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The heavy reliance on live feeds is currently restricting the growth and sustainability of the aquaculture industry, therefore, the overall goal of this research was to improve the utilization of formulated dry diets at first feeding of larval fish. This was done with a specific focus on the production and provision of the optimal dietary protein form and composition. Chapter 2 aimed to provide an efficient protein source for larval fish by using same-species muscle and endogenous enzymes to produce hydrolysates and by providing a series of diets with increasing molecular weight protein fragments through larval development. Largemouth Bass (Micropterus salmoides) (LMB) muscle was mixed with the digestive enzymes from adult LMB and hydrolyzed for 1.5, 3, and 6 h, respectively. Five diets were produced, an intact diet containing non-hydrolyzed muscle and four diets with 37% muscle hydrolysate inclusion. The molecular weight profile of those diets were formulated to vary based on the inclusion level of each hydrolysate. To account for gut development, one group of larval LMB was fed a weekly series of diets with an increasing molecular weight profile. The initial inclusion of the hydrolysates significantly improved the total length of the larval LMB; however, neither the hydrolysate inclusion nor the series of dietary molecular weight profiles improved the overall growth of larval LMB. The inclusion of hydrolysates significantly decreased the occurrence of skeletal deformities. The results from this study suggest that the inclusion of same-species hydrolysates can improve the initial growth of first-feeding LMB, but further research is necessary to determine the optimal molecular weight profile, hydrolysate inclusion level, and physical properties of feeds to improve the overall growth performance during the larval stage. Chapter 3 compared the effect of dietary inclusion of a fish muscle hydrolysate produced from species-specific muscle and enzymes to hydrolysates produced from those of a different species, in diets for larval Walleye (Sander vitreus). Four intact and hydrolyzed protein products were produced from each combination of Walleye muscle and endogenous enzymes, and muscle and endogenous enzymes from Nile Tilapia (Oreochromis niloticus). The hydrolyzed products were continuously mixed for 3 h during the hydrolysis, (at 22oC and 28oC for Walleye and Tilapia enzymes, respectively), and the pH was adjusted throughout the process to mimic gastric and intestinal digestion conditions. Four diets were produced with the dietary protein supplied as a 50/50 ratio of the intact and hydrolyzed muscle from the respective muscle/enzyme combination. There was a significant interaction effect between muscle and enzyme source on the growth of larval Walleye. At the conclusion of the study, the larval Walleye that received the diet with muscle hydrolysate produced with Walleye muscle and Walleye endogenous enzymes had a significantly higher average weight than all other groups, and significantly higher postprandial levels of total free amino acids and indispensable amino acids in the muscle. Each hydrolysate-based diet led to a significant reduction in skeletal deformities and survival, compared to a group fed with a commercial diet. The results from this study suggest that species-specific muscle and enzymes produce a more optimal dietary protein source for larval fish than non-species-specific products, but further research should focus on improving the physical properties of the formulated diets to improve survival of fish larvae. Chapter 4 proposed a practical controlled hydrolysis method to utilize the endogenous enzymes within the fish body for the breakdown of tissues proteins, and to produce a species-specific meal that is tailored to the nutritional requirements and absorptive capacity of fish larvae. Four Zebrafish (Danio rerio) meals were produced from whole-body adult Zebrafish, three hydrolysates that were hydrolyzed for 1, 2, and 3 h, respectively, and an unhydrolyzed meal. From these meals, three diets were produced, each defined by their supply of dietary protein. The Unhydro diet was solely based on the unhydrolyzed Zebrafish meal. The 50% Hydro diet was based on 50% Zebrafish hydrolysate mix and 50% unhydrolyzed Zebrafish meal. The 100% Hydro diet was 100% based on the Zebrafish meal hydrolysate. The hydrolysate mix contained equal parts of the 1, 2, and 3 h hydrolysates. Proteomic analysis showed that the proposed hydrolysis method was able to efficiently hydrolyze the protein within Zebrafish body. The feeding trial found no significant differences in the final weight, total length, or survival between the Unhydro, 50% Hydro, and 100% Hydro groups, but the 50% Hydro group did express a significant upregulation of PepT1 at 24 h after feeding, compared to the Unhydro group. The growth results paired with PepT1 gene expression potentially indicate Zebrafish larvae to be adapted to dry feeds at first feeding and able to utilize dietary protein in different molecular forms efficiently for growth. Overall, the proposed hydrolysis method provides a practical and cost-effective approach to producing species-specific fishmeal hydrolysates. Further research is necessary to determine whether the produced hydrolysates can improve the growth of larval fish in other fish models. Further insight into behavioral and physiological responses in fish to imbalanced dietary amino acid profiles was provided in Chapter 5. The objective of this study was to determine how stomachless fish respond to diets deficient in the main limiting IDAA (lysine, methionine, and threonine), using Zebrafish as a model species. Six semi-purified diets were formulated for this study. The CG diet contained casein and gelatin as its only protein sources, while FAA50 diet had 50% of is dietary protein supplied with crystalline amino acids. Both were formulated to contain identical, balanced amino acid profiles. The remaining diets were supplied with the same amino acid mix as the FAA50 diet, but with minor adjustments to create deficiencies of the selected IDAA. The (-) Lys, (-) Met, and (-) Thr diets had lysine, methionine, and threonine withheld from the free amino acid (FAA) mix, respectively, and the Def diet was deficient in all three. The fish were fed to apparent satiation three