Le contrôle de la masse musculaire est sous la dépendance d'un équilibre entre les processus de synthèse et de dégradation. Sur le plan cellulaire, deux voies signalétiques majeures sont impliquées : la voie des facteurs de transcription de la famille FoxO qui contrôle l'expression des gènes impliqués dans les systèmes de dégradation (système ubiquitine-protéasome et autophagie), et la voie IGF-1/Akt/mTORC1 qui représente la voie majeure de la synthèse protéique. Nos travaux mettent en évidence, sur des cellules musculaires le rôle de la protéine kinase AMP-dépendante (AMPK) qui inhibe l'activité de la voie mTOR et régule les systèmes ubiquitine-protéasome et autophagiques de manière FoxO3 dépendante. Une nouvelle cible de l'AMPK a également été identifiée : la protéine Ulk1 qui possède une fonction clé dans l'activation de l'autophagie. Par ailleurs, nous avons montré le rôle centraldu facteur d'initiation à la traduction eIF3f dans l'induction de l'hypertrophie, et dans l'augmentation de l'activité de la voie mTORC1 associée. De plus, nous montrons que la surexpression d'un mutant d'eIF3f résistant à la dégradation est associée à une protection effective contre l'atrophie. / Skeletal muscle mass is depending upon a dynamic balance between anabolic and catabolic processes. At a cellular level, two major signaling pathways are involved: the transcription factors FoxO related pathway, implicated in the control of protein breakdown systems(ubiquitin-proteasome system and autophagy), and the IGF-1/Akt/mTORC1 pathway associated with the canonic pathway of protein synthesis. We show in muscle cells that theAMP-activated protein kinase (AMPK) decreases the mTORC1 pathway activity and simulate subiquitin-proteasome and autophagy systems in a FoxO3-dependant manner. Furthermore,we identify Ulk1 as a new interacting partner of AMPK, which plays a major role in the autophagy induction. Moreover, we demonstrate the key role of the eukaryotic translation initiation factor eIF3f in hypertrophy induction and in the associated increase of the mTORC1activity. In addition, we show that the overexpression of an eIF3f mutant resistant to the degradation is associated with a protection against muscle atrophy.

The Amadumbe crop [Colocasia esculenta (L.) Schott] is a traditional Southern African tuber crop which is rich in starch, mucilage and micronutrients. Amadumbe tubers have limited amount of proteins and as a result, amadumbe-processed foods lack adequate protein. The purpose of this study was therefore to develop protein-rich amadumbe-soya composite biscuits, which would be acceptable to consumers. Biscuits were prepared by combining amadumbe and soya flours at ratios: 90:10, 70:30 and 50:50. Functional properties of composite flours and the physical properties of composite biscuits were determined. The proximate composition, amino acid composition and protein digestibility of composite biscuits were determined. Consumer acceptability test of biscuits was performed using nine-point hedonic scale. The results indicated that the 90% amadumbe and 10% soya composite biscuits had high significant values of moisture, ash, carbohydrates contents and energy values. The 50% amadumbe and 50% soya composite biscuits had significantly high values of fat, crude protein contents and acid detergent fibre (ADF). The protein digestibility, amino acid contents, especially the lysine contents of composite biscuits increased significantly (p ≤ 0.05) with an increase in the percentage of soya. The mineral contents of composite biscuits; Ca, Mg, P, Zn, Cu, Mn and Fe increased significantly (p ≤ 0.05) with the increase of soya in the composite biscuits. There was a significant difference in the mean taste acceptability and mean overall acceptability when the soya concentration was increased to 50%. Soya was successfully used to produce amadumbe composite biscuits with better nutritional quality with respect to protein content, amino acid profile and selected mineral contents and which were acceptable to consumers. / National Research Foundation (South Africa) / Life and Consumer Science / M.Sc. (Agriculture)

