Dissertations / Theses on the topic 'Provitamin A'

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
As algas marinhas sÃo fontes de uma grande variedade de compostos benÃficos para o homem, dentre os quais se destacam os minerais, as fibras dietÃrias e as vitaminas (A, B, C e E). O objetivo deste trabalho foi verificar a existÃncia de variaÃÃo sazonal nos teores de α- e β-caroteno (carotenÃides provitamina A) e de α-tocoferol (vitamina E), em cinco espÃcies de macroalgas marinhas pertencentes ao gÃnero Caulerpa (FamÃlia Caulerpaceae, DivisÃo Chlorophyta), âin naturaâ e desidratada. Os conteÃdos desses nutrientes nas algas desidratadas foram comparados com aqueles nas algas âin naturaâ, com o objetivo de verificar se houve alteraÃÃo pelo processo de desidrataÃÃo. As espÃcies de macroalgas marinhas foram coletadas mensalmente, de janeiro a dezembro de 2006, durante as marÃs baixas na Praia do Pacheco, Caucaia- CE. As anÃlises de α- e β-caroteno e α-tocoferol foram realizadas a partir da extraÃÃo da alga em metanol-Ãgua (90:10) nas proporÃÃes 1:10 (p/v), nas amostras âin naturaâ, e 1:20 (p/v), nas desidratadas, saponificaÃÃo com hidrÃxido de potÃssio 5% por 30 min a 70ÂC e partiÃÃo em n-hexano que foi evaporado sob corrente de ar. O resÃduo foi suspenso em 1 mL de metanol no momento da anÃlise cromatogrÃfica e 100 μL foram injetados manualmente. O sistema cromatogrÃfico consistiu em uma coluna Waters Spherisorb-Hichrom S5ODS-2 (4,6 x 250 mm) e uma fase mÃvel constituÃda de metanol:tetrahidrofurano (90:10, v/v), com fluxo de 1,5 mL min-1. O detector foi ajustado em 450 nm e 292 nm e os cromatogramas registrados atravÃs do sistema UnicornTM versÃo 5.0. Todas as espÃcies de Caulerpa âin naturaâ e desidratadas analisadas no presente trabalho apresentaram tanto α-caroteno quanto β-caroteno e as suas distribuiÃÃes mostraram diferenÃas ao longo dos doze meses de coleta. De um modo geral, os teores de α-caroteno foram superiores aos de β-caroteno. As perdas nos conteÃdos de carotenÃides provitamina oscilaram entre 10% e 94%. Para que as algas analisadas neste trabalho fossem consideradas fontes excelentes de vitamina A seria necessÃrio que as porÃÃes consumidas diariamente variassem de 52 g a 689 g, quando consumidas âin naturaâ ou de 42 g a 469 g, quando desidratadas. As cinco espÃcies analisadas neste trabalho apresentaram α-tocoferol, tanto nas amostras âin naturaâ quanto nas desidratadas, com exceÃÃo de C. racemosa coletada em marÃo que apÃs ser submetida a secagem nÃo foi detectado α- tocoferol, e sua distribuiÃÃo foi variÃvel ao longo do ano. Nos teores de α- tocoferol foi observado perdas que variaram de 22 a 91%. As porÃÃes que deveriam ser consumidas diariamente para que as espÃcies de Caulerpa estudadas fossem capazes de fornecer 1/2 da IDR sÃo relativamente pequenas, devendo oscilar entre 11 g e 168 g, quando âin naturaâ, ou entre 13 g e 70 g, quando desidratadas. As quantidades de retinol equivalente e α-tocoferol equivalente nas algas analisadas no presente trabalho nÃo diferiram muito daquelas encontradas nos vegetais normalmente consumidos
Marine macroalgae are sources of a great variety of beneficial compounds such as minerals, dietary fibers and vitamins. The aim of this work was to verify seasonal variation upon both provitamin A carotenoids (α- and β-carotene) and vitamin E (α-tocopherol) contents in five species of the marine green macroalga Caulerpa both fresh and oven-dried at 40ÂC for 15 h. The contents in dried algae were compared to those in fresh algae to evaluate the losses after drying. Algal material was collected monthly from January to December 2006, in Pacheco Beach, Caucaia, CearÃ. Analyses of α- and β-carotene and α-tocopherol were carried out in extracts 1:10 (p/v) for fresh alga and 1:20 (p/v) for dried alga using aqueous methanol (90:10, v/v). They were saponified with 5% KOH and partitioned into n-hexane, which was then evaporated. The residues were suspended in 1 mL methanol prior to HPLC analyses. Aliquots of 100 μL were injected in a HPLC system consisting of a Waters Spherisorb-Hichrom S5 ODS-2 column (4.6 x 250 mm) and a mobile phase of methanol:tetrahydrofurane (90:10, v/v), delivered at 1.5 mL min-1. The detector was set at 450 nm for α- and β-carotene and 290 nm for α-tocopherol. Chromatograms were registered at UnicornTM version 5.0. All samples showed α- and β-carotene and α-tocopherol, but their distribution along the year was variable. In general, the contents of α-carotene were greater than those of β-carotene. The losses of α- and β-carotene varied between 10% and 94%. In order to be considered an excellent source of vitamin A, the daily consumption would be 52 g to 689 g of fresh alga or 42 g to 469 g of dried alga. α-Tocopherol was detected in all samples except in dried C. racemosa collected in March. Similar to the distribution of α- and β-carotene along the year, α-tocopherol contents varied too. Losses varied from 22% to 91%. Daily portions to supply 50% of the Recommended Daily Allowance (RDA) would be 11 g to 168 g of fresh alga or 13 g to 70 g of dried alga. Amounts of vitamin A (retinol equivalents) and vitamin E (tocopherol equivalents) in all algae analyzed were not very different from most vegetables normally consumed

PIRES, Kelma Maria dos Santos. Avaliação sazonal de carotenóides provitamina A (α– e β– caroteno) e vitamina E (α–tocoferol) em macroalgas marinhas pertencentes a família Caulerpacea (Divisão Chlorophyta). 2007. 94 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortalezza-CE, 2007
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Marine macroalgae are sources of a great variety of beneficial compounds such as minerals, dietary fibers and vitamins. The aim of this work was to verify seasonal variation upon both provitamin A carotenoids (α- and β-carotene) and vitamin E (α-tocopherol) contents in five species of the marine green macroalga Caulerpa both fresh and oven-dried at 40°C for 15 h. The contents in dried algae were compared to those in fresh algae to evaluate the losses after drying. Algal material was collected monthly from January to December 2006, in Pacheco Beach, Caucaia, Ceará. Analyses of α- and β-carotene and α-tocopherol were carried out in extracts 1:10 (p/v) for fresh alga and 1:20 (p/v) for dried alga using aqueous methanol (90:10, v/v). They were saponified with 5% KOH and partitioned into n-hexane, which was then evaporated. The residues were suspended in 1 mL methanol prior to HPLC analyses. Aliquots of 100 μL were injected in a HPLC system consisting of a Waters Spherisorb-Hichrom S5 ODS-2 column (4.6 x 250 mm) and a mobile phase of methanol:tetrahydrofurane (90:10, v/v), delivered at 1.5 mL min-1. The detector was set at 450 nm for α- and β-carotene and 290 nm for α-tocopherol. Chromatograms were registered at UnicornTM version 5.0. All samples showed α- and β-carotene and α-tocopherol, but their distribution along the year was variable. In general, the contents of α-carotene were greater than those of β-carotene. The losses of α- and β-carotene varied between 10% and 94%. In order to be considered an excellent source of vitamin A, the daily consumption would be 52 g to 689 g of fresh alga or 42 g to 469 g of dried alga. α-Tocopherol was detected in all samples except in dried C. racemosa collected in March. Similar to the distribution of α- and β-carotene along the year, α-tocopherol contents varied too. Losses varied from 22% to 91%. Daily portions to supply 50% of the Recommended Daily Allowance (RDA) would be 11 g to 168 g of fresh alga or 13 g to 70 g of dried alga. Amounts of vitamin A (retinol equivalents) and vitamin E (tocopherol equivalents) in all algae analyzed were not very different from most vegetables normally consumed
As algas marinhas são fontes de uma grande variedade de compostos benéficos para o homem, dentre os quais se destacam os minerais, as fibras dietárias e as vitaminas (A, B, C e E). O objetivo deste trabalho foi verificar a existência de variação sazonal nos teores de α- e β-caroteno (carotenóides provitamina A) e de α-tocoferol (vitamina E), em cinco espécies de macroalgas marinhas pertencentes ao gênero Caulerpa (Família Caulerpaceae, Divisão Chlorophyta), “in natura” e desidratada. Os conteúdos desses nutrientes nas algas desidratadas foram comparados com aqueles nas algas “in natura”, com o objetivo de verificar se houve alteração pelo processo de desidratação. As espécies de macroalgas marinhas foram coletadas mensalmente, de janeiro a dezembro de 2006, durante as marés baixas na Praia do Pacheco, Caucaia- CE. As análises de α- e β-caroteno e α-tocoferol foram realizadas a partir da extração da alga em metanol-água (90:10) nas proporções 1:10 (p/v), nas amostras “in natura”, e 1:20 (p/v), nas desidratadas, saponificação com hidróxido de potássio 5% por 30 min a 70°C e partição em n-hexano que foi evaporado sob corrente de ar. O resíduo foi suspenso em 1 mL de metanol no momento da análise cromatográfica e 100 μL foram injetados manualmente. O sistema cromatográfico consistiu em uma coluna Waters Spherisorb-Hichrom S5ODS-2 (4,6 x 250 mm) e uma fase móvel constituída de metanol:tetrahidrofurano (90:10, v/v), com fluxo de 1,5 mL min-1. O detector foi ajustado em 450 nm e 292 nm e os cromatogramas registrados através do sistema UnicornTM versão 5.0. Todas as espécies de Caulerpa “in natura” e desidratadas analisadas no presente trabalho apresentaram tanto α-caroteno quanto β-caroteno e as suas distribuições mostraram diferenças ao longo dos doze meses de coleta. De um modo geral, os teores de α-caroteno foram superiores aos de β-caroteno. As perdas nos conteúdos de carotenóides provitamina oscilaram entre 10% e 94%. Para que as algas analisadas neste trabalho fossem consideradas fontes excelentes de vitamina A seria necessário que as porções consumidas diariamente variassem de 52 g a 689 g, quando consumidas “in natura” ou de 42 g a 469 g, quando desidratadas. As cinco espécies analisadas neste trabalho apresentaram α-tocoferol, tanto nas amostras “in natura” quanto nas desidratadas, com exceção de C. racemosa coletada em março que após ser submetida a secagem não foi detectado α- tocoferol, e sua distribuição foi variável ao longo do ano. Nos teores de α- tocoferol foi observado perdas que variaram de 22 a 91%. As porções que deveriam ser consumidas diariamente para que as espécies de Caulerpa estudadas fossem capazes de fornecer 1/2 da IDR são relativamente pequenas, devendo oscilar entre 11 g e 168 g, quando “in natura”, ou entre 13 g e 70 g, quando desidratadas. As quantidades de retinol equivalente e α-tocoferol equivalente nas algas analisadas no presente trabalho não diferiram muito daquelas encontradas nos vegetais normalmente consumidos

