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Solieria chordalis (Rhodophyta, Gigartinales) s’échoue depuis de nombreuses années sur la presqu’île de Rhuys dans le sud de la Bretagne. À hauteur de plus de 2 000 tonnes par an, ces arrivées massives d’algues causent de réels problèmes écologiques et économiques pour les communes du littoral breton. L’objectif des travaux de recherche a été d’étudier S. chordalis, dans l’optique de proposer une gestion raisonnée voire durable de cette biomasse ainsi que des perspectives d’applications. Les résultats d’un suivi mensuel de l’algue d’octobre 2013 à octobre 2015 ont montré une variation saisonnière de la biomasse sur l’estran, de la morphologie macro- et microscopique, du taux de matière sèche et de la composition biochimique de l’espèce. En fonction des facteurs saisonniers environnementaux, il est désormais possible d’estimer la quantité et la composition biochimique de l’algue et de proposer des applications. Le procédé d’Extraction Assistée par Enzymes pour ses avantages écologiques, sa spécificité et pour son efficacité catalytique a été choisi pour la production d’extraits hydrosolubles antiviraux, antibactériens et immunostimulants. La protéase commerciale Alcalase permet d’augmenter significativement de 30 % la quantité de composés hydrosolubles disponibles, par rapport à une extraction sans enzyme. Près de 60 % de la matière sèche de l’algue a été liquéfiée et solubilisée après optimisation des paramètres d’extraction. Les extraits hydrosolubles produits en présence d’enzymes ont montré des activités anti-herpétiques (CE50 de 86,0 à 145,9 µg/mL), sans montrer de cytotoxicité sur les cellules saines. L’activité immunostimulante a été évaluée en utilisant le modèle de pathogénicité Caenorhabditis elegans – Pseudomonas aeruginosa. L’application d’extraits hydrosolubles de S. chordalis prolonge l’espérance de vie du nématode infecté par la bactérie pathogène, notamment en stimulant l’expression de certains gènes de son système immunitaire (zk6.7, spp-1, f28d1.3 et f38a1.5). Les extraits produits présentent donc des activités antivirales et immunostimulantes intéressantes qui permettent d’envisager des applications dans divers domaines. / These last decades, Solieria chordalis (Rhodophyta, Gigartinales), a red and proliferative macroalga appears every year in south of Brittany on the peninsula of Rhuys. It is a question of removing more than 2 000 tons of red seaweeds, mainly composed of S. chordalis. These proliferations represent a true ecological problem and an economic constraint for the affected communities. This project aims to study S. chordalis in order to find a way to add more value to this seaweed by proposing a rational and sustainable management as well as potential applications. From October 2013 to October 2015, S. chordalis was collected monthly. Results have shown seasonal variations of the biomass on the shores, in the macro- and microscopic morphologies, in the dry matter rate and in the biochemical composition. In relation with the seasonal environmental factors, it is now possible to estimate the quantity and the biochemical composition of the seaweed and to propose potential applications. With characteristics of high catalytic efficiency, specificity, the environmental-friendly process of Enzyme-Assisted Extraction was chosen to produce water- soluble extracts with antiviral, antibacterial and immunostimulant activities. The commercial protease Alcalase improved the extraction yield with a significant gain of 30 % of the water-soluble and free compounds compared to an extraction without the action of enzymes. Almost 60 % of the dry matter of algae was liquefied and water-solubilized after the optimization of extraction parameters. Water-soluble extracts produced after the action of enzymes have shown anti- herpetic activities (EC50 from 86.0 to 145.9 µg/mL) and no cytotoxicity was detected. The immunostimulant activity was evaluated by using the pathogenicity model Caenorhabditis elegans Pseudomonas aeruginosa. The life span of infected nematods by pathogen bacteria increased after the application of water-soluble extracts from S. chordalis. Some immune genes of C. elegans were stimulated (zk6.7, spp-1, f28d1.3 and f38a1.5). Produced extracts present antiviral and immunostimulant activities that allow to anticipate applications for diverse domains.

