Doctor of Philosophy / Department of Food, Nutrition, Dietetics, and Health / Brian L. Lindshield / Iron, vitamin A, and protein inadequacies are common in food-aid receiving countries, and maximizing nutrient intake and bioavailability are essential treatments. Fortified-blended foods (FBFs), are food-aid micronutrient-fortified legume-grain porridges distributed worldwide. FBFs have not consistently, effectively treated undernutrition, and it has been suggested that formulation and processing changes could improve their nutritional quality. Sorghum is a well suited FBF commodity, but high concentrations of ‘antinutritional’ tannin and phytic acid have limited its adoption. Iron bioavailability adaptation may be possible after long-term antinutritional factor consumption, but adaptive mechanisms are not well understood. In rats, salivary proline-rich proteins (PRPs) have been found to chelate tannins to improve iron bioavailability, this could be true for people as well. Several research design methods were employed to summarize FBF quality outcomes and the effect of tannin and phytic acid consumption on iron bioavailability. Extruded sorghum and corn FBFs were developed; protein quality, iron, and vitamin A outcomes were compared with a non-extruded corn-soy blend (CSB+) in rats. A narrative literature review and meta-analysis were conducted to determine tannin’s antinutritional effects on iron bioavailability, and the potential for adaptation through salivary PRPs. Two clinical trials examined the effect of long-term tannin or phytic acid consumption on iron bioavailability, salivary protein production, and correlations between PRPs and iron bioavailability. There were no differences between iron (hepatic iron 207-300 µmol/g *100), vitamin A (hepatic retinol 423-585.5 ng/mg), or protein quality (caloric efficiency: 101.3-113.3 g/kcal*100) between extruded FBFs regardless of commodity in rats. Compared to extruded FBFs, CSB+ caloric efficiency (49.0 ± 2.2 g/kcal*100) and growth (96.3 ± 3.4g vs. 208.6-236.6) were significantly reduced. A literature review suggested that there were differences in acute meal and long-term iron bioavailability with tannin consumption; tannic acid inhibited iron availability, while food-tannins did not. Meta-analysis suggested that tannin-PRP binding could protect iron bioavailability, that long-term tannin consumption did not significantly affect hepatic iron or non-heme iron absorption respectively in rats (d = -0.64-1.84; -2.7-0.13), and that PRP expression in rats during tannin consumption was correlated with improved iron bioavailability. There were no reductions in iron bioavailability or status based on long-term tannin (ps > 0.126) or phytic acid (ps > 0.08) consumption clinically, but basic PRP and cystatin subtypes were significantly correlated with improved iron bioavailability during tannin (ps < 0.03) and phytic acid (ps < 0.02) consumption. In vitro, it phytic acid-PRP binding did not occur, but phytic acid did specifically bind with cystatin SN, a non-enzymatic salivary protein. In conclusion, FBF formulation changes may improve protein quality, and provide needed macronutrients to food-aid receiving areas. Despite this, this research did not suggest that antinutritional factors affected iron bioavailability. In support of this finding, literature, and clinical studies presented here suggest that salivary proteins, including PRPs and cystatin, may serve as adaptive protective mechanisms against phytic acid and tannin consumption, and that further research may be warranted before further recommendations for their removal from food-aid are made.

