Forty-six barrows fitted with steered ileo-cecal valve cannulas were used in four experiments to evaluate the effects of supplementing swine diets with microbial phytase or beta-mannanase on the apparent ileal (AID) and/or apparent total tract digestibility (ATTD) of amino acids, N, Ca, P, DM and energy. In Exp. 1, the addition of phytase to low CP corn-soybean meal based diets increased the AID of Ca (P < .01), P (P < .001), and all amino acids (P < .10) measured except Leu, Ser, Pro, Met, His and Tyr. In Exp. 2, the addition of microbial phytase to corn-soybean meal, corn-soybean meal-wheat middlings, or corn-soybean meal-meat and bone meal based diets resulted in increased AID of Ca and P, but had no effect (P > .1) on amino acid digestibilities. Diet type affected all digestibility measurements, but did not affect the efficacy of supplemental phytase. In Exp. 3, the addition of microbial phytase to corn-wheat-soybean meal, corn-wheat-cannola, or sorghum-corn-soybean meal based diets led to an increased ( P <.05) AID of P, Asp, Thr, Ser, Ala, Tyr, Phe, Lys and Arg. In Exp. 4, the addition of beta-mannanase to corn-soybean meal based swine diets led to an increased AID of DM and ATTD of energy. In addition, the AID of all amino acids measured were increased numerically, with many of these values approaching significance. The results of these studies demonstrate that supplementing pig diets with phytase or beta-mannanase, results in an increased digestibility of certain dietary components due to the breakdown of anti-nutritive compounds in the diet. / Ph. D.

Durante a maturação da semente, a partir da fertilização do óvulo, há alterações inerentes à formação da semente, com destaque para as variações do tamanho, do teor de água, das massas da matéria fresca e seca e da germinação das sementes. Paralelamente, para os frutos carnosos há alterações visíveis nos frutos relacionados à forma, à cor e à senescência. Não há, todavia, relatos de quais mudanças (morfológica, anatômica, fisiológica e bioquímica) ocorrem nas sementes que se desenvolvem no interior de frutos carnosos mantidos em repouso por diferentes períodos de tempo. O objetivo dessa pesquisa foi avaliar a fase final da formação das sementes de pimentão, considerando as variações da morfologia externa dos frutos e do parâmetro fisiológico, da morfologia interna, da enzima endo-?-mananase, da anatomia e do acúmulo de substâncias de reservas e do ciclo celular das sementes, visando verificar se essas mudanças explicam as alterações fisiológicas das sementes de pimentão após o repouso pós-colheita do fruto e durante o armazenamento das sementes. As sementes foram extraídas de frutos em diferentes estádios de maturação, (cores verde, verde-avermelhada e vermelha), mantidos em repouso por 3, 7 ou 14 dias ou sem repouso, e avaliadas logo após a colheita e durante o armazenamento. Os frutos foram avaliados quanto à coloração, dimensões e massa. As sementes foram avaliadas quanto ao teor de água, massa de matéria seca, massa de 1000 sementes, germinação e vigor, além da avaliação de imagens de raios X para o estudo da morfologia interna das sementes, da atividade da enzima endo-?-mananase pelo método da difusão em gel, análises histoquímicas para o estudo da anatomia e de substâncias de reserva e a avaliação do ciclo celular. A colheita das sementes de pimentão pode ser caracterizada pela coloração dos frutos. Em função do repouso há alteração da cor do fruto, do ciclo celular das sementes hidratadas, da atividade da enzima endo-?-mananase e de algumas substâncias de reserva, interferindo positivamente na qualidade das sementes de pimentão. Entretanto, o repouso de frutos, que estão em estágio avançado de desenvolvimento, causa redução da qualidade das sementes. O teor de água, a massa de matéria seca e a morfologia interna da semente, a anatomia das células do endosperma e a presença de substâncias de reservas das sementes não são alterados durante o repouso do fruto, mas estão relacionados ao estádio de formação das sementes. Na medida em que há a deterioração natural das sementes durante o armazenamento, há redução da germinação e do vigor, da atividade da endo-?-mananase, da quantidade de lipídios, proteínas e polissacarídeos das células do embrião e do endosperma. Durante o repouso do fruto por 14 dias, as sementes extraídas de frutos verdes têm tolerância à desidratação, caracterizada principalmente pelo aumento da germinação após a secagem das sementes. A maturidade fisiológica das sementes é caracterizada pela coloração verde-avermelhada dos frutos e também pela estabilização do teor de água das sementes (30%), pela quantidade de matéria seca e pelo conteúdo de DNA 4C; não há coincidência entre maturidade fisiológica e máxima germinação. / During seed maturation there are some significant alterations in seed size, moisture content, fresh and dry matter and germination. Furthermore, for fleshy fruits, visible alterations occur as the fruit shape, color and senescence. There are not reports about which morphological, anatomical, physiological and biochemical alterations occur in seeds that develop inside fleshy fruits during the rest of them, for different periods of time. The objective of this research was to evaluate the final stage of the bell pepper seed formation and it was considered the variations of the external morphology of the fruit, and the physiological parameter, internal morphology, activity of endo-?