Establishing metrics of diversification can calibrate the observed scope of diversity within a lineage and the potential for further phenotypic diversification. There are two potential ways to calibrate differences between phenotypes. The first metric is based on the structure of the network of direct and indirect connections between elements, such as the genes, proteins, enzymes and metabolites that underlie a phenotype. The second metric characterizes the dynamic properties that determine the strength of the interactions among elements, and influence which elements are the most likely to interact. Determining how the connectivity and strength of interactions between elements lead to specific phenotypic variations provides insight into the tempo and mode of observed evolutionary changes. In this dissertation, I proposed and tested hypotheses for how the structure and metabolic flux of a biochemical network delineate patterns of phenotypic variation. I first examined the role of structural properties in shaping observed patterns of carotenoid diversification in avian plumage. I found that the diversification of species-specific carotenoid networks was predictable from the connectivity of the underlying metabolic network. The compounds with the most enzymatic reactions, that were part of the greatest number of distinct pathways, were more conserved across species’ networks than compounds associated with the fewest enzymatic reactions. These results established that compounds with the greatest connectivity act as hotspots for the diversification of pathways between species. Next, I investigated how dynamic properties of biochemical networks influence patterns of phenotypic variation in the concentration and occurrence of compounds. Specifically, I examined if the rate of compound production, known as metabolic flux, is coordinated among compounds in relation to their structural properties. I developed predictions for how different distributions of flux could cause distinct diversification patterns in the concentrations and presence of compounds in a biochemical network. I then tested the effect of metabolic network structure on the concentrations of carotenoids in the plumage of male house finches (Haemorhous mexicanus) from the same population. I assessed whether the structure of a network corresponds to a specific distribution of flux among compounds, or if flux is independent of network structure. I found that flux coevolves with network structure; concentrations of metabolically derived compounds depended on the number of reactions per compound. There were strong correlations between compound concentrations within a network structure, and the strengths of these correlations varied among structures. These findings suggest that changes in network structure, and not independent changes in flux, influence local adaptations in the concentrations of compounds. Lastly, the influence of carotenoid network structure in the evolutionary diversification of compounds across species of birds depends on how the structure of the network itself evolves. To test whether the carotenoid metabolic network structure evolves in birds, I examined the patterns of carotenoid co-occurrence across ancestral and extant species. I found that the same groups of compounds are always gained or lost together even as lineages diverge further from each other. These findings establish that the diversification of carotenoids in birds is constrained by the structure of an ancestral network, and does not evolve independently within a lineage. Taken together, the results of this dissertation establish that local adaptations and the evolutionary diversification of carotenoid metabolism are qualitatively predictable from the structure of an ancestral enzymatic network, and this suggests there is significant structural determinism in phenotypic evolution.

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.

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.