O feijão é um importante alimento na dieta alimentar do brasileiro. A cada colheita ocorrem perdas dos grãos de feijão devido ao ataque de insetos e roedores. Uma das maneiras de se preservar os grãos de feijão, mantendo suas características nutricionais, é o uso da radiação gama. O objetivo do trabalho é avaliar os efeitos de diferentes doses da radiação gama nas propriedades físico-químicas do feijão carioca submetido à cocção e que sofreu ou não o processo de maceração. Partes dos feijões cariocas crus foram utilizadas como controle e outra parte foram submetidos à radiação ionizante com doses de 1, 5 e 10 kGy. Após a irradiação uma parcela das amostras (controle e irradiadas) passou pela maceração e outra não, em seguida, todos os feijões foram cozidos. As amostras cozidas foram liofilizadas, moídas e depois acondicionas em potes e armazenadas a temperaturas de -23ºC. As análises de composição centesimal, determinação da digestibilidade proteica, teores de tanino condensado e ácido fítico foram feitas utilizando as amostras moídas. Utilizando grãos inteiros, foram feitas análises de capacidade expansão e hidratação, tempo de cocção e cor instrumental. A irradiação não alterou a composição centesimal das amostras maceradas e sem macerar. Os teores de taninos condensados não reduziram conforme o aumento das doses. As concentrações de ácido fítico foram reduzidas nas doses de 5 e 10 kGy, para amostras maceradas, enquanto que para os feijões sem macerar, a irradiação gama não influenciou nos teores de fitatos. Ocorreu diminuição da digestibilidade proteica, nas amostras maceradas, a partir dose de 1 kGy, e as demais doses a redução não foi significativa. Já para os feijões sem macerar, houve aumento da digestibilidade, na dose de 10 kGy. Conforme o aumento das doses foi observado redução nos tempos de cocção dos feijões macerados e sem macerar. Na dose de 10 kGy, a capacidade de expansão do feijão aumentou. A coloração das amostras não modificou significativamente conforme o aumento das doses. Desta forma conclui-se que a radiação ionizante não influencia na composição centesimal e nos teores de taninos. A irradiação influenciou nos teores de ácido fítico em altas doses aplicadas, mas somente nas amostras maceradas, além de atenuar a digestibilidade proteica de feijões macerados. Reduz os tempos de cocção. Eleva a capacidade de expansão do feijão na dose de 10 kGy, porém não altera a coloração do feijão, conforme o aumento das doses / The common bean is an important component in the diet of the average Brazilian person. Each harvest of beans, losses occur due to attacks of insects and rodents. One of the ways to preserve the beans, and at the same time keep its nutritional characteristics, is the use of gamma radiation. This study aimed to evaluate the effects of different doses of gamma radiation on the physical and chemical properties of the carioca variety of common beans subject to cooking of soaked and unsoaked beans. Portions of raw beans were used as control and the other was subject to ionizing radiation at doses of 1, 5 and 10 kGy. Following irradiation, a portion of the samples (control and irradiated) were soaked and the other was not, then all beans were cooked. The cooked samples were lyophilized, milled and then accommodated in pots and stored at -23ºC temperatures. The analysis of chemical composition, determination of protein digestibility, condensed tannin and phytic acid content were performed using the milled samples. Using whole grains, were performed analysis of expansion capability and hydration, cooking time and instrumental color. Irradiation did not alter the chemical composition of soaked and unsoaked samples. The condensed tannin levels did not reduce according to increased doses. The phytic acid concentrations were reduced at the doses of 5 and 10 kGy for soaked samples, whereas for the not soaked beans, gamma irradiation did not influence the phytate content. The protein digestibility decreased on soaked samples, at doses of 1 kGy and in the other doses, the reduction was not significant. As for not soaked beans, increases in digestibility were observed at dose of 10 kGy. As the doses increased, reduction in cooking time on soaked and unsoaked beans was noted. At a dose of 10 kGy, the bean expansion capability increased. The samples\' color did not change significantly, as the doses increased. Therefore, it is concluded that ionizing radiation has no effect on the chemical composition and tannin content. Irradiation influenced the phytates when applied in higher doses, but only in soaked samples, as well as mitigate protein digestibility on soaked beans. It reduces cooking time, raises bean expansion capability at a 10 kGy dose, but it does not change the bean color as doses increase