Le muscle squelettique est le réservoir principal d’acides aminés libres de l’organisme. Ainsi, l’atrophie musculaire induite par l’immobilisation peut entraîner un affaiblissement et un allongement des périodes de récupération générant des coûts de santé publique élevés. Une aggravation de l’atrophie caractérise de façon surprenante le muscle tibialis anterior (TA) après le déplâtrage, retardant la récupération. Mon objectif a été de comprendre les mécanismes à l’origine de l’aggravation de l’atrophie du TA pendant les phases précoces de récupération en étudiant i) la structure et le phénotype des muscles, ii) la composition de la matrice extracellulaire (MEC), iii) la protéolyse et l’apoptose, et iv) les processus de signalisation via les intégrines. Des rats ont été soumis à une immobilisation par plâtrage pendant 8 jours d’une des deux pattes arrière, l’autre servant de témoin, et placés en récupération pendant 10 jours. L’aggravation de l’atrophie du TA apparaît dès déplâtrage, corrélée avec i) une baisse de l’aire des fibres associée à leur déformation, ii) une redistribution des isoformes des chaines lourdes de myosines, iii) une augmentation de l’apoptose localisée dans le tissu conjonctif, iv) un épaississement de l’endomysium pendant la remobilisation, v) des adaptations au niveau des processus de remodelage des collagènes, et vi) une activation prononcée et persistante du système protéolytique ubiquitine-protéasome (UPS) et de l’apoptosome. Nous montrons également une élévation des niveaux ARNm dans le TA remobilisé vii) de la ténascine-C et de Sparc dès le déplâtrage, et viii) de marqueurs de l’autophagie à partir du moment où l’atrophie se stabilise. Enfin, nous montrons également une élévation des ARNm dans le TA immobilisé ix) des facteurs myogéniques, et x) des intégrines membranaires et de leurs partenaires pendant l’immobilisation et après le déplâtrage. En conclusion, mon travail de thèse a permis de montrer que l'aggravation de l’atrophie du TA est précoce, associée à un remodelage important de la structure et de la composition de la MEC et du phénotype des fibres musculaires, et pourrait résulter de l’augmentation persistante et prononcée de la voie UPS et de l’apoptose. Ce travail suggère que des modifications au niveau des molécules matricielles pendant la remobilisation pourraient influencer la signalisation dépendante des intégrines et la régénération musculaire. / Skeletal muscle is the main reservoir of body amino acids. Thus, muscle atrophy induced by immobilization can lead to a weakening and to a lengthening of recovery periods, leading to elevated healthcare costs. Surprisingly, a worsening of tibialis anterior (TA) muscle atrophy prevailed after cast removal and thus delayed recovery. The aim of my Ph.D was to understand mechanisms underlying the worsening of TA atrophy during early recovery by studying i) the muscle structure and phenotype, ii) the composition of the extracellular matrix (ECM), iii) proteolysis and apoptosis, and iv) the signaling pathways via integrins. Rats were subjected to hindlimb casting for 8 days of one hindllimb, the other leg served as control, and then were allowed to recover for 10 days. The worsening of TA atrophy appeared immediately after cast removal and correlated with i) a decrease in fiber crosssection area associated to fiber deformation, ii) a redistribution of myosin heavy chain isoforms, iii) an increase in apoptosis localized in the connective tissue, iv) a thickening of the endomysium during remobilization, v) some adaptations in collagen remodeling processes, and vi) a pronounced and sustained activation of the ubiquitin-proteasome proteolytic system (UPS) and of the apoptosome. We also showed an increase in the remobilized TA of mRNA levels vii) of tenascin-C and Sparc immediately after cast removal, and viii) of some autophagy markers, when atrophy stabilized. Finally, we showed an elevation of mRNA levels encoding ix) myogenic factors, and x) transmembrane integrins and their partners during TA immobilization and after cast removal. In conclusion, my Ph.D project showed that the worsening of the TA atrophy occurred early after cast removal, was associated with a significant remodeling of the structure and composition of the ECM and of the phenotype of muscle fibers, and may result from pronounced and sustained increase in the UPS and apoptosis. This work suggests that changes in the matricellular matrix molecules during remobilization could influence integrin-dependent signaling and muscle regeneration.

