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Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-29T12:45:12Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE FINAL JULIETT XAVIER FICHA CATALOGRAFICA.pdf: 7964494 bytes, checksum: 4f95a881e713dadfb7a0c571d8b86036 (MD5) / Made available in DSpace on 2016-06-29T12:45:12Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE FINAL JULIETT XAVIER FICHA CATALOGRAFICA.pdf: 7964494 bytes, checksum: 4f95a881e713dadfb7a0c571d8b86036 (MD5) Previous issue date: 2014-02-28 / CAPEs / Carcaças e vísceras constituem um importante resíduo de processamento de peixes, podendo representar cerca de 70% do peso corporal da tilápia (Oreochromis niloticus). Esse material é uma fonte de biomoléculas, dentre elas proteínas e proteases, com propriedades interessantes para processos biotecnológicos como a produção de hidrolisado proteico de peixe (HPP). O objetivo do primeiro estudo foi avaliar o uso de resíduos do processamento de tilápia para produzir HPP. Assim, três condições de produção foram avaliadas: duas utilizando hidrolise enzimática com enzimas extraídas do intestino da tilápia em diferentes concentrações (HPP100, 100 mg de tecido/mL e HPP600, 600 mg de tecido/mL) e o terceiro utilizando 0.5% (v/v) de Alcalase (HPPcom), uma enzima comercial. Os extratos experimentais revelaram a presença de proteases totaias, tripsina, quimotripsina e leucinoaminopeptidase. O teor de proteínas, aminoácidos e ácidos graxos foram calculados em matéria seca. Depois de 4 horas de reação, o grau de hidrolise máximo (GH) do HPPcom, HPP100, HPP600 foram 34.73 ± 1.44%, 29.21 ± 0.79%, e 41.66 ± 1.33%, respectivamente. O perfil eletroforetico demonstrou bandas de 190 a 20 kDa (HPP100), 54 kDa (HPPcom) e 53 a 20 kDa (HPP600). O resultados dos teores proteicos foram, 584.8 g/kg, 492.3 g/kg, e 508.2 g/kg para HPPcom, HPP100, e HPP600, respectivamente. Metionina e lisina foram identificados em níveis de 32.0 e 77.0 g/kg (HPPcom), 31.0 e 64.0 g/kg (HPP100), e 33.0 e 69.0 g/kg (HPP600), respectivamente. O conteúdo de ácidos graxos polisanturados do HPPcom, HPP100 e HPP600 foram 101.0 g/kg, 138.0 g/kg e 70 g/kg, respectivamente, com predominância do ácido linoleico (C18:2n-6). O valor do IAAI foi de 1066.07, 688.4 e 738.51 no HPPcom, HPP100 e HPP600, respectivamente. A composição de aminoácidos, perfil lipídico e escore químico sugerem que todos os HPPs testados podem ser empregados como fonte proteica em dietas para organismos aquáticos. O segundo estudo comparou a eficiência de enzimas do intestino de tilápia (200 mg de tecido/mL) bruto e parcialmente purificados por precipitação salina (0 – 80%, v/v) e etanólica (0-30%, v/v), (30 – 70%, v/v) com duas enzimas comerciais Alcalase 0.5% (v/v) e Flavourzyme 0.5% (v/v) na hidrolise da carcaça da tilápia. O extrato enzimático semi-purificado com (NH4)2SO4 mostrou rendimento de 62.6% (ativ. esp. de 9.3 U/mg de tecido) e com etanol um rendimento de 42.6% (ativ. esp. de 37.0 U/mg de tecido) e 68.4% (33.9 U/mg de tecido). Após 4 horas de reação o HPPEC(200) mostrou maior GH (37.8%), seguidos por HPPA (35.3%), HPPET (33.2%), HPPAS (24.6%) e HPPF (18.5%). O perfil eletroforético demosntrou que o peso molecular dos HPPs variaram entre 116.25 a 29.05 KDa. O extrato bruto e os extratos semi-purificados foram mais eficientes na hidrolise da carcaça de tilápia que a enzima comercial Flavourzime. O terceiro estudo testou os efeitos da esterilização térmica, acidificação e irradiação na conservação de HPP. Foram realizadas análises físico-quimicas (TBARS, pH, composição centesimal), microbiológicas (mesófilos, piscicrófilos e microorganismos específicos) e sensorial (coloração) durante 60 dias. As análises mostraram que os HPPs possuem alto teor proteico e lipídico e são susceptíveis a mudanças de pH, oxidação lipídica e descoloração. A partir do décimo dia de estocagem todos os HPPs tiveram teores de TBARS aumentados e sofreram descoloração. O conteúdo proteico e lipídico foi alterado, mas não comprometeu o valor nutricional