This study has assessed the effects of pulse type and processing (soaking and cooking) on antinutritional factors (α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor, lectins, phytic acid and oxalates) in a wide range of Canadian pulses, using soybean as a control. The contents of these antinutrients in Canadian pulses varied widely, but the levels were generally lower than those found in soybean. Analysis of variance indicated that both pulse type and processing had significant effects (P < 0.0001) on the investigated seeds. Soaking markedly decreased the contents of α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor, lectins and oxalates, but had no impact on phytic acid. Cooking of presoaked seeds appeared to be more effective; all proteinaceous antinutrients (α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor and lectins) were decreased by 80 – 100%, and significant reductions in phytic acid content (11 – 39%) were observed for all pulses, except common beans and soybean. / February 2016

Orientador: José Roberto Sartori / Resumo: Avaliou-se o efeito da suplementação de protease na digestibilidade e desempenho de frangos de corte em ambiente termoneutro e estresse cíclico pelo calor. Foram utilizados 320 pintos machos Cobb de um dia distribuídos casualmente em esquema fatorial 2x2x2 [ambiente termoneutro ou ambiente quente x dieta atendendo as exigências nutricionais (controle positivo, CP) ou reduzida 6% em proteína bruta e aminoácidos (controle negativo, CN) x protease: 0 ou 200ppm] com 8 repetições. De 1 a 7 dias de idade, a conversão alimentar (CA) foi melhor em frangos CN quando em ambiente termoneutro, e quando em ambiente quente, a CA melhorou com a dieta CP. Frangos em ambiente termoneutro apresentaram melhor CA e maior ganho de peso no período de 1 a 14 dias. Maior consumo de ração e ganho de peso foram observados em frangos mantidos em ambiente termoneutro nos períodos de 1 a 21 dias e 1 a 35 dias de idade. No período de 1 a 35 dias de idade houve interação entre dieta e protease, sendo que frangos que receberam dietas CP apresentaram melhor CA quando receberam protease. Frangos que receberam protease melhoraram a conversão alimentar quando consumiram CP comparados aos que receberam CN. A suplementação de protease favoreceu balanço de nitrogênio, coeficiente de metabolizabilidade da proteína bruta e da matéria seca e a energia metabolizável aparente e energia metabolizável aparente corrigida pelo nitrogênio em frangos de corte em ambiente quente com dieta controle positivo. Concluindo, o estr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of the current study was to evaluate the effects of protease supplementation on diet digestibility and growth performance of broilers raised under thermoneutral condition and cyclic heat stress. In a 2x2x2 factorial system [thermoneutral condition or heat stress vs. diets formulated to meet nutritional (positive control, PC) or diets with crude protein and amino acids below requirements by 6% (negative control, NC) x Protease: 0.200ppm], 320 one-day-old male Cobb broilers were distributed in 8 replicates. From 1 to 7 days of age, feed conversion ratio (FCR) was better in thermoneutral condition, and in heat stress, feed conversion ratio (FCR) was better when consume PC. Broilers in thermoneutral environment presented best FCR and greater weight gain during the period from 1 to 14 days. Increased feed consumption and weight gain were observed in broilers kept in thermoneutral environment in periods of 1 to 21 days and 1 to 35 days old. During the period from 1 to 35 days of age there was interaction between diet and protease, and broilers that were given diets CP presented best FCR when they received protease. Broilers receiving protease improved feed conversion when consumed PC compared to those receiving NC. Protease supplementation favored nitrogen balance, metabolizable crude protein, dry matter, apparent metabolizable energy and nitrogen-corrected apparent metabolizable energy in broiler chickens in heat stress with positive control diet. In conclusion, heat... (Complete abstract click electronic access below) / Mestre

Orientador: Luiz Edvaldo Pezzato / Banca: Wilson Massamitu Furuya / Banca: Fabiana Pilarski / Resumo: O farelo de pinhão manso (Jatropha curcas) é um ingrediente protéico que se apresenta como possível substituto alternativo do farelo de soja nas rações. Entretanto, faltam informações a respeito de seu valor nutritivo e da ação dos fatores antinutricionais presentes, para peixes. Por 90 dias foi realizado experimento para avaliar o efeito da inclusão do farelo de pinhão manso (FPM) em dietas para tilápia do Nilo. Foram utilizados 240 peixes com 13,82 ± 0,39 g de peso médio inicial e foram distribuídos em 30 aquários de 250L num delineamento inteiramente casualizado. Os peixes foram alimentados com dietas (isoprotéicas = 32,00% PB, isoenergéticas = 4000 kcal ED/kg e isofibrosas = 8,7% de FB), contendo níveis crescentes de inclusão de FPM (0,0; 1,0; 2,0; 4,0; 8,0 e 16,0%). A composição química do FPM, o consumo de ração (CR), ganho de peso (GP), conversão alimentar (CA), porcentagem de mortalidade (MORT%) e índice hepatossomático (IHS) foram avaliados. Aos 45 e 90 dias todos os peixes foram anestesiados e pesados. Um peixe por aquário foi sacrificado para determinar a proteína bruta (PB), extrato etéreo (EE), umidade (UM) e cinzas das carcaças. Os valores de UM, cinzas e EE diminuíram com a inclusão de FPM na ração (P<0,05). Peixes alimentados com 2, 4 e 8% de FPM apresentaram maior teor de PB. Esta diferença foi provavelmente devido ao menor teor de EE desses peixes. Observou-se efeito quadrático negativo para as variáveis GP e CR e efeito quadrático positivo para a CA. A equação de regressão demonstrou que valores acima de 5,55% de inclusão de farelo de pinhão manso são significativamente prejudiciais para o desenvolvimento dos peixes. O IHS apresentou efeito linear negativo de acordo com o aumento do nível de FPM na dieta. Os peixes do tratamento 16%FPM apresentaram menor tempo de vida e 100% de mortalidade, indicando a alta toxicidade do FPM... