Thesis--University of Jyväskylä. / Text in English; abstract and summary also in Finnish. Thesis t.p. inserted. Includes bibliographical references (p. 69-84).

Thesis--University of Jyväskylä. / Text in English; abstract and summary also in Finnish. Thesis t.p. inserted. Includes bibliographical references (p. 69-84).

O objetivo deste trabalho foi investigar o efeito da castração, tempo de confinamento, metabolismo celular e regiões do musculo sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. Oitenta e quatro bovinos (castrados e inteiros) Nelore, confinados por diferentes períodos, foram usados para conduzir estudos em músculos Longissimus dorsi e Biceps femoris. O segundo musculo foi dividido em duas porções: origem (PO) e inserção (PI). No estudo com L. dorsi, bifes foram embalados sob condições de aerobiose (PVC) e anaerobiose (vácuo) e maturados por 1, 3, 5, 7 e 9, e 1, 7, 14 e 21 dias, respectivamente. Para este músculo, nenhuma diferença na estabilidade oxidativa [tióis, carbonilas e Substancias Reativas ao Ácido Tiobarbiturico (TBARS)] e cor entre as carnes dos animais inteiros e castrados foram encontradas. Isto poderia ser explicado pela falta de diferença no status oxidativo inicial, mensurados através da atividade de enzimas antioxidantes, conteúdo de glutationa total e composição de ácidos graxos, entre as condições sexuais. Os resultados também indicaram que a oxidação dos bifes embalados a vácuo leva o dobro de dias para iniciar, em comparação aos bifes em aerobiose. No estudo com o Bíceps femoris, os animais foram abatidos com 59 e 129 dias de confinamento e os bifes da PO e PI foram maturados por 1, 30, 60 e 100 dias. Os resultados da atividade das enzimas lactato desidrogenase e citrato sintase mostraram que a PO tem metabolismo mais oxidativo (aeróbio) e a PI glicolítico (anaeróbio). A carne dos animais inteiros tiveram menor TBARS e maior luminosidade (L*), perda de peso por cocção (PPC) e força de cisalhamento (FC) em comparação aos animais castrados. A PO foi mais susceptível a oxidação proteica (menor tióis) em comparação a PI. A carne dos animais confinados por 129 dias tiveram maiores PPC e oxidação proteica (menores tióis) em comparação a carne dos animais confinados por 59 dias. Diferenças de estabilidade oxidativa entre a carne de animais castrados e inteiros confinados por menor período desapareceram quando os animais foram confinados por maior período. Valores de pH e tióis na carne dos animais castrados e inteiros foram afetados pelo tempo de maturação. Ambas as condições sexuais tiveram carne com maior valores de pH no dia 30 de maturação e este, se manteve ao longo do tempo. A PO teve maiores valores de TBARS no dia 60, PPC no dia 100 e FC nos dias 30 e 60 de maturação em comparação a PI. Foi observada uma interação entre tempo de confinamento e tempo de maturação para tióis, TBARS, metamioglobina, pH, L* e FC. Quando comparado aos animais confinados por 59 dias, os animais confinados por 129 dias tiveram: maior oxidação (maior TBARS e menor tióis) nos dias 60 e 100 de maturação; oxidação da mioglobina (metamioglobina) mais tardia, sendo o maior valor obtido no dia 100; menor luminosidade (L*) em todos os tempos de maturação; maior maciez (menor FC) aos 100 dias de maturação. Os animais confinados por 59 dias tiveram: maior oxidação proteica (menor tióis) e maciez (menor FC) aos 30 dias de maturação. De forma geral, todos os efeitos testados tais como castração, tempo de confinamento, metabolismo celular e regiões do musculo pareceram influenciar sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. / The objective of this work was to investigate the effect of the castration, time on confinement, cellular metabolism and muscle region on the protein and lipid oxidation, and other traits of beef quality. Eight-four Nellore cattle (steers and bulls), confined for different periods, were used to conduct studies in Longissimus dorsi and Biceps femoris muscles. The latter muscle was divided in two portions: origin (OP) and insertion (IP). In the study of L. dorsi muscle, steaks were packaged under aerobiosis (PVC) and anaerobiosis (vacuum) conditions and aged for 1, 3, 5, 7 and 9, and 1, 7, 14 and 21 days, respectively. For this muscle, no differences in oxidative stability [thiols, carbonyls and Thiobarbituric Acid Reactive Substances (TBARS)] and color between the meat from bulls and steers were found. This could be explained by the lack of differences in initial oxidative status, measured through the activity of the antioxidants enzymes, content of total glutathione and composition of fatty acids, between the sexual conditions. The results also indicated that the oxidation of the steaks vacuum-packaged took about