Dissertations / Theses on the topic 'Carotenoids'

Orientador: Delia B. Rodriguez-Amaya
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A biodiversidade brasileira em fontes de carotenóides foi demonstrada no Capítulo 1, focalizando a riqueza dos alimentos brasileiros nativos ou pouco cultivados nos seis carotenóides (alfa-caroteno, B-caroteno, B-criptoxantina, licopeno, luteína e zeaxantina) considerados importantes à saúde. No Capítulo 2, dentre os produtos in natura analisados, frutas de clima temperado apresentaram menores teores de carotenóides do que as frutas tropicais, sendo a manga ¿Palmer¿ a maior fonte de -caroteno. Vegetais folhosos altamente consumidos como alface roxa e rúcula são ricos em luteína e -caroteno. Banana e tangerina são frutas comumente consumidas que apresentaram teores apreciáveis de carotenóides. No Capítulo 3, foi observado que a composição qualitativa de carotenóides em alimentos processados e preparados reflete a matéria-prima utilizada e a degradação de carotenóides. Entre os alimentos processados e preparados analisados, somente a goiabada em pasta apresentou licopeno com teor altíssimo. A seleta de legumes possui o maior teor de -caroteno. O teor de B-criptoxantina foi maior em pêssego enlatado. O cereal de milho com açúcar apresentou como carotenóide majoritário a zeaxantina. Os maiores teores de B-caroteno e luteína foram encontrados na catalonha e espinafre refogados. A comparação entre os dados obtidos por análise direta do alimento preparado e por cálculo utilizando dados tabelados dos ingredientes revelaram diferenças marcantes para as três receitas investigadas
Abstract: Brazilian biodiversity in sources of carotenoids is demonstrated in Chapter 1, higlighting the richness of native and uncultivated Brazilian foods in the six carotenoids (alpha-carotene, B-carotene, B-cryptoxanthin, lycopene, lutein and zeaxanthin) considered important to human health. In Chapter 2, among the raw foods analyzed, fruits of temperate climates had lower levels of carotenoids than tropical fruits, the mango ¿Palmer¿ being the major source of -carotene. Highly consumed leafy vegetables such as purple lettuce and roquette are rich in lutein and B-carotene. Banana and tangerine are commonly consumed fruits that present appreciable amounts of carotenoids. In Chapter 3, it was observed that the qualitative composition reflected the raw material used and degradation of carotenoids. Among the processed and prepared foods analyzed, only ¿goiabada¿ had lycopene at high level. A mixture of vegetables had the highest concentration of B-carotene. B-cryptoxanthin was higher in canned peach. Cereal with sugar presented zeaxanthin as the principal carotenoid. The highest levels of -carotene and lutein were found in stir-fried ¿catalonha¿ and New Zealand spinach. A comparison between calculated values and those obtained by direct analysis of three prepared foods revealed marked differences
Mestrado
Mestre em Ciência de Alimentos

Carotenoid pigments in W. dermatitidis, the first pathogenic, dematiaceous fungus in which carotenoid pigments nave been reported, are located primarily (81%) in lipid organelles which floated on the surface of the supernatant fraction of lysed cells. Pigment in this fraction could be extracted with ethyl ether without prior treatment with acetone indicating the pigment is unbound in the lipid organelle. Eight percent remains after exhaustive ether extraction and is recovered after the sample is treated with acetone indicating this fraction is non-covalently bound to proteins in the membranes associated with the lipid organelle. The remaining pigment (about 12%) represents contamination of the supernatant with the lipid organelles.

Two experiments were completed to develop methods for extracting xanthophylls from corn industry co-products, post fermentation (PF) corn oil and corn gluten meal (CGM). A solid phase extraction (SPE) method was used to fractionate a xanthophyll-rich portion of PF corn oil by varying conditioning and eluting solvents used with a diol SPE column. Conditioning with dichloromethane yielded highest xanthophyll fractionation, 86.5%. The elution solvent selected did not impact fractionation based on a two-way ANOVA. Supercritical fluid extraction of xanthohpylls from CGM was modeled using a Box-Behnken design, varying temperature, pressure, and co-solvent ratio. The optimum conditions were determined to be 40 ?C, 6820 psi, and 15% co-solvent, which would extract 85.4 ?g lutein/g CGM, 2.6 times more lutein than an ethanol and chloroform: dichloromethane solvent extraction. Co-solvent was the most influential extraction parameter and increasing it further could yield higher xanthophyll recovery. With further studies, this work has industrial potential.
Golden Growers; North Dakota Corn Council

Mestrado em Biotecnologia, ramo de Biotecnologia Industrial e Ambiental
Microalgae have been attracting a crescent attention regarding either the potential use of their biomass or the wide range of compounds of interest that they have in their constitution, which can be extracted and applied in several fields of industry, namely carotenoids and in particular fucoxanthin.. Fucoxanthin is a photosynthetic pigment, with numerous applications and benefits, namely in the human healthrelated domains, due to its high antioxidant properties, essential in the prevention and treatment of several diseases, such as cancer and cardiovascular diseases. Hereupon, the development of a feasible route for the production of carotenoids rich in fucoxanthin which could allow its commercialization is of utmost scientific, industrial and even social relevance. The Phaeodactylum tricornutum species is being reported as a relevant producer of fucoxanthin. Still, there are no economic feasible extraction methods for the carotenoids, and in particular fucoxanthin, considering their efficient obtainment with high purity levels. Thus, this work is aiming at the development of a sustainable and feasible production and extraction platform for the extraction of carotenoids (and fucoxanthin) from Phaeodactylum tricornutum. In this sense, a conventional methodology was optimized, as well as a novel extraction methodology based on alternative tensioactive solvents, exclusively in aqueous solution, namely common surfactants, copolymers and tensioactive ionic liquids. Regarding the alternative method developed, it was possible to achieve a final extraction of 32.67 mgcarotenoids/gbiomass with the ionic liquid [C18mim]Cl and 10.69 mgfucoxanthin/gbiomass with [C14mim]Cl, similar values or even superior when compared with those found for the conventional methodology. At the end, the achievement of fucoxanthin in its purest form is foreseen for further industrial application.
As microalgas têm vindo a despertar cada vez mais interesse pelas potencialidades de utilização da biomassa como um todo, quer pela diversidade de compostos de interesse que as constituem, com aplicações variadas, como é o caso dos carotenóides e, em particular, da fucoxantina. Fucoxantina é um pigmento fotossintético com inúmeras aplicações e benefícios, nomeadamente na área da saúde, pelasua elevada atividade antioxidante, importante na prevenção e tratamento de várias doenças, como o cancro e doenças cardiovasculares. Desta forma, é de interesse científico, industrial e mesmo social, o desenvolvimento de uma plataforma economicamente eficiente de produção e extração destes carotenóides que permitam o seu uso em larga escala, visando assim a sua aplicação industrial. Existem diversos estudos que apontam as microalgas como grandes produtoras deste pigmento, nomeadamente a espécie Phaeodactylum tricornutum. No entanto, os métodos de extração e purificação de carotenóides e em particular de fucoxantina que permitam a recuperação destes compostos com elevado grau de pureza são ainda algo deficitários, no que diz respeito ao seu elevado impacto económico e ambiental. Assim, o objetivo deste trabalho recai no desenvolvimento de uma plataforma rentável para produção e extração de carotenóides (e fucoxantina) a partir da microalga Phaeodactylum tricornutum. Para tal, o método de extração convencional foi otimizado, assim como foi desenvolvido um método de extração alternativo recorrendo ao uso de solventes alternativos com natureza tensioativa exclusivamente em solução aquosa, nomeadamente surfactantes comuns, copolímeros e líquidos iónicos tensioativos. A partir deste método alternativo foi possível extrair um total de 32.67 mgcarotenoides/gbiomassa com o [C18mim]Cl e 10.69 mgfucoxantina/gbiomassa com [C14mim]Cl, valores semelhantes ou mesmo superiores aos obtidos pelo método convencional.

Thesis (Ph.D.)--Tufts University, 2006.
Submitted to the School of Nutrition Science and Policy. Adviser: Xiang-Dong Wang. Includes bibliographical references. Access restricted to members of the Tufts University community. Also available via the World Wide Web;

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
O aumento da produÃÃo de biodiesel tem causado um aumento repentino na produÃÃo de glicerol residual, criando um excesso desse produto no mercado. O glicerol pode ser utilizado como fonte de carbono em processos biotecnolÃgicos, incluindo a produÃÃo de carotenoides. Os carotenoides microbianos tÃm sido estudados e seu potencial reconhecido ao longo dos anos. O gÃnero Rhodotorula à conhecido por sua capacidade de biossintetizar carotenoides, tais como β-caroteno, toruleno e torularrodina, em diferentes proporÃÃes. Este gÃnero tem sido estudado devido a seu potencial para produÃÃo industrial de carotenoides, uma vez que oferecem vantagens sobre os outros gÃneros em termos de taxa de crescimento elevada e uso de substratos de baixo custo. Neste contexto, o principal objetivo deste trabalho foi demonstrar a aplicabilidade do glicerol bruto como fonte de carbono na produÃÃo de pigmentos carotenoides por linhagens do gÃnero Rhodotorula atravÃs de fermentaÃÃo submersa. O meio de cultura utilizado nos processos fermentativos apresentou a seguinte composiÃÃo em (g.L-1): glicerol: 20,0; extrato de levedura: 1,0; K2HPO4: 1,0; (NH4)2SO4: 5,0 e MgSO4.7H2O: 0,5. Para tanto foram preparados 100 mL de meio, inoculados com 0,1 g.L-1 massa seca, e incubado a 30ÂC, 150 rpm por 24 h em agitador orbital. Posteriormente o meio fermentado foi transferido para um frasco de 2L contendo 400 mL de meio e incubado por 48 horas. O meio de cultura contendo 500 mL foi utilizado para inocular o fermentador, onde o crescimento prosseguiu por 240 horas a uma taxa de aeraÃÃo de 1,0 vvm-1. As amostras foram retiradas em intervalos regulares de 24 horas para determinaÃÃo de biomassa, glicerol, produtividade, cromaticidade a* e carotenoides. ApÃs os testes preliminares da investigaÃÃo, selecionaram-se trÃs linhagens do gÃnero Rhodotorula (R. lactosa CCT 2057, R. glutinis URM 5724 e R. aurantiaca URM 5726) capazes de produzir biomassa e sintetizar carotenoides atravÃs do glicerol P.A como fonte de carbono. Ensaios de aumento volumÃtrico do meio de fermentaÃÃo realizados com Rhodotorula mucilaginosa CMIAT 164 e Rhodotorula aurantiaca URM 5726 compararam o desempenho de glicerol bruto e P.A na produÃÃo de biomassa. Os testes em Erlenmeyers de 2 L revelaram o melhor desempenho de glicerol bruto na produÃÃo de biomassa rica em carotenoides, registrando-se 6,2  0,1 g.L-1 nos cultivos com Rhodotorula mucilaginosa CMIAT 164 e produtividade em biomassa de 0,069  0,007 g.L-1.h-1 utilizando glicerol bruto como fonte de carbono. No teste de inÃculo realizado em biorreator, selecionaram-se os tempos de 48 horas de fermentaÃÃo (fase exponencial) e 120 horas (fase estacionÃria) para as duas cepas. Nas fermentaÃÃes realizadas em biorreatores de 3 L com Rhodotorula mucilaginosa CMIAT 164 empregando o glicerol bruto como fonte carbono, observou-se que a inoculaÃÃo da suspensÃo proveniente de um cultivo de 48 h de propagaÃÃo influenciou maiores valores de produÃÃo de biomassa (6,35  0,3 g.L-1) e produtividade (0,080  0,004 g.L-1h-1). Os testes em biorreatores tambÃm revelaram que a temperatura de incubaÃÃo de 30 ÂC e controle do pH em 6 foram as melhores condiÃÃes para a sÃntese de biomassa e carotenoides. Os cultivos mantidos a 30 ÂC registraram 6,5  0,3 g.L-1 de biomassa e 0,546  0,02 mg.g-1 de carotenoides. As fermentaÃÃes mantidas em pH 6 apresentaram os maiores valores de produÃÃo de carotenoides (0,576  0,02 mg.g-1). O aumento da concentraÃÃo do inÃculo e as estratÃgias de alimentaÃÃo do biorreator, nas condiÃÃes avaliadas, nÃo impactaram consideravelmente no aumento da produÃÃo de biomassa e carotenoides por R. mucilaginosa CMIAT 164.
The increased production of biodiesel has caused a sudden increase in the production of glycerol, creating an excess of this product on the market. Glycerol can be used as biotechnological processes carbon source, including the carotenoids production. The microbial carotenoids have been studied and its potential recognised over the years. The genus Rhodotorula is renowned for its ability to biossintetizar carotenoids, such as β-carotene, toruleno and torularhodin, in different proportions. This genus has been studied due to their potential for industrial production of carotenoids, once that offer advantages over other genres in terms of high growth rate and use of low cost substrates. In this context, the main objective of this work was to demonstrate the applicability of crude glycerol as a carbon source on production of carotenoids pigments for the genus Rhodotorula by submerged fermentation. The culture medium used in fermentation processes performed as follows in (g. L-1): glycerol: 20.0; yeast extract: 1.0; K2HPO4:1.0; (NH4) 2SO4:5.0 and 4.7 MgSO.H2O: 0.5. For both were prepared 100 mL of medium, inoculated with 0.1 g L-1 dry mass, and incubated at 30Â C, 150 rpm for 24 hours in orbital shaker. Subsequently the fermented medium was transferred to a 2 L bottle containing 400 mL and incubated for 48 hours. The culture medium containing 500 mL was used to inoculate the fermentor, where growth continued for 240 hours under aeration rate of 1.0 vvm-1. The samples were taken at regular intervals of 24 hours for determination of biomass, glycerol, productivity, the chromaticity a* and carotenoids. After the preliminary tests of research, selected three strains of Rhodotorula genus (R. lactosa CCT 2057, R. glutinis URM 5724 e R. aurantiaca URM 5726) capable of producing biomass and synthesize carotenoids via glycerol. Testing of volumetric expansion of the fermentation medium with Rhodotorula mucilaginous CMIAT 164 and Rhodotorula aurantiaca 5726 URM compared the performance of raw glycerol and biomass production. In tests of 2 L Erlenmeyer flasks revealed the best performance of crude glycerol in the production of biomass rich in carotenoids, registering 6.2 Â 0.19 g.L-1 in Rhodotorula mucilaginous CMIAT 164 crops and biomass productivity of 0.069 Â 0.007 g.L-1.h-1 using crude glycerol as a carbon source. Inoculum test held in bioreactor, selected the times of 48 hours of fermentation (exponential phase) and 120 hours (stationary phase) for the two strains. In the fermentations conducted in 3 L bioreactors with Rhodotorula mucilaginous CMIAT 164 employing crude glycerol as carbon source, it was observed that the inoculation of the suspension from a 48 h culture propagation influenced higher biomass production values (6.35 Â 0.3 g L-1) and productivity (0.080 Â 0.004 g.L-1.h-1). The tests also revealed that the bioreactors in incubation temperature of 30 ÂC and pH control in 6 were the best conditions for the synthesis of biomass and carotenoids. The cultures kept at 30 ÂC registered biomass 6.5 Â 0.3 g.L-1 and 0.546 Â 0.02 mg.g-1 of carotenoids. Fermantations kept at pH 6 showed higher carotenoids production values (0.576 Â 0.02 mg.g-1). Increase in the concentration of the inoculum and the feeding strategies of bioreactor, under the conditions evaluated no impact considerably on increasing the production of biomass and carotenoid by R. mucilaginosa CMIAT 164.