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Jatropha curcas meal is a vegetable protein-rich ingredient which could be used in animal diets as a soybean meal substitute. Otherwise, information is lacking regarding to its nutritional value and the effect of antinutritional factors for fish. A 90-day feeding trial was performed to evaluate the effect of jatropha meal (JM) inclusion in diets for Nile tilapia. Two hundred and forty fish with 13.82 ± 0.39g mean initial weight were randomly assigned to 30 250l-aquaria in a completely randomized design. Fish were fed diets (isonitrogenous = 32.00% DP, isoenergetic = 4000 kcal GE/kg diet and same fiber level = 8.7% CF) containing graded JM levels (0.0, 1.0, 2.0, 4.0, 8.0 and 16.0%). Jatropha meal chemical composition, feed intake (FI), weight gain (WG), feed conversion ratio (FCR), mortality and hepatosomatic index (HSI) were evaluated. At 45 and 90 days all fish were anesthetized and weighed. One fish per aquarium was killed to determine dry matter (DM), ash, crude protein (CP) and ether extract (EE) content of fish carcass. Ash, DM and EE content reduced according to JM inclusion levels (P<0.05). Fish fed 2, 4 and 8% JM had higher CP content. This difference was probably due to the lower EE content of these fish. A negative quadratic effect was observed for WG and FI while positive quadratic effect was observed for FCR. The regression equation demonstrated that inclusion JM levels above 5.55% depressed fish growth. HSI showed negative linear effect according to increasing JM level in the diet. Fish fed 16% JM showed the lowest life span and completely died before the end of the experiment, indicating the high toxicity of JM. Tilapia fed JM showed anorexia signs, erratic swimming... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:27:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-06Bitstream added on 2014-06-13T18:48:01Z : No. of bitstreams: 1 santos_vg_me_botfmvz.pdf: 160598 bytes, checksum: 2fe58f8db5442f8ac8dc644c76ac3c0a (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Várias oleaginosas estão sendo estudadas para a produção de biocombustíveis. O processamento do grão para a obtenção do óleo resulta numa diversidade de subprodutos, entre eles o farelo de nabo forrageiro (Raphanus sativus) que, por possuir alto teor protéico, se apresenta como possível sucedâneo do farelo de soja nas rações. Entretanto, faltam informações de seu valor nutritivo e da ação dos fatores antinutricionais presentes. Este estudo foi realizado na Unesp - Universidade Estadual Paulista, da Faculdade de Medicina Veterinária e Zootecnia, Campus de Botucatu, para determinar os coeficientes de digestibilidade aparente (CDA) da matéria seca (MS), proteína bruta (PB), energia bruta (EB), aminoácidos (AA) e a disponibilidade do fósforo do farelo de nabo forrageiro, pela tilápia-do-Nilo. Empregouse uma ração purificada (referência) e uma ração composta de 60% da ração referência e 40% do farelo de nabo forrageiro. O farelo de nabo forrageiro apresentou 91,28% de MS; 42,07% de PB, 4256 kcal/kg de EB, 3,47% de EE, 7,37% de FB, 1,25% de cálcio e 1,0% de fósforo. Os CDA foram de 55,92% para MS, 82,10% para PB, 75,26% para EB e 85,23% do fósforo. Os CDA dos AA estiveram entre 81,12% para a glicina e 95,11% para o ácido glutâmico. Os resultados demonstram que o farelo de nabo forrageiro apresenta potencial para ser usado como fonte protéica alternativa para compor a ração da tilápia do Nilo. / Oil seeds have been studied to produce bio combustibles. In order to obtain oil, seed processing results in several byproducts, among them, the fodder radish meal (Raphanus sativus) which has high protein content. This byproduct is a potential substitute to soybean meal in feed manufacture. However, there is a lack of information regarding its nutritional value and the anti-nutritional content and effects. This study was conduced at the Veterinary Medicine and Animal Science College from Sao Paulo State University, Botucatu, to determine the apparent digestibility coefficient (ADC) of dry matter (DM), crude protein (CP), raw energy (RE), amino acids (A) and phosphorus availability. The fish were fed a purified feed (base diet) and a 60% base diet plus 40% of fodder radish meal feed. Radish meal presented 91.98% DM, 42% CP, 4256 kcal/kg RE, 3.47% Ether extract, 7.37% crude fiber, 1.25% calcium and 1.0% phosphorus. The ADC values were DM: 55.92%, CP: 82.10%, RE: 75.26% and phosphorus: 85.23%. The ADC value for AA was 81.12% for glycine and 95.11% for glutamic acid. The results presented a potential utility for radish meal as an alternative protein source for Nile tilapia feeds.