Master of Science
Food Science Institute - Food, Nutrition, Dietetics and Health
Weiqun Wang
Sorghum is a staple crop consumed in certain regions of Africa and Asia, where vitamin A deficiency is prevalent. However, the correlation of sorghum intake and vitamin A deficiency is inconsistent. The objective of this study was to identify and quantify the carotenoids and pro-vitamin A in the selected sorghum accessions with various pericarp pigments by using LC-MS. Among of total five carotenoids (α-carotene, β-carotene, lutein, zeaxanthin, and β-cryptoxanthin) that were identified and quantitated, three (α-carotene, β-carotene and β-cryptoxanthin) are precursors of vitamin A. The highest content of total carotenoids was detected in the sorghum accessions with yellow pericarp (PI656096, PI585374, PI563448, PI585351), while the highest β-carotene content was found in the accessions with brown or yellow pericarp (PI655996, PI656096, PI585374, PI563448, PI585351). The lowest carotenoids were found in the accessions with white pericarp (PI533943, PI656112, PI565121, PI560493). The pro-vitamin A was 584.9 ± 38.9 ng/g DW in yellow pericarp, 250.6 ± 28.9 ng/g DW in brown pericarp, and 89.0 ± 12.3 ng/g DW in white pericarp, respectively. It appeared the phenotypic diversity of sorghum pericarp colors was strongly associated with the contents of carotenoids and pro-vitamin A, indicating a different impact of various sorghum varieties on vitamin A deficiency and suggesting a possible prevention of vitamin A deficiency by breeding certain sorghum varieties with pericarp pigments.

Vitamin A deficiency (VAD) is a serious problem in developing countries, affecting approximately 127 million children of preschool age and 7.2 million pregnant women each year. However, this deficiency is readily treated and prevented through adequate nutrition. This can potentially be achieved through genetically engineered biofortification of staple food crops to enhance provitamin A (pVA) carotenoid content. Bananas are the fourth most important food crop with an annual production of 100 million tonnes and are widely consumed in areas affected by VAD. However, the fruit pVA content of most widely consumed banana cultivars is low (~ 0.2 to 0.5 ìg/g dry weight). This includes cultivars such as the East African highland banana (EAHB), the staple crop in countries such as Uganda, where annual banana consumption is approximately 250 kg per person. This fact, in addition to the agronomic properties of staple banana cultivars such as vegetative reproduction and continuous cropping, make bananas an ideal target for pVA enhancement through genetic engineering. Interestingly, there are banana varieties known with high fruit pVA content (up to 27.8 ìg/g dry weight), although they are not widely consumed due to factors such as cultural preference and availability. The genes involved in carotenoid accumulation during banana fruit ripening have not been well studied and an understanding of the molecular basis for the differential capacity of bananas to accumulate carotenoids may impact on the effective production of genetically engineered high pVA bananas. The production of phytoene by the enzyme phytoene synthase (PSY) has been shown to be an important rate limiting determinant of pVA accumulation in crop systems such as maize and rice. Manipulation of this gene in rice has been used successfully to produce Golden Rice, which exhibits higher seed endosperm pVA levels than wild type plants. Therefore, it was hypothesised that differences between high and low pVA accumulating bananas could be due either to differences in PSY enzyme activity or factors regulating the expression of the psy gene. Therefore, the aim of this thesis was to investigate the role of PSY in accumulation of pVA in banana fruit of representative high (Asupina) and low (Cavendish) pVA banana cultivars by comparing the nucleic acid and encoded amino acid sequences of the banana psy genes, in vivo enzyme activity of PSY in rice callus and expression of PSY through analysis of promoter activity and mRNA levels. Initially, partial sequences of the psy coding region from five banana cultivars were obtained using reverse transcriptase (RT)-PCR with degenerate primers designed to conserved amino acids in the coding region of available psy sequences from other plants. Based on phylogenetic analysis and comparison to maize psy sequences, it was found that in banana, psy occurs as a gene family of at least three members (psy1, psy2a and psy2b). Subsequent analysis of the complete coding regions of these genes from Asupina and Cavendish suggested that they were all capable of producing functional proteins due to high conservation in the catalytic domain. However, inability to obtain the complete mRNA sequences of Cavendish psy2a, and isolation of two non-functional Cavendish psy2a coding region variants, suggested that psy2a expression may be impaired in Cavendish. Sequence analysis indicated that these Cavendish psy2a coding region variants may have resulted from alternate splicing. Evidence of alternate splicing was also observed in one Asupina psy1 coding region variant, which was predicted to produce a functional PSY1 isoform. The complete mRNA sequence of the psy2b coding regions could not be isolated from either cultivar. Interestingly, psy1 was cloned predominantly from leaf while psy2 was obtained preferentially from fruit, suggesting some level of tissue-specific expression. The Asupina and Cavendish psy1 and psy2a coding regions were subsequently expressed in rice callus and the activity of the enzymes compared in vivo through visual observation and quantitative measurement of carotenoid accumulation. The maize B73 psy1 coding region was included as a positive control. After several weeks on selection, regenerating calli showed a range of colours from white to dark orange representing various levels of carotenoid accumulation. These results confirmed that the banana psy coding regions were all capable of producing functional enzymes. No statistically significant differences in levels of activity were observed between banana PSYs, suggesting that differences in PSY activity were not responsible for differences in the fruit pVA content of Asupina and Cavendish. The psy1 and psy2a promoter sequences were isolated from Asupina and Cavendish gDNA using a PCR-based genome walking strategy. Interestingly, three Cavendish psy2a promoter clones of different sizes, representing possible allelic variants, were identified while only single promoter sequences were obtained for the other Asupina and Cavendish psy genes. Bioinformatic analysis of these sequences identified motifs that were previously characterised in the Arabidopsis psy promoter. Notably, an ATCTA motif associated with basal expression in Arabidopsis was identified in all promoters with the exception of two of the Cavendish psy2a promoter clones (Cpsy2apr2 and Cpsy2apr3). G1 and G2 motifs, linked to light-regulated responses in Arabidopsis, appeared to be differentially distributed between psy1 and psy2a promoters. In the untranscribed regulatory regions, the G1 motifs were found only in psy1 promoters, while the G2 motifs were found only in psy2a. Interestingly, both ATCTA and G2 motifs were identified in the 5’ UTRs of Asupina and Cavendish psy1. Consistent with other monocot promoters, introns were present in the Asupina and Cavendish psy1 5’ UTRs, while none were observed in the psy2a 5’ UTRs. Promoters were cloned into expression constructs, driving the â-glucuronidase (GUS) reporter gene. Transient expression of the Asupina and Cavendish psy1 and psy2a promoters in both Cavendish embryogenic cells and Cavendish fruit demonstrated that all promoters were active, except Cpsy2apr2 and Cpsy2apr3. The functional Cavendish psy2a promoter (Cpsy2apr1) appeared to have activity similar to the Asupina psy2a promoter. The activities of the Asupina and Cavendish psy1 promoters were similar to each other, and comparable to those of the functional psy2a promoters. Semi-quantitative PCR analysis of Asupina and Cavendish psy1 and psy2a transcripts showed that psy2a levels were high in green fruit and decreased during ripening, reinforcing the hypothesis that fruit pVA levels were largely dependent on levels of psy2a expression. Additionally, semi-quantitative PCR using intron-spanning primers indicated that high levels of unprocessed psy2a and psy2b mRNA were present in the ripe fruit of Cavendish but not in Asupina. This raised the possibility that differences in intron processing may influence pVA accumulation in Asupina and Cavendish. In this study the role of PSY in banana pVA accumulation was analysed at a number of different levels. Both mRNA accumulation and promoter activity of psy genes studied were very similar between Asupina and Cavendish. However, in several experiments there was evidence of cryptic or alternate splicing that differed in Cavendish compared to Asupina, although these differences were not conclusively linked to the differences in fruit pVA accumulation between Asupina and Cavendish. Therefore, other carotenoid biosynthetic genes or regulatory mechanisms may be involved in determining pVA levels in these cultivars. This study has contributed to an increased understanding of the role of PSY in the production of pVA carotenoids in banana fruit, corroborating the importance of this enzyme in regulating carotenoid production. Ultimately, this work may serve to inform future research into pVA accumulation in important crop varieties such as the EAHB and the discovery of avenues to improve such crops through genetic modification.