dos HPPs. A esterilização térmica com adição de ácidos e esterlização por irradiação foram eficientes na à eliminação dos microorganismos garantindo a segurança do produto por 60 dias de armazenamento. No quarto trabalho obteve-se uma patente referente à separação da parte lipídica da proteica do hidrolisado proteico de peixe a partir da irradiação. / Fish viscera and carcasses represent about 70% of body weight of tilapia (Oreochromis nilotic), and are known sources of biomolecules such as protein and proteases. These enzymes can be employed in various biotechnological processes, e.g. preparation of fish protein hydrolysates. In this way, the aims of the first study were to evaluate the use of processing waste from Nile tilapia (Oreochromis niloticus) as a source of protein and proteases to produce FPH. Three FPH production conditions were evaluated: two conditions used autolysis with enzymes extracted from the tilapia intestine at different concentrations (FPH100, 100 mg of tissue/mL and FPH600, 600 mg of tissue/mL) and the third used 0.5% (v/v) Alcalase (FPHcom), a commercial protease preparation. The experimentais extracts showed total proteolytic activities, trypsin, chymotrypsin, and leucine aminopeptidase activities proteases. Protein, amino acids and fatty acids content were calculated as DM basis. After a 4- h reaction, maximum hydrolysis percentages (DH) from FPHcom, FPH100, and FPH600 systems were 34.73 ± 1.44%, 29.21 ± 0.79%, and 41.66 ± 1.33%, respectively. The protein content in the resulting FPS were 584.8 g/kg, 492.3 g/kg, and 508.2 g/kg for FPHcom, FPH100, and FPH600, respectively. Methionine and lysine were found at levels of 32.0 and 77.0 g/kg (FPHcom), 31.0 and 64.0 g/kg (FPH100), and 33.0 and 69.0 g/kg (FPH600), respectively. Polyunsaturated fatty acid contents of FPHcom, FPH100, and FPH600 were 101.0 g/kg, 138.0 g/kg, and 70 g/kg, respectively, with a predominance of linoleic acid (C18:2n-6). IAAI reached a value of 1066.07 in FPHcom, 688.4 in FPH100 and 738.51 in FPH600. Amino acid composition, lipid profile, and amino acid score suggested that all of the experimental FPHs could be employed as a protein source in diets for aquatic organisms and other farmed animals. The second study compared the efficiency of the enzymes from tilapia intestine (200 mg tissue/mL) crude and partially purified by salting-in (0 - 80% v/v) and ethanol (0-30%, v/v), (30 - 70% v/v) with two commercial enzymes Alcalase 0.5% (v/v) and 0.5% Flavourzyme (v/v) hydrolysis of the carcass tilapia. The partial purified enzyme extract with (NH4)2SO4 showed a yield of 62.6% (act. esp. of 9.3 U/mg of tissue) and ethanol a yield of 42.6% (act. esp. of 37.0 U/mg tissue ) and 68.4% (33.9 U/mg tissue). After 4 hours the reaction, FPHEC (200) showed a higher DH (37.8%), followed by FPHA (35.3%), FPHET (33.2%), FHPAS (24.6%) and FPHF (18.5%). The electrophoretic profile of FPHs showed molecular weight ranged from 116.25 to 29.05 kDa. The crude extract and partial purified extracts were more effective in the hydrolysis of tilapia carcasses than commercial enzyme Flavourzime. The third study tested the effects of heat sterilization, irradiation and acidification in the conservation of FPH. They were carried out physical-chemical analysis (TBARS, pH, chemical composition), microbiological (mesophilic, piscicrophilic and specific microorganisms) and sensorial (color) for 60 days. Analyzes have shown that FPHs have high protein and lipid content and FPHs are susceptible to changes in pH, lipid oxidation and discoloration. From the 10th day of storage all FPH had increased TBARS levels and discoloration. The protein and lipid content has changed, but did not compromise the nutritional value of FPHs. Heat treatment with citric acid and gamma irradiation were effective in the removal of microorganisms pathogenic securing of the product for 60 days of storage. In the fourth work we obtained a patent for the separation of the lipid portion of the protein fish protein hydrolyzate from the irradiation.

