Copper is one of the most vital trace elements in ruminant nutrition. It is required for several metabolic activities and it is also an essential component of several physiologically important metalloenzymes. Thus copper deficiency in ruminants results in distinctive pathologies, and hence in significant economic losses to farmers. Copper deficiency results from very low copper in diet (primary copper deficiency) and interference with Cu absorption in the animal due to Mo and S in food or water (secondary copper deficiency). The molybdenum-induced copper deficiency that affects ruminants can be attributed to the formation of thiomolybdates (TMs)from molybdate and sulfide in the rumen. The TMs formed then react irreversibly with copper to form insoluble Cu-TM complex which ultimately end up being excreted, thus reducing copper bioavailability to the ruminant. <p>In this study, an attempt has been made to use computer simulations to model speciation of copper in rumen fluid in the presence of TMs with the aim of understanding the extent to which TMs affects the levels of copper in the rumen. <p>This was done by initially refining the computer model of copper speciation with respect to low molecular mass (LMM) ligands in bovine rumen with the aim of correcting the discrepancy that was observed during experimental validation of the computer model in a previous study. To this end, mass balance equations which describes the distribution of Cu(II) amongst the different ligands were encoded into a spreadsheet to calculate equilibrium concentration of all species. Formation constants obtained from literature as well as those obtained from studies in our group were used as input values in the spreadsheet. Results show that at average ruminal pH, the metal would be present mostly as carbonate and phosphate complexes. The results obtained from the computer model in the present study were validated using 1H NMR experiments on simulated rumen fluid as well as actual rumen fluid containing Cu(II); using acetic acid chemical shift as the probe for monitoring the speciation pattern. Excellent agreement was observed between the computer model and experimental results. Discrepancy was however observed upon introduction of copper lysine as copper source into the model. Incorporation of a mixed ligand complex of Cu(II), acetate and lysine into the computer model gave an excellent agreement between the computer model and experimental results. <p>The study was extended to include glycine, histidine, methionine and EDTA complexes as the copper source in both rumen saliva (McDougalls solution) and rumen fluid. Results show that only the histidine and EDTA complexes persist to any significant extent, in spite of the large number of competing ligands present in these matrices.<p>In this study, success has also been achieved in the integration of the slow (kinetically controlled) formation of TMs and copper-tetrathiomolybdate (TM4) complexation into the previously developed model for the rapidly equilibrating copper-ligand speciation. To simulate the formation of the TMs and Cu-TM4 complex with respect to time, the differential equations representing rate expressions for each chemical species were solved to obtain an analytical solution using the Laplace transform method. The analytical solutions obtained were encoded in a spreadsheet and calculated as function of time to obtain time dependent concentrations of TMs and Cu-TM4 complex. This was then integrated with previously developed model for the rapidly equilibrating copper-ligand speciation in the rumen. The kinetic data used in the simulation of the formation thiomolybdates was obtained fron literature wheras that for Cu-TM4 complexation was obtained from our lab using Cu(II) - Ion Selective Electrode. The results show that that in the presence of TM4 the, Cu(II) bound to low molecular ligands in the rumen is drastically reduced confirming the effect TM4 on Cu(II) observed in several in vitro studies.<p>The study shows that in thiomolybdate contaminated rumen environment, the bioavailability of copper is considerably reduced. Though metal bioavailabilities are hard to predict this approach could help better our understanding of this process.