Made available in DSpace on 2014-06-11T19:22:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-08Bitstream added on 2014-06-13T19:27:41Z : No. of bitstreams: 1 fernandes_rn_me_jabo.pdf: 2674353 bytes, checksum: 1df586cc790a3f677f20fa5c520ce6d9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / O farelo de pinhão manso (Jatropha curcas) é um ingrediente protéico que se apresenta como possível substituto alternativo do farelo de soja nas rações. Entretanto, faltam informações a respeito de seu valor nutritivo e da ação dos fatores antinutricionais presentes, para peixes. Por 90 dias foi realizado experimento para avaliar o efeito da inclusão do farelo de pinhão manso (FPM) em dietas para tilápia do Nilo. Foram utilizados 240 peixes com 13,82 ± 0,39 g de peso médio inicial e foram distribuídos em 30 aquários de 250L num delineamento inteiramente casualizado. Os peixes foram alimentados com dietas (isoprotéicas = 32,00% PB, isoenergéticas = 4000 kcal ED/kg e isofibrosas = 8,7% de FB), contendo níveis crescentes de inclusão de FPM (0,0; 1,0; 2,0; 4,0; 8,0 e 16,0%). A composição química do FPM, o consumo de ração (CR), ganho de peso (GP), conversão alimentar (CA), porcentagem de mortalidade (MORT%) e índice hepatossomático (IHS) foram avaliados. Aos 45 e 90 dias todos os peixes foram anestesiados e pesados. Um peixe por aquário foi sacrificado para determinar a proteína bruta (PB), extrato etéreo (EE), umidade (UM) e cinzas das carcaças. Os valores de UM, cinzas e EE diminuíram com a inclusão de FPM na ração (P<0,05). Peixes alimentados com 2, 4 e 8% de FPM apresentaram maior teor de PB. Esta diferença foi provavelmente devido ao menor teor de EE desses peixes. Observou-se efeito quadrático negativo para as variáveis GP e CR e efeito quadrático positivo para a CA. A equação de regressão demonstrou que valores acima de 5,55% de inclusão de farelo de pinhão manso são significativamente prejudiciais para o desenvolvimento dos peixes. O IHS apresentou efeito linear negativo de acordo com o aumento do nível de FPM na dieta. Os peixes do tratamento 16%FPM apresentaram menor tempo de vida e 100% de mortalidade, indicando a alta toxicidade do FPM... / Jatropha curcas meal is a vegetable protein-rich ingredient which could be used in animal diets as a soybean meal substitute. Otherwise, information is lacking regarding to its nutritional value and the effect of antinutritional factors for fish. A 90-day feeding trial was performed to evaluate the effect of jatropha meal (JM) inclusion in diets for Nile tilapia. Two hundred and forty fish with 13.82 ± 0.39g mean initial weight were randomly assigned to 30 250l-aquaria in a completely randomized design. Fish were fed diets (isonitrogenous = 32.00% DP, isoenergetic = 4000 kcal GE/kg diet and same fiber level = 8.7% CF) containing graded JM levels (0.0, 1.0, 2.0, 4.0, 8.0 and 16.0%). Jatropha meal chemical composition, feed intake (FI), weight gain (WG), feed conversion ratio (FCR), mortality and hepatosomatic index (HSI) were evaluated. At 45 and 90 days all fish were anesthetized and weighed. One fish per aquarium was killed to determine dry matter (DM), ash, crude protein (CP) and ether extract (EE) content of fish carcass. Ash, DM and EE content reduced according to JM inclusion levels (P<0.05). Fish fed 2, 4 and 8% JM had higher CP content. This difference was probably due to the lower EE content of these fish. A negative quadratic effect was observed for WG and FI while positive quadratic effect was observed for FCR. The regression equation demonstrated that inclusion JM levels above 5.55% depressed fish growth. HSI showed negative linear effect according to increasing JM level in the diet. Fish fed 16% JM showed the lowest life span and completely died before the end of the experiment, indicating the high toxicity of JM. Tilapia fed JM showed anorexia signs, erratic swimming... (Complete abstract click electronic access below)