La protéolyse est une fonction clé de la cellule pour le maintien de l'intégrité du protéome, pour le métabolisme et pour la régulation de nombreux processus physiologiques. Le travail présenté dans cette thèse porte sur une famille de complexes peptidases cytosoliques auto-compartimentés et énergie indépendants découverts chez les Archées, les aminopeptidases TET. Chez l'Archée hyperthermophile Pyrococcus horikoshii, organisme modèle de cette étude, il existe 3 peptidases TET présentant chacune des spécificités de substrats différentes. Les caractérisations structurales des différents membres connus de cette famille de peptidases ont révélé un assemblage dodécamériques creux en forme de tétraèdre d'environ 450 kDa. Des études récentes ont montré l'existence de complexes adoptant la même conformation que les TET dans les 3 domaines du vivant. La première partie du travail présenté a permis d'identifier des marqueurs structuraux caractéristiques de l'assemblage tétraédrique afin de déterminer sans ambiguïté l'appartenance de ces complexes à la famille des TET. La seconde partie de l'étude a conduit à élucider la question de la multiplicité des TET chez les Archées hyperthermophile mise en évidence grâce à une étude phylogénétique initiée pendant la thèse. L'étude en co-expression de PhTET2 et PhTET3 révèle que ces aminopeptidases sont capable de former un hétéro-oligomère présentant une activité enzymatique accrue vis-à-vis des homo-oligomères. La dernière partie du travail porte sur les relations oligomérisation-fonction chez les peptidases TET. L'étude d'un mutant de l'oligomérisation de PhTET2 via une stratégie intégrative alliant biochimie, enzymologie, biophysique (SAXS et AUC) et des études in vivo a permis de mettre en évidence un processus d'assemblage contrôlé permettant d'augmenter l'efficacité de la peptidase. Enfin, la méthode de variation de contraste en diffusion de neutrons aux petits angles (SANS) appliqué à l'étude de l'hétéro-oligomère a permis de révéler une topologie rationalise du complexe hétéro-oligomérique favorisant la formations de poches multi-catalytique. L'ensemble de ce travail contribue à mieux comprendre l'importance et le rôle physiologique des machines TETs dans les cellules. / Proteolysis is a key function in the cell for the maintenance of the proteome integrity, the metabolism and for the regulation of many physiological processes. The thesis work is focused on a family of self-compartmentalized energy-independent cytosolic peptidases discovered in Archaea, the TET aminopeptidases. Three different TET showing contrasted enzymatic specificities co-exist in the cytosol of the hyperthermophilic archaeon Pyrococcus horikoshii, which is the model organism for this study. The structural characterization of the known members of this family shows that they self-assemble in a unique 450 kDa hollow tetrahedral structure . Recent studies have revealed the existence of peptidases complexes that adopt the same conformation in the three domains of life. The first part of this work allowed identifying structural markers to assign without any ambiguity uncharacterized peptidases to the TET family. The second objective of the work was to understand the multiplicity of TET peptidases in hyperthermophilic archaeon that was highlighted by a phylogenomic study presented in this work . The co-expression of PhTET2 and PhTET3 in E. coli revealed that the two proteins form a hetero-oligomeric complex with enhanced enzymatic activity compared to the homo-oligomers. The last part of the work addressed the question of oligomerization-function relationship in TET particles. A mutagenesis strategy was used to slow down the oligomerization process of PhTET2, and, using an integrative strategy combining biochemistry, enzymology, biophysics (SAXS and AUC) and in vivo studies we were able to dissect the oligomerization pathway of the TET particles and to demonstrate that it is a highly controlled process aim to enhance the activity of the peptidases. Finally, the contrast variation technique in small angle neutron scattering studies (SANS) allowed us to unravel the rational topology of the TET hetero-oligomers that favored the formation of multi-catalytic enzymatic pockets in the complex. All theses studies contributed to specify the biological importance of the TET molecular machines in the cells.

Orientador: Ana Lúcia Barretto Penna / Banca: Ariene G. F. Van Dender / Banca: Elisa Helena Giglio Ponsano / Resumo: O queijo Prato é fabricado por coagulação enzimática, adicionado de corante e complementado ou não pela ação de bactérias láticas específicas, podendo ser classificado como gordo e de média umidade. Atualmente, a procura por produtos lácteos com baixo teor de gordura vem aumentando, pois o consumo de gordura de origem animal apresenta relação com doenças coronárias e carcinogênicas. Além disso, para indivíduos que buscam a manutenção ou perda de peso corporal, o consumo de gordura deve ser restrito. Por outro lado, queijos com teor reduzido de gordura apresentam defeitos nas características sensoriais, no rendimento e na maturação, quando comparados aos queijos com teor integral. Para minimizar as alterações decorrentes da retirada da gordura, é necessário utilizar uma tecnologia que não altere a qualidade do produto final. Neste contexto, no presente trabalho estudou-se o uso da enzima proteolítica, fastuosaína, extraída do fruto verde do gravatá (Bromelia fastuosa) na maturação do queijo Prato com teor reduzido de gordura. Foram realizados 4 processamentos: um pelo método tradicional - sem adição de enzima fastuosaína (Processamento A, controle) e três pelo método modificado, nos quais se utilizaram diferentes concentrações de fastuosaína com diferentes atividades enzimáticas. O leite utilizado na fabricação dos queijos foi submetido a análises de densidade, gordura, extrato seco total, acidez, presença de antibiótico, Salmonella, contagem padrão em placas, coliformes totais e fecais, e os queijos foram submetidos a análises microbiológicas de bactérias aeróbias mesófilas, bolores e leveduras, Escherichia coli, coliformes totais e fecais, Estafilococos coagulase positiva, Salmonella e Listeria monocytogenes. Durante a maturação os queijos foram submetidos a análises de:...