twice more days to start than the steaks under aerobiosis. In the study of Biceps femoris muscle, the animals were slaughtered after 59 and 129 days on confinement and the steaks from OP and IP were aged for 1, 30, 60 and 100 days. The results of the lactate dehydrogenase and citrate synthase enzymes activity showed that the OP has a more oxidative metabolism and the IP has a more glycolytic metabolism. The meat from bulls had lower TBARS and higher lightness (L*), cooking loss (CL) and shear force (SF) in comparison with steers. The OP was more susceptible to protein oxidation (lower thiols) than the IP. Animals confined for 129 days had meat with higher CL when compared to those ones confined for 59 days. The meat from animals confined for 129 days had higher CL and protein oxidation (lower thiols) in regard to the meat from animals confined for 59 days. Differences in oxidative stability between the meat from steers and bulls confined for shorter period disappeared when the animals were confined for larger period. Values of pH and thiols in meat from steers and bulls were affected by the time of aging. Both the sexual conditions had meat with higher pH values at the day 30 of aging and this was kept across the time. The OP had higher values of TBARS at the day 60, CL at the day 100 and SF at the days 30 and 60 of aging when compared to the IP. It was observed an interaction between confinement time and aging time for thiols, TBARS, metmyoglobin, pH, L* and SF. When compared to the animals confined for 59 days, the animals confined for 129 days had: higher oxidation (higher TBARS and lower thiols) at the days 60 and 100 of aging; oxidation of myoglobin (metmyoglobin) slower, since the higher value was obtained at the day 100; lower lightness (L*) in all the times of aging; tender meat (lower SF) at 100 days of aging. The animals confined for 59 days had: higher protein oxidation (lower thiols) and tender meat (lower SF) at 30 days of aging. Overall, all the effects tested such as castration, time on confinement, cellular metabolism and muscle region seemed to influence on the protein and lipid oxidation and other traits of beef quality.

Orientador: Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T08:13:02Z (GMT). No. of bitstreams: 1 Bassalo_MarceloColika_M.pdf: 8530833 bytes, checksum: 675d7e6b45b1284552a7e3f9939d929a (MD5) Previous issue date: 2013 / Resumo: A sociedade atual é fundamentalmente dependente do petróleo, recurso natural inserido na grande maioria dos setores da economia. Entretanto, fatores como a limitada disponibilidade deste recurso, sua instabilidade no mercado devido a problemas de natureza geopolítica e a emissão de dióxido de carbono ocasionada pela utilização deste combustível, acentuaram as iniciativas para substituir o petróleo por fontes alternativas e renováveis de matéria prima. A bactéria Propionibacterium acidipropionici surge como uma excelente candidata para a substituição de compostos petroquímicos, através da produção do ácido propiônico. No entanto, antes de transformar esta bactéria em uma plataforma industrial, é necessário aprofundar a compreensão do metabolismo deste microrganismo e desenvolver ferramentas de manipulação genética. No que diz respeito à compreensão do metabolismo, poucos estudos avaliaram o perfil aeróbico desta bactéria, considerada anaeróbica estrita até recentemente. No presente trabalho, foi identificada nesta bactéria a presença de todos os componentes de uma cadeia transportadora de elétrons. No entanto, a citocromo c oxidase identificada apresenta-se mutada e os testes realizados confirmaram a não funcionalidade deste complexo. A existência de uma oxidase alternativa, a citocromo bd oxidase, caracterizada pela alta afinidade ao oxigênio, surge então como uma hipótese promissora acerca da microaerofilia desta bactéria. O trabalho também avaliou o perfil fermentativo dessa bactéria em condições aeróbicas com diferentes fontes de carbono, o que ressaltou a enorme flexibilidade metabólica apresentada por P. acidipropionici, capaz de redirecionar o fluxo de carbono para diferentes produtos finais a depender da necessidade de manutenção do balanço redox. Este estudo também revelou uma propriedade bastante peculiar e industrialmente relevante do xarope de cana-de-açúcar. A fermentação aeróbica com este substrato, ao contrário de todas as outras fontes de carbono, apresentou um crescimento superior ao das condições anaeróbicas e, adicionalmente, exibiu um perfil fermentativo próximo ao observado em ausência de oxigênio. A identificação do composto presente no xarope de cana-de-açúcar, responsável por simular o metabolismo anaeróbico, poderia viabilizar a produção do ácido propiônico em dornas