Biocatalytic membranes (BMs) have promising applications in a diversity of fields including food, pharmaceutical and water treatment industries. Of particular relevance, Alcalase is a commercially important protease that has been applied for the production of peptides from the hydrolysis of proteins. In this study, two different approaches were applied for the modification of electrospun polyacrylonitrile nanofibrous membranes (EPNMs) for Alcalase immobilization. The first approach is alkali modification of EPNMs followed by EDC/NHS coupling for covalent bonding with Alcalase, whereas the other is based on polydopamine coating with or without glutaraldehyde grafting as a covalent linker. Immobilized Alcalase on these prepared BMs were studied and compared with free enzymes. It was found that the stabilities of Alcalase on BMs created using both approaches were improved, which enabled their reuse of 10 cycles with significant retention of enzymatic activity. A continuous reactor housing BMs were tested for hydrolysis of both model substrate, azo-casein and soybean meal protein (SMP). It was found that decreasing flux could improve the extent of hydrolysis and that a single-layer reactor can hydrolyze about 50% of the substrate to peptides with the molecular weight of 10 kDa or less. Hydrolysis of SMPs was demonstrated in a continuous five-layer BM reactor and both BMs showed excellent hydrolysis capacity. This study provides the groundwork for the development of high-efficiency BM for continuous and cost-effective protein hydrolysis for the production of value-added peptides.

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Orientador: Míriam Dupas Hubinger / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-17T11:13:22Z (GMT). No. of bitstreams: 1 RodriguezDiaz_JulioCesar_M.pdf: 3275088 bytes, checksum: 4baa128a0e7dc07566683d567b97b97c (MD5) Previous issue date: 2011 / Resumo: O presente trabalho teve como objetivo a obtenção de hidrolisado protéico de pele de cação em pó pelos processos de hidrólise enzimática e secagem por atomização. Na primeira etapa, foi estudado o processo de hidrólise utilizando a enzima comercial ProtamexTM, visando a otimização do processo para a recuperação de peptídeos de baixo peso molecular. A influência da temperatura, do pH e da relação Enzima/Substrato sobre o grau de hidrólise, recuperação de proteína e cinética enzimática foram avaliadas. As condições ótimas de processo foram fixadas em: 51 °C, 4,0% p/p de enzima e pH 7,1. Nessas condições, o hidrolisado obtido foi caracterizado em termos de composição química, composição de aminoácidos e distribuição de tamanho de peptídeo. O grau de hidrólise atingiu 19,3%, recuperou-se 90,3% do total da proteína presente na matéria-prima e o peso molecular foi inferior a 6,5 kDa. Na segunda etapa do projeto, foi avaliada a influência das variáveis do processo de secagem por atomização sobre as propriedades físico-químicas e antioxidantes do hidrolisado protéico de pele de cação em pó, usando maltodextrina (DE=10) como agente carreador. A temperatura do ar e a concentração de agente carreador foram variadas. As respostas analisadas foram a umidade final, a atividade de água, a densidade aparente, a higroscopicidade, o tamanho de partícula e a capacidade antioxidante, determinada pelos métodos de redução do íon férrico (FRAP) e captura do radical catiônico ABTS+0 (TEAC). Os resultados mostraram que a adição de maltodextrina contribuiu significativamente para obtenção de partículas com menor umidade e higroscopicidade e maiores tamanhos de partícula e densidade aparente. Além disso, quanto maior foi a concentração de maltodextrina adicionada, maiores foram os valores de capacidade antioxidante, provavelmente devido à formação de compostos de Maillard durante a secagem / Abstract: The objetive of the present work was to obtain a shark skin protein hydrolysate powder by enzymatic hydrolysis and spray drying. In the first part of this work was studied the enzymatic hydrolysis process by using the commercial enzyme ProtamexTM, aiming the process optimization for recovery of low molecular weight peptides. The influence of temperature, pH and Enzyme/Substrate ratio on the degree of hydrolysis, protein recovery and enzymatic kinetics was evaluated. Optimal conditions were established at: 51°C, 4.0% p/p of enzyme and pH of 7.1. At these conditions, protein hydrolysate was characterized with respect to chemical composition, amino acid composition and peptide size distribution. Degree of hydrolysis achieved 19.3%, 90.3% of total protein was recovered and peptide size was lower than 6,5 kDa. In the second part of the project, the influence of spray drying variables process on physicochemical and antioxidant properties of shark skin protein hydrolysate powders, using maltodextrin (DE=10) as a carrier agent, was evaluated. Air temperature and maltodextrin concentration were varied. The responses analyzed were the final moisture content, water activity, bulk density, hygroscopicity, particle size distribution and antioxidant capacity determined by ferric reduction antioxidant power (FRAP) and ABTS+0 radical cation capture (TEAC) assays. Results showed that maltodextrin addition significantly contributed to obtaining particles with less moisture content, lower hygroscopicity and higher particle size and bulk density. Besides, antioxidant capacity was increased as increasing of maltodextrin concentration, probably due to the formation of Maillard compounds during drying process / Mestrado / Mestre em Engenharia de Alimentos