Protoplasts were prepared from two yeast strains P. rhodozyma (ATCC 24202) and K. fragilis (ATCC 8455). Protoplasts prepared from P. rhodozyma were facilitated by prior growth of the cells in a media containing S-(2-aminoethyl)-L-cysteine. Protoplasts from these two yeast genera were fused either by the use of electrofusion or polyethylene glycol treatment. Stable carotenoid producing cell lines were selected by growth at 30°C on yeast nitrogen base plus galactose. Selected single fusants display taxonomic characteristics common to both genera with a cellular morphology and a carotenoid composition similar to that of P. rhodozyma.
Land and Food Systems, Faculty of
Graduate

Com a demanda cada vez maior por corantes alimentícios não tóxicos e sustentáveis, fontes naturais têm emergido como uma potencial alternativa aos corantes sintéticos artificiais. O urucum, planta tropical, tem sido cada vez mais explorado para suprir as demandas por corantes naturais, visto que sua semente apresenta uma alta concentração de carotenoides com poder colorífico. O Brasil que já era grande produtor de urucum tem sido destaque na produção de suas sementes para extração de pigmentos, devido à alta na demanda por corantes de origem natural. Das sementes do urucum é possível a extração de dois dos principais compostos para fabricação de corantes - bixina e norbixina. Visando contribuir para o melhor entendimento das potencialidades das sementes de urucum, além de fornecer evidência experimental sobre as propriedades antioxidantes e conteúdo de compostos fenólicos, flavonoides e carotenoides, foram determinados o perfil químico, o potencial antioxidante a partir de extratos de sementes de urucum, bem como a caracterização do carotenoide bixina por HPLC-PDA e ESI-Orbitrap. Sendo assim possível observar que o perfil químico e os teores de compostos bioativos em sementes de urucum não apresentam diferença independente da região produtora. Por fim, o projeto de pesquisa focou na conversão de bixina (carotenoide lipossolúvel que representa 80% do total de carotenoides presentes na semente) em norbixina (carotenoide solúvel em água) para suprir o aumento de demanda por corantes hidrossolúveis, visto que a indústria de alimentos tem visado a redução de gorduras nos alimentos.
With the increasing demand for non-toxic and sustainable food coloring, natural sources have appeared as a potential alternative to artificial synthetic dyes. The tropical plant known as annatto has been increasingly exploited to attend the demands for natural colorings as its seed are rich in carotenoids with high coloring potential. Brazil already considered a big producer of annatto seeds has been prominent in the production of its seed for extraction of its pigments, due to increasingly demand for natural-source food coloring. From annatto seeds it is possible to extract two of the main compounds for fabrication of coloring - bixin and norbixin. In order to a better understanding of the potential of annatto seeds as well as providing experimental evidence on the antioxidant properties and content of phenolic, flavonoids and carotenoids, the chemical profile, antioxidant potential as well as the characterization of bixin carotenoid by HPLC-PDA and ESI-Orbitrap, were determined from annatto seeds extract. It was found that the chemical profile and the content of bioactive compounds in annatto seeds did not differ independently of its producing site. In addition, the research foccused on the conversion of bixin (liposoluble carotenoid, responsible for 80% of the seeds\' carotenoids) into norbixin (water soluble carotenoid) to attend the demand for water-soluble coloring, as the food industry has been aiming a reduction in food fat content.

Los isoprenoides son una de las familias más grande de metabolitos en la naturaleza y son especialmente diversos en el reino vegetal. Entre ellos, los carotenoides, los tocoferoles y la filoquinona tienen un interés especial por su papel como vitaminas. Las plantas sintetizan estos compuestos en cloroplastos para contribuir a la fotosíntesis y fotoprotección. En el caso de los carotenoides, sus niveles más altos se encuentran en plastos especializados llamados cromoplastos, que se encuentran típicamente en órganos pigmentados no verdes como pétalos de flores y frutos maduros, pero solo ocasionalmente en hojas. El objetivo general de esta tesis ha sido probar nuevas estrategias para el enriquecimiento de verduras en vitaminas isoprenoides a través de un sistema desarrollado en nuestro laboratorio para inducir la conversión de cloroplastos en cromoplastos en hojas. La herramienta se basa en la gran capacidad de la enzima crtB de la bacteria Pantoea ananatis para producir fitoeno y la capacidad de las hojas para convertir este fitoeno adicional en carotenoides posteriores con cambios concomitantes en la ultraestructura de los plástos. La disponibilidad de este sistema permitió definir dos objetivos específicos: (1) caracterizar el contexto fisiológico del fenotipo inducido por crtB en hojas de Nicotiana benthamiana y (2) probar diferentes estrategias aprovechando este sistema basado en crtB para mejorar la biofortificación de hojas. En la primera parte de la tesis, demostramos que el fenotipo no reversible de diferenciación de cloroplasto a cromoplasto desencadenado por crtB en plastos se asocia con una rápida pérdida de actividad fotosintética causada por la acumulación de fitoeno. Este fenómeno hace que el cloroplasto pase a ser competente para la cromoplastogénesis. Luego, la transición se completa una vez que el fitoeno se convierte en carotenoides posteriores mediante enzimas endógenas. También demostramos que los tratamientos que causan un equilibrio redox alterado de los fotosistemas y un estrés oxidativo facilitan la diferenciación de los cromoplastos. En la segunda parte de la tesis, caracterizamos los cambios estructurales asociados con la diferenciación de cromoplastos mediada por crtB en hojas. Durante el proceso, los plastoglóbulos aumentan en número y tamaño y se utilizan para almacenar fitoeno y otros isoprenoides, incluidos β-caroteno (pro-vitamina A), tocoferoles (vitamina E) y filoquinona (de vitamina K). Los plastoglobulos son el sitio de localización y acción de la proteína crtB y demostramos que las condiciones que estimulan la proliferación de plastoglóbulos (como la luz intensa) se pueden utilizar para promover aún más la acumulación de vitaminas isoprenoides. Nuestros resultados muestran que la combinación de crtB con genes involucrados en la biosíntesis de dichas vitaminas puede aumentar aún más sus niveles. Por último, mostramos que β-caroteno se puede acumular aún más combinando la cromoplastogénesis mediada por crtB con una vía extraplastidial sintetica. También demostramos que las optimizaciones del sistema crtB se pueden aplicar para biofortificar vegetales de verduras como la lechuga, contribuyendo así al desarrollo de nuevos alimentos.
Els isoprenoides són una de les famílies més gran de metabòlits en la naturalesa i són especialment diversos en el regne vegetal. Entre ells, els carotenoides, els tocoferols i la filoquinona tenen un interès especial pel seu paper com vitamines. Les plantes sintetitzen aquests compostos en cloroplasts per contribuir a la fotosíntesi i fotoprotecció. En el cas dels carotenoides, els seus nivells més alts es troben en plastidis especialitzats anomenats cromoplastos, que es troben típicament en òrgans pigmentats no verds com pètals de flors i fruits madurs, però només ocasionalment en fulles. L’objectiu general d’aquesta tesi ha estat provar noves estratègies per a l’enriquiment de verdures en vitamines isoprenoides a través d’un sistema desenvolupat en el nostre laboratori per induir la conversió de cloroplasts en cromoplastos en fulls. L’eina es basa en la gran capacitat de l’enzim crtB del bacteri Pantoea ananatis per produir fitoe i la capacitat de les fulles per convertir aquest fitoe addicional en carotenoides posteriors amb canvis concomitants a la ultraestructura dels plastidis. La disponibilitat d’aquest sistema va permetre definir dos objectius específics: (1) caracteritzar el context fisiològic de l’fenotip induït per crtB en fulls de Nicotiana benthamiana i (2) provar diferents estratègies aprofitant aquest sistema basat en crtB per millorar la biofortificación de fulles. A la primera part de la tesi, vam demostrar que el fenotip no reversible de diferenciació de cloroplast a cromoplast desencadenat per crtB en plastidis s’associa amb una ràpida pèrdua d’activitat fotosintètica causada per l’acumulació de fitoe. Aquest fenomen fa que el cloroplast passi a ser competent per a la cromoplastogénesis. Després, la transició es completa una vegada que el fitoe es converteix en carotenoides posteriors mitjançant enzims endògenes. També vam demostrar que els tractaments que causen un equilibri redox alterat dels fotosistemes i un estrès oxidatiu faciliten la diferenciació dels cromoplastos. A la segona part de la tesi, caracteritzem els canvis estructurals associats amb la diferenciació de cromoplastos intervinguda per crtB en fulls. Durant el procés, els plastoglóbuls augmenten en nombre i mida i s’utilitzen per emmagatzemar fitoe i altres isoprenoides, inclosos β-carotè (provitamina A), tocoferols (vitamina E) i filoquinona (de vitamina K). Els plastoglobuls són el lloc de localització i acció de la proteïna crtB i vam demostrar que les condicions que estimulen la proliferació de plastoglóbuls (com la llum intensa) es poden utilitzar per promoure encara més l’acumulació de vitamines isoprenoides. Els nostres resultats mostren que la combinació de crtB amb gens involucrats en la biosíntesi d’aquestes vitamines pot augmentar encara més els seus nivells. Finalment, vam mostrar que β-carotè es pot acumular encara més combinant la cromoplastogénesis intervinguda per crtB amb una via extraplastidial sintètica. També vam demostrar que les optimitzacions de sistema crtB es poden aplicar per biofortificar vegetals de verdures com l’enciam, contribuint així a el desenvolupament de nous aliments.
Isoprenoids are one of the largest families of metabolites in nature, and they are especially diverse in the plant kingdom. Among them, carotenoids, tocopherols and phylloquinone are interesting for their role as vitamins. Plants synthesize these compounds in chloroplasts to contribute to photosynthesis and photoprotection. In the case of carotenoids, their highest levels are found in specialized plastids named chromoplasts, which are typically found in non-green pigmented organs such as flower petals and ripe fruits but only occasionally in leaves. The general goal of this thesis has been testing new strategies for the enrichment of leafy vegetables in isoprenoid vitamins through a system developed in our lab to induce the conversion of chloroplasts into chromoplasts in leaves. The tool is based on the capacity of the crtB enzyme from the bacterium Pantoea ananatis to boost the production of phytoene and the ability of leaves to convert this extra phytoene into downstream carotenoids with the concomitant changes in plastid ultrastructure. The availability of this system allowed to define two specific objectives: (1) to characterize the physiological context of the crtB-induced phenotype in Nicotiana benthamiana leaves and (2) to test different strategies exploiting this crtB-based system to improve leaf biofortification. In the first part of the thesis, we demonstrate that the non-reversible chloroplast-to-chromoplast differentiation phenotype triggered by plastid-localized crtB is associated with a rapid loss of photosynthetic activity caused by the accumulation of phytoene. This phenomenon makes the chloroplast competent for chromoplastogenesis. The transition is then completed once phytoene is converted into downstream carotenoids by endogenous enzymes. We also demonstrate that treatments that cause altered redox balance of the photosystems and oxidative stress facilitate chromoplast differentiation. In the second part of the thesis, we characterize the structural changes associated with crtB-mediated chromoplast differentiation in leaves. During the process plastoglobules increase in number and size. Plastoglobules are used to store phytoene and other isoprenoids (including pro-vitamin A β-carotene, vitamin E tocopherols, and vitamin K phylloquinone. We also show that plastoglobules are the site of localization and action of the crtB protein and demonstrate that conditions that promote plastoglobule proliferation (such as high light) can be used to further promote the accumulation of isoprenoid vitamins. Our results show that the combination of crtB with genes involved in the biosynthesis of such vitamins can further increase their levels. Lastly, we show that β-carotene can be further accumulated by combining the crtB-mediated chromoplastogenesis with an engineered extraplastidial pathway. We also show that the optimizations of the crtB system can be applied to biofortify edible green leafy vegetables such as lettuce, hence contributing to the development of new functional foods.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia i Biotecnologia Vegetal