Made available in DSpace on 2015-03-26T13:42:29Z (GMT). No. of bitstreams: 1 texto completo.pdf: 546340 bytes, checksum: c6ef1899560e5249142f8d7f9943743d (MD5) Previous issue date: 2006-09-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Soybean is one of the most important crops in the world extensively used as a food and feed source. However, the proteins present in soybean seeds are not considered ideal because they contain low amounts of the essential amino acids methionine and lysine. Adverse nutritional and other effects following consumption of raw soybean meal have been attributed to the presence of endogenous inhibitors of digestive enzymes and allergenic factors. Among these proteins are the P34 protein and the Bowman-Birk protease inhibitors. The absence of these factors would increase the nutritional quality of the soybean seeds, as well as the availability of this product to large number of consumers. Recent advances in plant biotechnology have enabled the silencing of specific genes. One of the techniques used to achieve this goal is RNA interference (RNAi). This method of posttranscriptional gene silencing is highly efficient, especially when the construct results in the formation of a intron-spliced hairpin RNA. It is proposed that RNAi works by enzymatic RNA degradation induced by double-stranded RNA. The main goal of this work was to construct expression cassettes for RNAi-based silencing of genes encoding antinutritional factors in soybean seeds. To achieve seed-specific expression, specific primers were designed for the amplification of the promoter region of the alpha-subunit of the beta-conglycinin gene. Appropriate restriction sites - Sac I and Xho I - were added to the primers which were used to amplify a 634 bp fragment. This product was cloned into a pGEMTEasy vector and cloning was verified by PCR, enzymatic digestion and sequencing. The sequenced clone was analyzed in silico for identification of cis-elements related to seed specific expression. The elements TATA box, CAAT box, RY LEG box and RY FLEB box were found. The fragment of interest was isolated from vector pGEM-TEasy by cleavage with enzymes Sac I and Xho I and inserted in the expression vector pKANNIBAL. This new vector was designated pBKN. For the construction of the expression cassettes, primers were designed according to published sequences deposited in the GenBank. The fragments of interest were isolated by the RT-PCR method using RNA isolated from soybean seeds. RT-PCR was also used to analyze the expression of the gene of interest in the seed and in other organs of the plant (root, stem and leaves). These analyses showed that the expression of these genes was continuous and abundant during all stages of seed development, and the corresponding transcripts were present in all organs analyzed. The fragments obtained by RT-PCR were sequenced and their identity was confirmed by BLAST analysis. To clone the sense sequence, the vectors pKANNIBAL and pBKN, and the fragments of interest were cleaved with enzymes Xho I and Kpn I. The ligation reaction was catalyzed by T4 DNA ligase and competent cells of Escherichia coli were transformed by heat shock. Cloning was verified by PCR and enzymatic digestion. The cloning of the antisense fragment was done using restriction enzymes Xba I and Cla I and vectors containing the sense fragments. The complete expression cassettes were cloned into the binary vector pCAMBIA 3301 and verified by PCR. / A soja é uma das mais importantes culturas do mundo e como apresenta um alto teor protéico, ela tem sido muito utilizada na alimentação animal e humana. Apesar disso, as proteínas encontradas na semente desta leguminosa não são consideradas ideais por apresentarem baixo teor de metionina e lisina, além de fatores alergênicos e de inibidores de proteases. Dentre estes fatores antinutricionais estão a proteína P34 e os inibidores de protease do tipo Bowman-Birk. A ausência destes fatores aumentaria a qualidade da semente de soja, bem como a tornaria disponível para uma faixa mais ampla de consumidores. Recentes avanços na biotecnologia vegetal têm possibilitado o silenciamento completo ou em altos níveis de genes específicos. Uma destas técnicas é conhecida como interferência por RNA ou RNA interference. Este método é altamente eficiente e a clivagem do mRNA alvo é induzida pela presença de pequenos RNAs dupla fita na célula. O objetivo deste trabalho foi a construção de cassetes de expressão para silenciamento gênico, via interferência por RNA, dos inibidores de protease do tipo Bowman-Birk e da proteína P34 em sementes de soja. Para a expressão semente-específica dos transgenes, primers específicos foram desenhados para a amplificação da região promotora do gene da subunidade [alfa] da [beta]-conglicinina. A estes oligonucleotídeos foram adicionados sítios de restrição Sac I e Xho I - adequados para sua clonagem em vetores de expressão em plantas. Foram clonados 634 pb no vetor pGEM-TEasy. A clonagem foi confirmada por PCR, clivagem enzimática e sequenciamento do clone isolado. A seqüência clonada foi analisada in silico para a identificação de cis-elementos relacionados à expressão semente específica de genes