Microbiological oil solutions of provitamin A (β-carotene) as a dietary supplement are not widely used due to their low oxidation stability. The aim of research is determination of the effect of sesame antioxidants on the oxidative stability of provitamin A in oil solutions. The peroxide number of vegeTable oils is determined by the standard method by the titrimetric method. The value of the period of induction of oil oxidation is determined graphically from the growth curves of peroxide numbers. The content of tocopherols in oils is determined by spectrophotometric method. The content of sesamol and sesamoline in oils is determined by the colorimetric method. The oxidative stability of oils is determined using the accelerated "active oxygen" method. To plan the experiment and process the data, mathematical methods are applied using the software Microsoft Office Excel 2003. The oxidation stability of blended oils (a mixture of sesame, high oleic, sunflower and corn refined oils) and its components is investigated. Blended oil has a lower oxidative stability than sesame oil, but higher than corn and high oleic sunflower. The oxidative stability of the blend is enhanced by the antioxidant content of sesamol and sesamoline. The content of the above antioxidants, as well as the amount of tocopherols, is studied in blended oils. The oxidation stability of the obtained solution of provitamin A in blended oils is investigated. The period of induction of oxidation of blended oils with the addition of 0.2 % β-carotene increases by 1.3 times compared with the period of induction of the original blended oil. Blended long-life oil is recommended to be used to stabilize fat-soluble biologically active compounds.

In Uganda, a significant proportion of the population depends on the micronutrient poor East African highland banana as a food staple. Consequently, micronutrient deficiencies such as vitamin A deficiency are an important health concern in the country. To reach most vulnerable rural poor populations, staple crops can be biofortified with essential micronutrients though conventional breeding or genetic engineering. This thesis provided proof of concept that genetically modified East African highland bananas with enhanced provitamin A levels can be generated and fully characterised in Uganda. In addition, provitamin A levels present in popular banana varieties was documented.

Com o objetivo de determinar os parâmetros de cor e carotenóides, assim como as mudanças no teor pró-vitamínico A de batatas-doces cruas e cozidas, foram analisados cultivares desta raiz procedentes do Instituto Agronômico de Campinas e do Centro Nacional de Pesquisa de Hortaliças da EMBRAPA de Brasília. Foram identificados sete carotenóides na análise de batatas-doces, a saber: 5,6,5\',6\'-diepoxi-β-caroteno; 5,6-monoepoxi-β-caroteno; β-caroteno; luteocromo; α-zeacaroteno; β-zeacaroteno e aurocromo. O β-caroteno foi o principal carotenóide nos cultivares Centennial(148,81 J-Ig/g de batata-doce crua), Heart Gold (51,79 µg/g), Anápolis (14,24 µg/g), Acadian (218,15 µg/g), Morada Inta (10,61 µg/g), Vineland Bush (22,60 µg/g) e clone CNPH (4,87 µg/g).O luteocromo foi o principal carotenóide nos cultivares Monalisa (1,55 J-Ig/g de batata-doce crua), IAC-2-71 (0,77 J-Ig/g) e SRT-252 (0,92 µg/g). Com relação as raízes cruas, os valores de vitamina A variaram de 1 equivalente de retinol/100g para o cultivar IAC-2-71 até 3.703 equivalentes de retinol /100g para cultivar Acadian. Para as raízes cozidas o cultivar apresentou maior atividade pró-vitamínica A foi o Acadian com 4021 equivalentes de retinol/100g. A perda de β-caroteno pelo cozimento variou de 4,5% a 42,6% conforme o cultivar. Os parâmetros de cor (L Hunter, a Hunter, b Hunter, croma e ângulo do tom) não diferiram para os três cortes transversais das raízes (parte superior, central e inferior). Quanto ao cozimento, houve diminuição significativa dos parâmetros L Hunter (luminosidade), +a Hunter (vermelho) e aumento nos parâmetros -a Hunter (verde) e ângulo do tom. O parâmetro b Hunter (amarelo) e croma diminuíram pelo cozimento nos cultivares Centennial, SRT-252, Monalisa e Heart gold. Foram obtidas boas correlações do logaritmo neperiano dos carotenóides totais com os parâmetros de cor. A melhor correlação foi com Lab (R2= 0,9944) para a batata-doce crua e Lab (R2= 0,9936) para a batata-doce cozida. As equações para se estimar o teor de . carotenóides através dos parâmetros de cor foram: c (µg/g/ ) e(-1.2051 + 0,01075a + 0,1013b) para a batata doce crua e C (µg/g) = e (3,1094 - 0,0476L + 0,1164a + 0,0667b) para a batata-doce cozida.
With the purpose of determining the color and carotenoid parameters,as well as the changes in the provitamin A value of sweet potatoes raw and cooked, ten cultivars from \"Instituto Agronômico de Campinas\" and \"Centro Nacional de Pesquisa de Hortaliças da EMBRAPA de Brasilia\" were analysed. Seven carotenoids were identified in the sweet potatoes analysis, as follows: 5.6.5\' ,6\'- β-carotene-diepoxide; 5,6-β- carotene epoxide; β-carotene; luteochrome; µ-zeacarotene; β-azeacarotene and aurochrome. β-carotene was the main carotenoid of the followinq cultivars: Centennial (148.81 ~g/g of sweet potato); Heart Gold (51. 79 µg/g); Anapolis (14.24 µg/g); Acadian (218,15 µg/g) ; Morada Inta (10.61 µg/g); Vineland Bush (22.60 µg/g) clone CNPH (4.87 µg/g). Luteochrome was the main carotenoid of Monalisa (1.55 µg/g of raw sweet potato), IAC-2-71 (0.77 µg/g) and SRT-252 (0.92 µg/g) cultivars. For raw roots-the vitamin A values varied from 1 retinol equivalent/l00q for IAC-2-71 cultivar up to 3,703 retinol equivalents/l00g for Acadian cultivar. For cooked roots Acadian cultivar was the one that presented more provitamin A activity, with 4,021 retinol equivalent/100g. The loss of β-carotene due to cooking varied from 4.51% to 42.6%, in accordance with the kind of cultivar. The color parameters (L Hunter-, a Hunter-, b Hunter-, chroma and hue angle) did not show any change for the three transversal cuts in the root (top, middle and bottom). Regarding cooking, there were significant decreases in the parameters L Hunter(luminosity) and +a Hunter (red) and an increase of -a Hunter (green) and hue angle parameters. The b Hunter parameter (yellow) and chroma decreased due to cooking in the Centennial, SRT-252, Monalisa and Heart Gold cultivars. There were obtained good correlations of total carotenoid neperiane logarithim with the color parameters. The best correlation was with \"Lab\" (R2= 0,9944) for- r-aw sweet potato and \"Lab\" (R2= 0,9936) for cooked sweet potato. The equation used to estimate carotenoids value through color parameters was the following: C (µg/g) = e(- 1,2051 + 0,01075a + 0,1013b) for raw sweet potato and C (µg/g = e(3,1094 - 0,0476L + 0,1164a + 0,0667b) for cooked sweet potato.