El meu programa de recerca està enfocat a identificar els passos limitants de la biosíntesi de carotenoids en arròs (Oryza sativa) de forma que mitjançant enginyeria genètica amb múltiples gens es puguin dividir els passos i elucidar el mecanisme de acumulació de la pro-vitamina A i dels carotenoids en arròs, un cereal molt important en alimentació. Per tal d’assolir el meus objectius, he generat varies línies transgèniques de arròs que expressen combinacions específiques de gens diferents. Las plantes transgèniques presenten perfils carotenogènics diferents en funció de la combinació dels gens integrats. Durant les meves investigacions he desenvolupat un experiment que facilita la caracterització funcional de gens que representen una ruta metabòlica en un sistema de calls embriogènics d’arròs. A mes, he descobert que variïs promotors específics de l’endosperma són molt actius en calls, tot i que la seva activitat tindria que ser restringida a la llavor. El sistema d’expressió en calls proporciona una oportunitat única per a predir l’impacte de la enginyeria genètica de rutes complexes i la seva quantificació, i es converteix en el punt d’inici per al disseny racional d’estratègies d’enginyeria de rutes biosintètiques complexes utilitzant biologia sintètica. També he identificat i caracteritzat un nou ketocarotenoid, el 4 keto-alfa–carotè, com un subproducte no esperat als experiments en els quals el meu objectiu era la biosíntesi d’astaxantina mitjançant la co-expressió de BrCRTW/sCrBKT, ZmPSY1 i PaCRTI en calls d’arròs. En un altre conjunt d’experiments he introduït la ruta biosintètica per a la producció de astaxantina en l’endosperma d’arròs. Amb les anàlisis que he dut a terme, he detectat una acumulació de cantaxantina i adonirubina, més que de astaxantina, essent aquests dos els carotenoids més abundants en l’endosperma d’arròs, la qual cosa suggereix l’existència de un pas limitant en la capacitat endògena de hidroxilació de l’endosperma d’arròs. La caracterització específica del metaboloma de l’endosperma d’arròs co-expressant combinacions binàries (ZmPSY1 i PaCRTI) o terciàries (ZmPSY1 i PaCRTI i AtDXS o AtOR) de gens demostra que la quantitat subministrada de precursors isoprenoics derivats de la ruta metabòlica MEP, o la creació de un sistema efectiu de retenció o de deposició de carotenoids son dos factors clau limitants de l’acumulació de carotenoids en l’endosperma d’arròs.
My research project focused on the rice (Oryza sativa) carotenoid biosynthesis pathway. I used multigene engineering to identify and characterize bottlenecks in the pathway leading to β-carotene (pro-vitamin A) and other important carotenoids. I created transgenic lines expressing distinct combinations of transgenes and investigated the resulting diverse carotenoid profiles to determine how the integrated transgene complement influenced carotenoid production. I also developed a simple assay based on embryogenic rice callus for the functional characterization of carotenogenic transgenes. Remarkably, I discovered that diverse endosperm-specific promoters were highly active in callus tissue despite their restricted activity in mature plants. The callus system offers a unique opportunity to predict the impact of metabolic engineering in complex pathways and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. I identified and characterized the novel ketocarotenoid 4-keto-α-carotene as an unexpected byproduct during experiments targeting the biosynthesis of astaxanthin, which involved the co-expression of ZmPSY1, PaCRTI and two carotenoid ketolases (BrCRTW/sCrBKT) in rice callus. In separate experiments, I introduced the ketocarotenoid biosynthesis pathway to astaxanthin into rice endosperm. These transgenic lines accumulated canthaxanthin and adonirubin as major ketocarotenoids rather than astaxanthin, indicating the presence of a bottleneck caused by insufficient endogenous hydroxylation activity in rice endosperm tissue. Targeted metabolomics in rice endosperm co-expressing binary (ZmPSY1 and PaCRTI) or tertiary (ZmPSY1, PaCRTI and AtDXS or AtOR) transgene combinations demonstrated that carotenoid accumulation in rice endosperm is inhibited by the limited supply of isoprenoid precursors derived from the MEP pathway and/or the absence of an effective carotenoid sink.
Mi programa de investigación está enfocado a identificar los pasos limitantes de la biosíntesis de carotenoides en arroz (Oryza sativa) de forma que mediante ingeniería genética con múltiples genes pueda dividir los pasos y elucidar el mecanismo de acumulación de la pro-vitamina A y de los carotenoides en arroz, un cereal muy importante en alimentación. Para conseguir mis objetivos, he generado diversas líneas transgénicas de arroz que expresan combinaciones específicas de genes diferentes. Las plantas transgénicas presentan perfiles diferentes en función de la combinación de genes integrados. Durante mis investigaciones he desarrollado un experimento que facilita la caracterización funcional de genes que de una ruta metabólica en un sistema de callos embriogénicos de arroz. Además he descubierto que varios promotores específicos del endospermo son muy activos en callos a pesar de que su actividad tendría que estar restringida a la semilla. El sistema de expresión en callos proporciona una oportunidad única para predecir el impacto de la ingeniería genética de rutas complejas y su cuantificación, y se convierte en el punto de inicio para el diseño racional de estrategias de ingeniería de rutas biosintéticas complejas utilizando biología sintética. También he identificado y caracterizado un ketocarotenoide nuevo, el 4 keto-alfa –caroteno, como un subproducto inesperado en los experimentos donde mi objetivo era la biosíntesis de astaxantina mediante la co-expresión de BrCRTW/sCrBKT, ZmPSY1 y PaCRTI en callos de arroz. En otro conjunto de experimentos he introducido la ruta biosintética para la producción de astaxantina en el endosperma de arroz. Con mis análisis he detectado una acumulación de cantaxantina y adonirubina, más que de astaxantina, siendo estos dos los carotenoides mas abundantes en el endosperma de arroz, lo cual sugiere la existencia de un paso limitante en la capacidad endógena de hidroxilación del endosperma de arroz. La caracterización específica del metaboloma del endosperma de arroz co-expresando combinaciones binarias (ZmPSY1 y PaCRTI) o terciarias (ZmPSY1 i PaCRTI y AtDXS o AtOR) de genes demuestra que la cantidad suministrada de precursores isoprenoicos derivados de la ruta metabólica MEP, o la creación de un sistema efectivo de retención o de deposición de carotenoides son dos factores clave limitantes de la acumulación de carotenoides en el endosperma de arroz.

Structure elucidation of charge delocalised carotenoid mono- and dications by NMR and VIS/NIR spectroscopy. Studies of the nucleophilic reactions of these cations. Studies of the β,β-carotene-iodine complex. Isolation and anmalysis of new carotenoid glucoside esters from extremophilic bacteria.

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As frutas cítricas são muito consumidas e apreciadas, não só devido ao seu paladar agradável como também ao valor nutricional que possuem. O Brasil é o maior produtor e exportador mundial de laranjas, suco de laranja concentrado congelado e de suco de laranja pasteurizado (NFC - Not From Concentrated), sendo o estado de São Paulo o maior produtor. Já outras frutas cítricas como limão Siciliano, Grapefruit e Lima da Pérsia possuem uma produção nacional ainda pequena e parte do consumo interno é importada. Outro grupo de laranjas ainda pouco estudado é o das laranjas de polpa vermelha que apresentam o carotenóide licopeno na sua composição. Os compostos antioxidantes (carotenóides, compostos fenólicos, flavonóides) apresentam atividade anti-radical livre e alguns deles também são responsáveis pela cor das frutas. Considerando a importância das frutas cítricas com relação aos seus benefícios para a saúde, os objetivos deste trabalho foram avaliar a atividade anti-radical livre (DPPH• e ABTS•), compostos bioativos (carotenóides, flavanonas e ácido ascórbico) e parâmetros de cor de quatro variedades de frutas cítricas de polpa clara (Lima da Pérsia, Grapefruit, Limão Siciliano e Hamlin); comparar estes mesmos parâmetros entre duas variedades de polpa vermelha (Sanguíneas de Mombuca e baía Cara Cara) e uma de polpa clara (Pera Rio), colhidas no início, meio e fim de safra; e verificar o efeito da pasteurização nos compostos bioativos presentes no suco de laranja de polpa vermelha e de clara. As variedades Baía Cara Cara (CN486) e Pera Rio foram cultivadas na cidade Cordeirópolis/SP e a Sanguínea de Mombuca (CV 93) em três diferentes cidades do Estado de São Paulo, São Bento do Sapucaí, Cordeirópolis e Mogi Mirim. A variedade limão Siciliano apresentou o maior teor de ácido ascórbico (78,86mg), Grapefruit e Hamlin apresentaram o maior conteúdo...
Citrus fruits are widely consumed and enjoyed, not only because of its pleasant taste but also the nutritional value they have. Brazil is the largest producer and exporter of oranges, frozen concentrated orange juice (FCOJ) and orange juice NFC (Not from Concentrated), and the state of Sao Paulo largest producer. Yet other citrus fruits like lemon, grapefruit and Sweet Lime still have a small domestic production and share of domestic consumption is imported. Another group of oranges still less studied are the red pulp of oranges that have the carotenoid lycopene in its composition. The antioxidants (carotenoids, phenolic compounds, flavonoids) have anti-free radical activity and some of them are also responsible for the color of the fruit. Considering the importance of citrus fruits in relation to their health benefits, the objectives were to evaluate the anti-free radical (DPPH • and ABTS •), bioactive compounds (carotenoids, ascorbic acid, and flavanones) and parameters of color four varieties of citrus pulp clear (Sweet Lime, Grapefruit, Lemon and Hamlin), to compare these same parameters between two varieties of red pulp (of Blood and Mombuca Baía Cara Cara) and a clear pulp (Pera Rio) taken at the beginning, middle and end of harvest, and check the effect of pasteurization on bioactive compounds in orange juice pulp and red light. Varieties Baía Cara Cara (CN486) and Pear River were grown in the city Cordeirópolis / SP and Blood of Mombuca (CV 93) in three different cities of São Paulo, São Bento do Sapucaí, Cordeirópolis and Mogi Mirim. The Sicilian lemon variety had the highest content of ascorbic acid (78.86 mg), Grapefruit and Hamlin had the highest content of total carotenoids of the four fruits (13.58 and 11.32 mg / mL) and the antioxidant capacity was higher in fruit that had best levels of carotenoids and phenolic compounds. The hesperidin was present in three... (Complete abstract click electronic access below)