colocados sob controle deste promotor. Os elementos TATA box, CAAT box, RY LEG box e RY FLEB box encontrados. O fragmento de interesse foi, então, isolado do vetor pGEM-TEasy por clivagem com as enzimas Sac I e Xho I e inseridos em vetores de expressão em plantas (pKANNIBAL) originando o vetor pBKN. Para a construção dos cassetes de expressão, a escolha da região do cDNA de cada gene de interesse a ser amplificada foi determinada com o auxílio do programa BLOCK-iTTM RNAi Designer (INVITROGEN), e os primers específicos foram desenhados de acordo seqüências depositadas no banco de dados público GenBank. Inicialmente foi feita, por meio de RTPCR, uma análise da expressão dos genes de interesse durante o desenvolvimento da semente e em demais órgãos da planta (raiz, caule e folha). Essa análise evidenciou expressão contínua e abundante desses genes durante todo o desenvolvimento da semente bem como a presença de transcritos correspondentes em todos os órgãos vegetais analisados. Após sua amplificação, todos os fragmentos obtidos a partir de cDNA de semente foram sequenciados e sua identificação foi confirmada por meio de alinhamento utilizando o programa BLAST. Para a clonagem da seqüência sense, os vetores pKANNIBAL e pBKN, além dos fragmentos amplificados referentes aos genes de interesse, foram clivados com as enzimas Xho I e Kpn I. A reação de ligação foi realizada utilizando T4 DNA ligase e células de Escherichia coli ultracompetentes foram transformadas por meio de choque térmico. Os transformantes foram analisados por PCR e reação de clivagem enzimática. A clonagem do fragmento antisense foi realizada da mesma forma, utilizando as enzimas Xba I e Cla I e os clones contendo o fragmento sense como vetores. Em seguida, as construções foram transferidas para o vetor binário pCAMBIA 3301 e confirmadas por PCR.

O feijão é um importante alimento na dieta alimentar do brasileiro. A cada colheita ocorrem perdas dos grãos de feijão devido ao ataque de insetos e roedores. Uma das maneiras de se preservar os grãos de feijão, mantendo suas características nutricionais, é o uso da radiação gama. O objetivo do trabalho é avaliar os efeitos de diferentes doses da radiação gama nas propriedades físico-químicas do feijão carioca submetido à cocção e que sofreu ou não o processo de maceração. Partes dos feijões cariocas crus foram utilizadas como controle e outra parte foram submetidos à radiação ionizante com doses de 1, 5 e 10 kGy. Após a irradiação uma parcela das amostras (controle e irradiadas) passou pela maceração e outra não, em seguida, todos os feijões foram cozidos. As amostras cozidas foram liofilizadas, moídas e depois acondicionas em potes e armazenadas a temperaturas de -23ºC. As análises de composição centesimal, determinação da digestibilidade proteica, teores de tanino condensado e ácido fítico foram feitas utilizando as amostras moídas. Utilizando grãos inteiros, foram feitas análises de capacidade expansão e hidratação, tempo de cocção e cor instrumental. A irradiação não alterou a composição centesimal das amostras maceradas e sem macerar. Os teores de taninos condensados não reduziram conforme o aumento das doses. As concentrações de ácido fítico foram reduzidas nas doses de 5 e 10 kGy, para amostras maceradas, enquanto que para os feijões sem macerar, a irradiação gama não influenciou nos teores de fitatos. Ocorreu diminuição da digestibilidade proteica, nas amostras maceradas, a partir dose de 1 kGy, e as demais doses a redução não foi significativa. Já para os feijões sem macerar, houve aumento da digestibilidade, na dose de 10 kGy. Conforme o aumento das doses foi observado redução nos tempos de cocção dos feijões macerados e sem macerar. Na dose de 10 kGy, a capacidade de expansão do feijão aumentou. A coloração das amostras não modificou significativamente conforme o aumento das doses. Desta forma conclui-se que a radiação ionizante não influencia na composição centesimal e nos teores de taninos. A irradiação influenciou nos teores de ácido fítico em altas doses aplicadas, mas somente nas amostras maceradas, além de atenuar a digestibilidade proteica de feijões macerados. Reduz os tempos de cocção. Eleva a capacidade de expansão do feijão na dose de 10 kGy, porém não altera a coloração do feijão, conforme o aumento das doses / The common bean is an important component in the diet of the average Brazilian person. Each harvest of beans, losses occur due to attacks of insects and rodents. One of the ways to preserve the beans, and at the same time keep its nutritional characteristics, is the use of