Enzimas são proteínas utilizadas em processos tecnológicos diversos. Estas enzimas dependendo do tipo e grau de pureza são geralmente caras. Comumente as enzimas exigem controle contínuo do processo no que se refere à temperatura, pH, agitação, entre outros, e após o uso são descartadas, o que torna o custo do processo mais elevado. Em decorrência disto, a imobilização de enzimas em suportes insolúveis e inertes, vem sendo proposta com resultados promissores de manutenção e até mesmo aumento da atividade enzimática, resistência mecânica, térmica e de pH, bem como por apresentar maior facilidade de remoção da enzima do sistema e possibilitar sua reutilização. Por causa disto, diferentes tipos de suportes vêem sendo estudados, dentre estes, os materiais poliméricos, tem recebido atenção especial. A quitosana é um polímero natural, biocompatível, biodegradável e atóxico. É obtida de fontes renováveis provenientes do descarte de cascas de crustáceos da indústria de alimentos, o que constitui um fator ambiental importante atualmente. Neste trabalho a enzima pepsina foi imobilizada em membranas liofilizadas de quitosana e O-carboximetilquitosana reticuladas ou não com glutaraldeído. A pepsina imobilizada na membrana de quitosana reticulada com glutaraldeído manteve sua atividade enzimática e o suporte apresentou propriedades físico-químicas de resistência a solubilização em pH ácido, o qual é necessário para atividade da pepsina. O processo de liofilização preservou a estrutura do suporte e não comprometeu a atividade enzimática. Demonstrando que o processo de liofilização é viável para secagem e incorporação de enzimas. / Enzymes are proteins used in a wide variety of biotechnological processes. Commonly, enzymes require stringent conditions, such as a particular pH, temperature, stirring, etc. In chemical and biochemical reactions, purified enzymes can be rather costly and additionally, must be discarded after each use, which is still less economical. As a result of this, enzyme immobilization on insoluble and inert supports has been studied as a manner to overcome these problems and optimize enzymes use. Promising results of greater immobilized enzyme activity and stability over a broader range of pH and temperature have been reported. As well, immobilized enzymes can be easily removed from the system and reused. Various materials have been employed as enzymes supports, among then, the polymers have received special attention. Chitosan is a natural polymer that presents biocompatibility, biodegradability and nontoxicity. Chitosan is obtained from crustacean shell wastes discarded by the food industry, and recover this material constitutes an important environmental factor nowadays. In this work the enzyme pepsin was immobilized on freezedried chitosan and O-Carboxymethylchitosan membranes crosslinked or not with glutaraldehyde. Pepsin immobilized on chitosan membrane crosslinked with glutaraldehyde maintained its enzymatic activity and the polymer support provided physicochemical properties such resistance to dissolution in acid pH. Acid pH is required for pepsin activity. The freeze-drying process preserved the support structure and did not compromise the enzymatic activity. Demonstrating that, freeze drying process, is viable for drying and enzymes incorporation.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to evaluate the relationship between the digestive enzyme activity and the centesimal composition of the contents of the gastrointestinal tract and food habits of four teleost two food habits in summer and winter. Two omnivorous species Pimelodus maculatus and R. quelen and two detritivorous species Hypostomus commersoni and Loricariichthys anus were chosen. Fish were collected with the aid of the trawl during March and July 2013 in São Gonçalo channel, Pelotas - RS. The digestive tract was divided into stomach, anterior and posterior intestine. Stomach was assayed the activity of pepsin and the two portions of the intestine were assayed the activities of trypsin, chymotrypsin and lipase. The protein and lipid content of the contents of each portion of the digestive tract was determined. Digestive enzyme activity is not related to the feeding habits and the centesimal composition of the contents of the gastrointestinal tract. Detritivorous species showed higher activity of alkaline proteases, which may be an adaptation to better utilize the low protein content found in the gastrointestinal tract of these species. / O objetivo desse estudo foi avaliar a relação entre a atividade enzimática digestiva e a composição centesimal do conteúdo do trato gastrintestinal e hábitos alimentares de quatro teleósteos de dois hábitos alimentares no verão e no inverno. Foram escolhidas duas espécies onívoras Rhamdia quelen e Pimelodus maculatus e duas espécies detritívoras Hypostomus commersoni e Loricariichthys anus. Os peixes foram coletados com o auxílio de rede de arrasto nos meses de março e julho de 2013, no canal São Gonçalo, Pelotas RS. O trato digestório foi dividido em estômago, intestino anterior e posterior. No estômago foi ensaiada a atividade da pepsina e nas duas porções do intestino foram ensaiadas as atividades da tripsina, quimotripsina e lipase. Foi determinado o teor proteico e lipídico do conteúdo de cada porção do trato digestório. A atividade enzimática digestiva não está relacionada com o hábito alimentar e a composição centesimal do conteúdo do trato gastrintestinal. Espécies detritívoras apresentaram maior atividade das proteases alcalinas, o que pode ser uma adaptação para utilizar melhor o baixo teor proteico encontrado no conteúdo gastrintestinal dessas espécies.