de fermentação aeróbicas, o que traria enormes benefícios para a produção economicamente viável do ácido propiônico e na implementação de P. acidipropionici como uma plataforma industrial / Abstract: The dependence of contemporary society on petroleum is axiomatic, and this natural resource could be found intrinsically embedded in the vast majority of economic sectors. Nonetheless, the limited availability of this natural resource, the instability in the stock market due to geopolitical problems, and also the carbon dioxide emissions associated with the use of fossil fuels have highlighted the need to search for renewable energy sources. The bacteria Propionibacterium acidipropionici arises as an excellent strategy for the substitution of petrochemical compounds, through the production of propionic acid. Before we could implement this bacterium as an industrial platform, however, it becomes necessary to enhance the knowledge regarding the metabolism of P. acidipropionici, and thus create a backbone for the development of genetic manipulation tools. Regarding the metabolism of this bacterium, there aren't comprehensive studies about its aerobic metabolism, thus being considered strict anaerobes until recently. In the present work, it was identified that P. acidipropionici has all required components for a functional electron transport chain. However, the cytochrome c oxidase of this bacterium has a frameshift mutation, and the functional studies proved that this complex is not operative. The presence of an alternative oxidase of high oxygen affinity, called cytochrome bd oxidase, is then suggested as a hypothesis to explain the microaerofilic habit of this bacterium. This work has also shed light into the fermentative profile showed by this bacterium under aerobic cultivation with different carbon sources, bringing attention to the highly flexible metabolism of P. acidipropionici. This bacterium has shown to be capable of completely changing its carbon flux to different end products, as a strategy to maintain the redox balance. In addition, this work has also unveiled an interesting and industrially-relevant property of the sugar cane syrup. It was demonstrated that the aerobic cultivation of P. acidipropionici with sugar cane syrup increased the culture growth, as well as it changed the fermentation end products in a way more similar to the anaerobic cultivation. It was hypothesized that this unusual property found in the sugar cane syrup was due to the presence of a mineral compound that could be used as a final electron acceptor by P. acidipropionici. The identification of this specific compound would allow the aerobic production of propionic acid in industrial conditions, and thus could be a major breakthrough to turn its industrial production into an economically viable process / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

O objetivo deste trabalho foi investigar o efeito da castração, tempo de confinamento, metabolismo celular e regiões do musculo sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. Oitenta e quatro bovinos (castrados e inteiros) Nelore, confinados por diferentes períodos, foram usados para conduzir estudos em músculos Longissimus dorsi e Biceps femoris. O segundo musculo foi dividido em duas porções: origem (PO) e inserção (PI). No estudo com L. dorsi, bifes foram embalados sob condições de aerobiose (PVC) e anaerobiose (vácuo) e maturados por 1, 3, 5, 7 e 9, e 1, 7, 14 e 21 dias, respectivamente. Para este músculo, nenhuma diferença na estabilidade oxidativa [tióis, carbonilas e Substancias Reativas ao Ácido Tiobarbiturico (TBARS)] e cor entre as carnes dos animais inteiros e castrados foram encontradas. Isto poderia ser explicado pela falta de diferença no status oxidativo inicial, mensurados através da atividade de enzimas antioxidantes, conteúdo de glutationa total e composição de ácidos graxos, entre as condições sexuais. Os resultados também indicaram que a oxidação dos bifes embalados a vácuo leva o dobro de dias para iniciar, em comparação aos bifes em aerobiose. No estudo com o Bíceps femoris, os animais foram abatidos com 59 e 129 dias de confinamento e os bifes da PO e PI foram maturados por 1, 30, 60 e 100 dias. Os resultados da atividade das enzimas lactato desidrogenase e citrato sintase mostraram que a PO tem metabolismo mais oxidativo (aeróbio) e a PI glicolítico (anaeróbio). A carne dos animais inteiros tiveram menor TBARS e maior luminosidade (L*), perda de peso por cocção (PPC) e força de cisalhamento (FC) em comparação aos animais castrados. A PO foi mais susceptível a oxidação proteica (menor tióis) em comparação a PI. A carne dos animais confinados por 129 dias tiveram maiores PPC e oxidação proteica (menores tióis) em comparação a carne dos animais confinados por 59 dias. Diferenças de estabilidade oxidativa entre a carne de animais castrados e inteiros confinados por menor período desapareceram quando os animais foram confinados por maior período. Valores de pH e tióis na carne dos animais castrados e inteiros foram afetados pelo tempo de maturação. Ambas as condições sexuais tiveram carne com maior valores de pH no dia 30 de maturação e este, se manteve ao longo do tempo. A PO teve maiores valores de TBARS no dia 60, PPC no dia 100 e FC nos dias 30 e 60 de maturação em comparação a PI. Foi observada uma interação entre tempo de confinamento e tempo de maturação para tióis, TBARS, metamioglobina, pH, L* e FC. Quando comparado aos animais confinados por 59 dias, os animais confinados por 129 dias tiveram: maior oxidação (maior TBARS e menor tióis) nos dias 60 e 100 de maturação; oxidação da mioglobina (metamioglobina) mais tardia, sendo o maior valor obtido no dia 100; menor luminosidade (L*) em todos os tempos de maturação; maior maciez (menor FC) aos 100 dias de maturação. Os animais confinados por 59 dias tiveram: maior oxidação proteica (menor tióis) e maciez (menor FC) aos 30 dias de maturação. De forma geral, todos os efeitos testados tais como castração, tempo de confinamento, metabolismo celular e regiões do musculo pareceram influenciar sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. / The objective of this work was to investigate the effect of the castration, time on confinement, cellular metabolism and muscle region on the protein and lipid oxidation, and other traits of beef quality. Eight-four Nellore cattle (steers and bulls), confined for different periods, were used to conduct studies in Longissimus dorsi and Biceps femoris muscles. The latter muscle was divided in two portions: origin (OP) and insertion (IP). In the study of L. dorsi muscle, steaks were packaged under aerobiosis (PVC) and anaerobiosis (vacuum) conditions and aged for 1, 3, 5, 7 and 9, and 1, 7, 14 and 21 days, respectively. For this muscle, no differences in oxidative stability [thiols, carbonyls and Thiobarbituric Acid Reactive Substances (TBARS)] and color between the meat from bulls and steers were found. This could be explained by the lack of differences in initial oxidative status, measured through the activity of the antioxidants enzymes, content of total glutathione and composition of fatty acids, between the sexual conditions. The results also indicated that the oxidation of the steaks vacuum-packaged took about twice more days to start than the steaks under aerobiosis. In the study of Biceps femoris muscle, the animals were slaughtered after 59 and 129 days on confinement and the steaks from OP and IP were aged for 1, 30, 60 and 100 days. The results of the lactate dehydrogenase and citrate synthase enzymes activity showed that the OP has a more oxidative metabolism and the IP has a more glycolytic metabolism. The meat from bulls had lower TBARS and higher lightness (L*), cooking loss (CL) and shear force (SF) in comparison with steers. The OP was more susceptible to protein oxidation (lower thiols) than the IP. Animals confined for 129 days had meat with higher CL when compared to those ones confined for 59 days. The meat from animals confined for 129 days had higher CL and protein oxidation (lower thiols) in regard to the meat from animals confined for 59 days. Differences in oxidative stability between the meat from steers and bulls confined for shorter period disappeared when the animals were confined for larger period. Values of pH and thiols in meat from steers and bulls were affected by the time of aging. Both the sexual conditions had meat with higher pH values at the day 30 of aging and this was kept across the time. The OP had higher values of TBARS at the day 60, CL at the day 100 and SF at the days 30 and 60 of aging when compared to the IP. It was observed an interaction between confinement time and aging time for thiols, TBARS, metmyoglobin, pH, L* and SF. When compared to the animals confined for 59 days, the animals confined for 129 days had: higher oxidation (higher TBARS and lower thiols) at the days 60 and 100 of aging; oxidation of myoglobin (metmyoglobin) slower, since the higher value was obtained at the day 100; lower lightness (L*) in all the times of aging; tender meat (lower SF) at 100 days of aging. The animals confined for 59 days had: higher protein oxidation (lower thiols) and tender meat (lower SF) at 30 days of aging. Overall, all the effects tested such as castration, time on confinement, cellular metabolism and muscle region seemed to influence on the protein and lipid oxidation and other traits of beef quality.