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
O género Capsicum annuum L. inclui plantas de frutos picantes e adocicados vulgarmente conhecidos como pimentos ou pimentões. De entre os diferentes cultivares existem frutos com diferentes cores, sendo os mais conhecidos o verde e o vermelho. Porém existem outras variedades mais exóticas, como o amarelo, laranja, roxo e branco. A cor desenvolvida pelos vegetais está diretamente relacionada com a presença de certos metabolitos sintetizados, responsáveis por muitas ações farmacológicas atualmente reconhecidas. As propriedades fitoterápicas, aliadas ao elevado valor mercadológico do pimento impulsionam estudos mais aprofundados e direcionados sobre os aspetos biológicos da planta, necessários para a sua futura aplicação na indústria farmacêutica. O género Capsicum é reconhecido pelos seus elevados teores em vitamina C, vitaminas do complexo B, vitamina A e vitamina E. Carotenoides como o β-caroteno e a β-criptoxantina também são encontrados. Existem inúmeras aplicações deste material vegetal, destacando-se o seu uso como corantes naturais, na forma de extratos concentrados e de extratos e óleos vegetais na cosmética. No entanto, uma planta de uso tradicional e tão rica em fitoquímicos deve ser estudada de forma mais aprofundada permitindo dar a conhecer as suas propriedades nutricionais, composição química, funções terapêuticas e possíveis reações adversas. Face às diferenças de cor encontradas nos pimentos, crê-se que as suas propriedades poderão estar diretamente relacionadas com a diferença de concentração destes fitoquímicos, muitos deles responsáveis pela pigmentação natural do material vegetal e, consequentemente, nas propriedades terapêuticas. O presente trabalho teve como objetivo avaliar o teor das carotenoides dos pimentos de diferentes colorações e, consequentemente, a elaboração de sabonetes artesanais com incorporação dos extratos obtidos dos diferentes pimentos. Foi realizada a quantificação do teor de carotenoides presentes em seis variedades de pimentos (branco, amarelo, laranja, vermelho, verde e roxo), nomeadamente, teores de clorofila a, clorofila b, β-caroteno e licopeno por método colorimétrico. Nos sabonetes artesanais com incorporação dos extratos obtidos dos diferentes pimentos, foi avaliado o teor de carotenoides presentes após o processo de saponificação a frio, determinação do índice de espuma, Ph e textura. The genus Capsicum annuum L. includes hot and sweetened fruit plants commonly known as chilies or peppers. Among the different fruit varieties there are fruits with different colors, however, the green and red fruits are the most common. There are other exotic varieties, such as yellow, orange, purple and white. The color developed by the plants is directly related with the presence of certain metabolites synthesized, responsible for many pharmacological actions currently recognized. The fruits properties, coupled with the high marketing value of pepper, have been the focus of several studies, focused on biological aspects for their application in the pharmaceutical industry. The genus Capsicum is recognized for its high levels of vitamin C, vitamins from the complex B, vitamin A and vitamin E. Carotenoids in particular β-carotene and β-criptoxanthin are also found. There are many uses of plant material, especially its use as natural dyes in the form of concentrated extracts and extracts and vegetable oils in cosmetic. However, a traditional use of plants with high content of s should be studied in more detail in order to evaluate their nutritional properties, chemical composition, therapeutic functions and possible adverse reactions. Due to the color differences in peppers, some biological properties may be directly related to the phytochemical compounds concentration and therefore in their therapeutic properties. This study aimed to evaluate the carotenoid contents of different peppers and the development of handmade soaps with the incorporation of peppers aqueous extracts. The carotenoids contents presented in six varieties of peppers (white, yellow, orange, red, green and purple) was performed, in particular, chlorophyll a, chlorophyll b, β-carotene and lycopene contents. Also the carotenoids contents presented in handmade soaps were quantified, as well as the foam index, pH and texture of soaps.

Corynebacterium poinsettiae mutant strains blocked in carotenoid biosynthesis were obtained by treatment with the mutagen N-methyl-N1-nitro-N-nitrosoguanidine. Additional carotenoid (Crt) mutant strains were obtained from a previous study conducted in our laboratory. Fifty-nine Crt mutants affected in carotenoid biosynthesis were examined by a normal phase high performance liquid chromatography (HPLC) system. Pigment extracts of Crt mutants and C. poinsettiae wild type strains were resolved by an isocratic system with hexane:acetone:dicholoromethane, 11.35:1.73:1.00 (by vol.) as the eluting solvent. In addition to the five major peaks, twelve minor peaks were observed in the wild type C. poinsettiae strain used in this study. Crt mutant and wild type strain peak heights were measured from the individual chromatograms and the peak height data set created was analyzed using the Statistical Analysis System program to perform a cluster analysis. The cluster analysis revealed five carotenoid mutant groups. Carotenoid pigments which accumulated or were absent in each of the cluster groups are reported. Cluster group 1 mutants (CrtA) are blocked in the dehydrogenase(s) which is(are) responsible for the dehydrogenations between phytoene and lycopene. Cluster group 2 mutants (CrtB) appear to be blocked at a second dehydrogenase specific for the dehydrogenation from C.p. 470 to C.p. 496. Cluster group 3 mutants (CrtC) are blocked at a cyclization step in the pathway which involves cyclization of C.p. 496 to C.p. 470 and which may cyclize C.p. 473 to C.p. 450. The genes CrtA and CrtB map only 0.5 map units from each other while CrtA and CrtC map 2.1 map units from one another. Mutants which accumulate end products but which lack certain precursors indicate a branched pathway for pigment biosynthesis exists in this organism. Media for the formation, fusion and regeneration of C. poinsettiae protoplasts are reported and a protocol for the use of these media in genetic crosses of strains blocked in carotenoid biosynthesis is described. While isolating antibiotic resistant mutants useful in genetic analyses, novobiocin resistant mutants were observed to have a distinctly different colony pigment phenotype as compared to the wild type strain. HPLC chromatograms of a novobiocin resistant strain showed a distinctly different carotenoid pigment profile. The results provide evidence for differential gene expression in the carotenoid biosynthetic pathway when these mutants are grown in the presence of novobiocin.

El panís HC (de l'anglès high-carotenoid) va ser modificat genèticament per acumular alts nivells de carotenoides, utilitzant com a base un panís blanc sud-africà (M37W). Durant tres collites consecutives (2013, 2014 i 2015), es van cultivar el panís HC i la seva línia isogènica (M37W) en un camp experimental a Lleida (Catalunya, nord-est d'Espanya). Fusarium spp. va infectar la majoria de grans de panís d'ambdós tipus, el que va originar que es donés contaminació per fumonisines en les dues varietats de panís en tots els anys d'estudi, tot i que la proporció de grans contaminats va ser substancialment més gran en el panís M37W. El panís collit cada any també va servir com a matèria primera per elaborar pinsos a base de panís i productes derivats del panís. Els pollastres alimentats amb la dieta HC van tenir paràmetres de productivitat i salut similars als pollastres alimentats amb les dietes M37W i comercial (amb pigments), i també van desenvolupar una pigmentació similar als pollastres alimentats amb la dieta comercial (amb pigments). Els carotenoides provitamina A del panís HC van ser biodisponibles, almenys en la mateixa mesura que en els additius sintètics i naturals, i van contribuir als nivells de retinol hepàtic en pollastres. La carn obtinguda a partir de pollastres alimentats amb la dieta HC va tenir una bona qualitat i vida útil sensorial, així com una pigmentació groga-ataronjada de llarga durada. Finalment, els purés elaborats amb panís HC han demostrat no només conservar el contingut inicial de carotenoides, sinó també augmentar-lo a causa de l'extracció de carotenoides de la matriu alimentària.
El maíz HC (del inglés high-carotenoid) fue modificado genéticamente para acumular altos niveles de carotenoides, utilizando como base un maíz blanco sudafricano (M37W). Durante tres cosechas consecutivas (2013, 2014 y 2015), se cultivó el maíz HC y su línea isogénica (M37W) en un campo experimental en Lleida (Cataluña, noreste de España). Fusarium spp. infectó la mayoría de granos de maíz de ambos tipos, lo que originó que se diera contaminación por fumonisinas en ambas variedades de maíz en todos los años de estudio, aunque la proporción de granos contaminados fue sustancialmente mayor en el maíz M37W. El maíz cosechado cada año también sirvió como materia prima para elaborar piensos a base de maíz y productos derivados del maíz. Los pollos alimentados con la dieta HC tuvieron parámetros de productividad y salud similares a los pollos alimentados con las dietas M37W y comercial (con pigmentos), y también desarrollaron una pigmentación similar a los pollos alimentados con la dieta comercial (con pigmentos). Los carotenoides provitamina A del maíz HC fueron biodisponibles, al menos en la misma medida que en los aditivos sintéticos y naturales, y contribuyeron a los niveles de retinol hepático en pollos. La carne obtenida de pollos alimentados con la dieta HC tuvo una buena calidad y vida útil sensorial, así como una pigmentación amarilla-anaranjada de larga duración. Por último, los purés elaborados con maíz HC han demostrado no sólo conservar el contenido inicial de carotenoides, sino también aumentarlo debido a la extracción de carotenoides de la matriz alimenticia.
High-carotenoid (HC) maize was genetically engineered to accumulate high levels of carotenoids, using as a basis a South African white maize (M37W). During three consecutive harvest seasons (2013, 2014 and 2015), HC maize and its near isogenic line (M37W) were cultivated in an experimental field in Lleida (Catalonia, Northeastern Spain). Fusarium spp. infected most maize kernels, subsequently, fumonisin contamination was found in both maize varieties in all the years of study, but the proportion of contaminated grains was substantially higher in the M37W maize. Maize grains harvested each year also served as raw material to elaborate maize-based feed and maize-derived products. Chickens fed on the HC diet had similar productivity and health parameters to those fed on the M37W and commercial (plus color additives) diets, and they also developed similar pigmentation to those fed on the commercial (plus color additives) diet. Provitamin A carotenoids from HC maize were bioavailable, at least to the same extent than in synthetic and natural additives, and contributed to liver retinol levels in chickens. Meat obtained from chickens fed on the HC diet had a good quality and sensory shelf life as well as a long-lasting golden pigmentation. Finally, HC maize-based porridges showed not only to preserve the initial carotenoid content, but also to enhance it due to the carotenoid extractability from the food matrix.

This investigation has been performed on Norflurazon (NF) treated young wheat plants with

emphasis on their capacity to perform light-driven photosynthesis, as an effect of carotenoid

deficiency. The wheat seeds were soaked for approximately 17 hour in tap water or in water containing NF (28mg l-1 ca 10-4 M). Seedlings were grown for 7 days in a greenhouse (50 μmol m-2 s-1) or in weak light (ca 0.1 μmol m-2 s-1) in order to avoid photodestruction, before chloroplast and thylakoid isolation. NF-treatment and the low light condition also affected chlorophyll content. Isolated chloroplasts from NF-grown plants contained approximately 55 and 16 % of the chlorophyll content as compared to untreated plants grown in weak light or in greenhouse light, respectively.

In addition, protein separation by polyacryl amide electrophoresis (SDS-PAGE) on isolated thylakoids showed that the apoproteins of the LHC II (i.e. LHCP) were undetectable after NF-treatment. Despite a large number of attempts to measure PS II activity in isolated thylakoids, this was proven to give unreliable data and these were therefore excluded from this report.

The photosynthetic assays were therefore focused on PS I activity, which were compared between plants grown in greenhouse (untreated) or in weak light, with or without NF. As expected, PSI activity is greatly affected by light intensity, during growth as well as during the photosynhtetic assays. In untreated wheat, grown in the greenhouse, the highest activity was found at the strongest light intensity used at distance of 0.25 m from the light source (corresponding to approximately 1440µmol m-2s-1). The PS I activity then declines with decreasing intensity. A similar, albeit not so clear (lower maximum activity at strong light intensities), trend is observed in untreated wheat grown in weak light.

Thylakoids isolated from NF-treated plants show a clear photoinhibition at strong light but a clear increase in activity at subsequently lower light intensities.

Thesis (M.S. in food science)--Washington State University, December 2009.
Title from PDF title page (viewed on Jan. 19, 2010). "School of Food Science." Includes bibliographical references (p. 43-57).

The purpose of this study was to develop a deeper understanding of the nature of carotenoid metabolism in the human and primate retina. We have sought to do this from two perspectives (1) through preparation and study of a carotenoid diketone that is a candidate metabolic product and (2) through measurement of the carotenoid distribution in the retinas of neonatal macaques. In this thesis we report the synthesis, purification, and characterization of the product using HPLC, UV/Vis, MS, 1H-NMR. The data obtained are all consistent with the proposed β,β-carotene-3,3'-dione. There has been no thorough study of the development of the macular pigment during the earliest stages of life immediately following birth. In this study 30 macaque retinas ranging from 148 days of gestation to 15 years in age were analyzed. The amounts of the carotenoids, lutein, R,R-zeaxanthin, and meso-zeaxanthin were determined using C 18 reversed-phase column and chiral, normal-phase column HPLC.