-mannanase, anatomy and the reserve substances accumulation, and cell cycle of the seeds to verify if these changes explain the physiological changes of the bell pepper seeds after the fruit resting and during the seed storage. The seeds were extracted from fruits at different maturation stages (colors green, reddish-green and red), with (3, 7 or 14 days) or without fruit resting, and during the seed storage. The fruits were evaluated by color, and the measurements and weight. The seeds were evaluated for moisture content, dry mass, mass of 1000 seeds, germination and vigor and X-ray imaging to study the internal morphology of the seeds, endo-?-mannanase activity by gel diffusion assay, histochemical analyzes to study seed anatomy and reserve substances, and flow cytometry to assess the cell cycle. The harvest of bell peppers seeds can be made by the color of the fruits. The fruit resting alters the fruit color, germination, vigor, endo-?-mannanase activity, some storage substances and cell cycle improving bell pepper seed quality. However it has a negative role when the fruits are in advanced stage of development (red fruits and resting time more than 7 days). The moisture content, dry matter and the internal morphology of the seeds do not change by the fruit resting, but because of the seed maturation stage. It was observed reduction of all the physiological parameters, including the amount of lipids in the cells and endo-?-mannanase activity. During fruit resting, the endo-?-mannanase activity and the 4C DNA content of the cells decrease and there is an increased rate of germination and vigor. The water content, dry matter, internal morphology and anatomy of the cells of the endosperm and the presence of reserves substances in the seeds are not changed due to the rest of the fruit, but are related to the seed formation stage. There is natural seed deterioration during storage and a reduction of germination and vigor, endo-?-mannanase activity, amount of lipids, proteins and polysaccharides in the embryo and endosperm cells. During the fruit resting (14 days), the seeds extracted from green fruits have desiccation tolerance, characterized by increased of the seed germination after drying. The physiological maturity is reached when the fruits are reddish-green, characterized by stabilizing the water content of the seeds (30 %), amount of dry matter and the 4C DNA content. There is not a coincidence between physiological maturity and maximum germination.

Durante a maturação da semente, a partir da fertilização do óvulo, há alterações inerentes à formação da semente, com destaque para as variações do tamanho, do teor de água, das massas da matéria fresca e seca e da germinação das sementes. Paralelamente, para os frutos carnosos há alterações visíveis nos frutos relacionados à forma, à cor e à senescência. Não há, todavia, relatos de quais mudanças (morfológica, anatômica, fisiológica e bioquímica) ocorrem nas sementes que se desenvolvem no interior de frutos carnosos mantidos em repouso por diferentes períodos de tempo. O objetivo dessa pesquisa foi avaliar a fase final da formação das sementes de pimentão, considerando as variações da morfologia externa dos frutos e do parâmetro fisiológico, da morfologia interna, da enzima endo-?-mananase, da anatomia e do acúmulo de substâncias de reservas e do ciclo celular das sementes, visando verificar se essas mudanças explicam as alterações fisiológicas das sementes de pimentão após o repouso pós-colheita do fruto e durante o armazenamento das sementes. As sementes foram extraídas de frutos em diferentes estádios de maturação, (cores verde, verde-avermelhada e vermelha), mantidos em repouso por 3, 7 ou 14 dias ou sem repouso, e avaliadas logo após a colheita e durante o armazenamento. Os frutos foram avaliados quanto à coloração, dimensões e massa. As sementes foram avaliadas quanto ao teor de água, massa de matéria seca, massa de 1000 sementes, germinação e vigor, além da avaliação de imagens de raios X para o estudo da morfologia interna das sementes, da atividade da enzima endo-?-mananase pelo método da difusão em gel, análises histoquímicas para o estudo da anatomia e de substâncias de reserva e a avaliação do ciclo celular. A colheita das sementes de pimentão pode ser caracterizada pela coloração dos frutos. Em função do repouso há alteração da cor do fruto, do ciclo celular das sementes hidratadas, da atividade da enzima endo-?-mananase e de algumas substâncias de reserva, interferindo positivamente na qualidade das sementes de pimentão. Entretanto, o repouso de frutos, que estão em estágio avançado de desenvolvimento, causa redução da qualidade das sementes. O teor de água, a massa de matéria seca e a morfologia interna da semente, a anatomia das células do endosperma e a presença de substâncias de reservas das sementes não são alterados durante o repouso do fruto, mas estão relacionados ao estádio de formação das sementes. Na medida em que há a deterioração natural das sementes durante o armazenamento, há redução da germinação e do vigor, da atividade da endo-?