Made available in DSpace on 2016-06-02T19:56:48Z (GMT). No. of bitstreams: 1 4557.pdf: 2660137 bytes, checksum: e6d326cb231ca1671c56c791ae984575 (MD5) Previous issue date: 2012-05-30 / Soy protein concentrate (SPC) is used as an ingredient for food and animal feed. It is obtained from defatted soybean meal by extracting sugars and other soluble compounds. The main consequence of the extraction process is removal or inactivation of most anti-nutritional factors of soybean, which makes this product can be used in place of animal proteins in special diets. This work aimed to study the process of protein hydrolysis as an alternative to increase the value of the SPC through the improvement of their nutritional value. Hydrolysis process has been done many years ago with the purpose of improving physico-chemical, organoleptic and nutritional value of food proteins. Enzymatic hydrolysis is the most appropriate when the objective is to improve the nutritional value, because the chemical methods cause many unwanted effects. We studied two commercial alkaline proteases, Alcalase® and Novo-ProD®. Novo-ProD® showed superior activity than Alcalase® in all pH conditions tested. The process conditions that maximized efficiency of the hydrolysis of SPC by Novo-ProD® were: 55°C, pH 9 and enzyme/substrate ratio of 0.5% (enzyme mass / protein mass). The maximum degree of hydrolysis obtained in three hours reaction under these conditions was approximately 15.2% in bench scale and 12.8% in pilot scale. Increasing solid concentration of the reaction medium from 10 to 30%, maximum degree of hydrolysis of 11.2% was obtained in the same conditions. Digestibility trial was conducted with SPC hydrolyzate with 3,3% against SPC unhydrolyzed in adult dogs and turkeys in the initial growth phase. In dogs, hydrolysis increased apparent digestibility of dry matter from 76.5 to 86.2%, crude protein from 83.9 to 90.6% and metabolizable energy increased by approximately 5%. In turkeys, the increase was from 54.6 to 62.9% for dry matter digestibility and 9% increase in metabolizable energy. / O concentrado proteico de soja (SPC) é utilizado como ingrediente para alimentos e ração animal. É obtido a partir do farelo de soja desengordurado através da extração de açucares e outros compostos solúveis. A principal consequência do processo de extração é a remoção ou inativação de grande parte dos fatores antinutricionais da soja, o que faz com que este produto possa ser utilizado em substituição de proteínas de origem animal em dietas especiais. Este trabalho teve o objetivo de estudar o processo de hidrólise proteica como alternativa de aumentar a valor agregado do SPC através do melhoramento do seu valor nutricional. O processo de hidrólise tem sido feito há muitos anos com finalidade de melhorar propriedades físico-químicas, organolépticas e valor nutricional de proteínas alimentares. A hidrólise enzimática é a mais adequada quando o objetivo é melhorar o valor nutricional, pois os métodos químicos causam inúmeros efeitos indesejados. Foram estudadas duas proteases alcalinas comerciais: Alcalase® e Novo-Pro D®. A Novo-Pro D® apresentou atividades superiores às da Alcalase® em todas as condições de pH testadas. As condições de processo que maximizaram a eficiência da hidrólise do SPC pela Novo-Pro D® foram: 55°C, pH 9 e relação enzima/substrato de 0,5% (massa de enzima/massa de proteína). O grau de hidrólise máximo obtido, em três horas de reação nestas condições, foi de aproximadamente 15,2% em escala de bancada e 12,8% em escala piloto. Aumentando-se a concentração de sólidos do meio de reação de 10 para 30%, o grau de hidrólise máximo obtido foi de 11,2%, nas mesmas condições citadas. Avaliou-se a digestibilidade de nutrientes e metabolizabilidade da energia de um SPC hidrolisado com grau de hidrólise de 3,3% contra um SPC não hidrolisado em cães adultos e perus em fase inicial. Em cães, a hidrólise aumentou os coeficientes de digestibilidade aparente da matéria seca de 76,5 para 86,2%, da proteína bruta de 83,9 para 90,6% e a energia metabolizável aumentou em aproximadamente 5%. Em perus, o aumento foi de 54,6 para 62,9% para a digestibilidade da matéria seca e 9% de aumento da energia metabolizável.