times a day, and each feeding was carefully observed to ensure all feed added to the tanks was consumed. The results showed that although the singular deficiency of the three main limiting amino acids did not induce significant changes in feed intake, the combined deficiency of the three IDAA significantly increased the feed intake of juvenile Zebrafish. This increased feed intake prevented the IDAA deficiencies from significantly reducing growth, however, the feeding efficiency was also reduced. There was also an observed upregulation of neuropeptide Y (NPY), an orexigenic hormone, in the Def group, compared to the FAA50 group. The outcomes of this study provide insight into the behavioral and physiological response to dietary amino acid imbalances of stomachless fish and suggests stomachless fish increase their feed intake when challenged with IDAA-deficient diets, and that the regulation of NPY might play a role in this response. Chapter 6 assessed the postprandial FAA dynamics in the plasma, liver, and muscle of three species; 1) Largemouth Bass – warm-water, stomach-possessing carnivorous species; 2) Walleye – cool-water, stomach-possessing carnivorous species; and 3) Zebrafish– tropical, stomachless omnivorous species. Two diets were formulated for this study, a diet based on intact casein and gelatin (CG), and a diet with 50% of its protein supplied in FAA form (FAA50). Forty-two fish from each species were utilized, with one group of 21 receiving the CG diet, and the other 21 receiving the FAA50 diet. All fish were starved for 24 hours prior to the final feeding before sampling. Three fish were sampled at each time point, with three samples (plasma, liver, and muscle) taken from each fish. Samples were taken prior to feeding (0 h) and then at 0.5, 1, 2, 3, 6, and 12 h after feeding, for all species. A significant three-way interaction was observed between the diet, species, and postprandial time on the total FAA, IDAA, and DAA levels in the plasma, liver, and muscle, indicating that the postprandial FAA patterns were significantly different between species and in response to the different diets. In stomach-possessing species, dietary amino acids from the FAA50 diet were absorbed more rapidly than those from the CG diet, resulting in fewer correlations with the dietary IDAA profiles. The absorption of FAA in cool-water Walleye was more gradual and prolonged than the warm-water LMB, leading to more significant correlations with the dietary IDAA and more sustained peaks. The postprandial peaks of FAA typically occurred at the same time in the stomachless Zebrafish fed with the CG or FAA50 diet. The levels of FAA were noticeably lower after feeding with the FAA50 diet in Zebrafish, compared to the CG diet. These results provide a reference for differences in the FAA dynamic patterns of three species with differing physiological characteristics, when fed diets with intact protein or supplemented with FAA. The findings presented in this dissertation provide support and novel methods for the production and inclusion of species-specific protein hydrolysates as an ideal protein source in formulated diets for first-feeding larval fish. This research contributes to the development of larval diets that can release the limitations of growth placed on the aquaculture industry by the reliance on live feeds, particularly within the hatchery sector. This research also provides further understanding of dietary protein utilization and delivers new fish nutrition knowledge that will benefit the aquaculture industry as a whole.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / There are many studies showing that the salinity may directly affect the nitrate uptake, from their osmotic effect, nature of the salt and its ionic composition. However, little is known about the mechanisms related to the salt ability to inhibit the nitrate acquisition indirectly. This study was carried with aim to induce inhibition of NO3- influx in cowpea roots of indirect form triggered by a negative feedback mechanism, caused by the increase in the pool of free amino acids in the tissue, induced by salt stress.For this, were done three isolated studies and continuous.The exogenous glutamine application promoted an increase in the free amino acids content.The presence of glutamine decreased significantly the nitrate acquisition.The free ammonium can also be listed as a key-compound in the role of nitrate influx regulation, since use of the MSX (Methionine sulfoximine) promoted the increase of NH4+ content and also reduced nitrate influx, but, in a lesser degree when compared to treatment with AZA (Azaserine). Salt stress caused a reduction in NO3- influx by decrease in the growth of plants induced by salt. The data indicated which this reduction in the influx is triggered by increase of amino acids content, mainly the glutamine, that is main likely compound to act as signal in the N-feedback regulation. / Existem muitos estudos mostrando que a salinidade pode afetar a absorÃÃo de nitrato de forma direta, a partir do seu efeito osmÃtico, da natureza do sal e de sua composiÃÃo iÃnica. Entretanto, pouco se sabe sobre os mecanismos relacionados com a capacidade do sal em inibir a aquisiÃÃo de nitrato de forma indireta. O presente estudo teve como objetivo induzir a inibiÃÃo do influxo de NO3- em raÃzes de feijÃo-caupà de forma indireta desencadeada por um mecanismo de feedback negativo, provocado pelo aumento no pool de aminoÃcidos livres no tecido, induzido por estresse salino. Para isso, foram realizados trÃs estudos isolados e contÃnuos. A aplicaÃÃo de glutamina exÃgena promoveu um aumento no conteÃdo de aminoÃcidos livres. A presenÃa de glutamina reduziu significativamente a aquisiÃÃo de nitrato. O amÃnio livre tambÃm pode ser listado como um composto-chave no papel da regulaÃÃo do influxo de nitrato, pois a utilizaÃÃo do MSO (Metionina sulfoximina) promoveu o aumento do conteÃdo de NH4+ e tambÃm reduziu o influxo de nitrato, porÃm em menor grau quando comparado ao o tratamento com AZA (Azaserina). O estresse salino causou uma reduÃÃo no influxo de NO3-, pela diminuiÃÃo no crescimento das plantas induzida pelo sal. Adicionalmente, estes dados indicaram que esta reduÃÃo no influxo està ligada ao aumento do teor de aminoÃcidos, principalmente a glutamina, que à o principal componente para atuar como sinal na regulaÃÃo por N-feedback.