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Prato cheese is manufactured by enzymatic coagulation, with addition of colorings and complemented or not by the action of specific lactic bacteria, and it can be classified as semihard and fatty cheese. Currently, the demand for dairy products with low-fat content is increasing, therefore the consumption of animal fat is related to coronary and carcinogenic diseases. Moreover, for individuals that search for the maintenance or the loss of body weight, the intake of fat must be restricted. On the other hand, cheeses with reduced fat content present defects in sensory characteristics, both in the yield of low-fat cheese and in its ripening, when compared with full-fat product. To minimize the alterations due to the removal of fat, it is necessary to use a technology that does not modify the final quality of the product. In this context, in this work the addition of the proteolytic enzyme, fastuosain, extracted of the green fruit of gravatá (Bromelia fastuosa) on the ripening of the low fat Prato cheese was studied. Four batches of cheese were carried out. One of them was not modified - without addition of fastuosain enzyme (Process A, control) and three batches were added with different concentrations of fastuosain with different enzymatic activities. The milk used in the manufacture of the cheeses was submitted to the analyses of density, fat, total dry matter, acidity, presence of antibiotic, total and fecal coliforms, Salmonella, counting standard in plates and the cheeses were submitted to microbiological analyses of aerobic mesophilic bacteria, total and fecal coliforms, yeasts and molds, Escherichia coli, coagulase positive staphylococci, Salmonella and Listeria monocytogenes. During the ripening the cheeses were submitted to the analyses of: total dry matter, acidity, fat, fat in the dry matter, ashes, nitrogen and total protein, soluble nitrogen (NS)...(Complete abstract click eletronic access below) / Mestre

Made available in DSpace on 2015-03-26T13:47:10Z (GMT). No. of bitstreams: 1 texto completo.pdf: 859271 bytes, checksum: bc073f10b3e793f3a635f6735a9b85b7 (MD5) Previous issue date: 2012-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The application of refrigeration as a tool to preserve the microbiological quality of goat milk during storage and transportation determined the selection of a microbiota typically psychrotrophic in this product. Psychrotrophic microorganisms are potential producers of proteolytic enzymes, usually characterized by being heat resistant and responsible for the deterioration of milk casein even after heat treatments commonlymused at dairies, such as pasteurization and ultra-high-temperature. Pseudomonas spp. are considered the main psychrotrophic and proteolytic microorganisms present in milk. This study aimed to evaluate the microbiological quality of goat milk, obtain autochthonous Pseudomonas spp. isolates of this product and characterize their proteolytic activity. Twelve farms with activity of dairy goat were selected in regions of Muriaé and Viçosa, Minas Gerais, of which 61 samples of goat milk were collected and subjected to microbiological analysis for mesophilic aerobes counts, enterobacteriaceae, total coliforms, Escherichia coli, and proteolytic psychrotrophs, and estimatives of total and proteolytic Pseudomonas. The mean counts obtained for the samples were: 5.04 log CFU/mL for mesophilic aerobes, 3.34 log CFU/mL for enterobacteriaceae, 2.85 log CFU/mL for total coliforms, 0.22 log CFU/ml for E. coli, 3.62 log CFU/mL for total psychrotrophs, 2.73 log CFU/mL for proteolytic psychrotrophs, 3.87 log CFU/mL for Pseudomonas, and 3.43 log CFU/mL for proteolytic Pseudomonas. Although the average of mesophilic aerobes was below the limit recommended by the present Brazilian legislation (5.70 log CFU/mL), the samples of goat milk showed high counts of hygiene indicator microorganisms, which shows inadequate hygienic procedures during the different stages of production and storage. In 22 samples of raw milk the counts of proteolytic psychrotrophs accounted for over 50% of the counts of psychrotrophic, indicating the proteolytic potential of this group in goat milk. The same relationship was observed concerning the presence of proteolytic activity of Pseudomonas spp. which in 27 samples this ratio was greater than 50% of the estimated total counts of Pseudomonas. Considering the plates used for enumeration of Pseudomonas, 496 colonies that showed proteolytic activity were selected, purified and the cultures obtained were subjected to the following tests for preliminary identification:morphology by Gram staining, oxidase and glucose fermentation. Based on these results, 107 isolates were initially identified as Pseudomonas