Der Sprengstoff PETN ist äußerst schwer zu detektieren. Ein verbesserter anti-PETN-Antikörper wurde durch Anwendung des Bioisosterie-Konzepts entwickelt. Diese polyklonalen IgGs sind sehr selektiv und sensitiv. Die Nachweisgrenze des ELISAs beträgt 0,15 µg/L. Der Messbereich des Immunoassays liegt zwischen 1 und 1000 µg/L. Die Antikörper sind recht pH-stabil als auch robust gegen Lösungsmittelzusätze. Für die Umweltanalytik von TNT wurde eine HPLC-kompatible Affinitätssäule mit porösem Glas als Trägermaterial hergestellt. Um die anti-TNT-Antikörper selektiv aus den TNT-Seren zu isolieren, wurde eine Trennung an einer Dinitrophenyl-Affinitätssäule durchgeführt. Zur Optimierung der Kopplungsmethode wurden orangefarbene Dabsyl-Proteine synthetisiert und auf der Oberfläche gebunden. Die Färbung wurde als Indikator für die Ligandendichte verwendet. Wegen der hohen Affinitätskonstanten der anti-TNT-IgGs lässt sich TNT nicht reversibel von der TNT-Affinitätssäule eluieren. Daher wurde eine neuartige Elutionsmethode entwickelt, die thermische Online-Elution. Die maximale Kapazität einer TNT- Affinitätssäule betrug 650 ng TNT bzw. 10 µg/mL Säulenvolumen. Um die Ligandendichte der TNT-Affinitätssäulen zu bestimmen, wurde ein neues Verfahren entwickelt, da die spektroskopischen Proteinbestimmungsmethoden nicht geeignet waren. Zur Proteinbestimmung wurde eine HPLC-Trennung der Aminosäuren Tyr und Phe ohne vorherige Derivatisierung entwickelt. Die Proteinhydrolysezeit wurde durch Einsatz einer Mikrowelle von 22 h auf 30 min verkürzt. Zur internen Kalibrierung wurden HTyr und FPhe verwendet. Die Nachweisgrenze bei 215 nm ist sowohl für Tyr als auch für Phe 0,05 µM (~ 10 µg/L). Dieses neue Verfahren, das als Aromatische Aminosäureanalyse (AAAA) bezeichnet werden kann, wurde zur Proteinbestimmung von homogenen Proben mit NIST-BSA validiert, wobei die Nachweisgrenze für Proteine 16 mg/L (~ 300 ng BSA) ist. Die relative Standardabweichung incl. der Hydrolysestufe beträgt 5%. / The explosive Pentaerythritol tetranitrate (PETN) is extremely difficult to detect. An improved antibody against PETN was developed by using the bioisosteric concept. These polyclonal antibodies are highly selective and sensitive. The limit of detection (LOD) of the ELISA was determined to be 0.15 µg/L. The dynamic range of the assay was found to be between 1 and 1000 µg/L. The antibodies are sufficiently pH-stable and resistant to solvent additives. An HPLC-compatible TNT-affinity column with porous glass as support material was prepared for the environmental analysis. In order to isolate the anti-TNT antibodies of the TNT sera a separation was carried out on a dinitrophenyl-affinity column. To optimize the immobilization method, orange-coloured dabsyl proteins were synthesized and bound to the surface. The colour intensity was found to be an indicator for the immobilization rate. In consequence of the high affinity constants of the anti-TNT antibodies, TNT can''t elute by a typical acidic elution step. Therefore, a novel separation approach, the thermal online-elution was developed. The maximum capacity of an affinity column was 650 ng TNT or 10 µg/mL of column volume. To quantify the immobilization rate of proteins, a new method has been developed, because the usual protein determination methods were unsuitable. Therefore an HPLC separation method of Tyr and Phe was developed without prior derivatization. Two internal standard compounds, HTyr and FPhe, were used for calibration. The LOD was estimated to be 0.05 µM (~ 10 µg/L) for Tyr and Phe at 215 nm. The protein hydrolysis time was reduced from 22 h to 30 min using microwave technique. This procedure, that was termed aromatic amino acid analysis (AAAA), has been validated for protein determination of homogeneous samples with NIST-BSA. The LOD for proteins was calculated to be below 16 mg/L (~ 300 ng BSA absolute). The relative standard deviation, including the hydrolysis step, is 5%.