Though numerous valuable compounds from red algae already experience high demand in medicine, nutrition, and different branches of industry, these organisms are still recognized as an underexploited resource. This study provides a comprehensive characterization of the chemical composition of 15 Arctic red algal species from the perspective of their practical relevance in medicine and the food industry. We show that several virtually unstudied species may be regarded as promising sources of different valuable metabolites and minerals. Thus, several filamentous ceramialean algae (Ceramium virgatum, Polysiphonia stricta, Savoiea arctica) had total protein content of 20–32% of dry weight, which is comparable to or higher than that of already commercially exploited species (Palmaria palmata, Porphyra sp.). Moreover, ceramialean algae contained high amounts of pigments, macronutrients, and ascorbic acid. Euthora cristata (Gigartinales) accumulated free essential amino acids, taurine, pantothenic acid, and floridoside. Thalli of P. palmata and C. virgatum contained the highest amounts of the nonproteinogenic amino acid β-alanine (9.1 and 3.2 μM g−1 DW, respectively). Several red algae tend to accumulate heavy metals; although this may limit their application in the food industry, it makes them promising candidates for phytoremediation or the use as bioindicators.

A Annona crassiflora Mart. é uma espécie frutífera nativa do cerrado que possui alto potencial de uso e ampla distribuição. O presente trabalho tem os seguintes objetivos: identificar e quantificar aminoácidos livres, carboidratos solúveis e ácidos graxos da polpa e das sementes, visando ampliar os conhecimentos sobre potencial econômico de A. crassiflora; avaliar qual o nível de variações quantitativas e qualitativas de alcalóides de A. crassiflora de amostras provenientes de diferentes regiões do cerrado; avaliar qual a magnitude da diversidade genética entre diferentes populações de A. crassiflora, utilizando marcadores isoenzimáticos. Com relação às propriedades nutricionais, as análises de aminoácidos livres, carboidratos e lipídeos foram feitas por CLAE/F, por CLAE/CTI - DPA e por CG/EM, respectivamente. O teor total de aminoácidos livres na polpa variou de 1101,99 g/g a 1975,7 g/g de massa seca, enquanto nas sementes variou de 636,3 μg/g a 17.445,83 μg/g de massa seca. A glutamina foi majoritária em todas as amostras. O teor total dos carboidratos solúveis da polpa variou de 11,5 g/100g a 17,03 g/100g e das sementes variou de 13,69 g/100g a 40,47 g/100g de matéria seca. Foram detectadas a frutose, a glicose e a sacarose em todas as amostras de polpa e de sementes. A quantidade de lipídeos totais da polpa variou de 7g/100g a 16,2 g/100g de massa seca e das sementes variou de 34,36 g/100g a 35,98 g/100g de massa seca. O ácido oléico e/ou petroselínico (18:1) foram majoritários em todas as amostras de polpa e de sementes de diferentes origens geográficas. Certamente os dados obtidos permitiram ampliar os conhecimentos sobre potencial de aproveitamento dessa espécie. Para avaliar a magnitude da variação dos alcalóides das folhas de A. crassiflora, a quantificação e identificação dos alcalóides foram feitas, respectivamente, por CG/FID e CG/EM. Foi verificado que o teor total de alcalóides variou de 221,1 ± 17,14 μg/g a 2986,89 ± 367,1 μg/g de massa seca. Foram identificados os alcalóides anonaína, anoretina, romucosina e xilopina que mostraram diferenças entre as regiões. Essa variação na concentração e no perfil alcaloídico das populações de A. crassiflora sugere ampla plasticidade fenotípica desses metabólitos em resposta a fatores bióticos e/ou abióticos, além de alta variabilidade genética relacionada à expressão dos genes envolvidos na biossíntese desses metabólitos. A avaliação da estrutura genética foi feita através da análise isoenzimática. Os dados isoenzimáticos mostraram baixa variabilidade genética. Duas hipóteses são apontadas para explicar o baixo nível de polimorfismos verificado em A. crassiflora, uma delas relacionada à técnica e outra relacionada ao próprio processo de degradação do ambiente. Diante do exposto, os dados obtidos no presente trabalho sugerem formas de agregar mais valor econômico e ecológico à A. crassiflora e podem proporcionar uma base para o uso mais eficiente e racional dos recursos dessa espécie, contribuindo para exploração sustentável do cerrado, o que permite também o desenvolvimento de comunidades locais. / Annona crassiflora Mart. is a native fruit tree species from the cerrado, possessing high use potential and a wide distribution. The proposed aims were to identify and quantify free amino acids, free sugars and fatty acids in the pulp and seeds of this plant, with a view to widening knowledge on economical potentiality, as well as to evaluate the level of alkaloid quantitative and qualitative variation in samples coming from different regions of the cerrado, and finally define the size of genetic diversity among these different populations by using isoenzymatic markers. As to nutritional properties, analyses of free amino acids, free sugars and fatty acids were undertaken with, respectively, CLAE/F, CLAE/CTI DPA and CG/MS. The total rate of free amino acids in the pulp varied from 1101,99 ug/g to 1975,7 ug/g of dry mass, whereas in the seeds this variation was from 636,3 μg/g to 17.445,83 μg/g. Glutamin was preponderant in all the samples. Total free sugar rate in pulp varied from 11,5 g/100g to 17,03 g/100g and in seeds from 13,69 g/100g to 40,47 g/100g of dry matter. Fructose, glucose and sacarose was detected in all samples. The amount of total lipids in pulp varied from 7 g/100g to 16,2 g/100g of dry weight and in seeds from 34,36 g/100g to 35,98 g/100g. Oleic and/or petroselinic