A abóbora se destaca como uma hortaliça de grande potencial nutritivo e tecnológico. Para que se possa aumentar a variabilidade dos produtos de origem vegetal disponíveis no mercado, a abóbora deve ser submetida a processos térmicos que visam prolongar a sua vida de prateleira, além de realizar as modificações necessárias para a sua comercialização. Neste contexto, o objetivo principal da presente pesquisa foi avaliar a aplicação da tecnologia de aquecimento ôhmico em diferentes etapas a serem realizadas durante o processamento da abóbora híbrida Tetsukabuto. Na primeira etapa do trabalho, objetivou-se investigar os efeitos do branqueamento convencional e ôhmico (realizados a 80 ºC durante 4 minutos e 163 V no aquecimento ôhmico) sobre a inativação da enzima peroxidase e sobre a cor das abóboras. Para o ajuste dos dados relativos à inativação enzimática, diferentes modelos cinéticos foram avaliados, sendo o modelo de distribuição de Weibull o escolhido para descrever a cinética de inativação da peroxidase. Com este estudo, foi possível observar que o branqueamento com campo elétrico afetou significativamente os parâmetros cinéticos de inativação enzimática, tornando esse processo mais rápido. A aplicação de ambas as tecnologias não causou modificações significativas dos parâmetros de cor da abóbora. Na segunda etapa do trabalho, creme de abóbora foi pasteurizado via tratamento térmico convencional e ôhmico realizados a 90 ºC por 2 horas e 47 V no aquecimento ôhmico. O objetivo dessa etapa foi avaliar os efeitos de ambas as tecnologias sobre a variação de cor, degradação de carotenoides totais e mudanças nos parâmetros reológicos do creme. Foi possível observar que a cor do creme foi levemente afetada durante a aplicação dos processos, tornando-se ligeiramente mais escura e perdendo o tom amarelado. Além disso, foi verificada perda no teor de carotenoides totais presentes no creme. Ainda, não foi observado um efeito não-térmico adicional do campo elétrico sobre a cor e os carotenoides presentes no produto. O creme de abóbora apresentou comportamento pseudoplástico e que se ajustou satisfatoriamente ao Modelo da Lei da Potência. Com a aplicação dos tratamentos, foi observado um aumento significativo no índice de consistência do creme, sendo que ambas as tecnologias não apresentaram diferenças representativas entre si. O conjunto de resultados obtidos pelo presente trabalho sugere que o aquecimento ôhmico é uma tecnologia promissora a ser aplicada em diferentes operações envolvidas durante o processamento de alimentos. / Pumpkins stand out as vegetable of great nutritional and technological potential. In order to increase the variability of vegetal products available on the market, pumpkins must be subjected to thermal process which can prolong its shelf life, and imposing also the necessary modifications for its commercialization. In this context, the main objective of the present research was to evaluate the application of ohmic heating technology in different stages carried out during the processing of the hybrid pumpkin Tetsukabuto. In the first part of the work, the objective was to investigate the effects of conventional and ohmic blanching (performed at 80 ºC during 4 minutes and 163 V in the ohmic heating) on the inactivation of peroxidase enzyme and on the color of the pumpkins. In order to adjust the data related to enzyme inactivation, different kinetic models were evaluated, being the Weibull distribution model chosen to describe the inactivation kinetics of peroxidase. With this study, it was possible to observe that the electric field blanching significantly affected the kinetic parameters of enzymatic inactivation, turning this process faster. The application of both technologies did not cause significant modifications of pumpkins color parameters. In the second part of the work, pumpkin cream was pasteurized via conventional and ohmic thermal treatment carried out at 90 ºC for 2 hours and 47 V in the ohmic heating. The objective of this stage was to evaluate the effects of both technologies on the color variation, the degradation of total carotenoids and the changes in rheological parameters of the cream. It was possible to observe that the color of the cream was slightly affected during the application of the processes, becoming slightly darker and losing its yellowish tone. In addition, it was verified loss of total carotenoid content in the cream. Furthermore, no additional non-thermal effect of the electric field on the color and on carotenoids present in the product was observed. The pumpkin cream presented a shear thinning behavior and was satisfactorily adjusted to the Power Law Model. It was observed a significant increase in the consistency index of the cream after the application of both treatments, and both technologies did not present significative differences between them. The overall results obtained in the present work suggests that ohmic heating is a promising technology to be applied in different operations involved during food processing.

Genetically enhanced high-carotene Beta III hybrid carrot was compared to a commercial carrot cultivar, Nantes Careless, for carotene content and macronutrient composition. Beta III contained more than twice the total carotene content of Nantes Careless, with α-carotene/β-carotene ratios higher in Beta III. Total solids from protein, lipids, and carbohydrate were greater in Beta III than in Nantes Careless. Beta III contained about five times the amount of sucrose and one-fifth the amount of glucose and fructose than Nantes Careless or reported literature values. Comparison of the bioavailability of carotenes in Beta III and Nantes Careless to purified β-carotene and retinyl acetate standards was made using two bioassays: the liver storage, slope-ratio assay and a curative growth study. Beta III provided liver retinol stores equivalent to stores provided by Nantes Careless, β-carotene, and retinyl acetate at dosage 300 RE. At 600 RE, only retinyl acetate increased liver retinol storage with dose. Carotene sources did not increase liver retinol storage, presumably owing to the mechanisms that prevent carotene vitamin A toxicity. Growth study results indicated vitamin A potency of Beta III was not equivalent to that provided by retinyl acetate. Slopes of the growth response curves for Nantes Careless and β-carotene were not significantly different from retinyl acetate; the slope of the Beta III growth response curve was significantly different. All carotene sources were similar to retinyl acetate in the curvilinear response of the growth curves. The appropriate fit of the quadratic function to the growth response curves suggested 12 RE/d was greater than requirement. The differences in results of the two bioassays used to determine bioavailability of carotenes in Beta III may be explained by a number of factors. The utilization of β-carotene from Beta III may be affected by a-carotene or other carrot constituents for absorption or conversion to vitamin A, or both. Stored liver retinol may be a combination of retinol and a-retinol with reduced vitamin A activity. The growth assay may be more sensitive to external variables that cannot be controlled.