Epidemiological studies suggest that both retinoid and carotenoid intakes are inversely correlated with the incidence of human cancers. Animal studies show that both retinoids and carotenoids inhibit tumor cell growth. Both retinoids and carotenoids activate the cytotoxicity function of macrophages in animal experiments. The purpose of this study is to evaluate the molecular mechanism for 13-cis retinoic acid (13-cRA) and beta-carotene (BC) induced immunomodulation which could explain their anti-cancer affects. The effects of 13-cRA and BC were studied on various subpopulations of T-lymphocytes both in vitro and in vivo. For in vitro studies, peripheral blood mononuclear cells (PBMC) were incubated with test compounds at clinically achievable concentrations (10⁻⁸M) for three days. Then the cells were stained with monoclonal antibodies followed by the analysis of flow cytometer. For in vivo studies, PBMC were collected from Barrett's esophagus or oral leukoplakia patients during treatment with 13-cRA (1mg/kg/day) or BC (30 mg/day), respectively. Then the cells were analyzed with monoclonal antibodies and flow cytometry. Both compounds showed the capability of stimulating different subpopulations of T-lymphocytes. 13-cRA predominantly increased the number of T-helper cells, their interleukin 2 (IL-2) receptors and their response to mitogens. Whereas, BC elevated the number of Natural Kill (NK) cells, their IL-2 receptors and their cytotoxicity against K562 target cells. Though these immunomodulatory effects appeared to be unaffected by the presence and cytotoxic functions of macrophages, cytokines seemed to have an important role in the retinoid- and carotenoid-induced immunomodulation. Plasma levels of IL-2 and tumor necrosis factor (TNF) measured by ELISA procedures were increased in patients treated for two months with 13-cRA and BC respectively. Anti-IL-2 and anti-TNF antibodies blocked the retinoic- and carotenoid-induced immunomodulation in in vitro studies. These results indicate that 13-cRA, activating T-helper cells with IL-2 production, and BC, activating NK cells with TNF release, induced immunostimulation which might be able to provide the anti-cancer affects in part seen in epidemiological studies.

Orientador: Adilma Regina Pippa Scamparini
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Este trabalho teve como objetivo isolar e selecionar leveduras produtoras de carotenóides de ecossistemas brasileiros. As leveduras pigmentadas foram isoladas de amostras de solos, flores, folhas, frutos da região de Campinas-SP e de alimentos processados. As amostras foram colocadas em frascos de erlenmeyer de 50 mL, contendo 20 mL de meio de Extrato de Malte e Levedura (YM), e incubadas a 30° C por 48 horas. Após 48 horas, as amostras foram inoculadas em placas de petri contendo meio ágar-YM e incubadas a 30°C, por 120 horas. As colônias de leveduras que apresentaram coloração entre amarelo e vermelho, foram transferidas para tubos de ensaio contendo meio ágar YM inclinado e incubadas a 30°C até crescimento satisfatório. Estas leveduras foram reisoladas, pelo método de estrias de esgotamento, em placas de petri contendo meio ágar YM (30°C por 72 horas) e, posteriormente, transferidas para tubos de ensaios contendo ágar GYMP inclinado. As culturas pigmentadas foram codificadas do seguinte modo: L12, isolada como contaminante em massa de tomate; L108, isolada de solo da região da Universidade Estadual de Campinas; L125, isolada a partir de folhas da cana-de-açúcar; L135 e L137 isoladas de solo em Holambra-SP. Através das características morfológicas, de reprodução, testes fisiológicos e bioquímicos as leveduras foram identificadas como: L12, L108, L135 e L137 como Rhodotorula mucilaginosa, e L125 como Rhodotorula graminis. A composição de carotenóides, das leveduras isoladas no Brasil, foi estudada. As culturas de leveduras foram cultivadas em 200 mL de meio YM a 200 rpm em shaker, a 25°C por 5 dias. Cromatografia de coluna aberta, cromatografia de camada delgada e cromatografia líquida de alta eficiência foram utilizadas para separar os carotenóides obtidos das leveduras, a fim de identificá-los e quantificá-los. A linhagem de Rhodotorula glutinis foi a que apresentou maior concentração total de carotenóide (881 mg/L), seguido por Rhodotorula graminis (594 mg/L), Rhodotorula mucilaginosa-137 (590 mg/L) e Rhodotorula mucilaginosa-135 (545 mg/L). Rhodotorula minuta e Sporobolomyces tiveram a menor concentração de carotenóides (168 mg/L and 237 mg/L, respectivamente). Os principais pigmentos encontrados nestas linhagens foram toruleno e b-caroteno. b-Caroteno foi o carotenóide predominante em Rhodotorula grarninis-125, Rhodotorula glutinis e Sporobolornyces, enquanto que o toruleno foi o carotenóide principal nas leveduras de Rhodotorula rnucilaginosa. Em termos de produção específica de cartenóides (_g/g de células secas), Rhodotorula glutinis foi a que obteve maior concentração de carotenóides 132 mg/g. Duas linhagens foram selecionadas para otimização da produção de carotenóides, R. mucilaginosa-137 e R. glutinis. Estas duas culturas foram cultivadas em shaker a 200 rpm, a 25°C por 5 dias, sem iluminação. Utilizou-se planejamento experimental e análise de superfície de resposta para estudar o efeito do pH inicial, concentração de glicose, extrato de levedura, sais de fosfato e sulfato de magnésio na produção de carotenóides, de biomassa e proteína celular. Para cada linhagem, foram realizados 2 planejamentos fatoriais, sendo 1 fracionário e 1 completo.Para a linhagem de R. mucilaginosa-137, o extrato de levedura foi a variável de maior influência na produção de carotenóides, enquanto que os sais de sulfato e fosfato tiveram efeito negativo. O pH inicial não teve efeito significativo tanto na produção de carotenóides como na biomassa. Através dos resultados obtidos pelo planejamento completo, observou-se que a máxima concentração de carotenóides foi de 745 mg/L com 15 g/L de extrato de levedura e 20 g/L de glicose. Em relação a produção específica de carotenóides, a máxima concentração foi de 152 mg/g com 5 g/L de extrato de levedura e 15 g/L de glicose. A concentração de extrato de leveduras e glicose também foram importantes na produção da biomassa, que atingiu o valor máximo de 8 g/L, na faixa de concentração de 15 a 17,1 g /L de extrato de levedura e de 15 a 20 g/L de glicose. Para a linhagem de Rhodotorula glutinis as variáveis de maior influência na produção de carotenóides foram pH inicial, extrato de levedura e glicose. Os sais de sulfato e fosfato não tiveram efeito significativo. Através do planejamento fatorial completo 23 com três pontos centrais, observou-se que na produção de carotenóides, apenas a glicose teve efeito positivo significativo. Na produção específica de carotenóides, o pH inicial, glicose e extrato de levedura tiveram efeito positivo. A máxima concentração de carotenóides obtida foi de 1.269 mg/L com pH inicial 4, 4 g/L de extrato de levedura e 17 g/L de glicose. Na produção específica de carotenóides, a máxima concentração foi de 337 mg/g com pH inicial 4, a 4 g/L de extrato de levedura e 7 g/L de glicose. O crescimento celular foi afetado pelo pH inicial, concentração de extrato de levedura e glicose. Entretanto, o modelo matemático referente a biomassa não apresentou uma regressão satisfatória, devendo ser utilizado apenas para estabelecer tendência da resposta
Abstract: Pigmented yeasts were collected from soils, flowers, leaves, fruits from Campinas-SP region and industrialized foods. The samples were put in 50 mL erlenmeyers flasks, containing YM broth, and they were incubated at 30°C for 48 hours. After 48 hours, these samples were inoculated in Petri plates with YM agar, and incubated at 30°C for 120 hours. The yeasts colonies that had color between red and yellow were transferred to tubes, containing YM agar, and incubated at 30°C. These yeasts were reisolated by screening in Petri plates with YM agar (30°C for 72 hours) and then, transferred into tubes containing GYMP agar. After the selection, the pigmented yeasts were identified by a code: L12, was isolated from tomato sauce; L108, from soils of State University of Campinas; L125, from leaves of sugar cane; L135 e L137, from soils of Holambra-SP. The yeasts were identified by their morphology characteristics, reproduction characteristics, physiology and biochemical tests. The yeasts L12, L108, L135 and L137 were identified as Rhodotorula mucUaginosa and L125 as Rhodotorula graminis. The carotenoid composition of yeasts isolated in Brazil was studied. The yeasts were cultured in 200 mL broth yeast malt at 200 rpm in rotary shaker, 25°C for 5 days without illumination. Open column, thin layer chromatography and high performance liquid chromatography were used to separate, identify and determine carotenoid concentrations. The yeast Rhodotorula glutinis had the highest total carotenoid concentration (881 mg/L), followed by Rhodotorula graminis (594 mg/L), Rhodotorula mucUaginosa-137 (590 mg/L) and Rhodotorula mucilaginosa-135 (545 mg/L). Rhodotorula minuta and Sporobolomyces had the lowest carotenoid contents (168 mg/L and 237 mg/L, respectively). The principal pigments found in these yeasts were torulene and b-carotene. b-Carotene predominated in Rhodotorula graminis-125, Rhodotorula glutinis and Sporobolomyces, while torulene was the major carotenoid in Rhodotorula mucilaginosa. In specific carotenoid production (mg/g of dried cells), Rhodotorula glutinis had a total carotenoid concentration of 132 mg/g. Two of these strains were selected to optimize the carotenoid production, Rhodotorula mucilaginosa-137 and Rhodotorula glutinis. The cultures were cultivated into 200 mL broth yeast malt at 200 rpm in rotary shaker, 25°C for 5 days without illumination. Response surface design was used to study the effects of initial pH and concentrations of glucose, yeast extract, magnesium sulfate and potassium phosphate. Two statistical designs were used for each strain. For the strain of Rhodotorula mucilaginosa-137, the yeast extract the most important variable in terms of enhancing carotenoid formation; magnesium sulfate and potassium phosphate had a negative influence. The initial pH had no significant effect on carotenoid formation or on cell production. Analysis of the quadratic surfaces showed that after 5 days of cultivation at 25°C, the maximum carotenoid concentration of 745 mg/L appeared at 15 g/L of yeast extract and 20 g/L of glucose. The maximum concentration of specific carotenoid production was 152 mg/g at 5 g/L of yeast extract and 10 g/L of glucose. The concentrations of yeast extract and glucose were also important on biomass production, which reached maximum value of 8.0 g/L at a range of 15 to 17.1 glL of yeast extract and 15 to 20 g/L of glucose. The variables that had most influence on carotenoid production by Rhodotorula glutinis were initial pH, yeast extract and glucose. Magnesium sulfate and potassium phosphate had no influence. The carotenoid production was described by second order p01ynomial equation. Analysis of the 23 factorial design surfaces showed that after 5 days of cultivation at 25°C, the maximum carotenoid concentration of 1,269 mg/L with initial pH 4, 4 g/L of yeast extract and 17 g/L de glucose. The maximum specific carotenoid production was 337 j..tglg with initial pH 4, 4 g/L of yeast extract and 7 g/L of glucose. Moreover, carotenoid production in mg/g per liter was more sensitive to changes in yeast extract than to changes in glucose concentrations, in the vicinity of the optimum point of carotenoid production. The growth of the microorganism was affected by initial pH and concentration of yeast extract and glucose. However, the model obtained for biomass from the experimental designs had not a good correlation and because of that it should be used only to study the tendency of response
Doutorado
Doutor em Ciência de Alimentos