La pyocyanine (PYO) est une phénazine de couleur bleu-vert, produite spécifiquement par la bactérie pathogène opportuniste Pseudomonas aeruginosa (Pa). La toxicité aérobie de la PYO envers les cellules de mammifères, les levures et les bactéries a été décrite de longue date, mais la compréhension des mécanismes d’action est encore lacunaire, en particulier en conditions de limitation en O2 (conditions rencontrées dans le contexte infectieux). De plus, il a récemment été montré que la PYO peut apporter des effets bénéfiques pour la souche productrice en hypoxie. Au cours de ce travail, nous avons réexaminé les effets de la PYO sur un large panel de bactéries dont son propre producteur (Pa) ainsi que sur un modèle cellulaire eucaryote Saccharomyces cerevisiae exposées à différentes tensions en O2. Nos données suggèrent que la toxicité aérobie de la PYO envers S. cerevisiae est multifactorielle, impliquant à la fois une interaction avec le complexe III de la chaîne respiratoire et l’induction d’un stress oxydatif. Pour la première fois, nous avons mis en évidence une toxicité de la PYO exacerbée en anaérobiose chez un eucaryote (S. cerevisiae). Le mécanisme d’action impliquerait le PYO radical. Nous avons également montré que la PYO peut inhiber la croissance aérobie et anaérobie des microorganismes concurrents, plus particulièrement S. aureus en bloquant le complexe III de la chaîne respiratoire. A l’inverse, la PYO peut stimuler la respiration de Pa surtout dans les conditions mimant le contexte infectieux (hypoxie, vie ralentie). Le complexe III et/ou les oxydases terminales cbb3 serait impliqué favorablement. En conclusion, la PYO jouerait à la fois un rôle de poison hypoxique mais aussi un rôle de navette redox bénéfique pour la survie et la virulence de Pa en hypoxie. / Pyocyanin (PYO) is a blue-green phenazin, specifically produced by the opportunistic bacterium Pseudomonas aeruginosa (Pa). Aerobic toxicity of PYO toward mammalian cells, yeast and bacteria has been known for a long time, but the understanding of its mechanisms of action remains unclear, especially in conditions of limited O2 (conditions encountered during infection). In addition, it has recently been shown that PYO can bring benefits to the producer strain under hypoxia. In this study, we reexamined the effects of PYO toward a large panel of bacteria including its own producer Pa as well as a model of eukaryotic cells Saccharomyces cerevisiae exposed to different oxygen tensions. Our results suggest that the aerobic toxicity of PYO toward S. cerevisiae is multifactorial: involving both interaction with the respiratory chain at the level of complex III and induction of oxidative stress. For the first time, we have shown that PYO exerts an increased toxicity toward the eukaryotic cell, S. cerevisiae under anaerobiosis. The mechanism could involve the production of PYO radical. We have also shown that PYO can inhibit the aerobic and anaerobic growth of competing microorganisms, especially S. aureus by blocking the complex III of the respiratory chain. Conversely, PYO can stimulate the respiration of Pa, in mainly in conditions similar to those encountered during infection (hypoxia, slowed growth). The complex III and/or the cbb3 oxidases could be favorably involved. To conclude, PYO could act as a hypoxic poison as well as a redox shuttle beneficial for the survival and the virulence of Pa under hypoxia.