Fish and fish waste can be used to produce various value added by products such as proteins, oil, omega-3 fatty acids, biodiesel, amino acids, peptides, collagen, gelatin and silage, each of which has various applications in the food industry, renewable energy and medicinal purposes. Fish protein contains amino acids and many bioactive peptides . Fish proteins are found in the flesh, head, frames, fin, tail, skin and guts in varying quantities. After removing the flesh, all other parts are considered waste which is not properly utilized. The aim of this study was to evaluate the enzymatic extraction of amino acids from fish protein for use as substrates in the microbial production of jadomycin, an antimicrobial agent and potential anti-cancer drug. In this study, enzymatic extraction of proteins was carried out using Alcalase enzyme at three enzyme concentrations (0.5, 1 or 2%) and four time intervals (1, 2, 3 and 4 h). The fish protein hydrolysate was dried using spray dryer to obtain protein powder. The highest protein yield (76.30% from whole fish and 74.53% from the frame) was obtained using 2.0% enzyme concentration after 4 h of hydrolysis. The enzymatic extraction of amino acids were carried out using the enzymes Alcalase and Neutrase (individually and in combination) and the effect of reaction time (24 and 48 h) on the hydrolysis of proteins was studied. The profiling of amino acids was carried out using gas chromatography. Fourteen amino acids were extracted from fish proteins of which twelve amino acids have been used by researchers for the production of jadomycins. These are: alanine (7.59%), glycine (5.82%), histidine (3.59%), isoleucine (5.30%), leucine (9%), lysine (7.34%) methionine (2.2%), phenylalanine (4.2%), serine (4.3%), threonine (5.40%), tyrosine (3.17%) and valine (7.2%). Tryptophan which is suitable of producing jadomycin was not present in the fish protein. No reports were found in the literature for jadomycin production from glutamic acid. Therefore, glutamic acid (9.85%), and proline (0.98%) which are present in the fish protein should be investigated for possible production of jadomycins.

Made available in DSpace on 2016-06-02T19:55:29Z (GMT). No. of bitstreams: 1 DoutPWT.pdf: 1538718 bytes, checksum: a2cfebb6aac530f7f09069137eaebc60 (MD5) Previous issue date: 2003-08-22 / Universidade Federal de Minas Gerais / High value food protein hydrolysates can be obtained by sequential hydrolysis of proteins with trypsin, chymotrypsin, carboxypeptidase A (CPA) and Alcalase® (commercial preparation of subtilisin). For the process to be economically feasible, immobilized and stabilized enzymes should be used, and the kinetics of the reactions with this kind of biocatalyst must be known. To contribute to the development of such a process, this work focused on preparing stable CPA and Alcalase® derivatives, and on studying the kinetics of hydrolysis of polypeptides. These polypeptides were produced after the sequential hydrolysis of cheese whey proteins with trypsin and chymotrypsin. Cross-linked agarose beads (6% w/w for CPA, and 10% w/w for Alcalase®) were used as immobilization support, and different methods of activation and immobilization conditions were studied. A highly activated glyoxyl-agarose support (75 and 210 µeqv of aldehyde groups per milliliter of support, respectively for CPA and Alcalase®), 25oC, pH 10.05, and longer contact time (48 hours for CPA and 96 hours for Alcalase®), provided the best derivatives. CPA-glyoxyl agarose-6% and Alcalase®-glyoxyl agarose-10% derivatives were ca. 213- and 515-fold more stable than the soluble enzymes. These stabilized derivatives retained 42% (for CPA-glyoxyl agarose- 6%) and 54% (for Alcalase®-glyoxyl agarose-10%) of the immobilized activity, assessed with small substrates (hippuryl-L-Phe for CPA, and Boc-Ala-ONp for Alcalase®) and large substrates (Phe carboxy-terminal polypeptides for CPA, and casein for Alcalase®). These results showed that all activity losses were caused by the distortion of the immobilized enzyme molecule, due to the enzyme-support multi-interaction. Derivatives prepared using glutaraldehyde-agarose presented spatial hindrances when hydrolysis of macromolecular substrates was taking place. The amino acid analysis of acid hydrolysates of the soluble and immobilized enzymes (for the more stable derivatives) showed that ca. 30 and 40%, for CPA and Alcalase®, of the lysine residues were linked to the support, suggesting that there is intense multi-point interactions between enzyme and support, through covalent linkages. The temperatures for maximum hydrolysis rates, using respectively stabilized CPA and Alcalase® derivatives, were 20oC and 10oC