spp., and subjected to rep-PCR to characterize their genetic profiles. Considering similarity rates between 80 and 85%, 33 different genetic profiles were characterized; 36 isolates showed no genetic profile with the rep-PCR protocol used. The isolates were also subjected to PCR for identification in species level with specific primers for P. aeruginosa (38 isolates identified) and P. fluorescens (4 isolates); 61 isolates showed amplification products for both reactions, and 4 for none, and were considered as Pseudomonas spp. based on the laboratory tests during screening. All isolates were subjected to PCR for detection of the apr gene, responsible for encoding the alkaline metalloprotease enzyme, identifying 41 (38.3%) positives. Even isolates that showed no apr gene were capable of producing proteolytic enzymes at 7, 25 or 35 °C; all isolates were able to produce proteases at 25 °C, 72 at 7° and 85 at 35°C. The results made it possible to determine hygienic shortcomings in the initial stages of the production chain of goat milk, besides the relevant participation of psychrotrophic and proteolytic microorganisms in the microbiota of this product. The proteolytic activity of Pseudomonas spp. isolates of goat milk was characterized due to the presence of the apr gene and the proteolytic activity at different incubation temperatures. / A aplicação da refrigeração como ferramenta para conservação da qualidade microbiológica do leite caprino durante a estocagem e transporte determinou a seleção de uma microbiota tipicamente psicrotrófica nesse produto. Micro-organismos psicrotróficos são potenciais produtores de enzimas proteolíticas, caracterizadas usualmente por serem termo-resistentes, e responsáveis pela deterioração da caseína do leite mesmo após os tratamentos térmicos mais utilizados em laticínios, como a pasteurização e a ultra-alta-temperatura. Pseudomonas spp. são considerados os principais micro-organismos psicrotróficos e proteolíticos presentes em leite. O presente estudo teve como objetivos avaliar a qualidade microbiológica de leite de cabra, obter isolados de Pseudomonas spp. autóctones desse produto e caracterizar sua atividade e potencial proteolítico. Doze propriedades rurais com atividade de caprinocultura leiteira foram selecionadas nas regiões de Viçosa e Muriaé, Minas Gerais, das quais 61 amostras de leite de cabra foram coletadas e submetidas a análises microbiológicas para contagens de aeróbios mesófilos, enterobactérias, coliformes, Escherichia coli, psicrotróficos totais e proteolíticos, e estimativas de Pseudomonas totais e proteolíticos. As médias das contagens obtidas para todas as amostras foram: 5,04 log UFC/mL para aeróbios mesófilos, 3,34 log UFC/mL para enterobactérias, 2,85 log FC/mL para coliformes, 0,22 log UFC/mL para E. coli, 3,62 log UFC/mL para psicrotróficos totais, 2,73 log UFC/mL para psicrotróficos proteolíticos, 3,87 log UFC/mL para Pseudomonas e 3,43 log UFC/mL para Pseudomonas proteolíticos. Embora a média de aeróbios mesófilos tenha sido inferior ao limite recomendado pela legislação brasileira vigente (5,70 log UFC/mL), as amostras de leite de cabra apresentaram altas contagens de micro-organismos indicadores de higiene, o que demonstra deficiências higiênicas durante as diferentes etapas de produção e estocagem desse produto. Em 22 amostras de leite cru as contagens de psicrotróficos proteolíticos representaram mais de 50% das contagens de psicrotróficos, indicando o potencial proteolítico desse grupo em leite de cabra. A mesma relação foi observada em relação à presença de atividade proteolítica de Pseudomonas spp., que em 27 amostras essa proporção foi maior que 50% das contagens totais estimadas de Pseudomonas. Considerando as placas utilizadas para enumeração de Pseudomonas, 496 colônias que apresentaram atividade proteolítica foram selecionadas, purificadas e as culturas obtidas submetidas aos seguintes testes para identificação preliminar: morfologia pela coloração de Gram, oxidase e fermentação de glicose. Baseado nesses resultados, 107 isolados foram inicialmente identificados como Pseudomonas spp., e submetidos a rep-PCR para caracterização de seus perfis genéticos. Considerando taxas de similaridade entre 80 e 85%, 33 perfis genéticos diferentes foram caracterizados; 36 isolados não apresentaram perfil genético com o protocolo de rep-PCR utilizado. Os isolados ainda foram submetidos a PCR para identificação de espécies com primers específicos para P. aeruginosa (38 isolados identificados) e P. fluorescens (4 isolados); 61 isolados apresentaram produtos de amplificação para ambas as reações, e 4 para nenhuma, e foram considerados como Pseudomonas spp. considerando os testes laboratoriais realizados na triagem. Todos os isolados foram submetidos a PCR para detecção do gene apr, responsável pelam codificação da enzima metaloprotease alcalina, sendo identificados 41 (38,3%) positivos. Mesmo isolados que não apresentaram gene apr foram capazes de produzir enzimas proteolíticas a 7, 25 ou 35 °C; todos os isolados foram capazes de produzir proteases a 25 °C, 72 a 7 °C e 85 a 35 °C. Os resultados obtidos permitiram determinar deficiências higiênicas nas etapas iniciais da cadeia produtiva do leite de cabra, além da relevante participação de micro-organismos psicrotróficos e proteolíticos na microbiota desse produto. A atividade proteolítica de isolados de Pseudomonas spp. de leite de cabra foi caracterizada devido a presença do gene apr e pela atividade proteolítica em diferentes temperaturas de incubação.

