Ce travail étudie l'intérêt de l'ajout d'hydrolysats de levure dans un procédé de culture de cellules CHO productrices d'anticorps en vue, d'une part, de déterminer leur condition d'utilisation et leur rôle, et, d'autre part, de caractériser les composés actifs. Pour répondre à ces objectifs, une démarche intégrant des études cinétiques, des stratégies de fractionnement et l'analyse biochimique des hydrolysats et de leurs fractions a été développée. En premier lieu, il a été montré que les hydrolysats de levure présentent des effets significatifs sur les cultures selon leur composition et les conditions d'ajout. De même, des effets synergiques ont été mis en évidence par le mélange d'hydrolysats générés à partir de différents procédés. D'autre part, des études cinétiques ont permis de corréler l'influence positive des hydrolysats sur la croissance cellulaire à l'amélioration du métabolisme énergétique. Dans un deuxième temps, la nature biochimique et le rôle des composés actifs ont été étudiés par la mise en oeuvre d'un procédé de nanofiltration membranaire et la reconstitution de mélanges de molécules contenues dans un extrait de levure (EXL). Ces résultats ont mis en évidence l'intérêt des di- et tri-peptides pour approvisionner le métabolisme énergétique et de molécules non nutritives, de poids moléculaire supérieur à 500 Da, pour stimuler la vitesse spécifique de croissance des cellules. Finalement, le rétentat issu de la nanofiltration de l'EXL a été fractionné à l'aide de divers procédés chromatographiques, unitaires ou associés, pour caractériser les propriétés physico-chimiques des composés actifs. L'effet des fractions sur la culture de cellules a alors souligné l'intérêt des molécules chargées positivement, et plus particulièrement, des peptides hydrophiles et cationiques pour stimuler la croissance des cellules. Ainsi, nos travaux permettent de mieux appréhender les mécanismes d'action des hydrolysats de levure sur les cellules CHO productrices d'anticorps et proposent des voies d'optimisation pour la simplification d'additifs complexes dans les milieux de culture dédiés à la culture de cellules animales / This work studies the interest of the addition of yeast hydrolysates in culture medium of CHO cell producing antibody, to determine the operating conditions and their role, but also to improve the characterization of active compounds. In this way, an integrated approach including kinetic studies, fractionation strategies and biochemical analysis of hydrolysates and of their fractions was developed. First, we showed that yeast hydrolysates exhibited various properties depending on their composition and the operating conditions. In addition, synergistic effects were observed with different hydrolysate mixtures. Besides, kinetic studies underlined that the positive influence of hydrolysates on cell growth is correlated with energetic metabolism improvement. Then, the biochemical nature and the role of active compounds were studied by the implementation of a nanofiltration process and the reconstitution of mixtures of molecules contained in a yeast extract (YE). The results highlighted the interest of di- and tri-peptides to supply energetic metabolism, and of non-nutritive molecules, exhibiting a molecular weight greater than 500 Da, to stimulate the specific cell growth rate. Finally, the retentate fraction of nanofiltrated YE was fractionated by various chromatographic processes to characterize the physico-chemical properties of active compounds. The effect of fractions on cell culture emphasized the positive effect of positively charged molecules, especially hydrophilic and cationic peptides, to stimulate the cell growth. Thus, our work provides important insights in yeast hydrolysate mechanisms on CHO cells and suggests procedures to simplify such a complex additive of media dedicated to mammalian cell culture