acids (18:1) were the most abundant in all pulp and seed samples from the different geographic regions. The obtained results certainly permit widening knowledge on the potentiality for use of this plant species. In order to evaluate the size of alkaloid variation in A. crassiflora leaves, alkaloid quantification and identification was done with, respectively, CG/FID and and CG/EIMS. It was noted that the total alkaloid rate varied from 221,1 ± 17,14 ug/g to 2986,89 ± 367,1 μg/g of dry mass. The alkaloids anonain, anoretin, romucosin and xilopin were identified, these showing differences among regions. This variation in alkaloid concentration and profile in A. crassiflora populations suggests the wide phenotypic plasticity of these metabolites in response to biotic and/or abiotic factors, besides high genetic variability related to gene expression of those involved in their biosythesis. Genetic structure evaluation was done through isoenzymatic analysis. Isoenzymatic data revealed low genetic variability. Two hypotheses may be raised to explain this low polymorphism level. The first points to inefficient isoenzymatic techniques in reckoning species genetic diversity, whereas the second takes into consideration that excessive homozygosis (indicated by the low level of isoenzymatic variability) could be an indication of the process of species genetic erosion, possibly related to habitat (cerrado) degradation. In view of this, data obtained in the present study suggest forms of aggregating additional economical and ecological value to A. crassiflora, and could furnish a base for the more efficient and rational use of resources from this species, thus contributing to sustainable exploitation of the cerrado, and also allowing for local community development.

O arroz está entre os cereais mais consumidos do mundo. A desnutrição por micronutrientes como zinco (Zn) e ferro (Fe) é generalizada, especialmente em populações pobres, cujas calorias diárias são obtidas principalmente pelo consumo de cereais. Apesar de o arroz ser diferenciado por possuir maior quantidade de lisina em seus grãos em comparação ao trigo, milho ou cevada, a sua quantidade de proteínas representa somente de 8-9% do grão. Sendo o Zn é um micronutriente correlacionado com o funcionamento vital de plantas e animais, e estando relacionado direta e indiretamente com a síntese e funcionamento de várias proteínas, a proposta deste trabalho foi analisar a possível correlação da quantidade de Zn encontrado no grão com o perfil das proteínas de reserva, aminoácidos solúveis, enzimas envolvidas no metabolismo do ácido aspártico e do fator antinutricional ácido fítico, nos grãos de diferentes genótipos de arroz, avaliando primordialmente as diferenças inerentes de cada material genético a fim de agregar conhecimentos que possam ser utilizados como ferramenta para o melhoramento dessa espécie, visando ganho nutricional em seu grão. Para tanto, foram executados dois experimentos distintos. No primeiro foram avaliados três genótipos, os quais foram expostos à adubação foliar com sulfato de zinco, nas concentrações 0, 2 e 4 kg.ha-1 de ZnSO4.7H2O, buscando aumentar-se o nível de Zn nos grãos. No entanto não se verificou incremento significativo do metal na porção de interesse alimentar, sendo a variação encontrada nas concentrações de Zn em grãos integrais e polidos foram inerentes aos genótipos. Ainda assim, foram verificados os outros aspectos nutricionais, para os quais os resultados apontaram diferenças significativas entre os tipos de grãos analisados, sendo indicado o consumo de grão integral ao polido, destacando-se o genótipo Primavera, que apresentou maior concentração de proteínas e menor quantidade de fitato. Esse experimento ainda confirmou que o catabolismo da lisina é a principal via de modulação para o acréscimo desse aminoácido no grão. Para a execução do segundo experimento foram utilizados 16 genótipos cultivados no Brasil, buscando caracterizar a diversidade genética em relação às principais propriedades do grão relacionadas à qualidade nutricional humana. Os dados mostraram que apesar do Zn estar correlacionado positivamente com fitato, não há relação direta entre a concentração desse elemento com a síntese e concentração de aminoácidos e nem com a concentração total de proteínas de reserva. No entanto, com os resultados obtidos para grão integral em relação às características nutricionais, permite apresentar os genótipos Carolino e Goiano como os de melhor desempenho, além de ressaltar a importância da camada da aleurona do grão, já que está relacionada com todas as características nutricionais importantes para um projeto de bioforticação, indicando que estudos devem ser intensificados para entender o transporte de nutrientes até o interior do endosperma, ou ainda, que o consumo de arroz integral deve ser mais reforçado. / Human malnutrition due to low intake of the micronutrients zinc (Zn) and iron (Fe) is relatively common in poor population, who uses cereals as main sources of calories. Currently, rice is one of the most consumed cereals worldwide but, despite its high lysine concentration in comparison to other cereal crops, rice grains have low protein contents (8-9%). Therefore, the current work aimed to evaluate the influence of additional Zn supply on amino acid profile, storage proteins, macro- and micronutrients, as well as on phytate concentration, and on activity of enzymes from lysine metabolism in grains of different rice genotypes, primarily evaluating the inherent differences of each genetic material in order to aggregate knowledge like a tool for the improvement of this species, aiming nutritional gain in its grain. For this purpose, two different experiments were performed. First of all, three