Made available in DSpace on 2016-12-08T16:24:16Z (GMT). No. of bitstreams: 1 PGCA13MA125.pdf: 1115012 bytes, checksum: 736f2a82baac7129fc498949399b0b87 (MD5) Previous issue date: 2013-11-28 / The milk is rich in proteins, lipids, water and carbohydrates, making it an excellent environment for bacterial contamination from different origins and for its proliferation. Bacterial multiplication depends on its capacity of adaptation to the environment and time available. In dairy farms, milk can be storage under cold in bulk tanks models of two and four milking capacity, with a chilling capacity of 50% and 25% respectively of its total volume, in each milking process. The use of cold storage in milk production reduced the occurrence of acid milk, however, increased the proliferation of psychrotrophic microorganisms. These microorganisms presents proteolytic capacity over casein without modifing the milk acidity. This proteolytic capacity could be responsible for unstable non-acid milk (UNAM). The purpose of this study was to evaluate the effect of the cold storage length of time and temperature of raw milk in bulk tanks of two and four milking models on psychrotrophic bacterial count (PBC), casein stability in alcohol test and occurrence of UNAM. The study was conducted in 19 dairy farms in two rehearsal and each farm had a direct expansion bulk milk tank of two or four milking models. Dairy farms with an interval of four milking between milk transportation to the processing industry were selected, thus the milk had a 36 hours of cold storage in the dairy farm. Samples were collected before and after each milking for bacterial total count (BTC), physical-chemical analysis and PBC. Samples of 12 from a total of 19 dairy farms were collected for casein quantification. The length of time of cold storage did not affect the BTC and PBC (P > 0,05). The bulk tank model only affected the PBC (P < 0,05), presenting a lower mean tank s model of two milking when compared with models of four milking (3,61±0,104 and 4,00±0,120 CFU/mL(log10)). The PBC did not affected the casein fractions, however the length of time of cold storage affected the &#1179; and &#946; casein fraction concentrations. The reductions in these casein fractions did not affect the casein stability in the alcohol test. The length of time of cold storage affected the casein stability (P < 0,05) in the alcohol test without affecting the UNAM occurrence. Bulk tanks, when used properly in concern of raw milk volume to be chilled, allow the BTC and PBC rates to remain stable. The length of time of cold storage does not affect BTC and PBC but affects the casein concentration and casein stability in the alcohol test without affecting the UNAM occurrence / O leite é um meio nutritivo facilmente colonizado por bactérias de diferentes origens, com taxa de proliferação condicionada à capacidade do micro-organismo adaptar-se ao ambiente e do tempo disponível. Na propriedade rural o leite pode ser armazenado por até 48 horas em tanques de expansão direta modelo de duas ou quatro ordenhas, os quais diferem na capacidade de resfriamento a cada ordenha, respectivamente, esses equipamentos são capazes de resfriarem 50% e 25% de seu volume a cada ordenha. A conservação do leite sob resfriamento pode não ser suficiente para o controle de micro-organismos psicrotróficos, os quais apresentam atividade proteolítica sobre a caseína sem alterar a acidez do leite, podendo ser um fator desencadeador do leite instável não ácido (LINA). O objetivo do presente estudo foi avaliar o efeito do tempo e temperatura de estocagem do leite cru em tanques de expansão direta modelos de duas e quatro ordenhas sobre a contagem bacteriana total (CBT) e contagem de bactérias psicrotróficas (CBP), assim como, determinar o efeito do tempo e da CBP sobre a estabilidade da caseína ao teste do álcool e a ocorrência de LINA. O experimento foi conduzido em 19 propriedades leiteiras localizadas nas regiões Serrana e Meio-oeste Catarinense, as quais possuíam equipamentos de refrigeração do leite por expansão direta modelo duas ou quatro ordenhas com taxa de ocupação de no mínimo 60%. Nas propriedades rurais foram avaliadas informações referentes ao intervalo de 36 horas entre a captação de leite a granel para a indústria de laticínios, as quais incluíam monitoramento temperatura leite, temperatura ambiente e umidade relativa do ar, aplicação de questionários aos responsáveis pela ordenha e coletas de amostras de leite antes e após a cada ordenha para análise de CBT, CBP, composição e teste físico-químicos (pH, acidez titulável e concentração alcoólica), sendo que em 12 das propriedades as