Digestion of starch in humans starts in the mouth and progresses to the small intestine. Structures from salivary and pancreatic amylases hydrolysis can impact subsequent steps of digestion at the mucosa of the small intestine. However, structures of the starch digestion products along the gut from the mouth to the small intestines – products that impact glucose homeostasis are not well understood. This thesis focuses on the luminal step of starch digestion, i.e. impact of salivary and pancreatic amylase on the structure of hydrolysis products obtained from cooked starches from different botanical sources. Normal corn (NCS), wheat (NWS) and potato (NPS) starches were cooked at 1:0.7 (T0.7) or 1:2 (T2) starch:water ratios. Cooked starches were subjected to salivary amylase at conditions mimicking oral digestion. The composition of the hydrolysates was characterised by gel-permeation chromatography. Extent of hydrolysis was lower at T0.7 compared to T2, but the amount of carbohydrates in different fractions and the molecular weight profiles within each treatment were not different between starches from different botanical sources. However, debranching of the hydrolysates revealed structural differences in extent of amylose hydrolysis and amount and profile of lower molecular weight fractions between different starches. Cooked starches were also subjected to salivary and pancreatic amylases hydrolysis. Extent of 20 min hydrolysis was lower at T0.7 compared to T2 for all the starches. Oligosaccharide composition of 120 min hydrolysates differed in amounts of DP 2, 3, 5, 6 and 7 between processing treatments and starches. NCS (T2) was treated with saliva from six participants at equal activity. Salivary amylase activities ranged from 470 x 103 to 118 x 103 U/mL among the participants. While saliva from participant 2 (high amylase activity) greatly reduced the high molecular weight fraction, saliva from participant 6 (low amylase activity) more extensively hydrolysed the starch to small molecular weight fractions of oligosaccharides. These results show that different starch hydrolysates are produced during oral digestion by saliva from different individuals and are also different based on cooking condition or botanical source of starch. Further research is therefore needed to understand how these hydrolysate structures, impact glucose homeostasis.

Made available in DSpace on 2016-06-02T19:56:48Z (GMT). No. of bitstreams: 1 4595.pdf: 1437433 bytes, checksum: f83e0ea8c49064050b3f382c7d942d28 (MD5) Previous issue date: 2012-08-27 / Financiadora de Estudos e Projetos / Diseases caused by Streptococcus pneumoniae are one of the main problems of public health in the world. The pneumococcal surface protein A(PspA) is a potential canditate as carrier in a conjugate vaccine against this bacteria. Considering the inherent high losses of the purification and conjugation steps, it is fundamental to adopt a strategy of cultivation and expression that allows the obtainance of large quantities of protein. Thus, the use of Escherichia coli as expression system as well as its cultivation in complex medium constitutes promising alternatives for reducing the cost and increasing the productivity of the process. The goal of this work was to study the influence of the temperature and cultivation medium composition over the production of a PspA belonging to clade 4 protein fragment (PspA4Pro) during rE coli cultivations, aiming at to evaluate the possibility of employing vegetable-based nitrogen sources (soybean protein hydrolisates) instead of the Triptona, an animal-derived nitrogen source. The experiments were carried out in both shakers and benchscale bioreactor, using a complex medium which contained glucose and glycerol as carbon sources, lactose as inducer and Soytone, Phytone or Triptone as nitrogen sources, besides yeast extract. Samples were collected during the experiments to follow the cell growth (measurements of absorbance, dry cell weight and permittivity signal from biomass sensors), the carbon sources consumption and the production of organic acids by HPLC analysis. The stability of the plasmid (agar plates with or without kanamycin) and the production of recombinant protein (Bradford and SDS-PAGE electrophoresis followed by densitometry) were also evaluated. Preliminary experiments were performed in shake flasks, incubated at 300rpm and 37ºC, employing both complex and defined media. The highest productivity was achieved in complex medium, with a 42% superior protein production. Subsequently, nine complementary experiments were conducted in shake flasks with complex medium, under the agitation of 300rpm and temperatures of 37ºC (growth phase) and 25, 31 or 37ºC (induction phase). The largest specific production of soluble PspA4Pro was verified at 25ºC, reaching, respectively, 209±6, 192±5mg/g dry cell mass for Phytone and Triptone, with final absorbance values (after 12h of induction) of 9.0±0.4 and 8.5±0.4. The best protein production for Soytone (124±4mg/g dry cell