A modification of a linear diffusion test for measuring milk clotting enzymes at concentrations of 1 x 10^-4 to 1 x 10^-1 chymosin units/ ml was developed to permit quantitative assay of porcine pepsin in Cheddar cheese with a standard deviation of 6%. The amount of porcine pepsin retained in Cheddar cheese curd was dependent on pH of milk at setting. Milk at pH 6.6, 6.4, 6.2, and 6.0 was set with porcine pepsin, acidified with lactic acid and glucono-~-lactone and made into Cheddar cheese. After pressing the corresponding curd contained 0, 3.64+0 .12 %, 4.79+0.52%, and 5.94+0.30% of the pepsin activity added to the milk. Polyacrylamide gel electrophoresis of cheese revealed increasing degradation of the ~s-casein band with increasing residual pepsin in the curd. However, some degradation of the ~s-casein band was evident in curd set at pH 6.6 which showed no residual pepsin. Curd was made by ultrafiltration from whole milk followed by acidification to pH 5.2 with hydrochloric acid and glucono-6- lactone and vacuum evaporated to 39% moisture at 40 C (no clotting enzymes and no starter) . Degradation of the ~s- casein in this curd was similar to that observed in pepsin cheese set at pH 6.6. All degradation in ~s-casein cannot be attributed to milk clotting enzymes or starter bacteria. Porcine pepsin does not contribute to protein digestion in cheese curd during ripening unless the milk is below pH 6.6 at setting.

Orientador: Jaime Amaya-Farfan / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-09T15:53:11Z (GMT). No. of bitstreams: 1 Carvalho_IaraRibeiro_M.pdf: 1875369 bytes, checksum: 01034f576b4ca76425fe296fed74054e (MD5) Previous issue date: 2008 / Resumo: Estudos recentes sugerem que o consumo das proteínas do soro de leite, quando parcialmente hidrolisadas, resulta em efeitos fisiológicos diferentes daqueles produzidos pelas proteínas intactas. Desse modo, o objetivo do presente trabalho foi verificar as alterações metabólicas causadas nos tecidos do estômago e coração, e na resistência à exaustão, utilizando ratos Wistar alimentados com isolado do soro de leite, ou seu proteolisado enzimático, e submetidos a exercício físico. Foram investigadas possíveis alterações na atividade enzimática da pepsina, glicogênio sintase, glicogênio fosforilase, creatina quinase e glicogênio do miocárdio e a perfusão de peptídeos através do estômago. O ensaio biológico teve duração de 42 dias, com 120 ratos divididos em 12 grupos (n=10), sendo três fontes protéicas: caseína (C), proteína hidrolisada (H), proteína intacta do soro de leite (I) e quatro tipos de atividade física: treinado (T), treinado-exausto (TX), sedentário (S), sedentário-exausto (SX), em esteira rolante, por quatro semanas. Foram observados valores mais altos na atividade da enzima creatina quinase (sem diferença significativa) nos grupos T e TX, quando esses consumiram dieta H. Em relação ao treinamento, foram encontradas maiores quantidades de glicogênio miocárdico nos animais submetidos a menor atividade física. Em relação à dieta, as concentrações de glicogênio variaram aleatoriamente. Entretanto, os animais que consumiram a dieta H exibiram menor atividade da glicogênio sintase do que aqueles que receberam dieta I, e esses também menores que a dieta C. Na enzima glicogênio fosforilase, notou-se valores maiores de atividade enzimática nos grupos de animais do grupo T em relação aos outros grupos, sendo que a dieta H mostrou resultados menores em comparação com as outras dietas. Quanto à exaustão, foram comparados os grupos H e I, sendo que os grupos que foram submetidos a treinamento prévio (TX) mostraram-se mais resistentes à exaustão do que aqueles que foram mantidos sedentários (SX). Na atividade enzimática da pepsina, os resultados entre grupos foram semelhantes, notando-se aumento no grupo sedentário que consumiu o hidrolisado, mas desaparecendo com a exaustão. Estudo da possibilidade de perfusão de peptídeos através da parede estomacal encontrou evidências de que, ao menos, um peptídeo rico em valina é detectado no perfusado externo ao cabo de duas horas. Foi observada maior quantidade de picos eletroforéticos no conteúdo do estômago dos animais dos grupos SX e TX que receberam infusão do hidrolisado e teores de aminoácidos livres mais elevados no grupo S do que nos demais. Associando os valores encontrados para aminoácidos livres com os valores de aminoácidos totais, no interior e exterior do estômago, pode se afirmar que o conteúdo de peptídeos formados no estômago, no grupo que recebeu a infusão de hidrolisado, foi superior à que recebeu o isolado. Conclui-se que ambos o tipo de atividade física e a fonte protéica da dieta podem influenciar aspectos fisiológicos, tais como a atividade da pepsina, a facilidade