higher than the ones obtained using soluble enzymes. The most stable CPA-glyoxyl derivative could efficiently be used for polypeptides (cheese whey proteins hydrolyzed with trypsin and chymotrypsin) hydrolysis at high temperatures (e.g., 60oC), releasing ca. 2-fold more aromatic amino acids (Tyr, Phe and Trp) than the soluble enzyme, under the same operational conditions. The casein degree of hydrolysis, at 80oC, obtained using the most stable Alcalase®-glyoxyl derivative, was 2-fold higher than the one obtained with the soluble enzyme. Hence, the produced derivatives allow the design of a continuous process for the production of protein hydrolysates, which are composed of small peptides and have a low concentration of aromatic amino acids. This process can use higher temperature, avoiding microbial growth in the reaction medium. The C-terminal residues hydrolysis at 45oC (pH 7.0), catalyzed by CPA-glyoxyl, could be adequately represented by Michaelis-Menten kinetics, with substrate and product inhibition. The kinetic model was expressed in terms of C-terminal peptide bonds that can be hydrolyzed by CPA, regardless of the amino acid released. The concentration of each released amino acid as a function of the time of reaction could be well fitted by empirical models (hyperbolic or exponential decay). Hence, from the kinetics of total hydrolysis, it is possible to estimate the concentration of each amino acid as function of time. The hydrolysis catalyzed by the highly-loaded CPA-glyoxyl agarose-6% derivative was not limited by intra-particle diffusion resistance. The hydrolysis of peptides (long-time batch) at 50oC (pH 9.5), catalyzed by Alcalase®-glyoxyl agarose-10% derivative, could be adequately represented by Michaelis-Menten kinetics with product inhibition, and the kinetic parameters Vmax, KM e KI were correlated against the substrate initial degree of hydrolysis (total degree of hydrolysis obtained by previous action of trypsin and chymotrypsin on cheese whey proteins). Long-time batch hydrolyses, catalyzed by highly-loaded Alcalase-glyoxyl agarose-10% derivative, presented diffusion effects, with effectiveness coefficient, ηI, of ca. 0.5. / Hidrolisados protéicos de alto valor agregado podem ser obtidos através da hidrólise seqüencial de proteínas com tripsina, quimotripsina, carboxipeptidase A (CPA) e Alcalase® (preparação comercial de subtilisina). A viabilidade econômica do processo requer a utilização de enzimas imobilizadas e estabilizadas e o conhecimento da cinética das reações catalisadas com esse tipo de biocatalisador. Visando contribuir para o desenvolvimento de tal processo, os objetivos deste trabalho foram preparar derivados estáveis de CPA e Alcalase® e estudar a cinética da hidrólise de polipeptídios. Esses polipeptídios foram produzidos por hidrólise seqüencial de proteínas do soro de queijo com tripsina e quimotripsina. Utilizandose agarose entrecruzada (6% p/p para CPA e 10% p/p para Alcalase®) como suporte de imobilização, foram estudados diferentes métodos de ativação e condições de imobilização. Suportes glioxil-agarose altamente ativados (75 e 210 µeqv de grupos aldeídos por mililitro de suporte, respectivamente para CPA e Alcalase®) 25oC, pH 10,05 e tempo prolongado de contato (48 horas para CPA e 96 horas para Alcalase®) produziram os melhores derivados. Os derivados CPA-glioxil agarose-6% e Alcalase®-glioxil agarose-10% eram aproximadamente 213 e 515 vezes mais estáveis que as respectivas enzimas na forma solúvel. Esses derivados estabilizados retiveram 42% (para CPA-glioxil agarose-6%) e 54% (para Alcalase®-glioxil agarose-10%) da atividade imobilizada, medidas com substratos de menor massa molecular (hipuril-L-Phe para CPA, e Boc-Ala-ONp para Alcalase®) e substratos de maior massa molecular (polipeptídios com Phe carboxi-terminal para CPA, e caseína para Alcalase®). Esses resultados mostraram que toda a perda de atividade estava associada à distorção da molécula de enzima imobilizada, devido a multi-interação enzima-suporte. Derivados preparados em glutaraldeído-agarose-6% apresentaram impedimentos estéricos na hidrólise de substratos macromoleculares. A análise de aminoácidos de hidrolisados ácidos das enzimas solúveis e imobilizadas (para os derivados mais estáveis) mostrou que aproximadamente 30 e 40%, para CPA e Alcalase®, dos resíduos de lisina ligaram-se no suporte, sugerindo a existência de uma intensa ligação covalente multipontual entre a enzima e o suporte. As temperaturas de máximas taxas de hidrólise, usando respectivamente os derivados estabilizados de CPA e Alcalase®, foram 20oC e 10oC mais elevadas que aquelas obtidas para as respectivas enzimas solúveis. O derivado CPA-glioxil mais