weight) was observed at 31ºC, yielding a final absorbance 8.0±0.4. From the results obtained in the preliminary tests, the nitrogen source Phytone was selected for experiments in bioreactor. Four batch cultures were conducted in bench-scale bioreactor (5L), containing a modified auto-induction complex medium (10g/L glucose, 60g/L glycerol and 20g/L lactose), being three of them with Phytone and one with Triptone, for comparison. The best results in terms of protein production (245±7mg of PspA4Pro soluble/g dry mass) were obtained in the presence of Phytone, corresponding to an increase of 16% towards the maximum value achieved in the cultivation with Triptone. These results demonstrate the potential of vegetable-based nutrients as alternatives to animal-derived nitrogen sources in complex media, contributing to adequate these media formulations to the current guidelines of good manufacturing practices. / Doenças causadas por Streptococcus pneumoniae constituem um dos principais problemas de saúde pública mundial. A proteína A de superfície de pneumococo (PspA) é candidata em potencial a ser carreadora em vacina conjugada contra essa bactéria. Considerando as altas perdas inerentes às etapas de purificação e conjugação da proteína, é fundamental adotar uma estratégia de cultivo e expressão que permita obter grandes quantidades de proteína. Nesse sentido, o emprego da bactéria Escherichia coli como sistema de expressão e o cultivo da mesma em meio complexo se apresentam como alternativas promissoras para redução do custo e aumento da produtividade do processo. O objetivo do presente trabalho foi estudar a influência da temperatura e da composição do meio de cultivo sobre a produção do fragmento da proteína PspA do clado 4 (PspA4Pro) em cultivos de rE. coli, visando avaliar a viabilidade de utilização de fontes de nitrogênio de origem vegetal (hidrolisados protéicos de soja) em substituição à Triptona, de origem animal. Os experimentos foram realizados em câmara incubadora e em biorreatores de bancada, utilizando meio complexo contendo glicose e glicerol e lactose como fontes de carbono, lactose como indutor e Soytone, Phytone ou Triptona como fontes de nitrogênio, além de extrato de levedura. Amostras foram coletadas ao longo dos experimentos para acompanhamento do crescimento celular (medida de absorbância, massa seca e permissividade por sensor de biomassa), do consumo das fontes de carbono e da produção de ácidos orgânicos por análises em cromatografia líquida de alto desempenho. A estabilidade do plasmídeo (plaqueamento em meio contendo ou não canamicina) e a produção de proteína recombinante (Bradford e eletroforese SDS-PAGE seguida por densitometria) também foram avaliadas. Experimentos preliminares foram realizados em frascos agitados e incubados a 300rpm e 37oC, empregando tanto o meio complexo como o definido. A maior produtividade foi obtida em meio complexo, a qual foi 42% superior a alcançada com meio definido. Em seguida, nove experimentos complementares foram conduzidos em frascos agitados em meio complexo sob agitação de 300rpm e à temperatura de 37ºC (fase de crescimento) e de 25, 31 ou 37ºC (fase de indução). Verificou-se que a temperatura de 25ºC proporcionou a maior produção específica de PspA4Pro solúvel, alcançando-se, respectivamente, 209±6, 192±5mg/g massa seca para o Phytone e para a Triptona, com absorbâncias finais (após 12h de indução) de 9,0±0,4 e 8,5±0,4. Já para o Soytone, a melhor produção de proteína (124±4mg/g massa seca) foi observada à temperatura de 31ºC, obtendo-se uma absorbância de final de 8,0±0,4. A partir dos resultados obtidos nos ensaios preliminares, a fonte de nitrogênio de origem vegetal Phytone foi selecionada para experimentos em biorreator. Quatro cultivos em batelada foram conduzidos em biorreator de bancada (5L), contendo meio complexo de autoindução modificado (10g/L glicose, 60g/L glicerol e 20g/L lactose), sendo 3 com Phytone e um com Triptona, para comparação. Os melhores resultados em termos de produção de proteína (245±7mg de PspA4Pro solúvel/g massa seca) foram obtidos na presença de Phytone, correspondendo a um aumento de 16% em relação ao valor máximo alcançado no cultivo com Triptona. Esses resultados comprovam o potencial dos nutrientes de origem vegetal como alternativa às fontes de nitrogênio de origem animal em meios complexos, contribuindo para adequar as formulações desses meios às atuais diretrizes de boas práticas de fabricação.