L'acide succinique est une molécule linéaire, bi-fonctionnelle qui possède de nombreuses applications alimentaires, chimiques et pharmaceutiques etc. Les connaissances de la régulation des voies métaboliques d’organismes d’intérêt industriel, le génie génétique et le génie des procédés ont permis à des microorganismes recombinants (C. glutamicum) de produire jusqu'à 100 g.L-1 de succinate avec des rendements intéressants. C. glutamicum est largement connu comme l'un des meilleurs producteurs industriels de nombreux acides aminés (glutamate, lysine etc.). Cependant, des études de C. glutamicum ont démontré sa capacité à produire plusieurs acides organiques (succinate, lactate, acétate, etc.). Au cours de ce travail, nous avons supprimé le gène ldhA de C. glutamicum en utilisant le plasmide pk19mobsacBΔldhA. Nous avons démontré que la délétion de ce gène n’avait pas d’incidence sur la capacité de croissance de la bactérie. Par ailleurs, nous avons étudié les effets de l’oxygénation sur la réponse physiologique de C. glutamicum 2262ΔldhA à travers des expériences de cultures en fioles en verre lisses en imposant différentes conditions de kLa. Les résultats ont montré que des faibles kLa (<33 h-1) favorisaient la production d’acides organiques tandis que les kLa élevés amélioraient surtout l’accumulation de la biomasse. Nous avons également mis en œuvre un procédé de production très efficace avec une phase de transition aérobiose-anaérobiose basée sur la régulation de la concentration en oxygène dissous. Avec ce procédé, 327 mM de succinate avec un rendement de 0,94 mole par mole de glucose ont pu être produits avec le mutant ΔldhA. En outre, nous avons vérifié l’efficacité de ce nouveau procédé en l’appliquant à la souche sauvage qui normalement produit 10 fois plus de lactate que de succinate. Ces résultats ont montré une production de 793 mM (94 g.L-1) de succinate et 785 mM (71 g.L-1) de lactate. Ils soulignent ainsi, l'importance de la phase de transition aérobiose-anaérobiose lors des procédés de production de succinate par des bactéries aérobie facultatif. Enfin, des expériences en système bi-étagé ont montré que C. glutamicum 2262 pouvait s’adapter très facilement aux gradients et hétérogénéités en oxygène dissous dans les cultures à grande échelle / Succinic acid is a linear and bi-functional molecule that has several practical applications including food, chemical and pharmaceutical industries. Thanks to increased knowledge on metabolism and pathway regulation of industrially relevant organisms, to the development of performant genetic tools and process engineering, recombinants strains (Escherichia coli, Corynebacterium glutamicum etc.) have been reported to be able to produce up to 100 g.L-1 with interesting yields (> 1.5 mole per mole glucose). C. glutamicum is well known as one of the best industrial producers of numerous amino acids (glutamate, lysine etc.). However, recent studies of C. glutamicum revealed its capability to produce several organic acids (succinate, lactate, acetate, etc.). In this work, we have deleted the ldhA gene of C. glutamicum by using a plasmid vector pk19mobsacBΔldhA. We demonstrated that the mutant and the wild type presented similar growth kinetics with maximal growth rate of about 0.7 h-1. We studied also the effects of oxygenation on C. glutamicum 2262 ΔldhA through cultures at different kLa and it appeared that lower kLa (<33 h-1) favored organic acids production wile higher favored bacterial growth. Furthermore, we designed a tri-phasic process with transition phase by regulation of dissolved oxygen concentration which resulted in the production of 327 mM of succinic acid with a yield of 0.94 mole per mole glucose. The application of the designed process to C. glutamicum 2262 wild type that normally produces lactate with a lactate to succinate production ratio up to 13.3 mol.mol-1, resulted in succinate concentration up to 793 mM (94 g.L-1) and 785 mM (71 g.L-1) of lactate. The succinate production yield was 1.1 mole per mole glucose and acetate production was negligible. These results underlined the importance of aerobic to anaerobic transition in succinate production processes of facultative aerobes and the necessity to engineer not only the microorganism but also the process. Finally, scale-down study have demonstrated the robustness of C. glutamicum against the oxygen gradients in bioreactor