Copper is one of the most vital trace elements in ruminant nutrition. It is required for several metabolic activities and it is also an essential component of several physiologically important metalloenzymes. Thus copper deficiency in ruminants results in distinctive pathologies, and hence in significant economic losses to farmers. Copper deficiency results from very low copper in diet (primary copper deficiency) and interference with Cu absorption in the animal due to Mo and S in food or water (secondary copper deficiency). The molybdenum-induced copper deficiency that affects ruminants can be attributed to the formation of thiomolybdates (TMs)from molybdate and sulfide in the rumen. The TMs formed then react irreversibly with copper to form insoluble Cu-TM complex which ultimately end up being excreted, thus reducing copper bioavailability to the ruminant. <p>In this study, an attempt has been made to use computer simulations to model speciation of copper in rumen fluid in the presence of TMs with the aim of understanding the extent to which TMs affects the levels of copper in the rumen. <p>This was done by initially refining the computer model of copper speciation with respect to low molecular mass (LMM) ligands in bovine rumen with the aim of correcting the discrepancy that was observed during experimental validation of the computer model in a previous study. To this end, mass balance equations which describes the distribution of Cu(II) amongst the different ligands were encoded into a spreadsheet to calculate equilibrium concentration of all species. Formation constants obtained from literature as well as those obtained from studies in our group were used as input values in the spreadsheet. Results show that at average ruminal pH, the metal would be present mostly as carbonate and phosphate complexes. The results obtained from the computer model in the present study were validated using 1H NMR experiments on simulated rumen fluid as well as actual rumen fluid containing Cu(II); using acetic acid chemical shift as the probe for monitoring the speciation pattern. Excellent agreement was observed between the computer model and experimental results. Discrepancy was however observed upon introduction of copper lysine as copper source into the model. Incorporation of a mixed ligand complex of Cu(II), acetate and lysine into the computer model gave an excellent agreement between the computer model and experimental results. <p>The study was extended to include glycine, histidine, methionine and EDTA complexes as the copper source in both rumen saliva (McDougalls solution) and rumen fluid. Results show that only the histidine and EDTA complexes persist to any significant extent, in spite of the large number of competing ligands present in these matrices.<p>In this study, success has also been achieved in the integration of the slow (kinetically controlled) formation of TMs and copper-tetrathiomolybdate (TM4) complexation into the previously developed model for the rapidly equilibrating copper-ligand speciation. To simulate the formation of the TMs and Cu-TM4 complex with respect to time, the differential equations representing rate expressions for each chemical species were solved to obtain an analytical solution using the Laplace transform method. The analytical solutions obtained were encoded in a spreadsheet and calculated as function of time to obtain time dependent concentrations of TMs and Cu-TM4 complex. This was then integrated with previously developed model for the rapidly equilibrating copper-ligand speciation in the rumen. The kinetic data used in the simulation of the formation thiomolybdates was obtained fron literature wheras that for Cu-TM4 complexation was obtained from our lab using Cu(II) - Ion Selective Electrode. The results show that that in the presence of TM4 the, Cu(II) bound to low molecular ligands in the rumen is drastically reduced confirming the effect TM4 on Cu(II) observed in several in vitro studies.<p>The study shows that in thiomolybdate contaminated rumen environment, the bioavailability of copper is considerably reduced. Though metal bioavailabilities are hard to predict this approach could help better our understanding of this process.

Introdução. O rúmen bovino constitui um subproduto de abatedouro por ser rejeitado sensorialmente e culturalmente. Apesar disso, representa uma ótima fonte protéica, sendo desejável seu aproveitamento integral na alimentação humana. Objetivo geral. Desenvolver um produto à base de rúmen bovino, avaliando suas características físico-químicas e nutritivas e sua aplicação em alimentos. Metodologia. O rúmen bovino foi desengordurado a frio e extrusado em extrusora de rosca única. Utilizou-se a Metodologia de Superfície de Resposta para verificação dos efeitos da extrusão sobre as propriedades funcionais do rúmen: capacidade de retenção de água, capacidade de emulsificação, estabilidade da emulsão, solubilidade da proteína e hidrofobicidade da superfície protéica. Foi avaliada a composição nutritiva do rúmen (composição centesimal, colesterol, perfil de ácidos graxos e sua qualidade protéica através da composição de aminoácidos e digestibilidade real em ratos). Três produtos cárneos (lingüiça toscana, hambúrguer de frango e quibe) foram fabricados com extrusado ou com farinha de rúmen, analisados sensorialmente e comparados com produtos cárneos contendo extrusado de concentrado protéico de soja. Resultados. A capacidade de emulsificação, a solubilidade protéica e a hidrofobicidade da superfície protéica aumentaram após a extrusão, sendo que a capacidade de retenção de água e a estabilidade da emulsão foram reduzidas. A umidade mostrou ter influência sobre a variável resposta capacidade de retenção de água. As condições testadas de extrusão do rúmen foram fixadas em 35% de umidade (base seca) e 150ºC de temperatura de processamento. A avaliação da composição centesimal mostrou que o rúmen é uma excelente fonte protéica, seu colesterol foi reduzido consideravelmente após o desengorduramento e os ácidos graxos saturados foram extraídos totalmente. O escore de aminoácidos corrigido pela digestibilidade real da proteína foi de 1 ou 100% antes e após a extrusão. As análises sensoriais mostraram que para a lingüiça, a adição de rúmen deve ser feita em sua forma extrusada, sendo que no hambúrguer de frango e no quibe, a utilização de extrusado ou farinha de rúmen resultou em igual aceitação sensorial. Conclusão. A extrusão constitui uma forma de aproveitamento do rúmen bovino, mas não é essencial para sua aplicação em alimentos, já que dependendo do produto não se mostrou necessária. Por outro lado, a extrusão provocou mudanças nas propriedades funcionais do rúmen, favorecendo sua aplicação em determinados produtos embutidos e melhorando a aceitação sensorial. A qualidade nutritiva da proteína tanto da farinha como do extrusado foi alta e não foi afetada pela extrusão. / Introduction. Bovine rumen is a slaughterhouse by-product rejected by sensorial and cultural reasons. Nevertheless, it is a great proteic source and its utilization in human feed is necessary. Objective. Development of a product made with bovine rumen, analyzing its physical-chemical and nutritive characteristics and its use in human feed. Methodology. Bovine rumen was deffated and extruded in a single screw extruder. The Response Surface Methodology was applied to verify extrusion effects on functional properties of rumen: water holding capacity, emulsifying capacity, emulsion stability, protein solubility and surface hydrophobicity of protein. The nutritive composition of rumen (composition per 100g, cholesterol and fatty acid profile) was evaluated and also its proteic quality through its amino acid composition and in vivo true digestibility. Three meat products (sausage, chicken hamburger and quibe) were produced with extruded or flour rumen and compared with extruded of soy protein concentrated through sensory analysis. Results. After extrusion, emulsifying capacity, protein solubility and surface hydrophobicity of protein were improved, while water holding capacity and emulsion stability were reduced. Moisture had influence on water holding capacity. Extrusion of bovine rumen was fixed at 35% of moisture (dry basis) and 150oC of processing temperature. The evaluation of composition per 100g showed that bovine rumen is a great proteic source. After deffating, cholesterol was reduced and saturated fatty acids were totally removed. Amino acid scores corrected by true protein digestibility were 1 or 100% before and after extrusion. Sensory analysis showed that extruded bovine rumen should be added at sausage instead of flour rumen, but both extruded or flour rumen can be added at chicken hamburger and at quibe with equal sensory acceptance. Conclusion. Extrusion is a way of bovine rumen utilization, but it is not essential for its use in food, since it was not necessary in some cases. Nevertheless, extrusion changed functional properties of rumen, favoring its application in some sausage products and improving its sensory acceptance. Nutritive quality of protein, both raw and extruded rumen, was high and it was not affected by extrusion.

Introdução. O rúmen bovino constitui um subproduto de abatedouro por ser rejeitado sensorialmente e culturalmente. Apesar disso, representa uma ótima fonte protéica, sendo desejável seu aproveitamento integral na alimentação humana. Objetivo geral. Desenvolver um produto à base de rúmen bovino, avaliando suas características físico-químicas e nutritivas e sua aplicação em alimentos. Metodologia. O rúmen bovino foi desengordurado a frio e extrusado em extrusora de rosca única. Utilizou-se a Metodologia de Superfície de Resposta para verificação dos efeitos da extrusão sobre as propriedades funcionais do rúmen: capacidade de retenção de água, capacidade de emulsificação, estabilidade da emulsão, solubilidade da proteína e hidrofobicidade da superfície protéica. Foi avaliada a composição nutritiva do rúmen (composição centesimal, colesterol, perfil de ácidos graxos e sua qualidade protéica através da composição de aminoácidos e digestibilidade real em ratos). Três produtos cárneos (lingüiça toscana, hambúrguer de frango e quibe) foram fabricados com extrusado ou com farinha de rúmen, analisados sensorialmente e comparados com produtos cárneos contendo extrusado de concentrado protéico de soja. Resultados. A capacidade de emulsificação, a solubilidade protéica e a hidrofobicidade da superfície protéica aumentaram após a extrusão, sendo que a capacidade de retenção de água e a estabilidade da emulsão foram reduzidas. A umidade mostrou ter influência sobre a variável resposta capacidade de retenção de água. As condições testadas de extrusão do rúmen foram fixadas em 35% de umidade (base seca) e 150ºC de temperatura de processamento. A avaliação da composição centesimal mostrou que o rúmen é uma excelente fonte protéica, seu colesterol foi reduzido consideravelmente após o desengorduramento e os ácidos graxos saturados foram extraídos totalmente. O escore de aminoácidos corrigido pela digestibilidade real da proteína foi de 1 ou 100% antes e após a extrusão. As análises sensoriais mostraram que para a lingüiça, a adição de rúmen deve ser feita em sua forma extrusada, sendo que no hambúrguer de frango e no quibe, a utilização de extrusado ou farinha de rúmen resultou em igual aceitação sensorial. Conclusão. A extrusão constitui uma forma de aproveitamento do rúmen bovino, mas não é essencial para sua aplicação em alimentos, já que dependendo do produto não se mostrou necessária. Por outro lado, a extrusão provocou mudanças nas propriedades