Orientador: Flavia Maria Netto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T21:19:52Z (GMT). No. of bitstreams: 1 Silva_FernandaGuimaraesDrummonde_M.pdf: 1363127 bytes, checksum: 68b1a97c95798f4425019ee900814160 (MD5) Previous issue date: 2012 / Resumo: Existem evidências numerosas sobre o papel dos radicais livres em uma série de condições patológicas, incluindo envelhecimento, câncer, esclerose múltipla, doenças cardiovasculares. Hidrolisados protéicos de diferentes fontes têm sido estudados por seu potencial antioxidante. A atuação antioxidante da proteína, na maioria das vezes, encontra-se limitada devido à conformação espacial, que concentra resíduos capazes de neutralizar radicais livres no interior da molécula, dificultando o acesso das espécies reativas aos centros nucleofílicos. A hidrólise da proteína contribui para aumentar a exposição desses resíduos de aminoácidos, aumentando sua atuação como antioxidante. Compostos fenólicos podem estar presentes em hidrolisados proteicos de origem vegetal, devido a sua associação com as proteínas. Métodos in vitro que simulam as condições do trato gastrointestinal permitem estudar como a digestão pode interferir na atividade antioxidante de peptídeos e compostos fenólicos. O presente trabalho tem como objetivos obter hidrolisados proteicos com capacidade antioxidante a partir da farinha de linhaça e avaliar o efeito da digestão in vitro pode interferir nessa atividade. A farinha de linhaça marrom foi desengordurada, obtendo-se a farinha de linhaça marrom desengordurada (FLMD). O concentrado proteico de linhaça (CPL) foi obtido a partir da FLMD por extração alcalina e precipitação no ponto isoelétrico seguida de neutralização. Para obtenção dos hidrolisados proteicos (HPL), a partir do CPL, com Alcalase, foi realizado um delineamento composto central rotacional (DCCR) 2². As variáveis independentes foram pH que variou entre 7,5 a 9,5 e relação enzima: substrato (E:S) que variou de 1:150 a 1:30. As variáveis dependentes foram grau de hidrólise (GH), teor de substâncias redutoras do reagente de Folin-Ciocalteau e atividade antioxidante, determinada por FRAP e ORAC. Teor de substâncias redutoras e atividade antioxidante foram avaliados a partir dos extratos aquosos e metanólico (metanol 70%). Os hidrolisados de maior atividade antioxidante, a FLMD e o CPL foram submetidos à digestão in vitro, simulando as condições da digestão gastrintestinal. As amostras antes e após a digestão in vitro foram caracterizadas por eletroforese em sistema SDS-PAGE Tricina e por cromatografia liquida de alta eficiência de fase reversa (HPLC- RP). O teor de substâncias redutoras e da atividade antioxidante das amostras FLMD, CPL e HPL foram avaliados antes e após a digestão in vitro. As condições ótimas para obtenção de HPL de maior GH (21,0%) são pH entre 7,5 e 8,0 e E:S entre 1:60 e 1:30, indicando que a faixa de pH ótimo da enzima e a alta E:S favorecem maior hidrólise do CPL. Para obtenção de HPL com maior teor de substâncias redutoras para os extratos aquoso (24 mg EAG/ g HPL) e metanólico (20 mg EAG/ g HPL) as condições ótimas são pH ~ 8,5 /E:S 1:30. Este resultado parece estar relacionado à liberação de compostos fenólicos ligados a proteína e também de peptídeos durante a hidrólise. Açúcares e aminoácidos aromáticos presentes no hidrolisado podem interferir na reação e superestimar o teor de fenóis dos HPL. A maior atividade antioxidante determinada pelo método de FRAP para o extrato aquoso (42 mg SF/ g HPL) se dá nas condições de pH ~ 9,5/E:S ~1:150 e para o extrato metanólico (40 mg SF/ g HPL) pH entre 8,5 e 9,0/E:S entre 1:90 a 1:150. Para o método de ORAC, as condições ótimas para maior atividade antioxidante no extrato aquoso (300 µmol TE/ g HPL) são pH entre 7,5 a 9,5/E:S ~ 1:30 ou ~1:150 e para o extrato metanólico (330 µmol TE/ g HPL) são pH ~ 8,5/E:S entre 1:150 e 1:30. Os hidrolisados de maior atividade antioxidante foram os obtidos em pH 8,5/E:S 1:90, e em pH 9,2/E:S 