estável pôde ser eficientemente utilizado na hidrólise de polipeptídios (proteínas do soro de queijo hidrolisadas com tripsina e quimotripsina) a altas temperaturas (por exemplo, 60oC), liberando duas vezes mais aminoácidos aromáticos (Tyr, Phe e Trp) do que a enzima solúvel, sob as mesmas condições operacionais. O grau de hidrólise de caseína, a 80oC, obtido com o derivado Alcalase®-glioxil mais estável, foi duas vezes maior que aquele obtido com a enzima solúvel. Assim, os derivados produzidos permitem o projeto de um processo contínuo para a produção de hidrolisados protéicos, compostos de pequenos peptídios e com uma baixa concentração de aminoácidos aromáticos. Esse processo pode ser conduzido a alta temperatura, evitando-se assim problemas de contaminação microbiana do meio reacional. A hidrólise de resíduos carboxi-terminais a 45oC (pH 7,0), catalisada pelo derivado CPA-glioxil, pôde ser adequadamente representada por cinética de Michaelis-Menten, com inibição pelo substrato e produto. O modelo cinético foi representado em termos de ligações peptídicas carboxiterminais hidrolisáveis pela CPA, sem considerar-se a natureza do resíduo a ser liberado. A concentração de cada aminoácido liberado em função do tempo de hidrólise pôde ser ajustada por modelos empíricos (hiperbólico e decaimento exponencial). Assim, a partir da cinética de hidrólise total, é possível estimar-se a concentração de cada aminoácido em função do tempo de hidrólise. A hidrólise catalisada pelo derivado CPA-glioxil agarose-6%, com alta carga enzimática imobilizada, não foi limitada pela resistência difusional intrapartícula. A hidrólise de peptídios (bateladas de longa duração) a 50oC (pH 9,5), catalisada pelo derivado Alcalase®-glioxil agarose-10%, pôde ser adequadamente representada por cinética de Michaelis-Menten com inibição pelo produto, e os parâmetros cinéticos Vmax, KM e KI foram correlacionados com o grau de hidrólise inicial do substrato (grau de hidrólise total obtido pela prévia ação de tripsina e quimotripsina sobre as proteínas do soro de queijo). Hidrólises em batelada de longa duração, catalisadas por Alcalase®-glioxil agarose-10% com alta carga enzimática imobilizada, apresentaram efeitos de difusão, com um fator de efetividade, ηI, de aproximadamente 0,5.

Kidney bean proteins and peptides can be developed to serve as an important ingredient for the formulation of high quality foods or therapeutic products that may positively impact on body function and human health. The main goal of this thesis was to determine the in vitro structural and functional characteristics of major proteins and enzymatic protein hydrolysate of red kidney bean (Phaseolus vulgaris). Selective aammonium sulfate precipitation of the kidney bean proteins yielded 88% globulin and 7% albumin.The globulin and albumin are glycoproteins that contained ~4% and 45% carbohydrate contents, respectively. Physicochemical and functional characteristics of the globulin fraction, such as, gelation concentration, foam stability, emulsion capacity, and emulsion stability were superior to those of albumin. Reducing SDS-PAGE revealed vicilin with molecular weight of ~45 kDa as the major globulin in kidney beans. Circular dichroism spectroscopy of the purified vicilin showed reductions in α-helix, and β-pleated sheet conformations upon addition of NaCl or changes in pH. Likewise, the tertiary structures as observed from the near-UV CD spectra were also changed by shifts in pH conditions and NaCl addition. Far UV-CD showed increased β-sheet content up till 60oC from room temperature, but a steady loss in the tertiary structure as temperature was further increased; however, β-sheet structure was still detectable at 80oC. Differential scanning calorimetry thermograms showed a prominent endothermic peak with denaturation temperature at around 90oC, attributed to thermal denaturation of vicilin. Alcalase hydrolysis of kidney bean globulin produced multifunctional peptides that showed potential antihypertensive properties because of the in vitro inhibition of activities of renin and angiotensin I converting enzyme as well as the antioxidant properties. The <1 and 5-10 kDa peptide fractions exhibited highest (p<0.05) renin inhibition and the ability to scavenge 2, 2-Diphenyl-1-picrylhydrazyl free radical, inhibit peroxidation of linoleic acid and reduce Fe3+ to Fe2+. Based on this study, incorporation of kidney bean globulin as an ingredient may be useful for the manufacture of high quality food products. Likewise, the kidney bean protein hydrolysates, especially the <1 kDa fraction represent a potential source of bioactive peptides for the formulation of functional foods and nutraceuticals.