funcionais do rúmen, favorecendo sua aplicação em determinados produtos embutidos e melhorando a aceitação sensorial. A qualidade nutritiva da proteína tanto da farinha como do extrusado foi alta e não foi afetada pela extrusão. / Introduction. Bovine rumen is a slaughterhouse by-product rejected by sensorial and cultural reasons. Nevertheless, it is a great proteic source and its utilization in human feed is necessary. Objective. Development of a product made with bovine rumen, analyzing its physical-chemical and nutritive characteristics and its use in human feed. Methodology. Bovine rumen was deffated and extruded in a single screw extruder. The Response Surface Methodology was applied to verify extrusion effects on functional properties of rumen: water holding capacity, emulsifying capacity, emulsion stability, protein solubility and surface hydrophobicity of protein. The nutritive composition of rumen (composition per 100g, cholesterol and fatty acid profile) was evaluated and also its proteic quality through its amino acid composition and in vivo true digestibility. Three meat products (sausage, chicken hamburger and quibe) were produced with extruded or flour rumen and compared with extruded of soy protein concentrated through sensory analysis. Results. After extrusion, emulsifying capacity, protein solubility and surface hydrophobicity of protein were improved, while water holding capacity and emulsion stability were reduced. Moisture had influence on water holding capacity. Extrusion of bovine rumen was fixed at 35% of moisture (dry basis) and 150oC of processing temperature. The evaluation of composition per 100g showed that bovine rumen is a great proteic source. After deffating, cholesterol was reduced and saturated fatty acids were totally removed. Amino acid scores corrected by true protein digestibility were 1 or 100% before and after extrusion. Sensory analysis showed that extruded bovine rumen should be added at sausage instead of flour rumen, but both extruded or flour rumen can be added at chicken hamburger and at quibe with equal sensory acceptance. Conclusion. Extrusion is a way of bovine rumen utilization, but it is not essential for its use in food, since it was not necessary in some cases. Nevertheless, extrusion changed functional properties of rumen, favoring its application in some sausage products and improving its sensory acceptance. Nutritive quality of protein, both raw and extruded rumen, was high and it was not affected by extrusion.

Le microbiote du rumen bovin est un écosystème très diversifié et efficace pour la dégradation de substrats complexes, notamment issus de la biomasse végétale. Composé majoritairement de microorganismes non cultivés, il constitue un réservoir très riche de nouvelles enzymes d’intérêt potentiel pour les biotechnologies industrielles, en particulier les bioraffineries et la bioremédiation. Dans le cadre de cette thèse, nous avons mis en œuvre une approche de criblage fonctionnel du métagenome ruminal pour accélérer la découverte d’enzymes de dégradation des lignocelluloses, mais aussi de divers polluants synthétiques. En particulier, de nouvelles estérases capables de dégrader un insecticide de la famille des carbamates, le fenobucarb, ainsi qu’un polyuréthane commercial, l’Impranil DLN, ont pu être identifiées. De plus, le développement d’une nouvelle stratégie de criblage d’oxydoréductases nous a permis d’isoler trois enzymes bactériennes originales, très polyspécifiques, ne requérant ni cuivre ni manganèse pour dégrader différents substrats polycycliques tels que des polluants majeurs de l’industrie textile, mais aussi des dérivés de lignine. Enfin, le criblage de deux banques issues d’enrichissements in vivo et in vitro du microbiome du rumen sur paille de blé a permis d’isoler des cocktails d’enzymes lignocellulolytiques au profil fonctionnel et d’origine taxonomique différents, constitués de glycoside-hydrolases, estérases et oxydoréductases. Quinze nouveaux modules CAZy, correspondant à des familles enzymatiques jamais caractérisées, ont été identifiés. L’ensemble de ces résultats met en lumière l’immense potentiel d’innovation biotechnologique contenu dans les écosystèmes microbiens, en particulier dans le microbiote du rumen bovin / Bovine rumen microbiota is a highly diverse and efficient ecosystem for the degradation of complex substrates, especially those issued from plant biomass. Predominantly composed of uncultivated microorganisms, it constitutes a rich reservoir of new enzymes of potential interest for industrial biotechnologies, especially biorefineries and bioremediation. As part of this thesis, we used the functional screening of the ruminal metagenome to increase the discovery of enzymes able to degrade lignocelluloses, as well as different synthetic pollutants. In particular, new esterases able to degrade a carbamate insecticide, fenoucarb, and a commercial polyurethane, Impranil DLN, have been identified. Moreover, the development of a new screening strategy for oxidoreductases allowed the isolation of three original bacterial enzymes that are very polyspecific, and do not need copper nor manganese to degrade different polycyclic substrates, like major pollutants of the textile industry, as well as lignin derivatives. Finally, the screening of two libraries from in vivo and in vitro enrichments of the ruminal microbiome on wheat straw allowed the isolation of lignocellulolytic enzymatic cocktails, with different functional profiles and taxonomical origins, comprising glycoside-hydrolases, esterases and oxidoreductases. Fifteen novel CAZy modules, related to enzymatic families never characterized, were identified. All these results highlight the vast potential of microbial ecosystems, in particular the bovine rumen microbiota, for biotechnological innovation