Orientador : Carol Hollingworth Collins
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica
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Mestrado

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Há muito tempo que as características relacionadas à coloração do fruto e sua qualidade nutricional têm sido consideradas desejáveis para manipulação em citros. Entretanto, a produção de novas cultivares dessas plantas tem sido limitada pelos vários obstáculos que impedem o seu melhoramento genético. A biotecnologia, por meio das técnicas de cultura de tecidos e transformação genética de plantas, surge como uma opção viável para contornar esse problema. Os objetivos do presente estudo foram isolar e caracterizar alguns genes envolvidos na rota biossintética de carotenóides de Citrus paradisi (Macf.) e desenvolver metodologias de regeneração in vitro e transformação genética para manipulação dessa rota biossintética em citros, visando alterar o conteúdo de provitamina A, a coloração do fruto e o porte das plantas. Utilizando-se a técnica de RT-PCR, as seqüências de cDNAs dos genes da sintase do fitoeno (AF152892), desaturase do fitoeno (AF364515), desaturase do ζ-caroteno (AF372617), ciclase-β do licopeno (AF152246) e ciclase-ε do licopeno (AF486650) foram isoladas de C. paradisi. Em geral, essas seqüências apresentaram elevada homologia aos genes correspondentes em tomate, com identidade de 72-83% em nível de aminoácidos. Dois ou mais transcritos diferentes foram identificados para três dos cinco genes caracterizados nesse estudo. Em cada caso, um ou mais transcritos foram considerados aberrantes, em que a produção de polipeptídeos não-funcionais foi predita. Estudos de expressão gênica revelaram que a maioria dos genes isolados é transcricionalmente regulada durante o desenvolvimento do fruto. No entanto, a diferenciação da coloração do fruto entre as cultivares de C. paradisi pode ser causada por mutações na seqüência aberta de leitura e não por regulação transcricional diferencial, sendo a desaturase do fitoeno e/ou ciclase-ε do licopeno os genes candidatos pelas diferenças observadas. Na avaliação da morfogênese in vitro de tecidos derivados de epicótilos de limão ‘Cravo’ (C. limonia Osb.), pomelo ‘Foster’ (C. paradisi Macf.) e laranja ‘Pêra’ [C. sinensis (L.) Osb.], verificou-se diferentes respostas às condições de cultivo in vitro em função da cultivar, da região do epicótilo utilizada como fonte de explantes, da composição do meio de cultura e das condições de incubação. Um sistema eficiente de transformação genética via Agrobacterium tumefaciens foi desenvolvido para C. paradisi, examinando-se os efeitos de seis fatores na eficiência de transformação. A pré- cultura dos explantes e a composição do meio de co-cultivo foram os fatores que mais influenciaram a eficiência de transformação. O protocolo otimizado foi empregado na produção de plantas transgênicas contendo os genes da sintase do fitoeno, desaturase do fitoeno ou ciclase-β do licopeno sob expressão constitutiva.
Fruit color and its nutritional value have been considered desirable for manipulation in citrus for a long time. However, citrus breeding has been limited due to several factors that hinder its genetic improvement. Plant biotechnology appears to be a viable option for the improvement of citrus species by means of the plant tissue culture and genetic transformation techniques. The objectives of this study were to isolate and characterize some genes involved in the carotenoid biosynthetic pathway of Citrus paradisi (Macf.) and to develop protocols of in vitro regeneration and genetic transformation for manipulation of this pathway in citrus, aiming to change the provitamin A content, fruit color, and plant height. By using the RT-PCR technique, the cDNA sequences of the genes phytoene synthase (AF152892), phytoene desaturase (AF364515), ζ-carotene desaturase (AF372617), lycopene β-cyclase (AF152246), and lycopene ε-cyclase (AF486650) were isolated from C. paradisi. In general, they were highly homologous to the corresponding tomato genes at the amino acid level, with identity ranging from 72-83%. Two or more different transcripts were identifyed for three of the five genes characterized in this study. In each case, one or more of the transcripts were aberrant, so that the production of a nonfunctional protein would be predicted. The expression analysis of the isolated carotenoid biosynthetic genes indicated complex expression patterns during fruit development. However, the fruit color differentiation between the grapefruit cultivars may be caused by frame-shift mutation and not by differential transcriptional regulation, with phytoene desaturase and/or lycopene ε-cyclase being the candidate genes for fruit color differences. The in vitro responses of epicotyl explants from ‘Cravo’ rangpur lime (Citrus limonia Osb.), ‘Foster’ grapefruit (C. paradisi Macf.), and ‘Pêra’ sweet-orange [C. sinensis (L.) Osb.] varyed according to cultivar, region of the epicotyl used as source of explant, culture medium composition, and incubation conditions. An improved protocol for Agrobacterium-mediated transformation of epicotyl explants from ‘Duncan’ grapefruit was developed by examining the effects of six different factors on the efficiency of transformation and combining the best treatments for each factor. The preculturing of the explants and the composition of the cocultivation medium were the factors that most influenced transformation efficiency. The optimized protocol was successfully employed in the production of transgenic grapefruit plants containing the carotenoid biosynthetic genes phytoene synthase, phytoene desaturase, or lycopene β-cyclase under constitutive expression.
Tese importada do Alexandria

Orientador: Delia Rodriguez-Amaya
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Estratos de 4 diferentes tipos de frutas e seus produtos processados, de 5 diferentes Hortaliças folhosas e de sementes de urucum foram analisadas por cromatografia líquida de alta eficiência (CLAE) em coluna C-18 com acetonitrila/clorofórmio 92:8 como fase móvel. As provitarainas A e os carotenóides majoritárias foram quantificados através dos métodos de padronização externa e de adição de padrão. Os resultados obtidos foram equivalentes, com uma precisão ligeiramente maior no caso da padronização externa. O emprego de CLAE não mostrou ser vantajoso no caso da determinação dos carotenóides totais do urucum e o método da adição de padrão deve ser utilizado com restrições, sendo recomendado apenas para extratos não saponificados de folhas. Para os demais extratos, a padronização externa deve ser o método de escolha. No entanto, esta técnica requer purificação e manutenção de carotenóides padrões, uma tarefa difícil, considerando a diversidade dos carotenóides e sua susceptibilidade à degradação
Abstract: Extracts from 4 different kinds of fruits and their processed products, from 5 different leafy vegetables and from annatto seeds were analysed by high performance liquid chromatography (HPLC) on a C-18 column with acetonitrile/chloroform 92:8 as mobile phase. The provitamins and major carotenoids were quantified by means of external standardization and standard addition methods. Results showed the two techniques to be equivalent, but external standardization was a little more precise. The use of HPLC demonstrated no advantage in the quantitation of total annatto carotenoids. The standard addition method should be used with restrictions, being suitable only for unsaponified leaf extracts. For all the other samples, the external standardization should be the method of choice. This technique, however, requires the purification and maintenance of carotenoid standards, a difficult task considering the diversity of carotenoids and their susceptibility to degradation
Doutorado
Doutor em Engenharia de Alimentos

FundaÃÃo de Amparo à Pesquisa do Estado do CearÃ
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
O objetivo do presente trabalho foi investigar a existÃncia de variaÃÃo sazonal nos conteÃdos de carotenÃides provitamina A ( e caroteno) e vitamina E ( e tocoferol) em duas espÃcies de macroalgas marinhas vermelhas, Cryptonemia luxurians e C. crenulata. As algas foram coletadas mensalmente de janeiro a dezembro de 2007, durante as marÃs baixas na Praia do Pacheco, Caucaia, CearÃ. Em laboratÃrio as algas foram desidratadas em estufa a 40ÂC por 15 horas. O material desidratado foi triturado atà a obtenÃÃo de um pà fino e, em seguida, submetido aos procedimentos de extraÃÃo com metanol, saponificaÃÃo com hidrÃxido de potÃssio e partiÃÃo em n-hexano. Para a anÃlise simultÃnea de carotenÃides e tocoferÃis, por cromatografia lÃquida de alta eficiÃncia, uma coluna Waters Spherisorb-Hichrom S5 ODS-2 (4,6 x 250 mm) e fase mÃvel de MeOH: THF (95:5, v/v), com fluxo de 1,5 mL min -1 foram utilizadas, com detecÃÃo em 450 nm e 292 nm, respectivamente. Em ambas as espÃcies pertencentes ao gÃnero Cryptonemia foram detectados e caroteno ao longo dos meses de coleta. De uma maneira geral, as duas espÃcies apresentaram maiores teores de caroteno que caroteno. Apesar de as macroalgas analisadas pertencerem ao mesmo gÃnero, foi possÃvel verificar uma variaÃÃo nos teores de carotenÃides provitamina A ao longo do ano entre as espÃcies. C. luxurians pode ser eleita como melhor fonte de vitamina A que C. crenulata. Dentre os isÃmeros da vitamina E, tocoferol apresentou as maiores concentraÃÃes. C. luxurians exibiu os maiores teores de ambos os tocoferÃis. De acordo com os resultados obtidos no presente estudo, as macroalgas marinhas C. luxurians e C. crenulata desidratadas em estufa a 40ÂC por 15 horas possivelmente sÃo melhores fontes de vitamina E do que de vitamina A. Para avaliar o comportamento sazonal dos teores de  e caroteno e  e tocoferol, foi utilizada a anÃlise de agrupamentos. A partir dessa anÃlise constatou-se que os teores de tocoferÃis apresentaram variaÃÃo mais proeminente, tanto entre as estaÃÃes do ano como entre as espÃcies, quando comparados com os carotenÃides provitamina A, os quais demonstraram maior estabilidade nos teores por espÃcie e por estaÃÃo do ano. Entretanto, somente a partir de estudos posteriores serà possÃvel determinar quais fatores e sua influÃncia na variaÃÃo dos teores de carotenÃides provitamina A e vitamina E das espÃcies analisadas.
The objective of this research project was to investigate the existence of seasonal variation of both provitamin A carotenoids ( and carotene) and vitamin E ( and tocopherol) in two species of red marine macroalgae, Cryptonemia luxurians and C. crenulata. The alga samples were collected monthly from January to December of 2007, during the low tides of Pacheco Beach, Caucaia, CearÃ. The alga samples were dehydrated at 40ÂC for 15 h. The dried alga material was crushed into a fine powder and then submitted into the procedures of extraction with methanol, saponification with potassium hydroxide and partition with n-hexane. For simultaneous analyses of carotenoids and tocopherols the HPLC system consisted of a Waters Spherisorb-Hichrom S5 ODS-2 (4.6 x 250 mm) column and a mobile phase of MeOH: THF (95:5, v/v), delivered at 1.5 mL min -1 , with detection at 450 nm and 292 nm, respectively. In both species belonging to the genre Cryptonemia were detected  and carotene throughout all the sampling months. In general, both species contained larger concentrations of carotene than carotene. Despite the macroalgae analyzed belonged to the same genre, it was possible to verify a variation in the concentrations of provitamin A carotenoids throughout the year. C. luxurians was elected as a better source of vitamin A than C. crenulata. Among the isomers of vitamin E, tocopherol showed the higher concentrations. C. luxurians showed the highest levels of both tocopherols. According to these results, C. luxurians and C. crenulata oven-dried at 40ÂC for 15 h are possibly better sources of vitamin E than vitamin A. To verify the seasonal behavior of the concentrations of provitamin A carotenoids and vitamin E, grouping analysis was utilized. Based upon the results of this analysis it was established that tocopherol concentrations showed a more substantiated variation in the macroalgae within the seasons of the year as well as among the two species. On the other hand, the provitamin A carotenoids, exhibited more stable concentrations considering the species and the seasons. Nevertheless, only with subsequent studies it will be possible to determine the factors and its influence on the variation of provitamin A carotenoids and vitamin E contents of the analyzed species.