The effects of peptides derived from β-lactoglobulin and α-lactalbumin on metabolic syndrome were studied. α-lactalbumin and β-lactoglobulin were hydrolyzed with trypsin, alcalase, flavourzyme, or a combination of alcalase and flavourzyme and fractionated. Angiotensin coverting enzyme inhibition of the < 1 kDa fraction of alcalase hydrolyzed β-lactoglobulin was 95 %. Antioxidant activity of the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme was 18 %. Stimulated adipocytes incubated with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with either trypsin or alcalase produced 30 pg/mL of interleukin 6. Adiponectin and glucose transporter type 4 secretions increased 1.1 and 0.86 fold respectively during incubation with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme. Results indicate that β-lactoglobulin peptides formed with alcalase and a combination of alcalase and flavourzyme influence markers associated with metabolic syndrome and may be useful as functional foods or nutraceuticals.

Kidney bean proteins and peptides can be developed to serve as an important ingredient for the formulation of high quality foods or therapeutic products that may positively impact on body function and human health. The main goal of this thesis was to determine the in vitro structural and functional characteristics of major proteins and enzymatic protein hydrolysate of red kidney bean (Phaseolus vulgaris). Selective aammonium sulfate precipitation of the kidney bean proteins yielded 88% globulin and 7% albumin.The globulin and albumin are glycoproteins that contained ~4% and 45% carbohydrate contents, respectively. Physicochemical and functional characteristics of the globulin fraction, such as, gelation concentration, foam stability, emulsion capacity, and emulsion stability were superior to those of albumin. Reducing SDS-PAGE revealed vicilin with molecular weight of ~45 kDa as the major globulin in kidney beans. Circular dichroism spectroscopy of the purified vicilin showed reductions in α-helix, and β-pleated sheet conformations upon addition of NaCl or changes in pH. Likewise, the tertiary structures as observed from the near-UV CD spectra were also changed by shifts in pH conditions and NaCl addition. Far UV-CD showed increased β-sheet content up till 60oC from room temperature, but a steady loss in the tertiary structure as temperature was further increased; however, β-sheet structure was still detectable at 80oC. Differential scanning calorimetry thermograms showed a prominent endothermic peak with denaturation temperature at around 90oC, attributed to thermal denaturation of vicilin. Alcalase hydrolysis of kidney bean globulin produced multifunctional peptides that showed potential antihypertensive properties because of the in vitro inhibition of activities of renin and angiotensin I converting enzyme as well as the antioxidant properties. The <1 and 5-10 kDa peptide fractions exhibited highest (p<0.05) renin inhibition and the ability to scavenge 2, 2-Diphenyl-1-picrylhydrazyl free radical, inhibit peroxidation of linoleic acid and reduce Fe3+ to Fe2+. Based on this study, incorporation of kidney bean globulin as an ingredient may be useful for the manufacture of high quality food products. Likewise, the kidney bean protein hydrolysates, especially the <1 kDa fraction represent a potential source of bioactive peptides for the formulation of functional foods and nutraceuticals.