Gac (Momordica Cochinchinensis Spreng or Muricia Cochinchinensis Spreng) is an indigenous fruit of Vietnam, commonly used as a colourant in traditional dishes. Recent studies showed that this fruit had a high carotenoid content, especially lycopene and β-carotene. These carotenoids are currently in high demand as they are natural antioxidants to prevent and treat cancers. Over the last ten years some new products from Gac fruit have been found on the market but the cultivation and consumption of the fruit is still limited. Gac is harvested seasonally and the post harvest processing has not been well developed. To make the best use of the valuable substances in Gac, this project investigated some processing methods to make Gac powder and developed the product quality analyses, potentially applicable to local small-scale manufacture. In order to evaluate the efficiency of the Gac powder process and some of the most important qualities of the Gac powder products, the analytical methods to determine lycopene and β-carotene were developed and validated in this project. The new HPLC-based method included a liquid-liquid extraction, followed by a solid phase extraction before the analysis. Both newly- developed HPLC and SP methods exhibited a high extraction yield, good recovery and reproducibility. Compared with the HPLC, the SP method gives higher mean values and standard deviations, but it is quicker, simpler, less expensive and still reliable for industrial applications due to the use of less toxic solvents. The production of powder from Gac fruit included two main stages: seed removal and aril dehydration. Derived from the same drying operation, the enzymatic-treated Gac powder had a lower carotenoid content compared to the heat-treated powder. The Gac powder produced by the freeze-drying method had the highest carotenoid level and brightest colour. Finally the orange-red Gac powder with high carotenoid content was added to some foods as a colorant and nutrient supplement to illustrate its applications. The colours of foods were significantly improved and the carotenoid contents were increased after processing, indicating a potential market for the Gac powder. / Master of Science (Hons)

Total antioxidant activity and total carotenoid levels were evaluated for more than 100 common potato (Solanum tuberosum, L.) cultivars grown in the United States, advanced breeding lines from several Western U.S. breeding programs, and 47 related, tuber-bearing species. An initial assessment of variability for antioxidant activity provided baseline information to be used for potential potato promotion and for the development of new varieties with greater human health benefits. Wide variability in antioxidant levels provided evidence of genetic control of this trait, indicating that it could be possible to breed for enhanced levels of antioxidant compounds in potato. Accessions, varieties, and advanced breeding lines identified in the broad screen as having high antioxidant activity and high total carotenoid levels, were fine screened via HPLC to determine specific phenolic and carotenoid compounds present in potato. The objective of the study was to identify parents for use in the Texas breeding program to develop potato varieties containing increased levels antioxidant compounds. In the broad screen for total antioxidant activity, the 47 related, tuber-bearing species showed a wider range of variability than the cultivated varieties and breeding lines. Based on the DPPH assay, antioxidant activity ranged from 103-648 uM trolox equivalents in the cultivated varieties and advanced breeding lines, while that of the wild species was 42-892. HPLC analysis revealed that the phenolic content of the species, and their cultivated counterparts, was primarily composed of caffeic and chlorogenic acids. Other phenolics identified were p-coumaric acid, rutin hydrate, vanillic acid, epicatechin, t-cinnamic acid, gallic acid, and salicylic acid. The highest phenolic content discovered in the accessions was five-fold higher than the highest of the cultivated genotypes. Carotenoid analysis revealed lutein in the accessions, but the yellow-flesh breeding lines were much higher in carotenoids. In addition to the work conducted on antioxidants, an attempt was made to separate intraclonal variants of the potato cultivar Russet Norkotah. Eleven microsatellite primers and 112 AFLP primer combinations failed to produce any reproducible polymorphisms. The inability to detect differences between the clones could be due to the tetraploid nature of the clones or epigenetic differences not detected by the procedures utilized in this study.