genotypes were evaluated and exposed to foliar fertilization with zinc sulfate, in the concentrations 0, 2 and 4 kg.ha-1 of ZnSO4.7H2O, seeking to increase the level of Zn in the grains. However, there was no significant increase of the metal in the grain, and the variation found in Zn concentrations in brown and polished grains was according to the genotypes. Nevertheless, the other nutritional aspects were verified, for which the results indicated significant differences between the types of grains analyzed, being indicated the consumption of brown grain to instead polished one, and the genotype \'Primavera\' stood out with higher concentration of proteins and smaller amount of phytate. This experiment also confirmed that lysine catabolism is the main modulation pathway to increase this amino acid in the grain. For the second experiment 16 genotypes cultivated in Brazil were used to characterize the genetic diversity in relation to the major grain properties related to human nutritional quality. Zinc concentration was positively correlated to phytate, but no direct association between Zn and synthesis and concentration of amino acids or storage proteins was detected. Based on results, \'Carolino\' and \'Goiano\' rice cultivars presented the best performance. Moreover, due to the relevance of aleurone layer in retaining important nutritional features in rice grains, biofortification programs should investigate nutrient transportation from this region to endosperm in order to achieve better results.

A Annona crassiflora Mart. é uma espécie frutífera nativa do cerrado que possui alto potencial de uso e ampla distribuição. O presente trabalho tem os seguintes objetivos: identificar e quantificar aminoácidos livres, carboidratos solúveis e ácidos graxos da polpa e das sementes, visando ampliar os conhecimentos sobre potencial econômico de A. crassiflora; avaliar qual o nível de variações quantitativas e qualitativas de alcalóides de A. crassiflora de amostras provenientes de diferentes regiões do cerrado; avaliar qual a magnitude da diversidade genética entre diferentes populações de A. crassiflora, utilizando marcadores isoenzimáticos. Com relação às propriedades nutricionais, as análises de aminoácidos livres, carboidratos e lipídeos foram feitas por CLAE/F, por CLAE/CTI - DPA e por CG/EM, respectivamente. O teor total de aminoácidos livres na polpa variou de 1101,99 g/g a 1975,7 g/g de massa seca, enquanto nas sementes variou de 636,3 μg/g a 17.445,83 μg/g de massa seca. A glutamina foi majoritária em todas as amostras. O teor total dos carboidratos solúveis da polpa variou de 11,5 g/100g a 17,03 g/100g e das sementes variou de 13,69 g/100g a 40,47 g/100g de matéria seca. Foram detectadas a frutose, a glicose e a sacarose em todas as amostras de polpa e de sementes. A quantidade de lipídeos totais da polpa variou de 7g/100g a 16,2 g/100g de massa seca e das sementes variou de 34,36 g/100g a 35,98 g/100g de massa seca. O ácido oléico e/ou petroselínico (18:1) foram majoritários em todas as amostras de polpa e de sementes de diferentes origens geográficas. Certamente os dados obtidos permitiram ampliar os conhecimentos sobre potencial de aproveitamento dessa espécie. Para avaliar a magnitude da variação dos alcalóides das folhas de A. crassiflora, a quantificação e identificação dos alcalóides foram feitas, respectivamente, por CG/FID e CG/EM. Foi verificado que o teor total de alcalóides variou de 221,1 ± 17,14 μg/g a 2986,89 ± 367,1 μg/g de massa seca. Foram identificados os alcalóides anonaína, anoretina, romucosina e xilopina que mostraram diferenças entre as regiões. Essa variação na concentração e no perfil alcaloídico das populações de A. crassiflora sugere ampla plasticidade fenotípica desses metabólitos em resposta a fatores bióticos e/ou abióticos, além de alta variabilidade genética relacionada à expressão dos genes envolvidos na biossíntese desses metabólitos. A avaliação da estrutura genética foi feita através da análise isoenzimática. Os dados isoenzimáticos mostraram baixa variabilidade genética. Duas hipóteses são apontadas para explicar o baixo nível de polimorfismos verificado em A. crassiflora, uma delas relacionada à técnica e outra relacionada ao próprio processo de degradação do ambiente. Diante do exposto, os dados obtidos no presente trabalho sugerem formas de agregar mais valor econômico e ecológico à A. crassiflora e podem proporcionar uma base para o uso mais eficiente e racional dos recursos dessa espécie, contribuindo para exploração sustentável do cerrado, o que permite também o desenvolvimento de comunidades locais. / Annona crassiflora Mart. is a native fruit tree species from the cerrado, possessing high use potential and a wide distribution. The proposed aims were to identify and quantify free amino acids, free sugars and fatty acids in the pulp and seeds of this plant, with a view to widening knowledge on economical potentiality, as well as to evaluate the level of alkaloid quantitative and qualitative variation in samples coming from different regions of the cerrado, and finally define the size of genetic diversity among these different populations by using isoenzymatic markers. As to nutritional properties, analyses of free amino acids, free sugars and fatty acids were undertaken with, respectively, CLAE/F, CLAE/CTI DPA and CG/MS. The total rate of free amino acids in the pulp varied from 1101,99 ug/g to 1975,7 ug/g of dry mass, whereas in the seeds this variation was from 636,3 μg/g to 17.445,83 μg/g. Glutamin was preponderant in all the samples. Total free sugar rate in pulp varied from 11,5 g/100g to 17,03 g/100g and in seeds from 13,69 g/100g to 40,47 g/100g of dry matter. Fructose, glucose and sacarose was detected in all samples. The amount