frações de caseína foram quantificadas em duplicata. O tempo de estocagem do leite cru não afetou a CBT e CBP (P > 0,05). O modelo de tanque de expansão afetou somente a CBP (P < 0,05), com média inferior para amostras de leite de tanques de duas ordenhas em relação a quatro ordenhas (3,61±0,104 e 4,00±0,120 UFC/mL(log10)). A CBP não afetou as frações das caseínas e caseína total, entretanto, o tempo de estocagem do leite cru reduziu (P < 0,05) a concentração das frações &#1179; e &#946; da caseína e caseína total. A redução das frações de caseína não demonstrou efeito sobre a estabilidade do leite ao teste do álcool. O tempo de estocagem reduziu a (P < 0,05) a estabilidade do leite ao teste do álcool, entretanto não cursou com o aumentou a ocorrência de LINA. Conclui-se que tanques de resfriamento por expansão, utilizados dentro de suas especificações quanto ao volume de leite a ser resfriado, permitem a manutenção da CBT e CBP . O tempo de estocagem não afeta a CBT e CBP, entretanto reduz a concentração de caseína e a estabilidade do leite ao teste do álcool, sem alterar a ocorrência de LINA

Orientador: Paulo Roberto Rodrigues Ramos / Banca: Cledson Augusto Garcia / Banca: Cíntia Ludovico Martins / Banca: Luiz Artur Loyola Chardulo / Banca: Roberto de Oliveira Roça / Resumo: O trabalho objetivou identificar por eletroforese as mudanças nas frações das proteínas musculares, separadas no período post-mortem de bovinos de quatro diferentes grupos genéticos (Nelore, TRC-Brahman, Brangus e TRC-Pardo), submetidos ao modelo biológico superprecoce, assim como determinar os valores da força de cisalhamento destas amostras. Foram utilizadas 12 amostras do músculo Longissimus dorsi por grupo genético. De cada uma delas foram retiradas duas fatias, uma foi refrigerada por 24 horas e a outra foi maturada por 7 dias a 2oC. As amostras submetidas a 7 dias de maturação não diferiram quanto à maciez da carne nos grupos genéticos, mas todas apresentaram maciez aceitável, o que pode ser explicado pela baixa idade ao abate destes animais. Na análise das bandas da eletroforese, notou-se degradação da miosina (MHC) e da Troponina-T (TN-T) e o aparecimento do fragmento de 30 kDa para todos os grupos genéticos durante o período de 7 dias de maturação. / Abstract: This study aimed to identify by means of eletrophoresis the changes in fractions of the muscular proteins, separated within the post-mortem period from cattle from four different genetic groups ( Nelore, TRC- Brahman, Brangus and TRC- Pardo), submitted to the superprecoce biologic model, as well as to determine the values of those samples shear force. Twelve samples of the muscle Longissimus dorsi were used for each genetic group. From each of those there were taken two slices, one of them was kept cold along 24 hours and the other one was aged during 7 days at 2oC. The samples that came through a seven-day-aging process didn't show any changes concerning the meat tenderness in the genetic groups, even though all of them had acceptable tenderness, which can be explained due to the low age of these animals when slaughtered. In the analysis of the eletrophoresis' bands, it was noticed some degradation of the myosin ( MHC) and of the Troponin-T (TN-T) and the presence of the fragment of 30 kDa for all the genetic groups during the seven-day-ageing period. / Doutor

Orientador: Mirna Lucia Gigante / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T18:42:59Z (GMT). No. of bitstreams: 1 Vianna_PriscilaCristinaBizam_D.pdf: 1512890 bytes, checksum: cfe5342097a5fc9d5f272c136b132b45 (MD5) Previous issue date: 2010 / Resumo: O objetivo deste trabalho foi avaliar o efeito da adição de dióxido de carbono (CO2) sobre a qualidade dos leites cru e UHT. Inicialmente, o leite cru adicionado ou não de CO2 foi armazenado em garrafas de vidro a 4 e 7ºC e amostras foram avaliadas diariamente quanto à contagem padrão em placas, psicrotróficos e Pseudomonas spp. e a cada dois dias quanto à concentração de CO2, proteólise e lipólise, até que a contagem padrão em placas atingisse 7,5x105 ufc/mL. O delineamento experimental utilizado foi o de sub-sub-parcelas divididas e os resultados foram avaliados por análise de variância multivariada, pelo teste de médias de Tukey (p<0,05) e através do modelo matemático de Gompertz. A contagem padrão aumentou ao longo do tempo para todos os tratamentos e o tempo de conservação foi de 14 dias para o leite armazenado a 4ºC adicionado de CO2 e de 5 dias para o armazenado a 7ºC não adicionado de CO2. Independente da temperatura de armazenamento, a adição de CO2 estendeu o tempo de fase lag e de geração dos micro-organismos psicrotróficos e reduziu