com que peptídeos são gerados e acumulados no órgão, sendo que alguns peptídeos podem atravessar a parede estomacal. As implicações decorrentes destes fenômenos ainda aguardam maior investigação / Abstract: Recent studies suggest that the consumption of milk whey proteins, if partially hydrolyzed, result in different physiological effect from those produced by the ingestion of the unbroken proteins. Therefore, the objective of the present work was to verify eventual metabolic alterations caused in the stomach and heart tissues of exercising Wistar rats fed an enzymatic milk whey hydrolyzate, as compared to cohorts receiving the unhydrolyzed proteins. The enzymatic activity of pepsin and those of glycogen synthase, glycogen phosphorylase, creatine kinase, as well as glycogen stores of the myocardium were thus investigated. Additionally, the perfusion of peptides through the stomach wall using capillary electrophoresis and liquid chromatography was also verified. The biological assay was conducted with 120 rats, during 42 days. The animals were divided into 12 groups (n=10), as follows: three protein sources: casein (C), protein hydrolyzate (H) and the unbroken whey protein (I), in addition to four types of physical activity: trained (T), trained-exhausted (TX), sedentary (S), sedentary-exhausted (SX) for four weeks. For creatine kinase, higher activities were observed (without significant difference) in groups T and TX, when these consumed diet H. With regard to myocardial glycogen, higher stores were found in the animals with lesser physical activity, while glycogen concentrations varied randomly in response to the diet. However, the animals that consumed diet H exhibited lower glycogen synthase activity in comparison to those that received diet I, which in turn were lower than those that received diet C. As for glycogen phosphorylase, higher values were noticed in the groups subjected to training (T) in relation to the other groups. Similarly, the animals on diet H also responded with lower activities. With regard to exhaustion time, only those groups that underwent training (TX) appeared to be more resistant to exhaustion than those that remained sedentary (SX). The enzymatic activity of pepsin did not show significant differences among groups, except for the increase of the sedentary that consumed diet H. The increase, however, disappeared when the animals were brought to exhaustion. Study of the possibility of perfusion of whey protein peptides through the stomach wall suggested that at least one peptide rich in valine promptly perfused to the external fluid. A higher number of capillary electrophoretic peaks were also observed in the stomach contents of the animals of groups SX and TX that received the hydrolyzate infusion and, by liquid chromatography, it was possible to notice that the group S stomachs had greater levels of free amino acids than the other groups. Comparing the values found for free amino acids with the total amino acids, in both the inside and outside of the stomach, it could be stated that the content of peptides formed in the stomach was considerably greater in the group infused with the hydrolyzate than with the isolate. It is concluded that chronic consumption of the whey protein hydrolyzate as the only source of protein results in enzymatic changes consistent with a more efficient physiologic state, favorable to higher physical performance, such as higher myocardial creatine kinase and lower glycogen kinase and phosphorylase activities. No significant changes in pepsin activity in the rat stomach were observed, but the readiness with which the hydrolyzate peptides accumulate and traverse the organ wall was evident. Further data to provide a better understanding of the implications of consuming prehydrolyzed proteins await investigation / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

Orientadores: Nelson Adami Andreollo, Antônio Santos Martins / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T16:33:01Z (GMT). No. of bitstreams: 1 DelNegro_Andre_D.pdf: 3471885 bytes, checksum: 25c2d3d8936970ea8a29e110268320a4 (MD5) Previous issue date: 2011 / Resumo: Tabagismo e etilismo são os dois fatores mais importantes na gênese do carcinoma espinocelular do trato aero-digestivo superior (TADS). Outros fatores de risco podem estar envolvidos, como o refluxo faringolaríngeo (RFL). O objetivo desta pesquisa foi testar modelos experimentais de simulação do refluxo faringolaríngeo e gastro-esofágico e modelo de exposição à fumaça do cigarro em câmara fechada utilizando ratos Wistar, avaliando a injúria tecidual causada nas mucosas faringolaríngea e esofágica dos animais. Na Fase I, foram empregados cinquenta ratos Wistar divididos em 5 grupos submetidos à