of total lipids in pulp varied from 7 g/100g to 16,2 g/100g of dry weight and in seeds from 34,36 g/100g to 35,98 g/100g. Oleic and/or petroselinic acids (18:1) were the most abundant in all pulp and seed samples from the different geographic regions. The obtained results certainly permit widening knowledge on the potentiality for use of this plant species. In order to evaluate the size of alkaloid variation in A. crassiflora leaves, alkaloid quantification and identification was done with, respectively, CG/FID and and CG/EIMS. It was noted that the total alkaloid rate varied from 221,1 ± 17,14 ug/g to 2986,89 ± 367,1 μg/g of dry mass. The alkaloids anonain, anoretin, romucosin and xilopin were identified, these showing differences among regions. This variation in alkaloid concentration and profile in A. crassiflora populations suggests the wide phenotypic plasticity of these metabolites in response to biotic and/or abiotic factors, besides high genetic variability related to gene expression of those involved in their biosythesis. Genetic structure evaluation was done through isoenzymatic analysis. Isoenzymatic data revealed low genetic variability. Two hypotheses may be raised to explain this low polymorphism level. The first points to inefficient isoenzymatic techniques in reckoning species genetic diversity, whereas the second takes into consideration that excessive homozygosis (indicated by the low level of isoenzymatic variability) could be an indication of the process of species genetic erosion, possibly related to habitat (cerrado) degradation. In view of this, data obtained in the present study suggest forms of aggregating additional economical and ecological value to A. crassiflora, and could furnish a base for the more efficient and rational use of resources from this species, thus contributing to sustainable exploitation of the cerrado, and also allowing for local community development.

O arroz está entre os cereais mais consumidos do mundo. A desnutrição por micronutrientes como zinco (Zn) e ferro (Fe) é generalizada, especialmente em populações pobres, cujas calorias diárias são obtidas principalmente pelo consumo de cereais. Apesar de o arroz ser diferenciado por possuir maior quantidade de lisina em seus grãos em comparação ao trigo, milho ou cevada, a sua quantidade de proteínas representa somente de 8-9% do grão. Sendo o Zn é um micronutriente correlacionado com o funcionamento vital de plantas e animais, e estando relacionado direta e indiretamente com a síntese e funcionamento de várias proteínas, a proposta deste trabalho foi analisar a possível correlação da quantidade de Zn encontrado no grão com o perfil das proteínas de reserva, aminoácidos solúveis, enzimas envolvidas no metabolismo do ácido aspártico e do fator antinutricional ácido fítico, nos grãos de diferentes genótipos de arroz, avaliando primordialmente as diferenças inerentes de cada material genético a fim de agregar conhecimentos que possam ser utilizados como ferramenta para o melhoramento dessa espécie, visando ganho nutricional em seu grão. Para tanto, foram executados dois experimentos distintos. No primeiro foram avaliados três genótipos, os quais foram expostos à adubação foliar com sulfato de zinco, nas concentrações 0, 2 e 4 kg.ha-1 de ZnSO4.7H2O, buscando aumentar-se o nível de Zn nos grãos. No entanto não se verificou incremento significativo do metal na porção de interesse alimentar, sendo a variação encontrada nas concentrações de Zn em grãos integrais e polidos foram inerentes aos genótipos. Ainda assim, foram verificados os outros aspectos nutricionais, para os quais os resultados apontaram diferenças significativas entre os tipos de grãos analisados, sendo indicado o consumo de grão integral ao polido, destacando-se o genótipo Primavera, que apresentou maior concentração de proteínas e menor quantidade de fitato. Esse experimento ainda confirmou que o catabolismo da lisina é a principal via de modulação para o acréscimo desse aminoácido no grão. Para a execução do segundo experimento foram utilizados 16 genótipos cultivados no Brasil, buscando caracterizar a diversidade genética em relação às principais propriedades do grão relacionadas à qualidade nutricional humana. Os dados mostraram que apesar do Zn estar correlacionado positivamente com fitato, não há relação direta entre a concentração desse elemento com a síntese e concentração de aminoácidos e nem com a concentração total de proteínas de reserva. No entanto, com os resultados obtidos para grão integral em relação às características nutricionais, permite apresentar os genótipos Carolino e Goiano como os de melhor desempenho, além de ressaltar a importância da camada da aleurona do grão, já que está relacionada com todas as características nutricionais importantes para um projeto de bioforticação, indicando que estudos devem ser intensificados para entender o transporte de nutrientes até o interior do endosperma, ou ainda, que o consumo de arroz integral deve ser mais reforçado. / Human malnutrition due to low intake of the micronutrients zinc (Zn) and iron (Fe) is relatively common in poor population, who uses cereals as main sources of calories. Currently, rice is one of the most consumed cereals worldwide but, despite its high lysine concentration in comparison to other cereal crops, rice grains have low protein contents (8-9%). Therefore, the current work aimed to evaluate the influence of additional Zn supply on amino acid profile, storage proteins, macro- and micronutrients, as well as on phytate concentration, and on activity of enzymes from lysine metabolism in grains of different rice genotypes, primarily evaluating the inherent differences of each genetic material in order to aggregate knowledge like a tool for the