Aquesta tesi es centra principalment en l'anàlisi funcional i la caracterització dels gens que codifiquen per a alguns metabòlits secundaris i en l’estudi de la seva regulació en les plantes. Els objectius generals varen ser (a) entendre millor la regulació transcripcional del gen de la biosíntesi dels carotenoids, la β-carotè hidroxilasa 2 (BCH2) en el blat de moro i (b) l'anàlisi funcional de les dues isopentenil difosfat isomerasas (OsIPPI) d'arròs i determinar la seva localització subcel·lular. Simultàniament, es va estudiar com la llum afecta la via metabòlica i a la producció de pelargonidina en l'arròs; es van identificar també els gens essencials de la seva biosíntesi en Gentiana lutea L. var. aurantiaca. Les plantes de blat de moro i arròs es varen transformar amb els gens dels factors de transcripció ZmMYB i ZmPBF. Es va analitzar l’expressió gènica transitòria i es va realitzar transformació estable. Els resultats obtinguts indiquen que tant ZmPBF com ZmGAMYB poden transactivar l'expressió de ZmBCH2 a l’endosperm del blat de moro, i ZmPBF i ZmGAMYB transactiven independentment el promotor de ZmBCH2 en arròs. Els dos paràlegs de IPPI (OsIPPI1 i OsIPPI2) aïllats prèviament en arròs varen tenir un patró d'expressió diferent; l'ARNm de OsIPPI1 va ser més abundant que l'ARNm de OsIPPI2 en tots els teixits. Es va usar la microscòpia de fluorescència confocal i microscòpia inmunoelectrónica per determinar la localització de les dues proteïnes. Aquestes es localitzen en el reticle endoplasmàtic (RE), així com en els peroxisomes i les mitocòndries, mentre que només es va detectar OsIPPI2 en els plastidis. La detecció d'ambdues isoformes en el RE indica que DMAPP es pot sintetitzar de novo en aquest compartiment. Diferents tècniques com UPLC, GC-MS i qRT-PCR també es varen utilitzar per perfilar els metabòlits primaris i secundaris i l'expressió gènica relacionada en plàntules d'arròs des-etioladas. Els resultats varen revelar que el metabolisme primari i secundari i els gens corresponents estan regulats per la llum, especialment en la biosíntesi d'isoprenoides en fulles d'arròs. Onze derivats de pelargonidina es varen identificar en els pètals de G. lutea i es varen perfilar els gens de la seva via de biosíntesi, revelant que DFR, ANS i 3GT afecten principalment a l'acumulació dels glucòsids de pelargonidina. Tots aquests resultats suggereixen la idea que la biosíntesi dels carotenoids en plantes superiors és regulada a diferents nivells.
Esta tesis se centra principalmente en el análisis funcional y en la caracterización de los genes que codifican para algunos metabolitos secundarios y en el estudio de su regulación en las plantas. Los objetivos generales fueron (a) profundizar en el conocimiento de la regulación transcripcional del gen de la biosíntesis de los carotenoides, la β-caroteno hidroxilasa 2 (BCH2) en el maíz, y (b) analizar la función de las dos isopentenil difosfato isomerasas (OsIPPI) de arroz, determinando además su localización subcelular. Simultáneamente, se estudió cómo la luz afecta a la vía metabólica y a la producción de pelargonidina en el arroz; se identificaron también los genes esenciales de su biosíntesis en Gentiana lutea L. var. aurantiaca. Las plantas de maíz y arroz se transformaron con los genes de los factores de transcripción ZmMYB y ZmPBF. Se analizó la expresión génica transitoria y se realizó transformación estable. Los resultados obtenidos indicaron que tanto ZmPBF como ZmGAMYB pueden transactivar la expresión de ZmBCH2 en endospermo de maíz, y ZmPBF y ZmGAMYB transactivar independientemente el promotor de ZmBCH2 en arroz. Los dos parálogos de IPPI (OsIPPI1 y OsIPPI2) aislados previamente en arroz tuvieron un patrón de expresión diferente; el ARNm de OsIPPI1 fue más abundante que el ARNm de OsIPPI2 en todos los tejidos. Se usó la microscopía de fluorescencia confocal y microscopía inmunoelectrónica para determinar la localización de ambas proteínas. Estas se localizan en el retículo endoplásmico (RE), así como en los peroxisomas y las mitocondrias, mientras que solo se detectó OsIPPI2 en los plastidios. La detección de ambas isoformas en el RE indica que DMAPP se puede sintetizar de novo en este compartimiento. Diferentes técnicas como UPLC, GC-MS y qRT-PCR también se utilizaron para perfilar los metabolitos primarios y secundarios y la expresión génica en plántulas de arroz des-etioladas. Los resultados revelaron que los genes involucrados en la en el metabolismo primario y secundario están regulados por la luz, especialmente en la biosíntesis de isoprenoides en hojas de arroz. Once derivados de pelargonidina se identificaron en los pétalos de G. lutea y se perfilaron los genes de la vía de biosíntesis, revelando que DFR, ANS y 3GT afectan principalmente a la acumulación de los glucósidos de pelargonidina. Todos estos resultados contribuyen al conocimiento, a diferentes niveles, de la regulación de las rutas biosinteticas de los carotenoides en plantas superiores.
This thesis mainly focuses on functional analysis and characterization of a number of secondary metabolite biosynthetic genes and the regulation of the corresponding secondary metabolite biosynthetic pathway in plants. The overall aims were to elucidate the transcriptional regulation of β-carotene hydroxylase 2 gene (BCH2) in maize, the functional analysis of rice isopentenyl diphosphate isomerases (OsIPPI), and determine their subcellular localization. Simultaneously, the influence of light on the metabolic pathway in rice was studied and the pelargonidin quantification and essential pelargonidin biosynthesis genes in Gentiana lutea L. var. aurantiaca were identified. Maize and rice plants were transformed with transcription factor genes ZmMYB and ZmPBF, via transient gene expression and stable transformation respectively. The results indicated that both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm and ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter in rice. The two IPPI paralogs (OsIPPI1 and OsIPPI2) isolated previously in rice had a different expression pattern; OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues. Confocal fluorescence microscopy and immuno-electron microscopy were used to determine the localization of both proteins. These localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids. The detection of both isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. UPLC, GC-MS and qRT-PCR were used to profile the primary and secondary metabolites and gene expression in de-etioleted rice seedlings. The results revealed both primary and secondary metabolism and the corresponding genes are regulated by light, especially isoprenoids biosynthesis in rice leaves. Eleven pelargonidin derivatives were identified in the petals of G. lutea and the biosynthetic pathway genes were profiled, revealing DFR, ANS and 3GT mainly affect the accumulation of pelargonidin glucosides. Collectively my results provide novel insights of the regulation of carotenoid and flavonoid biosynthesis in higher plants at different levels.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
This study was carried out at the Poultry Science Laboratory (LAVIC) of Federal University of Santa Maria (UFSM). The objective was to evaluate the antioxidant effect of canthaxanthin on diets with or without addition of carotenoids to breeding males. It was used a total of 48 White Plymouth Rock breed hens from the 48th to the 59th week of age in a completely randomized design in a factorial arrangement, with two ingredients (corn or sorghum), and two levels of canthaxanthin (0 mg/kg or 6 mg/kg) totaling four experimental diets with 12 repetitions, being each hen considered a repetition. The experimental phase was divided into 3 periods of 28 days, to evaluate the performance of the hens. The evaluated parameters were: body weight, food consumption, semen volume, sperm motility, sperm vigor, sperm concentration and fertility. All parameters, except the sperm concentration, were not affected by the diets. The sperm concentration was significantly affected by the ingredient corn and by the canthaxanthin supplementation in the third period (56th to 59th week of age). The use of sorghum is suggested in the diets of roosters with no need of canthaxanthin supplementation. Researches in breeding males and the use of new antioxidants may help in better reproductive rates in poultry.
O presente estudo foi conduzido no Laboratório de Avicultura (LAVIC) da Universidade Federal de Santa Maria (UFSM). O objetivo do trabalho foi avaliar o efeito antioxidante da cantaxantina em dietas com ou sem adição de carotenoides para machos reprodutores avícolas. Foram utilizados 48 galos da raça White Plymouth Rock da 48ª a 59ª semana de idade, distribuídos em um delineamento inteiramente casualizado em arranjo fatorial, com dois ingredientes (milho ou sorgo), e dois níveis de cantaxantina (0mg/kg ou 6mg/kg), totalizando 4 dietas experimentais com 12 repetições, sendo que, cada ave foi considerada uma repetição. A fase experimental foi dividida em 3 períodos de 28 dias, para avaliar o desempenho das aves. Os parâmetros avaliados foram: peso corporal, consumo alimentar, volume do ejaculado, motilidade, vigor espermático, concentração espermática e fertilidade. Todos os parâmetros avaliados, exceto a concentração espermática, não foram afetados pelas dietas. A concentração espermática foi afetada significativamente pelo ingrediente milho e pela a suplementação de cantaxantina no terceiro período (56ª a 59ª semana de idade). Sugere-se o uso de sorgo nas dietas de reprodutores avícolas sem a necessidade de suplementação de cantaxantina. Pesquisas em reprodução de machos e o uso de novos antioxidantes poderá contribuir em melhores índices reprodutivos na avicultura.

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Rubrivivax gelatinosus é uma bactéria fototrófica natural de ambientes aquáticos que apresenta a capacidade de crescer em substratos contendo diversos compostos orgânicos. Neste trabalho, a biomassa da bactéria, produzida em águas residuárias provenientes de abate e processamento de pescado, foi caracterizada em relação a cor, composição centesimal, composição microbiológica e conteúdo de pigmentos carotenóides e aminoácidos, visando avaliar seu potencial como ingrediente para ração animal. Os resultados encontrados apontaram valores médios de 57% de proteína bruta, 23% de extrativo não nitrogenado e contaminantes, 11% de extrato etéreo, 5% de umidade e 4% de matéria mineral. Vários aminoácidos considerados essenciais para diversas espécies animais foram detectados no produto, tais como metionina (0,66g/100g), lisina (4,52g/100g), fenilalanina (3,43g/100g) e valina (4,39g/100g). Os resultados das análises microbiológicas indicaram contagens médias de 5,3 x 106 UFC/g para bactérias mesófilas aeróbias e facultativas viáveis, 20,27 NMP/g para coliformes a 35°C, <1,0 NMP/g para coliformes a 45°C e 1,2 x 103 UFC/g para bolores e leveduras. Não foram encontrados Aeromonas spp., Salmonella spp. e estafilococos coagulase positivo. O resultado da análise objetiva da cor da biomassa indicou valores médios de 25,48 para tonalidade, 14,22 para saturação da cor e 22,42 para luminosidade. A concentração média de carotenóides foi de 3,03 mg/g de biomassa. A determinação da composição e das características sensoriais da biomassa de Rubrivivax gelatinosus indicaram baixo nível de contaminação microbiana e alto potencial nutricional e pigmentante, apontando positivamente para sua utilização como ingrediente de rações na criação de animais
Rubrivivax gelatinosus is a photoautotrophic bacterium that inhabits aquatic environment in which it uses different organic substrates for growth. In this work, the biomass of the bacterium, grown in effluent from fish slaughter and processing industry was characterized for color, proximate composition, microbiological composition, carotenoids and amino acids contents, with the aim of evaluating its potential as a feed ingredient. Mean results showed 57% crude protein, 23% non nitrogen extract and contaminants, 11% ether extract, 5% moisture and 4% ashes. Many amino acids considered essential for different animal species were present in the product, like metionine (0.66g/100g), lysine (4.52g/100g), phenylalanine (3.43g/100g) and valine (4.39g/100g). Microbial analysis showed mean counts of 5.3 x 106 UFC/g for mesophilic bacteria, 20.27 NMP/g for coliforms at 35°C, <1.0 NMP/g for coliforms at 45°C and 1.2 x 103 UFC/g for yeasts and moulds. Aeromonas spp., Salmonella spp. and positive coagulase staphylococci were not detected in the product. Results from objective color evaluation showed means at 25.48 for hue, 14.22 for chroma and 22.42 for lightness. Mean carotenoids concentration detected was 3.03 mg/g biomass. The determination of composition and sensorial characteristics of Rubrivivax gelatinosus biomass indicated low contamination level and high nutritional and pigmenting potential, pointing out for a positive use of the product as an ingredient for animal feeds

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
O Brasil à um dos paÃses que mais produz resÃduos agroindustriais, como os resÃduos de frutas pelas indÃstrias de polpas, o que tem contribuÃdo para o aumento da produÃÃo do lixo orgÃnico provocando graves problemas ambientais. O Nordeste se destaca devido à enorme diversidade de frutos considerados exÃticos, sendo o cajà (Spondias mombin L.) um forte exemplo. Este fruto contÃm substÃncias antioxidantes, presenÃa de compostos fenÃlicos, tais como flavonoides, Ãcidos fenÃlicos, antocianinas, carotenoides, alÃm das vitaminas A e C. Desta forma, este estudo propÃs caracterizar e quantificar a pelÃcula comestÃvel de cajà a fim de direcionar o processo de extraÃÃo de carotenoides por maceraÃÃo enzimÃtica e assim determinar a melhor condiÃÃo, atravÃs de um planejamento experimental, alÃm de avaliar a influÃncia de fatores fÃsicos e fÃsico-quÃmicos na maceraÃÃo enzimÃtica de pelÃcula comestÃvel de cajÃ, e por fim realizar a maceraÃÃo enzimÃtica de pelÃcula de cajà em reator de bancada e obter um produto em pà atravÃs de secagem por atomizaÃÃo. A pelÃcula de cajà apresentou-se como uma boa fonte de carotenoides, onde a melhor condiÃÃo para recuperaÃÃo de carotenoides foi ao utilizar 300 ÂL do complexo enzimÃtico Pectinex XXL durante o perÃodo de 3 horas de incubaÃÃo, conseguindo o teor de 120,94 Âg/g. Dentre os fatores fÃsicos e fÃsico-quÃmicos, a utilizaÃÃo de preparaÃÃes enzimÃticas de carÃter celulolÃticos junto Ãs preparaÃÃes pectinolÃticas, para a recuperaÃÃo de carotenoides de pelÃcula comestÃvel de cajÃ, nÃo se mostrou eficiente e o ajuste do pH prÃximo à neutralidade (pH 6,0), antes de iniciar a maceraÃÃo mostrou-se eficiente para uma maior recuperaÃÃo de carotenoides, isto a nÃvel laboratorial. A quantidade de Ãgua adicionada no processo nÃo altera a recuperaÃÃo de carotenoides, podendo ser utilizada nas proporÃÃes pelÃcula: Ãgua de 1: 2, 1: 3 ou 1: 4. O emprego do ultrassom associado à maceraÃÃo enzimÃtica funcionou em escala laboratorial. A utilizaÃÃo de reator de bancada foi tecnicamente viÃvel especialmente pela melhora das condiÃÃes de homogeneizaÃÃo da mistura reacional, o que por sua vez levou à obtenÃÃo de maior teor de carotenoides recuperados na fase aquosa, nÃo sendo necessÃrio ajuste do pH do meio reacional. A secagem por atomizaÃÃo foi possÃvel sendo necessÃria uma adequaÃÃo dos fatores que influenciam a secagem para que se encontre a melhor condiÃÃo.
Brazil is one of the countries that most produces organic residues, such as residues of fruit pulp industries, which have contributed to the increased production of organic waste causing serious environmental problems. The Brazilian Northeast stands out due to the huge diversity of fruits considered exotic, where the yellow mombin (Spondias mombin L.) is a good example. This fruit contains antioxidants, phenolic compounds such as flavonoids, phenolic acids, anthocyanins, carotenoids, and also vitamins A and C. Thus, this study aimed to characterize and quantify the yellow mombin edible peel, in order to direct the process of extraction of carotenoids by enzymatic maceration and then determine the best condition, through an experimental design, and also to evaluate the influence of physical and physico-chemical factors on enzymatic maceration of the yellow mombin edible peel, and finally performing enzymatic maceration in the the yellow mombin edible peel in a batch reactor and obtain a product powder through spray drying. The yellow mombin edible peel presented himself as a good source of carotenoids, where the best condition for recovery of carotenoids was using 300 ÂL of the Pectinex enzymatic complex XXL during 3 hours of incubation, reaching the level of 120.94 Âg/g. Among the physical and physico-chemical factors, the use of enzyme preparations of cellulolytics status with the pectinolytic preparations for the recovery of carotenoids yellow mombin edible peel was not efficient, and adjusting the pH to neutrality (pH 6.0) before starting maceration proved efficient for a greater recovery of carotenoids, that in the laboratory level. The amount of water added in the process does not alter the recovery of carotenoids, used in proportions skin: water of 1: 2, 1: 3 or 1: 4. The use of ultrasound associated with enzymatic maceration worked at the laboratory scale. The use of batch reactor was technically feasible especially for the improvement of the conditions of homogenization of the mixture, which in turn led to obtaining higher levels of carotenoids recovered in the aqueous phase, not being necessary to adjust the pH of the reaction environment. Spray drying was possible being necessary suitability of the factors that influence drying to find the best condition necessary.