gamma radiation. This study aimed to evaluate the effects of different doses of gamma radiation on the physical and chemical properties of the carioca variety of common beans subject to cooking of soaked and unsoaked beans. Portions of raw beans were used as control and the other was subject to ionizing radiation at doses of 1, 5 and 10 kGy. Following irradiation, a portion of the samples (control and irradiated) were soaked and the other was not, then all beans were cooked. The cooked samples were lyophilized, milled and then accommodated in pots and stored at -23ºC temperatures. The analysis of chemical composition, determination of protein digestibility, condensed tannin and phytic acid content were performed using the milled samples. Using whole grains, were performed analysis of expansion capability and hydration, cooking time and instrumental color. Irradiation did not alter the chemical composition of soaked and unsoaked samples. The condensed tannin levels did not reduce according to increased doses. The phytic acid concentrations were reduced at the doses of 5 and 10 kGy for soaked samples, whereas for the not soaked beans, gamma irradiation did not influence the phytate content. The protein digestibility decreased on soaked samples, at doses of 1 kGy and in the other doses, the reduction was not significant. As for not soaked beans, increases in digestibility were observed at dose of 10 kGy. As the doses increased, reduction in cooking time on soaked and unsoaked beans was noted. At a dose of 10 kGy, the bean expansion capability increased. The samples\' color did not change significantly, as the doses increased. Therefore, it is concluded that ionizing radiation has no effect on the chemical composition and tannin content. Irradiation influenced the phytates when applied in higher doses, but only in soaked samples, as well as mitigate protein digestibility on soaked beans. It reduces cooking time, raises bean expansion capability at a 10 kGy dose, but it does not change the bean color as doses increase

Dietary protein quality depends on two key measures: true ileal protein digestibility and the metabolic utilization of absorbed amino acids (AA). The objectives of this study were to determine the influence of two dietary factors (antinutritional factors and peptides) on ileal endogenous protein flows; to validate the intubation technique used in humans for ileal digesta sampling; to determine the postprandial metabolic utilization of dietary AA depending on their delivery form and to assess the validity of the growing pig for predicting true ileal protein digestibility in the adult human. Investigations were undertaken in the growing rat, growing pig and adult human. Ileal digesta were collected from euthanised rats, post valve T-caecum cannulated pigs, and naso-ileal intubated conscious adult humans. Ileal endogenous nitrogen (N) and AA were measured using a protein-free (PF) diet, diets containing 15N-labelled casein in the intact (C) or hydrolysed (HC) form, or a diet based on free AA (diet A), for which some dispensable AA were omitted to allow a direct determination of their endogenous flows. Digesta centrifugation and ultrafiltration (diet HC) allowed for the determination of ileal endogenous protein flows and the extent of tracer (15N) recycling. Antinutritional factors from a crude extract of kidney beans (Phaseolus vulgaris), when given at amounts commonly ingested in practice, enhanced ileal endogenous protein flows (rats, PF diet). After adaptation to the diet, body N balance per se did not influence ileal endogenous protein flows (rats, diets PF and A) but dietary peptides led to greater ileal endogenous AA and N flows compared with a protein-free diet. Dietary peptides (HC), compared with peptides naturally released in the gut during protein digestion (C), did not enhance ileal endogenous protein flows (rats, pigs, and humans). The extent of tracer recycling, however, was maximal in frequently-fed rats, lower in meal-fed pigs and minimal in meal-fed humans (65, 21, and 11% of 15N-labelled ileal endogenous proteins, respectively). Naso-ileal intubation for ileal digesta sampling in humans was shown to be an accurate method and evidence was obtained supporting the growing pig as a valid model for predicting true ileal protein digestibility in the adult human. Finally, the form of delivery of dietary AA (from HC or C) influenced the postprandial metabolic fate of dietary AA, especially in terms of AA catabolism kinetics. However, the overall nutritional value of C and HC were similar.