improvement of this species, aiming nutritional gain in its grain. For this purpose, two different experiments were performed. First of all, three genotypes were evaluated and exposed to foliar fertilization with zinc sulfate, in the concentrations 0, 2 and 4 kg.ha-1 of ZnSO4.7H2O, seeking to increase the level of Zn in the grains. However, there was no significant increase of the metal in the grain, and the variation found in Zn concentrations in brown and polished grains was according to the genotypes. Nevertheless, the other nutritional aspects were verified, for which the results indicated significant differences between the types of grains analyzed, being indicated the consumption of brown grain to instead polished one, and the genotype \'Primavera\' stood out with higher concentration of proteins and smaller amount of phytate. This experiment also confirmed that lysine catabolism is the main modulation pathway to increase this amino acid in the grain. For the second experiment 16 genotypes cultivated in Brazil were used to characterize the genetic diversity in relation to the major grain properties related to human nutritional quality. Zinc concentration was positively correlated to phytate, but no direct association between Zn and synthesis and concentration of amino acids or storage proteins was detected. Based on results, \'Carolino\' and \'Goiano\' rice cultivars presented the best performance. Moreover, due to the relevance of aleurone layer in retaining important nutritional features in rice grains, biofortification programs should investigate nutrient transportation from this region to endosperm in order to achieve better results.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Amino acids are organic compounds abundant in animal tissues, especially in meat and meat products, in free form or polymers, participating in biochemical reactions and may be precursors of compounds of interest in the quality control of these foods. The aim of this study was to optimize and validate a method for determination of free amino acids by gas chromatography in Italian dry fermented sausage subjected to ultrasonic bath for 3, 6 and 9 min. Free amino acids were evaluated in salami pieces fermented/seasoned (initial time), at finished product (28 days) and 120 days after manufacture. The extraction was performed using 0.1 N HCl. An aliquot of samples were subjected to vacuum drying, followed by derivatization with MTBSTFA, which has been optimized with respect to time, temperature and reaction solvents (pyridine and acetonitrile). The instrumental and chromatographic conditions of injector temperature, constant pressure and column temperature program was optimized to ensure the method selectivity and efficiency. The use of a mixture of acetonitrile/MTBSTFA in the ratio 1:1 (v/v) using a temperature of 100 °C for 2 h promoted derivatizing all L-amino acids contained in the analytical standards.The injector temperature of 320 °C and 15 psi constant pressure gave the best efficiency in mass transfer of the analytes into the column, while also providing higher resolution and accuracy. The determination coefficients obtained for amino acids were higher than 0.99, except for glycine (0.98). Fourteen amino acids showed a linear range of from 1.0 to 160.0 mg L-1 with LOD and LOQ ranged between 0.3 and 10 g ml-1 and 1 and 20 mg L-1 and inter-day coefficients of variation accurately less than 15% for most analytes.The increase in the concentrations of most amino acids was observed over time processing/storage, indicating that there was an intense proteolytic activity with the release of free amino acids. In the finished product (28 days), treatment with ultrasound affected significantly (p <0.05) concentration of some amino acids. At this point, the amino acids alanine, glycine, valine, leucine, proline and methionine (non-polar) and tyrosine (polar uncharged) showed lower concentrations to control, on the other hand there was an increase of glutamic acid and arginine. The free amino acids found in salami are important compounds because through chemical reactions of decarboxylation or deamination can be precursors of volatile compounds responsible for characteristic processed meat product flavor/aroma. Thus, the employment of no conventional analytical techniques such as gas chromatography for determination of free amino acids allows obtaining information of great importance for the quality control and product development, correlating the technological and nutritional properties with the properties sensory involving amino acids as precursors. / Os aminoácidos são compostos orgânicos abundantes em tecidos animais, sobretudo em carne e produtos cárneos, na forma livre ou de polímeros, participando de reações bioquímicas, podendo ser precursores de compostos de interesse no controle de qualidade destes alimentos. O objetivo do presente trabalho foi otimizar e validar um método para determinação de aminoácidos livres por cromatografia em fase gasosa em salame tipo Italiano submetido a banho ultrassônico por 3, 6 e 9 min. Os aminoácidos livres foram avaliados na massa de salame não fermentada/maturada (tempo inicial), no produto acabado (28 dias) e após 120 dias da fabricação. A extração dos analitos foi realizada com a utilização de HCl 0,1 N. Uma alíquota das amostras foram submetidas à secagem a vácuo seguida de derivatização com MTBSTFA, a qual foi otimizada em relação ao tempo, temperatura e solventes de reação (piridina e acetonitrila). As condições instrumentais e cromatográficas de temperatura do injetor, pressão constante e programação de temperatura da coluna foram otimizadas para garantir a seletividade e eficiência do método. O emprego da mistura de acetonitrila/MTBSTFA na proporção de 