1:133 denominados HPL 0 e HPL 3, respectivamente. Para a FLMD, CPL e os hidrolisados, após a digestão in vitro, observou-se que o teor de substâncias redutoras totais aumentou (9 a 20 vezes) para todas as amostras. O teor de substâncias redutoras do CPL (~24 mg EAG/ g amostra), em ambos os extratos, após a digestão in vitro se igualou ao teor dos hidrolisados (~23 mg EAG/ g amostra). Este resultado sugere que tanto a hidrólise com Alcalase quanto o processo digestório liberam compostos redutores, dentre eles fenólicos da proteína de linhaça. A atividade antioxidante dos extratos de FLMD e CPL, determinada por FRAP, também aumentou (de 3 a 10 vezes) após a digestão, mas não se igualou à atividade antioxidante dos hidrolisados (48 mg SF/g amostra). No entanto, o CPL apresentou atividade antioxidante determinada por ORAC semelhante à dos hidrolisados no extrato aquoso (~420,24 µmol TE/ g amostra) e 10 % maior que o encontrado para os hidrolisados (~365 µmol TE/ g amostra) no extrato metanólico. Após a digestão in vitro, os hidrolisados apresentaram a maior atividade antioxidante medida por FRAP (50 mg SF/ g amostra), e o CPL, a maior atividade determinada pelo método de ORAC (~430 µmol TE/ g amostra). Estes resultados sugerem o processo digestório é tão ou mais eficiente que a Alcalase em liberar os compostos com atividade redutora no CPL. Uma vez que a metodologia de determinação da atividade antioxidante por ORAC tem maior proximidade com o mecanismo de oxirredução que ocorre in vivo, esses resultados sugerem o uso do CPL como melhor produto protéico da linhaça com maior potencial antioxidante para a formulação de nutracêuticos e alimentos funcionais / Abstract: There are several evidences which indicate the role of free radicals on a series of pathological conditions, including aging, cancer, multiple sclerosis and cardiovascular disease. Hydrolysates from different sources have been studied because of their antioxidant potential. The antioxidant activity of the protein, in most cases, is limited due to their conformation, which concentrates residues capable of neutralize free radicals in the molecule¿s core, hampering the access of the reactive species to nucleophilic sites. The protein hydrolysis contributes to increasing the exposure of these amino acid residues, increasing their role as antioxidants. Phenolic compounds may also be present in vegetable protein hydrolysates because of their association with proteins. In vitro methods that simulate the conditions of the gastrointestinal digestion are an important way to evaluate how the digestion affects the antioxidant activity of phenolic compounds and peptides. This study aims at obtaining hydrolysates with antioxidant capacity from defatted flaxseed flour and evaluate the effect of the in vitro digestion on this activity. The brown flaxseed flour was defatted, resulting in the brown defatted flaxseed meal (BDFM). The flaxseed protein concentrate (FPC) was obtained from the BDFM by alkaline extraction and precipitation at the isoelectric point followed by neutralization. To obtain the flaxseed protein hydrolysates (FPL), using FPC and Alcalase, a central composite rotational design (DCCR) was performed. The independent variables were pH ranging from 7.5 to 9.5 and enzyme: substrate ratio (E: S) that ranged from 1:150 to 1:30. The dependent variables were the degree of hydrolysis (DH), total phenolic content and antioxidant activity, determined by FRAP and ORAC. Phenolic and antioxidant activity were evaluated from the aqueous and methanol (70% methanol). The hydrolysates with the highest antioxidant activity, the CPL FLMD were submitted to the in vitro digestion. The samples obtained before and after the in vitro digestion were characterized by electrophoresis SDS-PAGE- tricine and HPLC. The total phenolic content and antioxidant activity of FLMD, CPL and HPL were evaluated before and after in vitro digestion. The optimum conditions to obtain HPL with the highest GDH (21.0%) are pH (7.5-8) and E:S ratio (1:60-1:30), which indicates that the Alcalase optimum pH and highest E:S ratio collaborates to highest hydrolysis of CPL. To obtain HPL with higher content of Folin-Ciocalteau reducing compounds content in aqueous (EAG 24 mg / g HPL) and methanol (20 mg EAG / g HPL) extracts, the optimum conditions were pH ~ 8.5 / E: S 1:30. This result seems to be related to the release of phenolic compounds bound to protein and also of peptides during hydrolysis. The highest antioxidant activity determined by the FRAP method in the aqueous extract (42 mg SF / g HPL) occurs under pH ~ 9.5 / E: S ~ 1:150 and the methanol extract (40 mg SF / g HPL) pH 8.5-9.0 / E: S 1:90-1:150. For the ORAC method, optimum conditions for increased antioxidant activity in aqueous extract (300 µmol TE / g HPL) are pH 7.5-9.5 / E: S ~ 1:30 or 1:150 and the methanol extract (330 µmol TE / g HPL) are pH ~ 8.5 / E: S 1:30-1:150. The hydrolysates with the highest antioxidant activities were obtained at pH 8.5 / E: S 1:90, and at pH 9.2 / E: S 1:133 denominated HPL ) and HPL 3, respectively. For FLMD, CPL and hydrolysates, after in vitro digestion, the content increased (9-20 times) for all samples. The Folin-Ciocalteau reducing capacity of the CPL (EAG ~ 24 mg / g sample) in both extracts after in vitro digestion equaled the content of hydrolysates (EAG ~ 23 mg / g sample). This result suggests that both hydrolysis with Alcalase and the digestion process are able to release phenolic compounds from the flaxseed products. The antioxidant activity of extracts of FLMD, CPL determined by FRAP, also increased (from 3 to 10 times) after digestion, but did not reached the antioxidant activity of hydrolysates (48 mg SF / g sample). However, when the activity was determined by ORAC, the FPC showed value similar to the hydrolysates, measured on the aqueous extract (~ 420.24 µmol TE / g sample) and 10% higher than on the methanol extract (~ 365 µmol TE / g sample). After in vitro digestion, hydrolysates showed the highest antioxidant activity measured by FRAP (SF 50 mg / g sample), and the FPC, the highest activity determined by ORAC method (~ 430 micromol TE / g sample). These results suggest that digestive process are equally or more effective than Alcalase in releasing peptides and phenolic compounds present in the FPC. Since the methodology for determining the antioxidant activity by ORAC utilizes a biologically relevant radical source, these results suggest the use of FPC as the best protein product of flaxseed with potential antioxidant in the formulation of nutraceuticals and functional foods / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

The physicochemical properties of tuna protein hydrolysates were enhanced by interaction with sodium alginate. The increase in emulsifying capacity and stability was from 50 to 150 m² g⁻¹ and from 36 to 49 min, respectively. The increase in foaming capacity and stability was from 100% to 140% and from 65% to 70%, respectively. The reason for the increased physicochemical properties was the reduced zeta potential level of tuna protein hydrolysates after interaction with sodium alginate. The change in internal structure of tuna protein hydrolysates after interaction with sodium alginate was determined by SEM and FTIR. The SEM results showed that a net cross-linking structure was formed from a sheet structure after the tuna protein hydrolysates interacted with sodium alginate. FTIR demonstrated that parts of the β-sheet of tuna protein hydrolysates were changed into an irregular coiled structure or α-helix after interaction with sodium alginate. In order to understand the interacted complex better, the rheological properties of interacted tuna protein hydrolysates with sodium alginate were further determined. In this study, the one-step was developed, easy-to-operate and cost-effective process that can further add value to tuna protein hydrolysates derived from tuna processing by-products.