NASCIMENTO, Rômulo Malta. Avaliação sazonal de Carotenóides provitamina A (alfa- e beta caroteno) e vitamina E ( alfa- tocoferol) em macroalgas marinhas pertencentes ao gênero Cryptonemia. 2009. 77 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2009
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The objective of this research project was to investigate the existence of seasonal variation of both provitamin A carotenoids ( and carotene) and vitamin E ( and tocopherol) in two species of red marine macroalgae, Cryptonemia luxurians and C. crenulata. The alga samples were collected monthly from January to December of 2007, during the low tides of Pacheco Beach, Caucaia, Ceará. The alga samples were dehydrated at 40°C for 15 h. The dried alga material was crushed into a fine powder and then submitted into the procedures of extraction with methanol, saponification with potassium hydroxide and partition with n-hexane. For simultaneous analyses of carotenoids and tocopherols the HPLC system consisted of a Waters Spherisorb-Hichrom S5 ODS-2 (4.6 x 250 mm) column and a mobile phase of MeOH: THF (95:5, v/v), delivered at 1.5 mL min -1 , with detection at 450 nm and 292 nm, respectively. In both species belonging to the genre Cryptonemia were detected  and carotene throughout all the sampling months. In general, both species contained larger concentrations of carotene than carotene. Despite the macroalgae analyzed belonged to the same genre, it was possible to verify a variation in the concentrations of provitamin A carotenoids throughout the year. C. luxurians was elected as a better source of vitamin A than C. crenulata. Among the isomers of vitamin E, tocopherol showed the higher concentrations. C. luxurians showed the highest levels of both tocopherols. According to these results, C. luxurians and C. crenulata oven-dried at 40°C for 15 h are possibly better sources of vitamin E than vitamin A. To verify the seasonal behavior of the concentrations of provitamin A carotenoids and vitamin E, grouping analysis was utilized. Based upon the results of this analysis it was established that tocopherol concentrations showed a more substantiated variation in the macroalgae within the seasons of the year as well as among the two species. On the other hand, the provitamin A carotenoids, exhibited more stable concentrations considering the species and the seasons. Nevertheless, only with subsequent studies it will be possible to determine the factors and its influence on the variation of provitamin A carotenoids and vitamin E contents of the analyzed species.
O objetivo do presente trabalho foi investigar a existência de variação sazonal nos conteúdos de carotenóides provitamina A ( e caroteno) e vitamina E ( e tocoferol) em duas espécies de macroalgas marinhas vermelhas, Cryptonemia luxurians e C. crenulata. As algas foram coletadas mensalmente de janeiro a dezembro de 2007, durante as marés baixas na Praia do Pacheco, Caucaia, Ceará. Em laboratório as algas foram desidratadas em estufa a 40°C por 15 horas. O material desidratado foi triturado até a obtenção de um pó fino e, em seguida, submetido aos procedimentos de extração com metanol, saponificação com hidróxido de potássio e partição em n-hexano. Para a análise simultânea de carotenóides e tocoferóis, por cromatografia líquida de alta eficiência, uma coluna Waters Spherisorb-Hichrom S5 ODS-2 (4,6 x 250 mm) e fase móvel de MeOH: THF (95:5, v/v), com fluxo de 1,5 mL min -1 foram utilizadas, com detecção em 450 nm e 292 nm, respectivamente. Em ambas as espécies pertencentes ao gênero Cryptonemia foram detectados e caroteno ao longo dos meses de coleta. De uma maneira geral, as duas espécies apresentaram maiores teores de caroteno que caroteno. Apesar de as macroalgas analisadas pertencerem ao mesmo gênero, foi possível verificar uma variação nos teores de carotenóides provitamina A ao longo do ano entre as espécies. C. luxurians pode ser eleita como melhor fonte de vitamina A que C. crenulata. Dentre os isômeros da vitamina E, tocoferol apresentou as maiores concentrações. C. luxurians exibiu os maiores teores de ambos os tocoferóis. De acordo com os resultados obtidos no presente estudo, as macroalgas marinhas C. luxurians e C. crenulata desidratadas em estufa a 40°C por 15 horas possivelmente são melhores fontes de vitamina E do que de vitamina A. Para avaliar o comportamento sazonal dos teores de  e caroteno e  e tocoferol, foi utilizada a análise de agrupamentos. A partir dessa análise constatou-se que os teores de tocoferóis apresentaram variação mais proeminente, tanto entre as estações do ano como entre as espécies, quando comparados com os carotenóides provitamina A, os quais demonstraram maior estabilidade nos teores por espécie e por estação do ano. Entretanto, somente a partir de estudos posteriores será possível determinar quais fatores e sua influência na variação dos teores de carotenóides provitamina A e vitamina E das espécies analisadas.

Pour l'industrie chimique et plus particulièrement pour la chimie fine, la photochimie est un outil de synthèse à forte potentialité. La lumière (photons) représente un réactif hautement spécifique, qui offre des voies de synthèse nouvelles, plus sélectives et moins agressives que les voies thermiques. Tout l'art duphotochimiste, pour mettre en œuvre de façon optimale un procédé et plus particulièrement pour privilégier la production d'un produit ciblé, consiste à bien appréhender les propriétés d'absorption des substances soumises au rayonnement photonique, de connaître leurs états excités et d'analyser finement leurs différentes voies de désactivation; toutes ces caractéristiques sont fonction de la structure des composés et surtout de leur environnement (nature du solvant, ordre du milieu, polarité, viscosité, etc. ). C'est dans cet esprit que nous avons développé de laphotochimie dans des milieux complexes pour modifier les propriétés d'environnement comparées à celles de solvants classiques: dans (i) soit des systèmes moléculaires organisés (SMO), pour lesquels l'organisation à l'échelle moléculaire crée un ordre local plus élevé et une répartition de concentration des produits différente et localisée, (ii) soit des fluides supercritigues (FSC), qui se différencient par leurs propriétés de faible viscosité et de grande diffusivité. Les photoréactions étudiées correspondent à la production de produits à haute valeur ajoutée: prévitamines D, rétrokétal, cis-jasmone. Pour ces composés, une des étapes clés de leur photoréactivité repose sur une isomérisation cis/trans, qui est paniculièrement sensible aux propriétés d'environnement. L'irradiation des provitamines (ergostérol, prokétal) dans des SMO améliore la production des prévitamines (précurseurs des vitamines D et du rétrokétal) en limitant l'isomérisation cis/trans (PVD/tachystérol). L'utilisation de FSC (dioxyde de carbone) qui augmente l'efficacité de l'isomérisation trans/cis de la jasmone permettrait de valoriser l'isomère trans-jasmone
For industrial chemistry and especially for fine chemistry, photochemistry is a powerful synthetic tool. Light (photons) represents a highly specific reagent, offering new synthetic pathways, more selective and less aggressive than thermal ones. Ln order to set up a new process in an optimal way and particularly to favor a target product, the photochemist bas to weIl know the absorption properties of the irradiated substances, their excited states and to analyze accurately their different deactivation pathways ; aIl these characteristics depend not only on the structure of the compounds but also on their environment (solvent nature, order of the medium, polarity, viscosity, etc. ). With these ideas in mind, photochemistry was developed in complex media, which modify the environment features by comparison to classical solvents : (i) organized molecular systems (OMS), which undergo a higher local order at molecular level and a different and local concentration distribution for products, (ii) supercritical fluids (SCF), which have low viscosity and high diffusivity properties. The involved photoreactions concern the production of high value products : previtamins D, retroketal, cis-jasmone. For these compounds, one of the photoreactivity key steps consists of a cis/trans isomerization, which is particularly sensitive to the environment properties. Provitamins D irradiation (ergosterol, proketal) in OMS improve the previtamins production (precursors of vitamins D and retroketal) by lowering the cis/trans isomerization rate (PVD/tachysterol). The SCF use (carbon dioxide) which increases the trans/cis isomerization efficiency of jasmone would valorize the trans-jasmone isomer