1:1 (v/v), utilizando a temperatura de 100 °C por 2 h promoveu a derivatização de todos os L- aminoácidos contidos nos padrões analíticos. A temperatura do injetor a 320 °C e pressão constante de 15 psi proporcionou a melhor na eficiência na transferência de massa dos analitos para coluna, proporcionando também maior resolução e precisão. Os coeficientes de determinação obtidos para os aminoácidos foram maiores que 0,99, com exceção da glicina (0,98). Quatorze aminoácidos apresentaram faixa linear entre 1,0 160,0 μg mL-1, com LOD e LOQ variando entre 0,3 e 10 μg mL-1 e 1 e 20 μg mL-1 e precisão intra-dia com coeficientes de variação menores que 15% para a maioria dos analitos. O aumento nas concentrações da maioria dos aminoácidos foi observado com decorrer do tempo do processamento/armazenamento, indicando que houve uma intensa atividade proteolítica com liberação de aminoácidos livres. No produto pronto (28 dias), os tratamentos com ultrassom afetaram significativa (p<0,05) a concentração de alguns aminoácidos. Nesse ponto, os aminoácidos alanina, glicina, valina, leucina, prolina e metionina (apolares) e tirosina (polar sem carga) apresentaram concentrações inferiores ao controle, por outro lado foi observado um aumento dos de ácido glutâmico e arginina. Os aminoácidos livres encontrados nas amostras de salame são compostos importantes, pois através de reações químicas de descarboxilação ou desaminação podem ser precursores de compostos voláteis responsáveis pelo sabor/aroma característicos do produto cárneo processado. Dessa forma, o emprego de técnicas analíticas não convencionais, como a cromatografia em fase gasosa, para a determinação de aminoácidos livres, permite a obtenção de informações de grande importância no controle de qualidade e desenvolvimento de produtos, correlacionando as propriedades tecnológicas e nutricionais com as propriedades sensoriais que envolvem os aminoácidos como precursores.

Soil carbon (C) and nitrogen (N) are predominantly in organic form. Proteins/ peptides, as an important organic form of N, constitute a substantial part of soil organic matter. On one hand, proteins/peptides are an important N source for plants and microorganisms, particularly in soils where inorganic N is limited. On the other hand, their stabilization in soils by forming organo-mineral associates or macromolecule complex reduces the C loss as CO2 into the atmosphere. Therefore, studies on the turnover, abundance, composition, and stability of proteins/peptides are of crucial importance to agricultural productivity and environmental sustainability. In the first part of this study, the bioavailability and distribution of amino acids, (building block of proteins/peptides), were investigated, in soils across the North-South and West-East transects of continental United States. The second part of this study aimed to understand the variations of organic C speciation in soils of continental United States. Previous investigations of the interactions between soil minerals and proteins/peptides were mostly limited to batch sorption experiments in labs, seldom of which gave the details at the molecular scales. Therefore, in the third part of this study, the molecular orientation of self-assembled oligopeptides on mineral surfaces was investigated by employing synchrotron based polarization-dependent Near Edge X-ray Adsorption Fine Structure Spectroscopy (NEXAFS) techniques. Specific aims of this study were: 1) to assess potentially bioavailable pool of proteinaceous compounds and the immediately bioavailable pool of free amino acids in surface and subsurface soils of various ecosystems; 2) to evaluate the relationship between environmental factors and levels/composition of the two pools; 3) to investigate the organic C speciation in soils of various land use; and 4) to understand molecular level surface organization of small peptides on mineral surfaces. The levels of free amino acids and hydrolysable amino acids which represent the potentially bioavailable pool of proteinaceous compounds in A-horizon soils were significantly high than in C-horizon soils due to the accumulation of organic matter in surface. On average, free amino acids accounted for less than 4 % of hydrolysable amino acids which represent the total proteinaceous compounds in soils. The composition of free amino acids was significantly different between surface soil and subsurface soil and was significantly influenced by mean annual temperature and precipitation. A relatively uniform composition of hydrolysable amino acids was observed irrespective of a wide range of land use. Significant variations were observed for the levels of free and hydrolysable amino acids along mean annual temperature and precipitation gradients, as well as among vegetation types of continental USA, suggesting levels of free and hydrolysable amino acids were associated with the above-ground biomass and root distribution. Organic C speciation investigation revealed the presence of carboxylic-C (38%), aliphatic-C (~ 22%), aromatic-C (~ 18%), O/N-alkyl-C (~ 16%), and phenolic-C (< 6%). Factors such as temperature and vegetation cover were revealed in this study to account for the fluctuations of the proportions of aromatic-C and phenolic-C, in particular. Phenolic-C may serve as a good indicator for the effect of temperature or vegetation on the composition of SOC. The average composition of soil organic C, over the continental scale, was relatively uniform over various soil ecosystems and between two soil horizons irrespective of surface organic C content. Polarization dependent NEXAFS analysis showed the oligopeptides tend to orient on mineral surface with an average tilt angle of 40 ° between the molecular chain and the mineral surface. / Ph. D.