The effects of peptides derived from β-lactoglobulin and α-lactalbumin on metabolic syndrome were studied. α-lactalbumin and β-lactoglobulin were hydrolyzed with trypsin, alcalase, flavourzyme, or a combination of alcalase and flavourzyme and fractionated. Angiotensin coverting enzyme inhibition of the < 1 kDa fraction of alcalase hydrolyzed β-lactoglobulin was 95 %. Antioxidant activity of the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme was 18 %. Stimulated adipocytes incubated with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with either trypsin or alcalase produced 30 pg/mL of interleukin 6. Adiponectin and glucose transporter type 4 secretions increased 1.1 and 0.86 fold respectively during incubation with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme. Results indicate that β-lactoglobulin peptides formed with alcalase and a combination of alcalase and flavourzyme influence markers associated with metabolic syndrome and may be useful as functional foods or nutraceuticals.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-11-29T17:12:51Z No. of bitstreams: 1 2013 - Renata Silva Cabral de Souza.pdf: 1753830 bytes, checksum: 2dff8752eb3f7974e34f8809d059f126 (MD5) / Made available in DSpace on 2018-11-29T17:12:51Z (GMT). No. of bitstreams: 1 2013 - Renata Silva Cabral de Souza.pdf: 1753830 bytes, checksum: 2dff8752eb3f7974e34f8809d059f126 (MD5) Previous issue date: 2013-05-23 / CAPES / The aim of this study was to evaluate the process of protein concentration in bovine whey proteins by ultrafiltration process and subsequently the protein hydrolysate obtained by enzymatic hydrolysis to produce bioactive peptides with potential antimicrobial and antioxidant activities. For concentration process was used a ceramic ultrafiltration membrane with a molecular range cut-off of 10-20 kDa, transmembrane pressure of 5 bar and, temperature of 30 ?C, 40 ?C and 50 ?C . The optimum temperature condition was at 40 ?C. The Volume Concentrate Factor (VCF) parameter was used as a end-point of the ultrafiltration process and fixed at 2, corresponding on concentrating the initial volume twice, in volume. At the temperature of 40 ?C, VCF had a correspondence on final protein concentration on the concentrated fraction by ultrafiltration and confirmed by Bradford method. Two commercial enzymes were tested Alcalase, Flavourzyme and an equivalent mixture of both 50:50 (w/w) in the hydrolysis reaction. The hydrolysis conditions were determined according to manufacturer instructions and confirmed by other studies: 60 ?C and pH 8 for Alcalase; 50 ?C and pH 7 for Flavourzyme; 50 ?C and pH 8 for enzyme mixture with enzyme / substrate ratio (w / w) 5/100 for all enzymes. The reaction was monitored by pH Stat method. The final Degree of Hydrolysis (DH) achieved was 15%, 52% and 63% for Flavourzyme, Alcalase and enzyme mixture, respectively. Five aliquots were collected along the hydrolysis for each enzyme reaction corresponding to differents DH in order to evaluatethe antioxidant activity by ORAC and ABTS assays with final values between 597- 1092 m? TE (ABTS) and from 1615 to 2920 ?M TE (ORAC) for Flavourzyme; 998-6290 ?M TE (ABTS) and 3092-7567 ?M TE ( ORAC) for Alcalase and finally 913-2678 ?M TE (ABTS) and 2547-5903 ?M TE (ORAC) for the enzyme mixture. The samples from all hydrolysates showed no antimicrobial activity against strains of Salmonella choleraesuis subsp. Enteritidis (ATCC 13076) and Listeria monocytogenes (ATCC 9117). / A proposta do presente trabalho foi avaliar a concentra??o das prote?nas do soro de leite bovino por ultrafiltra??o e posterior obten??o de hidrolisados proteicos deste concentrado via hidr?lise enzim?tica visando obter pept?deos bioativos com potencial atividade antimicrobiana e antioxidante. Para concentra??o das prote?nas do soro foi utilizada membrana cer?mica de ultrafiltra??o com massa molar de corte de 10-20 kDa, press?o aplicada ? membrana de 5 bar, temperaturas testadas (30 ?C, 40 ?C e 50 ?C) . A temperatura ?tima selecionada foi de 40 ?C. O Fator de Concentra??o Volum?trica foi o par?metro utilizado para indicar o final do processo de ultrafiltra??o sendo fixado em duas vezes o volume inicial da alimenta??o. Na temperatura de 40 ?C foi obtida correspond?ncia entre a concentra??o