simulação do RFL, exposição à fumaça do cigarro, simulação do refluxo gastro-esofágico e simulação combinada do RFL e exposição tabágica, durante 6 meses. Na Fase II, oitenta e dois ratos Wistar foram divididos em 7 grupos e submetidos à simulação do RFL com "aplicações bi e tri-semanais" de ácido clorídrico, pepsina e nitrato de sódio durante 6 meses. Ambas as fases possuíam grupo controle expostos à apenas água. Não se observou displasia ou neoplasia. Alterações inflamatórias em graus variados foram observadas, com infiltrado linfocitário e mastocitário, além de hiperplasia epitelial. Os modelos experimentais desenvolvidos mostraram-se eficientes, reprodutíveis e de baixo custo para a simulação do tabagismo e do refluxo em animais de pequeno porte / Abstract: Smoking and drinking are the two main causes of squamous cell carcinoma in upper aerodigestive tract (UADT) mucosa. Other risk factors can be involved, as laryngopharyngeal reflux (LPR). The objective of this study was to test experimental models that simulate laryngopharyngeal and gastroesophageal refluxes and a tobacco exposition chamber, analyzing the tissue injury caused to the pharyngolaryngeal and esophageal mucosa using Wistar rats. In Fase I fifty Wistar rats were divided in five groups submitted to LPR, smoke exposition, gastroesophageal reflux and combined reflux and smoke exposition, during 6 months. In Fase II eighty-two Wistar rats were divided in seven groups and submitted to 2 or 3 weekly applications of hydrochloric acid, pepsin and sodium nitrate during 6 months. Both fases had control groups exposed to water alone. Neither dysplasia nor neoplasia were detected. Inflammatory changes were observed in varying degrees with lymphocytes and mast cells, and focal epithelial hyperplasia was observed. The data of the current study could not corroborate the hypothesis that gastroesophageal and pharyngolaryngeal refluxes are carcinogenic factors to UADT mucosa. The experimental model developed, had a low cost, was efficient and reproducible for smoking simulation in small animals / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências

MSCAGR (Animal Science) / Department of Animal Science / Forage and browse legumes play an important role in sustaining livestock in small holder farming systems in the tropics, mainly as a result of their contribution to economic and environmental sustainability of livestock production. The study was conducted to determine the chemical composition, ruminal degradability and in vitro digestibility of dry matter (DM) and crude protein (CP) of Dichrostachys cinerea and Bauhinia thonningii leaves. The browse tree leaves were harvested in the wild in Shayandima, Limpopo province. The leaves were collected, oven-dried, milled to pass through a 1.0 mm sieve and analysed for chemical composition in the Animal Science Nutrition Laboratory, at the University of Venda. The browse tree leaves were analysed for DM nitrogen, neutral detergent fibre (NDF) and acid detergent fibre (ADF). Approximately 5 g of leaf sample milled to pass through through a 1 mm sieve were placed in nylon bags (external dimension: 6 × 12 cm, pore size of 41 μm) and incubated in duplicates for 0, 4, 8, 16, 24, 48, 72, 96 and 120 hours periods in the rumen of three cannulated Bonsmara steers. The residues were then analysed for DM and nitrogen. Parameters to describe the dynamics of ruminal degradability of DM and CP were obtained by fitting the data on the exponential equation P = a + b (1 - e-ct) using NEWAY computer program, where “a” is the rapid degradable fraction, “b” is the slow degradable fraction and “c” is the outflow rate. The in vitro DM and CP degradability of rumen undegradable residue collected after 24 and 48 hour incubation was determined by sequential in vitro digestion in pepsin (abomasal) and pancreatin (small intestine) solutions. DM and CP content differed significantly (P ˂ 0.05). D. cinerea leaves had higher levels of DM and CP content than B. thonningii leaves. DM and CP disappearance increased (P < 0.01) as the incubation period increased. There was no difference (P > 0.05) in soluble fraction ‘a’ and ‘b’ of DM of the two species. The CP components for both fraction ‘a’ and ‘b’ differed significantly (P < 0.01) for CP among the two species. There was significant difference (P < 0.01) in post-ruminal digestibility among the two species. CP digestibility of B. thonningii and D. cinerea leaves was reduced (P < 0.01). In conclusion, B. thonningii and D. cinerea leaves showed significant difference based on their fermentation kinetics and in vitro digestibility, suggesting a good nutritional quality which can be used as protein source for ruminants in dry season and supplement to low-quality diets. / NRF