Vitamin A deficiency (VAD) is a major public health problem in developing countries, including South Africa. The potential of provitamin A-biofortified maize for use as a complementary strategy to alleviate vitamin A deficiency in developing countries, where maize is the dominant staple food, is currently a subject of research. Although the nutritional composition of white maize is thought to be similar to that of biofortified maize, apart from the differences in provitamin A carotenoid content, the comparative nutritional composition of the two maize types seems not to have been subjected to a comprehensive scientific study. When setting the target level of provitamin A in the provitamin A-biofortified maize, it is important to consider the potential effect of processing on the final provitamin A carotenoid content of the biofortified food products, as the provitamin A carotenoids levels may decrease on processing. Furthermore, the yellow/orange provitamin A-biofortified maize may not be widely accepted by African consumers who are vulnerable to VAD, and are traditional consumers of white maize. This study firstly aimed to evaluate the nutritional composition, including provitamin A composition, and grain quality of provitamin A-biofortified maize varieties, compared to white maize. The second aim was to assess the effect of processing (milling and cooking) on the retention of provitamin A carotenoids and other nutrients in popular South African maize food products prepared with provitamin A-biofortified maize. Thirdly, the study aimed to assess the acceptability of maize food products prepared with provitamin A-biofortified maize by consumers of different age and gender in rural KwaZulu-Natal, South Africa. The grains of the provitamin A-biofortified maize varieties and grain of a white maize variety (control) were analysed for their nutritional composition using standard or referenced methods. The carotenoid content of the grains was analysed by High-Performance Liquid Chromatography (HPLC) and mass spectroscopy. The provitamin A carotenoids β-cryptoxanthin, and trans and cis isomers of β-carotene, and other unidentified cis isomers of β-carotene were detected in varying levels in the provitamin A-biofortified maize varieties. The total provitamin A content in the biofortified maize varieties ranged from 7.3-8.3 μg/g dry weight (DW), with total β-carotene ranging from 3.5-3.6 μg/g DW, and β-cryptoxanthin from 3.7-4.8 μg/g DW, whilst no carotenoids were detected in the white maize variety. Results of the evaluation of the content of other nutrients showed that, when compared with the white maize variety, the provitamin A-biofortified maize varieties had higher levels of starch, fat and protein but were lower in iron. The zinc and phosphorus levels in the white maize and the biofortified maize were comparable. The biofortified maize varieties were better sources of most of the essential amino acids relative to the white maize, but, similar to the white maize, they were deficient in histidine and lysine, indicating that further improvement is required. Selected quality attributes (grain density, susceptibility of kernels to cracking, milling quality and resistance of the kernels to fungal infection) of grains of 32 provitamin A-biofortified maize varieties and a white variety (control) were assessed. Overall, the quality of the grains of the provitamin A-biofortified maize varieties were found to be superior to that of the white maize grain, although the biofortified maize grains showed less resistance to fungi, including mycotoxin-producing types. This indicates that the trait of grain resistance to infection by fungi should also be incorporated in the provitamin A-biofortified maize varieties during breeding. To assess the retention of provitamin A carotenoids and other nutrients in maize food products, three selected provitamin A-biofortified maize varieties and the control (white maize variety) were milled into mealie meal and samp. The milled products were cooked into three products: phutu and thin porridge (from the mealie meal) and cooked samp. Nutrient retention during processing was determined. Milling resulted in either an increase or slight decrease in the provitamin A carotenoid levels, but there was no major decrease in the total provitamin A level. Most of the other nutrients were well retained during milling, but there were substantial losses of fibre, fat and minerals. Provitamin A carotenoid levels decreased on cooking. In phutu 96.6 ± 20.3% β-cryptoxanthin and 95.5 ± 13.6% of the β-carotene was retained after cooking. In thin porridge 65.8 ± 4.6% β-cryptoxanthin and 74.7 ± 3.0% β-carotene; and in samp 91.9 ± 12.0% β-cryptoxanthin and 100.1 ± 8.8% of the β-carotene was retained after cooking, respectively. Provitamin A retention seemed to be influenced by both maize variety and food form, indicating that suitable varieties and food forms should be found. There was generally a high retention of the other nutrients in all the three cooked products, except for the substantial losses of fat in thin porridge and iron and phosphorus in cooked samp. These findings indicate that an optimal delivery of provitamin A to the consumer can be achieved by processing provitamin A-biofortified maize into foods that have a good retention of provitamin A carotenoids, such as phutu and samp. These food products would be recommended in areas where VAD is prevalent. In order to assess consumer acceptability of provitamin A-biofortified maize, a total of 212 subjects aged 3-55 years from Mkhambathini Municipality, in KwaZulu-Natal province, South Africa, participated in the sensory evaluation of phutu, thin porridge and cooked samp prepared with provitamin A-biofortified maize varieties and a white variety (control). Preference for yellow maize food products was negatively associated with an increase in the age of the subjects. Overall, preschool children preferred yellow maize to white maize food products: phutu (81% vs. 19%), thin porridge (75% vs. 25%) and samp (73% vs. 27%). In contrast, primary school children preferred white maize to yellow maize food products: phutu (55% vs. 45%), thin porridge (63% vs. 38%) and samp (52% vs. 48%). Similarly, secondary school children and adults also displayed a similar preference for white maize food products. There was no association between gender and preference for maize variety. Focus group discussions revealed that participants had a negative attitude towards biofortified maize due to its colour, taste, smell and texture. However, the participants expressed a willingness to consume biofortified maize if it was cheaper than white maize and was readily available in local grocery stores. These findings indicate that there is a potential to promote the consumption of provitamin A-biofortified maize and its food products in this part of South Africa, thereby contributing to a reduction in the incidence of VAD. This study has shown that provitamin A-biofortified maize has a good potential to be used as an additional strategy to alleviate VAD in poor communities of South Africa, including similar environments in sub-Saharan Africa. However, the study has revealed that there are still challenges to be overcome in order to achieve the target provitamin A content of 15 μg/g in provitamin A-biofortified maize, set by HarvestPlus, an international challenge program. This may also explain why provitamin A-biofortified maize varieties with this level of provitamin A have been scarcely reported in the literature. Thus, more research is required to achieve the target provitamin A level in maize by conventional breeding. The results of this study indicate that besides provitamin A, the biofortified maize is also a good source of other nutrients including starch, fat, protein and zinc. However, improving the consumer acceptability of the provitamin A-biofortified maize remains a challenge, due to the negative attitudes towards the yellow/orange maize by African consumers. On the other hand, the results of this study indicate that there is an opportunity to promote the consumption of provitamin A-biofortified maize food products by preschool children, a finding which has not been previously reported in the literature. Nutrition education on the benefits of provitamin A-biofortified maize, as well as improved marketing are recommended, in this part of South Africa and also in similar environments in other sub-Saharan countries.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Introduction: Micronutrient malnutrition has been identified as a serious health problem globally and is on the rise in South Africa. This is evident from the escalating burden of vitamin A deficiency (VAD) in South Africa. Rural infants are the most affected, as their diets often lack micronutrients. Food fortification, vitamin A supplementation and dietary diversity are the strategies that have been employed in South Africa to alleviate VAD. However, these strategies have not been effective, for various reasons. Biofortification is the production of micronutrient dense staple crops to alleviate micronutrient deficiencies. This strategy could complement existing strategies in the alleviation of VAD in South Africa and in other countries, especially in sub-Saharan Africa (SSA), where VAD is prevalent. Aim: The aim of this study was to investigate the nutritional composition and acceptance of a complementary food (soft porridge) made with provitamin A-biofortified maize by female infant caregivers from the rural areas of Umgungundlovu District of KwaZulu-Natal, South Africa. Objectives: (i) To evaluate the nutritional composition of soft porridge made with provitamin A-biofortified maize compared to non-biofortified white maize porridge; (ii) To assess the sensory acceptability of soft porridge made with the biofortified maize by black African female infant caregivers of varying age; and (iii) To determine the perceptions of the black African infant caregivers about the biofortified maize relative to the non-biofortified white maize. Methods: A cross-sectional study was conducted. Grains of two provitamin A-biofortified maize varieties and one white variety (control) were used. Grain and soft porridge of each variety of maize were analysed for their nutritional composition. The sensory acceptability of the porridges were evaluated by black African female infant caregivers, using a five-point facial hedonic scale. Focus group discussions were conducted, using some of the study subjects, to determine their perceptions about the provitamin A-biofortified maize. Results: The results showed that the grains of the provitamin A-biofortified maize varieties and their soft porridges were more nutritious than the control white variety in terms of energy, fibre, fat, protein, iron, zinc and phosphorus content. The results of the sensory evaluation indicated that there was no significant difference in the sensory acceptability of the biofortified soft porridges and the white maize soft porridge, irrespective of the age of the sensory evaluation panellists. The female caregivers perceived the biofortified maize as nutritious and health-beneficial and thought that infants would like its unique yellow colour and taste. However, the black African female caregivers perceived the provitamin A-biofortified maize as an animal feed or food for the poor. Nevertheless, the female caregivers expressed a willingness to give their infants porridge made with provitamin A-biofortified maize if it was cheap, readily available and health-beneficial. Conclusion: This study suggests that provitamin A-biofortified maize has the potential to be used as a complementary food item. Biofortification of maize with provitamin A could be used as a possible complementary strategy to assist in the alleviation of VAD in SSA. Furthermore, the relatively higher energy, fibre, fat, protein, iron, zinc and phosphorus content of the biofortified maize could contribute to the alleviation of protein-energy malnutrition and mineral deficiencies, respectively, which are prevalent in children of SSA. Although the findings of this study, like other previous studies, indicate that there are some negative perceptions about the provitamin A-biofortified maize, this study shows that provitamin A-biofortified maize soft porridge is as acceptable as white maize soft porridge to female infant caregivers from the rural areas of Umgungundlovu District of KwaZulu-Natal, South Africa. The female caregivers are thus likely to accept the biofortified maize for use as an infant complementary food in the form of soft porridge. Further research is recommended to expand the study area and consumer sample size in order to increase the confidence of inferring these results for large rural populations.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2014.