volum?trica e a concentra??o proteica final na fra??o retida pela UF, que tamb?m foi o dobro da encontrada na fra??o alimenta??o, avaliada pelo m?todo de Bradford. Foram testadas duas enzimas comerciais: Alcalase, Flavourzyme e uma mistura equivalente de ambas, na propor??o 50:50 (m/m) na rea??o de hidr?lise. As condi??es de rea??o enzim?tica foram determinadas de acordo com instru??es do fabricante e corroboradas por outros estudos em: 60 ?C, pH 8 para Alcalase; 50 ?C, pH 7 para Flavourzyme; 50 ?C, pH 8 para mistura enzim?tica e rela??o enzima/substrato (g/g) foi de 5/100 para todas as enzimas. A rea??o de hidr?lise foi monitorada pelo m?todo pH Stat. Os Graus de Hidr?lise (GH) finais alcan?ados foram de 15 %, 52 % e 63 % para Flavourzyme, mistura enzim?tica e Alcalase, respectivamente. Foram coletadas cinco al?quotas correspondentes a diferentes GH ao longo da rea??o para cada condi??o enzim?tica utilizada e avaliadas quanto a atividade antioxidante pelos m?todos ABTS e ORAC tendo valores entre 597 a 1092 ?M TE (ABTS) e 1615 a 2920 ?M TE (ORAC) para Flavourzyme, 998 a 6290 ?M TE (ABTS) e 3092 a 7567 ?M TE (ORAC) para Alcalase e por fim, 913 a 2678 ?M TE (ABTS) e 2547 a 5903 ?M TE (ORAC) para a mistura enzim?tica. Nenhuma das amostras de hidrolisado com diferentes GH apresentou atividade antimicrobiana contra cepas de Salmonella choleraesuis subsp. Enteritidis (ATCC 13076) e Listeria monocytogenes (ATCC 9117).

Submitted by Viviane Lima da Cunha (viviane@biblioteca.ufpb.br) on 2016-02-22T15:25:54Z No. of bitstreams: 1 arquivototal.pdf: 1326791 bytes, checksum: 047e6cb846e3a375011e97377274ccc0 (MD5) / Made available in DSpace on 2016-02-22T15:25:54Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1326791 bytes, checksum: 047e6cb846e3a375011e97377274ccc0 (MD5) Previous issue date: 2015-03-24 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The reactive oxygen species (ROS) are generated through reactions physiologically normal in the human organism during the respiratory process and perform various functions such as signage and providing defense against infections. However, in excessive amounts the ROS cause cellular damage and are involved in the initiation or progression of degenerative chronic diseases. In contrast, the antioxidant agents play a vital role for reduce the processes oxidative in the organism. Among them are hydrolyzed peptides from protein sources and, that demonstrate antioxidant activity. However, it is necessary the hydrolytic process for release of small fragments of amino peptide with the capacity to reduce the ROS. However, the conditions of hydrolysis employed such as type of enzyme, enzyme concentration, pH, time and temperature can influence the formation of peptides with antioxidant properties. Thus, this study aimed to obtain hydrolyzed from okra seed proteins using Alcalase® hydrolytic enzyme in different conditions and evaluate the antioxidant capacity of the hydrolysates produced. Using a Central Composite Rotatable Design (CCRD), was evaluated the influence of independent variables: enzyme concentration (EC) and hydrolysis time (T), on the dependent variables: degree of hydrolysis (DH), protein recovery efficiency (PRE), total antioxidant capacity (TAC) and scavenging of radicals radicais 2.2’-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) e 2.2-diphenyl-1-picrilidrazil (DPPH). The results showed that the increase in EC and T influenced positively on DH and TAC and only increased T influenced on the PRE, the ability of eliminating radical ABTS and DPPH were not influenced by the independent variables. It was verified that the increased TAC is directly into proportional to the increase in DH. The electrophoretic profile revealed that okra seed protein concentrate (OSPC), presents protein bands with relative masses above 38 kDa and less than 8.5 kDa. It was also found, the efficiency of the process of hydrolysis by Alcalase®, evidenced by the disappearance of most of the protein bands and the appearance of peptides of molecular mass < 3.5 kDa in hydrolysates with a higher DH. The OSPC showed lower TAC and ability of the ABTS and DPPH radical scavenger in comparison to the hydrolysates obtained. The best operating conditions of the independent variables (EC and T) obtained in the