-mananase, da quantidade de lipídios, proteínas e polissacarídeos das células do embrião e do endosperma. Durante o repouso do fruto por 14 dias, as sementes extraídas de frutos verdes têm tolerância à desidratação, caracterizada principalmente pelo aumento da germinação após a secagem das sementes. A maturidade fisiológica das sementes é caracterizada pela coloração verde-avermelhada dos frutos e também pela estabilização do teor de água das sementes (30%), pela quantidade de matéria seca e pelo conteúdo de DNA 4C; não há coincidência entre maturidade fisiológica e máxima germinação. / During seed maturation there are some significant alterations in seed size, moisture content, fresh and dry matter and germination. Furthermore, for fleshy fruits, visible alterations occur as the fruit shape, color and senescence. There are not reports about which morphological, anatomical, physiological and biochemical alterations occur in seeds that develop inside fleshy fruits during the rest of them, for different periods of time. The objective of this research was to evaluate the final stage of the bell pepper seed formation and it was considered the variations of the external morphology of the fruit, and the physiological parameter, internal morphology, activity of endo-?-mannanase, anatomy and the reserve substances accumulation, and cell cycle of the seeds to verify if these changes explain the physiological changes of the bell pepper seeds after the fruit resting and during the seed storage. The seeds were extracted from fruits at different maturation stages (colors green, reddish-green and red), with (3, 7 or 14 days) or without fruit resting, and during the seed storage. The fruits were evaluated by color, and the measurements and weight. The seeds were evaluated for moisture content, dry mass, mass of 1000 seeds, germination and vigor and X-ray imaging to study the internal morphology of the seeds, endo-?-mannanase activity by gel diffusion assay, histochemical analyzes to study seed anatomy and reserve substances, and flow cytometry to assess the cell cycle. The harvest of bell peppers seeds can be made by the color of the fruits. The fruit resting alters the fruit color, germination, vigor, endo-?-mannanase activity, some storage substances and cell cycle improving bell pepper seed quality. However it has a negative role when the fruits are in advanced stage of development (red fruits and resting time more than 7 days). The moisture content, dry matter and the internal morphology of the seeds do not change by the fruit resting, but because of the seed maturation stage. It was observed reduction of all the physiological parameters, including the amount of lipids in the cells and endo-?-mannanase activity. During fruit resting, the endo-?-mannanase activity and the 4C DNA content of the cells decrease and there is an increased rate of germination and vigor. The water content, dry matter, internal morphology and anatomy of the cells of the endosperm and the presence of reserves substances in the seeds are not changed due to the rest of the fruit, but are related to the seed formation stage. There is natural seed deterioration during storage and a reduction of germination and vigor, endo-?-mannanase activity, amount of lipids, proteins and polysaccharides in the embryo and endosperm cells. During the fruit resting (14 days), the seeds extracted from green fruits have desiccation tolerance, characterized by increased of the seed germination after drying. The physiological maturity is reached when the fruits are reddish-green, characterized by stabilizing the water content of the seeds (30 %), amount of dry matter and the 4C DNA content. There is not a coincidence between physiological maturity and maximum germination.

Aspergillus fumigatus produces enzymes required for the hydrolysis of galactomannans like locust bean gum. Among these enzymes endo-beta-1,4 mannanase is also produced at high levels. However, the fungus is not safe for use in the food industry. Therefore, the gene encoding endo-beta-1,4-mannanase of A. fumigatus IMI 385708 was previously cloned in our laboratory into Aspergillus sojae ATCC11906 which is a safe microorganism for use in food applications. Altogether eight transformants were obtained. It was shown that some of these transformants overproduce the enzyme because of expression under the control of glyceraldehyde-3-phosphate dehydrogenase promoter and fusion to the glucoamylase signal and pro-peptide coding region of Aspergillus niger. In this study, mannanase production of these transformants was compared with A. fumigatus and A. sojae transformant AsT1 showed c. 12 fold increase with the maximum activity of 352 U/ml. The effects of initial medium pH and number of spores on activity were investigated and maximum activity was achieved at pH 7.0 and the number of spores was found as 3.6 &times / 106. Optimization of the growth conditions for maximum mannanase production in shake flasks by using the best mannanase producing transformant AsT1 was carried out by using Box-Behnken design under Response Surface Methodology. The highest beta-mannanase activity on the fourth day of cultivation at 30 &ordm / C was obtained as 363 U/ml in the optimized medium containing 7% sugar beet molasses, 0.43% NH4NO3, 0.1% K2HPO4, 0.05% MgSO4 as the weight/volume percentage at 207 rpm. On sixth day of cultivation under the optimized conditions, the highest mannanase activity was achieved as 482 U/ml which is 1.4 fold of 352 U/ml activity found on glucose medium previously. After 48 h of LBG hydrolysis by 40 U of mannanase, mannotriose, 61-galactosyl-beta-D-mannotriose and 63,64-di-alpha-galactosyl-beta-1,4-mannopentaose were found as the main products via HPLC analysis.