submitted in fulfilment of the academic requirements for the degree of Master of Applied Science in Food Science and Technology, Durban University of Technology, 2015.
Vitamin A deficiency (VAD) is a major health problem in sub-Saharan Africa where maize is a staple food. Amahewu, a fermented non-alcoholic,maize-based beverage is a popular drink in southern Africa.The aim of this study is to produce a provitamin A enriched and acceptable amahewu, using provitamin A biofortified maize which can be used to alleviate VAD. The optimal processing parameters for the production of amahewu using provitamin A-biofortified maize were determined. Amahewu samples were prepared with reference to a traditional method by boiling a mixture of maize meal and water (rato:1:7) at 90ᴼC, with occasional stirring, for 15 minutes. The resulting porridge was left to cool to approximately 40ᴼC, before inoculation and fermentation at 37oC. Processing parameters investigated were inoculum types (wheat bran (WB), maize malt (MM) and Lactobacillus mixed starter culture) and inoculum concentration (0.5,1 and 2% (w/w)) and varieties of provitamin A maize (PVAH 62 and PVAH 19). Wheat flour (at 2%) was used as reference inoculum to conform to the traditional practice. White maize amahewu samples processed in the same way as those of provitamin A-biofortified maize were used as references. Provitamin A amahewu samples were produced using the optimized processing parameters and then analysed for nutrient composition, including carotenoids, protein, ash, amino acids, mineral profile and invitro protein digestibility. The consumer acceptability of amahewu samples was evaluated using regular consumers of amahewu (n= 54), who rated the acceptability of the samples on a 9-point hedonic scale (1:disliked extremely, 9:liked extremely). The storage stability of the provitamin A biofortified amahewu samples was assessed by subjecting the samples to different storage conditions: 4ᴼC, 25ᴼC and 37ᴼC. The microbiological quality of the stored samples was monitored by taking samples every day for a period of five days to analyse for the presence of aerobic and anaerobic bacterial spore formers, E.coli and moulds. The provitamin A maize variety did not influence pH and Total titratable acidity (TTA) of amahewu samples during fermentation. As expected, there was a substantial drop in pH with fermentation time. After 24 hours, all the samples of amahewu, including those made with white maize, prepared using malted maize and wheat bran inocula reached a pH of 3.3-3.8 and TTA of 0.3-0.6, which were within acceptable range for amahewu. The addition of a starter culture substantially reduced fermentation time, from 24 to six hours. The inoculum of WB and MM, respectively, at a concentration of 0.5%, with or without starter culture (5%), were found to be suitable for the production of amahewu using provitamin A biofortified maize. The total provitamin A content of amahewu samples, produced using optimised parameters (i.e one variety of provitamin A biofortified maize, 0.5% MM, WB with or without starter culture), ranged from 3.3-3.8 μg/g (DW). The percentage retention of total provitamin A ranged from 79%- 90% (DW). The lowest percentage retention was observed in products fermented with the addition of starter culture. The gross energy of the amahewu samples was approx. 20 MJ/kg. There was a slight increase in the lysine content of amahewu after fermentation. The protein digestibility (approx. 91%) of amahewu samples was slightly higher than that of raw provitamin A maize (86%). Amahewu processed using starter cultures had a slightly higher iron content than those processed without a starter culture. Consumer acceptability data showed that amahewu samples made with provitamin A biofortified maize were slightly more acceptable (average rating for overall acceptability was 7.0 ± 1.2), compared to those made with white maize (average rating for overall acceptability was 6.4 ± 0.8). Principal component analysis (PCA) of Amahewu sensory data showed that 71% of variation was due to maize types and 18% of variation may be due to the inoculum used during fermentation. The use of a starter culture improves the taste and aroma acceptability of amahewu. Segmentation of consumers based on overall linking for amahewu revealed three clusters, named A, B and C. Cluster A consisted of most consumers (43%), who liked amahewu moderately. About 60% of these consumers were females. Cluster B consisted of most of the consumers (31%) who were undecided about their liking for the product. Approximately 52% of the consumers in this cluster were female. Cluster C consisted of consumers (26%) who liked amahewu very much. Sixty-four percent (64%) of these consumers were female. It appeared that gender may have some influence on overall liking for amahewu, as cluster B, consisting of undecided consumers, had more male consumers compared to clusters A and C. Age did not seem to be significantly associated with the liking of amahewu. Provitamin A biofortified amahewu samples stored under refrigerated conditions (4ᴼC) had better microbiological quality compared to those stored at 25ᴼC and 37ᴼC. Refrigeration effectively maintains the microbiological quality of amahewu for about three of days. Provitamin A biofortified maize can be used to produce β-carotene enriched amahewu that is acceptable to consumers following the processing method that is traditionally employed for white amahewu at both domestic and commercial level. Provitamin A biofortified amahewu has the potential to make a significant contribution towards alleviating VAD among rural communities, who are the most vulnerable to VAD.

Vitamin A deficiency is the leading cause of childhood blindness affecting over 190 million preschool children around the world where the highest rates are found in Sub-Saharan Africa (1). The coexistence of this deficiency with shortfalls in iron and zinc has resulted in a shift in intervention strategies from single targeted approach to broader diet diversification. As a result, food-based strategies leveraging local nutrient-dense plants as natural fortificants have gained significant interest for their potential to simultaneously address multiple micronutrient, and in some instances macronutrient, deficiencies. However, the efficacy of such approach depends upon several factors including knowledge on the nutritional composition of native plant materials as well as strategies for their incorporation into staple consumer products. Additionally, there is lack of information on impact of concurrent introduction of mineral and provitamin A rich plants on the stability and bioavailability of each individual nutrients including changes in these factors over extended periods of exposure. This is a key point considering that many of these materials are reported to have potential inhibitors of carotenoid absorption (minerals, fiber and phenolics).

To address these research gaps, this dissertation focuses on three areas including 1) micronutrient, phytochemical and polysaccharide characterization of three commercially available native micronutrient dense African plant materials [Adansonia digitata (baobab), Moringa Oleifera (moringa) and Hibiscus Sabdariffa (hibiscus)] that have been targeted for use as natural iron fortificants; 2) determination of the impact of these materials on the bioaccessibility and intestinal uptake of provitamin A carotenoids from model composite cereal products and 3) assess the effect of longer term exposure to baobab and moringa on provitamin A carotenoid absorption and cellular differentiation biomarkers of human intestinal Caco-2 cells to better understand the potential impacts of extended exposure periods on long term micronutrient uptake.