optimization study, using the methodology CCRD was possible to obtain a hydrolyzate considered optimal, the enzyme concentration was 2% and time of hydrolysis of 300 min, in this condition the hydrolyzed resulted in increased DH (19.32%), TAC (51.54%) and significant results for DPPH (52.60%) and ABTS (73.04%). The hydrolyzed great presented a considerable content of amino acids with ability to donate protons and electrons, especially acids (32.90 g/100 g of protein), hydrophobic (38.58 g/100 g of protein) and aromatic (9.43 g/100 g of protein). The results show that the protein hydrolyzate of okra seed emerges as a promising bioactive compound, with antioxid capacity. However, other studies are needed to assess the effects that the hydrolysate can produce in vivo, in order to verify the effectiveness of antioxidant activity in the human organism. / As espécies reativas de oxigênio (EROs) são geradas através de reações fisiologicamente normais no organismo humano durante o processo respiratório e exercem diversas funções como sinalização e fornecimento de defesa contra infecções. No entanto, em quantidades excessivas as EROs causam dano celular e estão envolvidas na iniciação ou progresso de doenças crônicas degenerativas. Em contrapartida, os agentes antioxidantes desempenham um papel vital para reduzir os processos oxidativos no organismo. Dentre os quais destacam-se hidrolisados e peptídeos oriundos de fontes proteícas, que demonstram atividade antioxidante. Porém, se faz necessário o processo hidrolítico para que ocorra a liberação dos pequenos fragmentos de peptídeos com capacidade em reduzir as EROs. Todavia, as condições de hidrólise empregadas tais como tipo de enzima, concentração enzimática, pH, tempo e temperatura podem influenciar a formação de peptídeos com propriedades antioxidantes. Desta forma, esse estudo objetivou obter hidrolisado a partir das proteínas de sementes de quiabo com uso da enzima Alcalase® em diferentes condições hidrolíticas e avaliar a capacidade antioxidante dos hidrolisados produzidos. Utilizando um delineamento composto central rotacional (DCCR), foi avaliada a influência das variáveis independentes: concentração enzimática (CE) e tempo de hidrólise (T), sobre as variáveis dependentes: grau de hidrólise (GH), rendimento da recuperação de proteínas (RRP), capacidade antioxidante total (TAC) e sequestro dos radicais 2,2’-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) e 2,2-diphenyl-1-picrilidrazil (DPPH). Os resultados mostraram que o aumento da CE e T influenciaram positivamente sobre o GH e TAC e apenas o aumento do T influenciou no RRP, a capacidade de eliminação dos radicais ABTS e DPPH não foram influenciadas pelas variáveis independentes. Foi verificado que o aumento da TAC é diretamente proporcional ao aumento do GH. O perfil eletroforético revelou que o concentrado proteico de sementes de quiabo (CPSQ) apresenta bandas proteícas com massas relativas acima de 38 kDa e menor que 8,5 kDa. Foi constatado ainda a eficiência do processo de hidrólise pela Alcalase®, através do desaparecimento da maioria das bandas proteicas e aparecimento de peptídeos de massa molecular < 3,5 kDa nos hidrolisados com maior GH. O CPSQ apresentou menor TAC e capacidade sequestrante dos radicais ABTS e DPPH em comparação aos hidrolisados obtidos. Operando-se as melhores condições das variáveis independentes (CE e T) obtidas no estudo de otimização, a partir da metodologia do DCCR foi possível obter um hidrolisado considerado ótimo, cuja concentração enzimática foi de 2% e tempo de hidrólise de 300 min, nesta condição o hidrolisado resultou em maior GH (19,32%), TAC (51,54%) e resultados expressivos para DPPH (52,60%) e ABTS (73,04%). O hidrolisado ótimo apresentou um teor considerável de aminoácidos com capacidade em doar prótons e elétrons, destacando-se os ácidos (32,90 g/100g de proteína), hidrofóbicos (38,58 g/100g de proteína) e aromáticos (9,43 g/ 100g de proteína). Os resultados evidenciam que o hidrolisado proteico de sementes de quiabo surge como um composto bioativo promissor, com capacidade antioxidante. No entanto, outros estudos são necessários para avaliar os efeitos que o hidrolisado pode produzir in vivo, no intuito de constatar a efetividade da atividade antioxidante no organismo humano.