Orientador: Elisa Helena Giglio Ponsano
Banca: Marcelo Vasconcelos Meireles
Banca: Otto Mack Junqueira
Resumo: Rubrivivax gelatinosus é uma bactéria fototrófica natural de ambientes aquáticos que apresenta a capacidade de crescer em substratos contendo diversos compostos orgânicos. Neste trabalho, a biomassa da bactéria, produzida em águas residuárias provenientes de abate e processamento de pescado, foi caracterizada em relação a cor, composição centesimal, composição microbiológica e conteúdo de pigmentos carotenóides e aminoácidos, visando avaliar seu potencial como ingrediente para ração animal. Os resultados encontrados apontaram valores médios de 57% de proteína bruta, 23% de extrativo não nitrogenado e contaminantes, 11% de extrato etéreo, 5% de umidade e 4% de matéria mineral. Vários aminoácidos considerados essenciais para diversas espécies animais foram detectados no produto, tais como metionina (0,66g/100g), lisina (4,52g/100g), fenilalanina (3,43g/100g) e valina (4,39g/100g). Os resultados das análises microbiológicas indicaram contagens médias de 5,3 x 106 UFC/g para bactérias mesófilas aeróbias e facultativas viáveis, 20,27 NMP/g para coliformes a 35°C, <1,0 NMP/g para coliformes a 45°C e 1,2 x 103 UFC/g para bolores e leveduras. Não foram encontrados Aeromonas spp., Salmonella spp. e estafilococos coagulase positivo. O resultado da análise objetiva da cor da biomassa indicou valores médios de 25,48 para tonalidade, 14,22 para saturação da cor e 22,42 para luminosidade. A concentração média de carotenóides foi de 3,03 mg/g de biomassa. A determinação da composição e das características sensoriais da biomassa de Rubrivivax gelatinosus indicaram baixo nível de contaminação microbiana e alto potencial nutricional e pigmentante, apontando positivamente para sua utilização como ingrediente de rações na criação de animais
Abstract: Rubrivivax gelatinosus is a photoautotrophic bacterium that inhabits aquatic environment in which it uses different organic substrates for growth. In this work, the biomass of the bacterium, grown in effluent from fish slaughter and processing industry was characterized for color, proximate composition, microbiological composition, carotenoids and amino acids contents, with the aim of evaluating its potential as a feed ingredient. Mean results showed 57% crude protein, 23% non nitrogen extract and contaminants, 11% ether extract, 5% moisture and 4% ashes. Many amino acids considered essential for different animal species were present in the product, like metionine (0.66g/100g), lysine (4.52g/100g), phenylalanine (3.43g/100g) and valine (4.39g/100g). Microbial analysis showed mean counts of 5.3 x 106 UFC/g for mesophilic bacteria, 20.27 NMP/g for coliforms at 35°C, <1.0 NMP/g for coliforms at 45°C and 1.2 x 103 UFC/g for yeasts and moulds. Aeromonas spp., Salmonella spp. and positive coagulase staphylococci were not detected in the product. Results from objective color evaluation showed means at 25.48 for hue, 14.22 for chroma and 22.42 for lightness. Mean carotenoids concentration detected was 3.03 mg/g biomass. The determination of composition and sensorial characteristics of Rubrivivax gelatinosus biomass indicated low contamination level and high nutritional and pigmenting potential, pointing out for a positive use of the product as an ingredient for animal feeds
Mestre

Els carotenoids són el major grup de pigments presents en la natura. Aquests compostos són molt apreciats al presentar la capacitat de prevenir malalties cardiovasculars, certs tipus de càncer i diversos processos degeneratius. Malgrat la necessitat d’aquests compostos els humans no són capaços de sintetitzar-los y s’han adquirir en la dieta. Essent el fruit del tomàquet la major font de carotenoids en la dieta occidental. El licopè i el β-carotè són els carotenoids que es produeixen en grans quantitats en el fruit del tomàquet al llarg del procés de maduració. L’acumulació de carotenoides provoca el característic color vermell del fruit i constitueix una de les principals característiques nutricionals del tomàquet madur. L’acumulació de licopè i β-caroteno se realitza dins d’un plast específic anomenat cromoplast. Actualment l’acumulació no ha estat extensivament estudiada, tot i que s’ha postulat que es realitza en entorns membranosos i mitjançat complexes lipoproteics (Vishnevetsky, et al. 1999). El coneixement dels mecanismes i proteïnes implicats en el procés és un dels passos claus per entendre l’acumulació i la informació pot ser utilitzada pera incrementar o modificar la quantitat de licopè i β-caroteno en el fruit, objectiu molt rellevant en els programes de millora del tomàquet. En aquest treball s’ha procedit a identificar i caracteritzar gens i proteïnes relacionades amb l’acumulació de carotenoids en el fruit de tomàquet des de dues aproximacions diferents. En la primera, es va determinar quins individus dins d’una població de línies de caràcter autofecunadatiu provinents del creuament dels tomàquet Solanum lycopersicum (Cv Money Maker) i Solanum pimpinellifollium presentaven nivells contrastants de carotenoids. Una cop detectades les línies contrastants, amb alts o baixos nivells de carotenoides, es van determinar quins eren els gens amb expressió diferencial en experiments de microarray, caracteritzant els més importants. I com a segona aproximació es va procedir a purificar, mitjançant tècniques proteòmiques, complexes lipoproteics i la posterior caracterització de las proteïnes detectades en ells.
Los carotenoides son el mayor grupo de pigmentos presentes en la naturaleza. Estos compuestos son muy apreciados ya que presentan la capacidad de prevenir enfermedades cardiovasculares, ciertos tipos de cáncer y diversos procesos degenerativos. A pesar de la necesidad de estos compuestos los humanos no son capaces de sintetizarlos y los deben adquirir por la dieta. Siendo el fruto de tomate la mayor fuente de carotenoides en la dieta occidental. El licopeno y β-caroteno son los carotenoides que se producen en mayores cantidades en el fruto del tomate durante el proceso de maduración. La acumulación de dichos compuestos provoca el característico color rojo del fruto y constituye una de las principales características nutricionales del tomate maduro. La acumulación de licopeno y β-caroteno se realiza dentro de un plasto específico llamado cromoplasto. Actualmente la acumulación no ha sido extensivamente estudiada, aunque se ha postulado que se realiza en entornos membranosos y mediante complejos lipoproteicos (Vishnevetsky et al. 1999). El conocimiento de los mecanismos y proteínas implicados en el proceso es unos de los pasos claves para entender la acumulación y la información puede ser usada para incrementar o modificar la cantidad de licopeno y β-caroteno en el fruto, objetivo muy relevante en los programas de mejora del tomate. En este trabajo se ha procedido a identificar y caracterizar genes y proteínas relacionadas con la acumulación de carotenoides en el fruto de tomate desde dos aproximaciones diferentes. En la primera aproximación, se determino que individuos en una población de líneas de carácter autofecunadativo provinentes del cruce de Solanum lycopersicum (Cv Money Maker) y Solanum pimpinellifollium presentaban niveles contrastantes de carotenoides. Una vez detectadas las líneas contrastantes, con altos o bajos niveles de carotenoides, se determinaron cuales eran los genes de expresión diferencial mediante experimentos de microarray, llegando a caracterizar las más importantes. Y Como segunda aproximación se procedió a purificar, mediante técnicas proteómicas, complejos lipoprotéicos i la posterior caracterización de las proteínas detectadas en los mismos.

Includes bibliographical references (leaves 178-194). Genetic and non-genetic factors affecting fat colour in cattle were examined in biopsy and carcass samples of Jersey and Limousin cattle in their F1 and backcross progeny.

