s) potentially involved in bixin synthesis. We used several approaches in order to discriminate the best candidates with CCDs genes. A reverse transcription-PCR (RT-qPCR) expression analysis was carried out in five developmental stages of two accessions of B. orellana seeds with different bixin contents: (P13W, low bixin producer and N4P, high bixin producer). The results showed that three BoCCDs (BoCCD4-1, BoCCD4-3, and BoCCD1-1) had an expression pattern consistent with bixin accumulation during seed development. Additionally, an alignment of the CCD enzyme family and homology models of proteins were generated to verify whether the newly proposed CCD enzymes were bona fide CCDs. The study confirmed that these three enzymes were well-preserved and belonged to the CCD family. In a second selection round, the three CCD genes were analyzed by in situ RT-qPCR in seed tissue. Results indicated that BoCCD4-3 and BoCCD1-1 exhibited tissue-specific expressions in the seed aril. To test whether the two selected CCDs had enzymatic activity, they were expressed in Escherichia coli; activity was determined by identifying their products in the crude extract using UHPLC-ESI-QTOF-MS/MS. The cleavage product (bixin aldehyde) was also analyzed by Fourier transform infrared. The results indicated that both BoCCD4-3 and BoCCD1-1 cleave lycopene in vitro at 5,6-5',6'.

SUBMITTER: Carballo-Uicab VM

PROVIDER: S-EPMC6592262 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Isolation and functional characterization of two dioxygenases putatively involved in bixin biosynthesis in annatto (Bixa orellana L.).

Carballo-Uicab Victor Manuel VM Cárdenas-Conejo Yair Y Vallejo-Cardona Alba Adriana AA Aguilar-Espinosa Margarita M Rodríguez-Campos Jacobo J Serrano-Posada Hugo H Narváez-Zapata José Alberto JA Vázquez-Flota Felipe F Rivera-Madrid Renata R

PeerJ 20190621

Carotenoid cleavage dioxygenases (CCDs) are enzymes that have been implicated in the biosynthesis of a wide diversity of secondary metabolites with important economic value, including bixin. Bixin is the second most used pigment in the world's food industry worldwide, and its main source is the aril of achiote (Bixa orellana L.) seeds. A recent transcriptome analysis of B. orellana identified a new set of eight CCD members (BoCCD4s and BoCCD1s) potentially involved in bixin synthes ...[more]

PMID: 31275744

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Project description:The Scientific Panel on Food Additives and Flavouring (FAF) provides a scientific opinion on the safety evaluation of annatto E and an exposure assessment of the annatto colouring principles bixin and norbixin (E 160b) when used as a food additive, taking into account new proposed uses and use levels. In 2016, the EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) adopted a scientific opinion on the safety of annatto extracts (E 160b) as a food additive. In that opinion, the Panel concluded that the toxicological database was sufficient to derive an acceptable daily intake (ADI) of 6 mg bixin/kg body weight (bw) per day and an ADI of 0.3 mg norbixin/kg bw per day. The Panel was not able to conclude on the safety of annatto E. Dietary exposure for annatto (E 160b), bixin- and norbixin-based annatto extracts was estimated taking into account the maximum permitted levels (MPLs) as set in the Annex II to Regulation (EC) No 1333/2008, the use levels provided by food industry as well as the proposed use levels from an applicant for an extension of use. Exposure estimates for bixin were below the ADI for all population groups and for all refined exposure scenarios, including the proposed extension of use. For norbixin, exceedance was observed for the extension of use at the 95th percentile for some population groups. In the 2017 EFSA statement related to the exposure assessment of colouring principles bixin and norbixin considering new proposed uses and use levels, the dietary exposure for bixin did not exceed the ADI. For norbixin, dietary exposure exceeded the ADI at the high level (95th percentile) for toddlers and children. In the current opinion, the Panel concluded that food colour annatto E does not raise concern for genotoxicity. Accordingly, the ADIs established in 2016 for the two colouring principles bixin and norbixin can be applied also to the annatto E. For bixin, none of the exposure estimates exceeded the ADI of 6 mg/kg bw per day. For norbixin, the ADI was reached at the high level (p95) for toddlers in the refined exposure assessment scenarios in one country. Considering the uncertainties and the very likely overestimation of the exposure, the Panel concluded that the level of exposure does not raise a health concern.

Dataset Information

Isolation and functional characterization of two dioxygenases putatively involved in bixin biosynthesis in annatto (Bixa orellana L.).

Publications

Isolation and functional characterization of two dioxygenases putatively involved in bixin biosynthesis in annatto (<i>Bixa orellana</i> L.).

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