Yellow pigment (YP) concentration is an important quality trait in durum wheat (<i>Triticum turgidum</i> L. var <i>durum</i>) and is comprised primarily of carotenoids. The main objective of our study was to measure the accumulation of carotenoids during the grain fill period to improve our understanding of the physiological basis for differences among durum wheat cultivars. Thirteen cultivars and breeding genotypes with large variation in total YP concentration (<6 µg g-1 to >15 µg g-1) were studied. Spikes were sampled from replicated field plots in 2007 and 2008 near Saskatoon and Swift Current, Saskatchewan, Canada, at 14, 21, 28 and 35 days after heading (DAH). The remainder of each plot was combined at grain maturity for YP and carotenoid analysis. Carotenoids were extracted with 1:1 methanol:dichloromethane (0.1% BHT) and quantified with HPLC. <i>Trans</i> (E)-lutein was the predominant carotenoid at maturity and was detected at 14 DAH in all genotypes. The rate and duration of E-lutein accumulation was variable among genotypes expressing high, intermediate and low YP. The accumulation of all carotenoids was lowest in genotypes expressing low YP, and suggests rate limitations early in the carotenoid biosynthetic pathway. E-zeaxanthin concentrations were highest in mature grain, but no significant differences were detected among genotypes. However, the ratio of E-zeaxanthin to E-lutein was inversely correlated with total YP, suggesting that the â,å branch of lycopene cyclization is favoured over the â,â branch in high-YP genotypes. These results provide insights to the regulation of the carotenoid biosynthetic pathway during grain fill stage in durum wheat and will facilitate breeding for higher carotenoid concentration.

Yellow pigment (YP) concentration is an important quality trait in durum wheat (<i>Triticum turgidum</i> L. var <i>durum</i>) and is comprised primarily of carotenoids. The main objective of our study was to measure the accumulation of carotenoids during the grain fill period to improve our understanding of the physiological basis for differences among durum wheat cultivars. Thirteen cultivars and breeding genotypes with large variation in total YP concentration (<6 µg g-1 to >15 µg g-1) were studied. Spikes were sampled from replicated field plots in 2007 and 2008 near Saskatoon and Swift Current, Saskatchewan, Canada, at 14, 21, 28 and 35 days after heading (DAH). The remainder of each plot was combined at grain maturity for YP and carotenoid analysis. Carotenoids were extracted with 1:1 methanol:dichloromethane (0.1% BHT) and quantified with HPLC. <i>Trans</i> (E)-lutein was the predominant carotenoid at maturity and was detected at 14 DAH in all genotypes. The rate and duration of E-lutein accumulation was variable among genotypes expressing high, intermediate and low YP. The accumulation of all carotenoids was lowest in genotypes expressing low YP, and suggests rate limitations early in the carotenoid biosynthetic pathway. E-zeaxanthin concentrations were highest in mature grain, but no significant differences were detected among genotypes. However, the ratio of E-zeaxanthin to E-lutein was inversely correlated with total YP, suggesting that the â,å branch of lycopene cyclization is favoured over the â,â branch in high-YP genotypes. These results provide insights to the regulation of the carotenoid biosynthetic pathway during grain fill stage in durum wheat and will facilitate breeding for higher carotenoid concentration.

Total antioxidant activity and total carotenoid levels were evaluated for more than 100 common potato (Solanum tuberosum, L.) cultivars grown in the United States, advanced breeding lines from several Western U.S. breeding programs, and 47 related, tuber-bearing species. An initial assessment of variability for antioxidant activity provided baseline information to be used for potential potato promotion and for the development of new varieties with greater human health benefits. Wide variability in antioxidant levels provided evidence of genetic control of this trait, indicating that it could be possible to breed for enhanced levels of antioxidant compounds in potato. Accessions, varieties, and advanced breeding lines identified in the broad screen as having high antioxidant activity and high total carotenoid levels, were fine screened via HPLC to determine specific phenolic and carotenoid compounds present in potato. The objective of the study was to identify parents for use in the Texas breeding program to develop potato varieties containing increased levels antioxidant compounds. In the broad screen for total antioxidant activity, the 47 related, tuber-bearing species showed a wider range of variability than the cultivated varieties and breeding lines. Based on the DPPH assay, antioxidant activity ranged from 103-648 uM trolox equivalents in the cultivated varieties and advanced breeding lines, while that of the wild species was 42-892. HPLC analysis revealed that the phenolic content of the species, and their cultivated counterparts, was primarily composed of caffeic and chlorogenic acids. Other phenolics identified were p-coumaric acid, rutin hydrate, vanillic acid, epicatechin, t-cinnamic acid, gallic acid, and salicylic acid. The highest phenolic content discovered in the accessions was five-fold higher than the highest of the cultivated genotypes. Carotenoid analysis revealed lutein in the accessions, but the yellow-flesh breeding lines were much higher in carotenoids. In addition to the work conducted on antioxidants, an attempt was made to separate intraclonal variants of the potato cultivar Russet Norkotah. Eleven microsatellite primers and 112 AFLP primer combinations failed to produce any reproducible polymorphisms. The inability to detect differences between the clones could be due to the tetraploid nature of the clones or epigenetic differences not detected by the procedures utilized in this study.