Thesis (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Plants cannot avoid stress and must therefore be capable of rapidly responding to extreme environmental changes. An inability to control and regulate the photosynthetic process during stress conditions will lead to the formation of highly reactive oxygen species that concomitantly causes photo-oxidative damage to the pigments and proteins of the photosynthetic apparatus. Since light is the primary source of energy for the photosynthetic process, it is clear that plants are continuously required to balance the light energy absorbed for the photochemical reactions against photoprotection in a dynamic way in order to survive. Carotenoids are precursors of abscisic acid, but more importantly structural components of the photosynthetic apparatus. During photosynthesis carotenoids function as accessory light-harvesting pigments, and also fulfil a photoprotective function by quenching the reactive molecules formed during conditions that saturate the photosynthetic process. Due to the importance of carotenoids to plant fitness and human health (as Vitamin A precursors) this study has attempted to isolate and characterise genes that are directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In total eleven full-Iength- and eight partial genes have been isolated, cloned and sequenced. These genes can be grouped into the following pathways: (i) the 1- deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) the mevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii) the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as a degradation product of carotenoids); and general isoprenoid biosynthetic pathways (as precursors of carotenoids). The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXP synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPP isomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoene synthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH), zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) have been isolated from cDNA. In addition, the full-length genomic copy and putative promoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated from genomic DNA by the construction and screening of sub-genomic libraries. Alignments of the genomic copies of these genes to the corresponding cDNA sequences have provided useful information regarding the genomic organisation of these genes, including the intron-exon junction sites in V. vinifera. The copy number of the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genome have been determined. DXS, PSY, BCH and lEP are single copy genes, whereas LBCY and NCED have two and three copies, respectively. The transcriptional activity of the putative promoters of six of the isolated genes (i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reporter gene assay. None of the putative promoters tested showed any transcriptional activity of the reporter gene. The transcription of these genes, has however been shown using northern blot analysis and/or RT-PCR. Preliminary expression profiles for PSY, LBCY, BCH, and lEP were determined in different plant organs and the expression of these genes was generally higher in photosynthetically active tissues. The expression of these genes following different treatments (abscisic acid, NaCI and wounding) was also assayed. The functionality of five of the isolated full-length genes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colour complementation assay. In silica analysis of the predicted protein sequences of all eleven isolated genes revealed that they are conserved and share a high degree of homology to the corresponding proteins in other plant species. The sequences were further analysed for conserved domains in the protein sequences, and these proteins typically demonstrated similar domain profiles to homologues in other species (plant, bacteria and algae). The predicted protein sequences were further analysed for transit peptides, the presence of which would provide evidence for the sub-cellular localisation of the mature peptides. Since these genes are involved in biosynthetic pathways that are active in discrete organelles, the sub-cellular localisation of most of these proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH and ZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathway genes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonate pathway gene, HMGS, was localised to both the cytosol and the mitochondria, and the general isoprenoid precursor genes, FPS and GGPS, were localised to the cytosol and the chloroplast, respectively. All these results are in agreement with the localisation of the respective pathways. In order to increase our understanding of carotenoid biosynthesis and functions in plants, we constitutively overexpressed one of the isolated genes (BCH) in the model plant, Nicotiana tabacum. Plants expressing the BCH gene in the sense orientation maintained a healthy photosynthetic rate under stress conditions that typically caused photoinhibition and photodamage in the untransformed control plants. This result was inferred using chlorophyll fluorescence and confirmed using CO2 assimilation rates and stomatal conductance. Chlorophyll fluorescence measurements indicated that the photo protective non-photochemical quenching ability of the BCH-expressing plants increased, enabling the plants to maintain photosynthesis under conditions that elicited a stress response in the untransformed control plants. An integral photosynthetic protein component, the D1 protein, was specifically protected by the additional zeaxanthin in the BCH sense plants. Plants expressing an antisense BCH proved the converse, i.e. lower levels of BCH resulted in decreased zeaxanthin levels and made the transgenic plants more susceptible to high-light induced stress. These results have shown the crucial role of carotenoids (specifically the xanthophylls) in the photoprotective mechanism in plants. The increased photoprotection provided by the BCH expressing plants suggests that the scenario in plants is not optimal and can be improved. Any improvement in the photoprotective ability of a plant will affect both the fitness and productivity of the plant as a whole and will therefore find application in a number of crop plants on a global scale. This study has resulted in the successful isolation and characterisation of genes involved in the direct, or indirect, carotenoid biosynthetic pathways. The further study and manipulation of these genes in model plants will provide useful insights into the physiological role of specific carotenoids in photosynthesis and in plants as a whole.
AFRIKAANSE OPSOMMING: Plante het nie die vermoë om stres te ontwyk nie en moet dus vinnig op veranderinge in hulomgewingstoestande kan reageer. Indien hulle nie die fotosinteseproses kan kontroleer en reguleer tydens streskondisies nie, sal dit tot die vorming van hoogs reaktiewe suurstofspesies lei, wat beide die pigmente en proteiene van die fotosintetiese apparaat sal beskadig. Lig is die primêre energiebron vir fotosintese en daarom is dit noodsaaklik dat plante deurgaans 'n dinamiese balans tussen fotosintese en fotobeskerming moet handhaaf. Karotenoiëde is voorlopers vir die vorming van absisiensuur, maar meer belangrik vir die plant, ook integrale komponente van die fotosintetiese apparaat. Tydens fotosintese word karotenoiëde vir die opneem van lig benodig, terwyl dit ook die fotosintetiese apparaat beskerm wanneer lig 'n versadigingspunt bereik vir fotosintese. Weens die belang van karotenoiëde vir plant- en menslike gesondheid (as Vitamiene A voorlopers), het hierdie studie beoog om gene te isoleer en karakteriseer wat direk of indirek 'n rol in karoteenbiosintese in Vitis vinifera speel. Elf vollengte- en agt gedeeltelike gene is geïsoleer, gekloneer, en gekarakteriseer. Hierdie gene kan in die volgende biosintetiese paaie gegroepeer word: (i) die 1- deoksi-D-xilulose 5-fosfaat (DOXP)/2-C-metiel-D-eritritol-4-fosfaat (MEP) pad (d.w.s. die plastiediese isopenteniel difosfaat biosintetiese pad); (ii) die mevalonaat pad (d.w.s. the sitosoliese/mitokondriale IPP biosintetiese pad); (iii) die karotenoiëd biosintetiese pad; (iv) die absisiensuur biosintetiese pad (as 'n afbraak produk van karotenoiëde) en die algemene isoprenoïed bisintetiese paaie (as voorlopers van karotenoiëde ). Die vollengte gene (d.w.s. vanaf die geskatte ATG tot die STOP kodon) van DOXP-sintase (DXS), 4-hidroksi-3-metielbut-2-eniel difosfaatreduktase (lytB), IPPisomerase (IPI), 3-hidroksi-3-metielglutariel koensiem A sintase (HMGS), fitoeën sintase (PSY), likopeen p-siklase (LBCY), p-karoteen hidroksilase (BCH), zeaxantien oksidase (ZEP), 9-cis-epoksi karotenoiëd dioksigenase (NCED), farnesiel difosfaat sintase (FPS)en geranielgeraniel difosfaat sintase (GGPS) is met behulp van. RTPKR vanaf eDNA geïsoleer. Die vollengte genomiese kopieë en die verwagte promotors van die DXS, PSY, LBCY, BCH, NCED and ZEP gene is ook geïsoleer d.m.v. die opstel en sifting van subgenomiese biblioteke. Vergelykende analises van die genoom- en eDNA kopieë het insiggewende data oor die genomiese rangskikking van die gene, insluitende die intron-ekson setels in V. vinifera gelewer. Die kopiegetalle van DXS, PSY, LBCY, BCH, NCED en ZEP is bepaal. DXS, PSY, BCH en ZEP is in die Vitis-genoom as enkel kopieë teenwoordig, terwyl LBCYen NCED twee en drie kopieë, repektiewelik, beslaan. Die transkipsionele aktiwiteit van die verwagte promotors van ses van die geïsoleerde gene (naamlik DXS, PSY, LBCY, BCH, ZEP en NCED) is d.m.v. 'n tydelike verklikkergeentoets ondersoek. Geeneen van die promotors het die transkripsie van die verklikkergeen bemiddel nie. Die transkripsie van die gene is egter wel bewys deur van northernhibridisasies en/of RT-PKR gebruik te maak. Die promotors van hierdie gene kan dus as transkipsioneel aktief beskou word. Voorlopige uitdrukkingsprofiele van PSY, LBCY, BCH, en ZEP is in verskillende plantorgane bepaal; die profiele was deurgaans hoër in fotosinteties aktiewe weefsels. Die uitdrukkingsprofiele van die gene is verder ook in reaksie op verskillende induktiewe behandelings (absisiensuur, NaCI en beskadiging) bepaal. Vyf van die vollengte gene (IPI, GGPS, PSY, LBCYen BCH) is funksioneel bewys in 'n bakteriese funksionele kleurkomplementasiesisteem. In silico analises van die afgeleide proteïene van al elf geïsoleerde gene het 'n hoë vlak van homologie met ooreenstemende proteiene van ander plantspesies getoon. Gekonserveerde domeine is ook in die proteïensekwense van die geïsoleerde gene teenwoordig. Hierdie proteïene het deurgaans dieselfde domeinprofiele vertoontoon as homoloë in ander spesies (bakterieë, alge en plante). Die sub-sellulêre teikening van die gene kon voorspel word deur die seinpeptiede in die proteiensekwense te eien. Aangesien hierdie gene betrokke is by biosintetiese paaie wat in diskrete kompartemente plaasvind; is die sub-selluiêre lokalisering van hierdie proteïene voorspelbaar. Die karotenoïed biosintetiese gene (PSY, LBCY, BCH en ZEP), die absisiensuur biosintetiese geen, NCED, sowel as die DOXP/MEP pad se gene (DXS, lytB en IPI) kom almal in die chloroplast voor. Die mevalonaatpadgeen, HMGS, word na beide die sitosol en die mitokondria geteiken, terwyl die algemene isoprenoïed voorlopergene, FPS en GGPS, onderskeidelik na die sitosol en die chloroplast geteiken word. Die verkreë voorspellings stem met die lokalisering van die biosintetiese paaie in die selooreen. Om ons kennis rakende karotenoïed biosintese en veral hulle funksie(s) in plante te verbreed, het ons een van die geïsoleerde gene, BCH, in die model plant, Nicotiana tabacum, konstitutief ooruitgedruk. Plante wat die BCH geen in die "sense" orientasie uitgedruk het, kon normale fotosintetiese aktiwiteit handhaaf onder kondisies wat foto-inhibisie en foto-osidatiewe skade in die ongetransformeerde kontrole plante veroorsaak het. Hierdie resultaat is met chlorofil fluoresensie analises aangetoon terwyl dit met CO2 assimilasie- en huidmondjie geleidingseksperimente bevestig is. Chlorofil fluoresensie metings het aangetoon dat die beskermingsvermoë van die transgeniese plante verhoog is, en dit dan die plante in staat stelom fotosintetese te handhaaf onder streskondisies van hoë lig. Proteïen analises het aangetoon dat 'n integrale fotosintetiese proteien, die 01 proteïen, word veral deur die verhoogde zeaxantien vlakke in die BCH transgeniese plante beskerm. Plante wat verminderde zeaxantien vlakke gehad het, weens die konstitutiewe ooruitdrukking van die BCH geen in die anti-"sense" orientasie, het die teenoorgestelde bewys. Met ander woorde. laer BCH vlakke (en dus laer zeaxantien vlakke) het tot plante wat meer vatbaar was vir hoë lig geïnduseerde stress gelei. Hierdie resultate het die essensiële beskermende rol wat karotenoiede tydens fotosintese speel, uitgelig. Die vermoë om hierdie beskermende meganisme te manipuleer in transgenies plante het aangetoon dat die sisteem in plante, alhoewel effektief, nie optimaal is nie. Enige verbetering in 'n plant se inherente vermoë om streskondisies te weerstaan sal die plant se algemene gesondheid en dus produktiwiteit beïnvloed. As sulks sal hierdie in meeste gewasspesies toepassing vind. Hierdie studie beskryf die isolering en karakterisering van gene wat direk, of indirek, by karotenoïedbiosintese betrokke is. Verdere studies, en veral die manipulering van hierdie gene in model plante, sal die fisiologiese rol van spesifieke karotenoïeede in fotosintese, en die plant as 'n geheel, ontrafel.

The mechanism of protection by carotenoids against photodynamic killing in Curtobacterium flaccumfaciens pathover poinsettiae (C. poinsettiae) was studied using pigment mutants isolated by treatment with nitrosoguanidine and DNA gyrase inhibitors. Growth rates, pigment composition, pigment levels and the ultrastructure of the wild-type streptomycin resistant strain of G. poinsettiae (wt-str) and all mutants were compared. One mutant, NTG-1, lacked colored carotenoids, and another, NTG-2, was a slow growing mutant containing low levels (14%) of wild-type carotenoid pigments. Except for NTG-1, the other pigment mutants had different proportions of the same pigments found in the wild type as determined by high performance liquid chromatography (HPLC). Only NTG-2 was morphologically distinct at the ultrastructural level.

Astaxanthin (3,3'-dihydroxy-β-carotene-4,4'-dione) is a ketocarotenoid that is beneficial for human health due to its ability of boosting immune function and preventing tumor formation. The biosynthesis of astaxanthin is, however, limited only to a few organisms. The burgeoning demand for natural astaxanthin has attracted much recent interest in extending the carotenoid pathway of higher plants to astaxanthin by expressing a microbial β-carotene ketolase (BKT). One major challenge of engineering an astaxanthin pathway in plants is the low astaxanthin content achieved. Five green microalgae including Chlamydomonas reinhardtii, Chlorococcum sp., Neochloris wimmeri, Protosiphon botryoides and Scotiellopsis oocystiformis were selected with enhanced function for astaxanthin biosynthesis. The products of the BKT cDNAs from the algae are similar in sequence to the BKT from Haematococcus pluvialis (ca 70% amino acid identity). Based on an Escherichia coli system, the BKT enzymes were shown to exhibit various efficacies in converting zeaxanthin into astaxanthin with Chlamydomonas BKT exhibiting the highest conversion rate (ca 85%). To investigate if the function-enhanced Chlamdomonas BKT (CRBKT) has advantages over other algal BKTs in triggering astaxanthin biosynthesis in higher plants, the CrBKT, together with the BKTs from Chlorella zofingiensis (CzBKT) and H. pluvialis (HpBKT3) was expressed in Arabidopsis thaliana. Transgenic Arabidopsis expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g-1 dry weight. In contrast, the expression of CzBKT resulted in much lower content of astaxanthin (0.24 mg g-1 dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in Arabidopsis. Similarly, overexpression of CrBKT in tobacco also resulted in the massive accumulation of astaxanthin in leaves (1.60 mg g-1 DW). Taken all together, it can be concluded that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic plants. Tomato is an important food crop with high amounts of carotenoids in its fruit. To investigate if tomato fruit can serve as a bio-factory for astaxanthin production, the CrBKT was overexpressed in three genotypes of tomato. All transgenic tomato plants developed brown red leaves that accumulated canthaxanthin rather than astaxanthin as a major carotenoid, resulting from the poor catalytic activity of the endogenous BHY1 toward canthaxanthin. To overcome this problem, CrBKT and HpBHY, the best pair of genes catalyzing the formation of astaxanthin in β- carotene-producing E. coli, were coexpressed in tomato. Canthaxanthin was efficiently converted to astaxanthin, resulting in a massive accumulation of astaxanthin in leaf (3.12 mg g-1) and fruit (16.1 mg g-1) with enhanced total carotenoid capacities of 1.7-fold in leaf and 16.6-fold in fruit. Moreover, the over-production of astaxanthin in fruit enhanced its antioxidant capacity 3-5-fold and vitamin C 2-fold, although it did not affect growth and development. In summary, the Chlamydomonas BKT is proven to be superior to other sources of BKT/CrtW enzymes in triggering astaxanthin biosynthesis in plants. By coexpressing a pair of well-cooperating BKT and BHY genes, the transgenic B-type tomato could accumulate commercially attractive amounts of the high-value astaxanthin in its fruit. This study highlights the potential of higher plants to be engineered as cell factories for producing the high-value astaxanthin.
published_or_final_version
Biological Sciences
Doctoral
Doctor of Philosophy