Characterization of the plant fortificants focused on understanding both nutritive components and potential limiters of carotenoid bioavailability. Baobab, moringa and hibiscus all were found to contain key phytochemical and polysaccharide components that could be leveraged as nutritional and function ingredients. The relatively higher levels of lutein (57  4.6 g/g), zeaxanthin (11  0.1g/g) and -carotene (20  2 g/g) in moringa leaf powder support the notion that this plant material can be used as a source of provitamin A and non-provitamin A carotenoids. Phenolic analysis revealed the presence of substantial amounts of flavan-3-ols (1234  16 mg/100g) in baobab, anthocyanins (2001  56 mg/100g) in hibiscus, and flavonols (5352  139 mg/100g) in moringa leaf powder. Polysaccharide analysis demonstrated that the primary monosaccharide in baobab was found to be xyloglucan (47 %) which is in agreement with the tentative identification Xyloglucans (hemicellulosic polysaccharide) based on linkage analysis. Hibiscus was found to contain similar amounts of xylose (20%) and galactose (27%) supporting the presence of similar proportions of xyloglucans and pectic polysaccharides (type I, type II AG, RG I). The main monosaccharide in moringa was found to be galactose (36%) followed by glucose (23%) and linkage analysis revealed the presence of high proportions of pectic polysaccharides (type I, type II AG, RG I). These results provide insight into presence of potential enhancer or inhibitors of target micronutrient (provitamin A carotenoids or iron/zinc) bioavailability when used as functional and nutritional food ingredients.

Subsequently, the impact of mineral-rich baobab formulated at levels relevant for iron fortification on the bioaccessibility of provitamin A carotenoids (proVAC) from composite millet porridges containing dried carrot and mango was assessed using in vitro digestion. Proportions of millet flour and plant materials were dry blended to deliver ~25% of the RDA for vitamin A(VA) and iron(Fe) as follows: decorticated extruded millet (Senegalese Souna var.) (40-60%), dried proVA rich carrot and mango blend (30%), and dried Fe and ascorbic acid rich Adansonia digitata (baobab) (0-25%). While there were no significant differences in proVAC bioaccessibility from porridge formulations with 5 and 15% baobab (18.8+/-2.0 and 18.8±2.0% respectively) as compared to control containing no mineral-rich plant (23.8 +/- 1.2%), 25% baobab resulted in a significant decrease (p<0.05) in bioaccessibility of proVAC (13.3+/-1.6%). However, baobab inclusion did not impact intestinal uptake efficiency of provitamin A carotenoids by Caco-2 human intestinal cells

(3.3-3.6% -carotene and 3.7-4.5% for -carotene) across all formulation. These results suggest that any potential negative effects of baobab inclusion may be limited to food matrix interactions and digestion. This was confirmed in separate experiments that with experiments on baobab and carotenoid blends showing that digested baobab did not affect carotenoid absorption by Caco-2 cells. Overall these data support the notion that that modest inhibition of carotenoid bioaccessibility by baobab may not significantly limit carotenoid delivery from composite porridges. Furthermore, bioaccessible provitamin A content of a serving (200 g) of composite porridges can provide 27 - 48% of the RDA of vitamin A for children 1-3 years of age.

Finally, we evaluated the impact of long-term exposure to baobab and moringa digesta on Caco-2 cell differentiation biomarkers and provitamin A uptake to gain insight into how inclusion of these materials in to a daily diet may alter absorption and transport of nutrients or otherwise have potential negative effects on the intestine. Based on NMR analysis of intracellular metabolites in differentiating Caco-2 monolayers, significant alterations in specific osmotic pressure regulators, particularly glycerophosphocholine, taurine and myo-inositol were observed with repeated exposure to all treatment groups including the control (digested 0.9% saline solution). Changes in these metabolites levels have been linked with specific cellular function including protection against hyperosmotic stress and regulation of paracellular permeability of Caco-2 cells. Evaluation of carotenoid uptake comparing acute and acute on repeated exposure to treatment groups demonstrated that there was an overall significant reduction in carotenoid uptake with repeated exposure across all treatment groups including the control. Despite the reduction in carotenoid uptake, mRNA and protein levels of carotenoid transporters (CD-36, SR-B1 and FABP1) were not significantly altered with exposure through differentiation (except for SR-B1 protein levels). Decrease in SR-B1 levels may be due to bile acid accumulation from the digesta matrix which is known to regulate its own biosynthesis by a mechanism that involves the down-regulation SR-B1 expression to protect cells from cytotoxicity. Our results provide some insight into the impact of simulated gastrointestinal fluids alone on provitamin A uptake in this model system which are usually not taken into consideration in most Caco-2 cell studies. However, overall, these findings indicate that the introduction of baobab and moringa at levels relevant for delivery of meaningful levels of iron (15-23% RDA) should not have negative impacts on human intestinal function or carotenoid uptake over chronic use.

Taken together, our findings indicate that the three native Africa plant materials selected for investigation in these studies can be important sources of key micronutrients (iron, zinc and provitamin A carotenoids) and have potential as natural fortificants with application in staple foods such as cereal porridges. Incorporation of these plant materials, do not appear to negatively affect carotenoid bioavailability although there is a potential for their interaction during micellarization of carotenoids during normal digestion. While in vivo studies evaluating the bioavailability of provitamin A carotenoids from such composite formulations are required, these data support the further exploration of such natural fortification strategies in addressing micronutrient deficiencies in local African communities.

Successful adoption of biofortified orange maize in developing countries requires careful consideration of factors across the chain from farm to fork. This includes consideration of post-harvest storage conditions optimal for the retention of both proviatamin A carotenoids and cooking quality critical to consumers. In these considerations, identification of economical storage methods is critical considering the limitations within specific countries that biofortified maize is being disseminated. To address these points, this dissertation research focused on evaluation of the utility of the Purdue Improved Crop Storage (PICS) bags as a post-harvest storage solution for biofortified maize. The specific focus of this research was to monitor retention of provitamin A and other carotenoids in two biofortified maize genotypes (OPVI and OPVII) as well as storage effect on flour functionality. Finally, a preliminary assessment of the impacts of storage on carotenoid bioaccessibility was completed to begin to translate findings to practice.

Maize grain from 2016 harvest was stored at ambient conditions for eight months in either PICS bags with or without an O₂ scavenger, (PICS-oxy) and (PICS-noxy), respectively and compared to storage in common polypropylene woven bags (control). After 4 months of storage carotenoid content was significantly higher (p<0.05) in PICS-oxy compared to PICS-noxy and woven bags demonstrating the importance of entrapped oxygen on maize carotenoid degradation. Furthermore, differences in carotenoid stability between maize genotypes were observed with OPVI having higher retention than OPVII. After 8 months, carotenoid retention remained dependent on storage bag and genotype with retention being greater in PICS-oxy and PICS-noxy compared to woven bags. However, final levels after 8 months were more similar between storage methods. Overall, oxygen content and genotype were found to be determining factors in the effectiveness of PICS to mitigate carotenoid degradation during post-harvest storage of maize.

While reducing the rate of carotenoid degradation during postharvest storage of biofortified maize is important, success of biofortified maize is also dependent on consumer adoption of these grains and their performance in traditional food preparation. Assessment of the rheological and functional properties of these two biofortified maize genotypes as a function of post-harvest storage was completed to assess the impact of post-harvest storage in PICS bags on flour functionality and rheological properties for the two biofortified orange maize genotypes and a control white maize genotype. Flour pasting profiles were assessed initially and at 4 and 8 months. After 8 month storage in woven and PICS bag, OPVI and OPVII produced porridges with similar viscosities to their initial viscosities regardless of postharvest storage type. White maize viscosities progressively decreased with storage and were significantly lower (p<0.05) in woven compared to PICS storage. Sequestration of oxygen (PICS-oxy) had modest but significant effects (p<0.05) on key pasting parameters including peak and final viscosities. These results suggest that oxygen sequestration has a critical effect on final flour functionality. DTT treatment partially restored flour pasting profiles suggesting disulfide linkages may modify pasting profiles of flour. There was also an increase in free ferrulic and p-coumaric acids during storage which may have contributed to observed decreases in porridge viscosities. Evidence of this was found through Raman spectroscopy with spectral intensity at both 478cm^-1 and 2911cm^-1 decreasing with storage suggesting the potential for structural changes induced by storage on starch polymer. While storage in PICS bags does not seem to adversely affect flour functionality it may provide some additional economic benefit resulting from requiring proportionally less flour to achieve similar final viscosities as flour from woven bag stored grains.

Finally, the effect of postharvest storage on bioaccessibility of carotenoids was explored using experimental wet cooked porridges made from ‘fresh’ and stored grains using an established three stage in-vitro digestion model. Relative carotenoid bioaccessibility (% micellarization) was generally higher in less viscous porridge made from grains stored in woven bags compared to porridge from initial or PICS bags stored grains suggesting that higher viscosity might partly explain lower relative bioaccessibility in porridge from grains stored in PICS bags. Absolute carotenoid bioaccessibility from experimental porridge was dependent on carotenoid species and storage system. Extrapolation of relative bioaccessibility (%) to absolute bioaccessibility (µg/g flour) suggests that fresh grains and their corresponding porridges would provide more absolute bioaccessible carotenoids compared to stored grains despite some improvement in relative accessibility. As such, storage losses remain the main factor impacting total available carotenoids and should continue to be an area of focus for future mitigation. With the potential to minimize post-harvest losses, improve carotenoid retention and provide a product with improved cooking performance, PICS bags do appear to offer a viable storage alternative to improve both food and nutrition security in developing countries.