sua taxa de crescimento. O leite adicionado de CO2 apresentou menor proteólise e lipólise quando comparado ao não adicionado devido ao menor desenvolvimento de psicrotróficos. Com base nesses resultados, definiu-se o armazenamento do leite cru adicionado ou não de CO2 a 4ºC por 6 dias antes do processamento UHT. Nesta etapa, o leite cru adicionado ou não de CO2 foi armazenado em tanques de expansão antes do processamento UHT (140ºC/5 s). O leite cru foi avaliado no dia da recepção e após 6 dias de armazenamento quanto à composição físico-química, proteólise, lipólise e contagens microbianas. Após o processamento, as amostras foram avaliadas durante 120 dias quanto à composição físico-química, lipólise e proteólise. O delineamento experimental utilizado foi o de parcelas subdivididas em blocos com três repetições. Os resultados foram avaliados por análise de variância e teste de médias de Tukey (p<0,05). Após 6 dias de armazenamento a 4 ºC o leite cru adicionado de CO2 manteve sua qualidade físico-química e microbiológica enquanto que o leite não adicionado sofreu perda significativa de qualidade. A taxa de aumento de proteólise foi 1,4 vezes maior no leite UHT produzido a partir de leite cru não adicionado de CO2 quando comparado à amostra produzida com adição de CO2. Em ambas as amostras, a proteólise foi decorrente tanto da ação de plasmina como de proteases microbianas. Entretanto, o leite UHT produzido a partir de leite cru não adicionado de CO2 apresentou maior ação de proteases microbianas, caracterizada pelo aumento de peptídeos menos hidrofóbicos. A lipólise aumentou para as duas amostras durante os 120 dias e foi maior no leite UHT produzido a partir de leite cru não adicionado de CO2. Os resultados mostraram que a adição de CO2 ao leite cru preservou sua qualidade físico-química e microbiológica durante o armazenamento refrigerado e afetou positivamente a manutenção da qualidade do leite UHT durante seu armazenamento / Abstract: The objective of this work was to evaluate the effect of the CO2 addition on the raw and UHT milk quality. First, raw milk with or without CO2 addition was stored in glass bottles at 4ºC and 7ºC and daily analyzed to standard plate count, psychrotrophic bacteria count and Pseudomonas spp. and every other day to CO2 concentration, proteolysis and lipolysis until standard plate count reached 7,5x105 ufc/mL. Split-split-plot design was used and the results were evaluated by multivariate variance analysis, Tukey¿s test (p<0,05) and by Gompertz model. The standard plate count increased throughout the time for all treatments and the preservation time was 14 days for CO2 added raw milk stored at 4ºC and 5 days for raw milk without CO2 addition stored at 7ºC. Independent of the storage temperature, CO2 extended the lag phase, increased the generation time and decreased the growth rate of psychrotrophic bacteria. Milk with CO2 addition presented lower proteolysis and lipolysis, related to the slower psychrotrophic bacteria development. Based on these results, it was defined a storage of raw milk with and without CO2 addition in bulk tanks at 4ºC during 6 days before UHT treatment (140ºC/5 s). Raw milk was evaluated to physical-chemical composition, proteolysis, lipolysis and microbial counts in the day of reception and after 6 days of storage. After processing, samples were evaluated to physical-chemical composition, lipolysis and proteolysis. Split-plot design was used with three replications. The results were evaluated by analysis of variance (ANOVA) and Tukey¿s test (p<0,05). After 6 days of storage at 4ºC, CO2 added raw milk kept its physical-chemical and microbiological quality, while raw milk without CO2 addition had significant losses. Proteolysis increased ratio was 1,4 higher in UHT milk produced with raw milk without CO2 addition when compared to UHT milk produced with CO2. In both samples proteolysis was a consequence of plasmin and microbial proteases action, but the sample produced with raw milk without CO2 addition presented higher microbial proteases action, characterized for the increase of less hydrophobic peptides. Lipolysis increased for both samples during the 120 days of storage and it was higher in UHT milk produced from raw milk without CO2 addition. The results had shown that CO2 addition to raw milk preserved its physical-chemical and microbiological quality during refrigerated storage and positively affected the quality maintenance of UHT milk during its storage / Doutorado / Doutor em Tecnologia de Alimentos

Made available in DSpace on 2014-07-29T15:13:53Z (GMT). No. of bitstreams: 1 Karyne_Coelho.pdf: 479024 bytes, checksum: d5d2ef481cb3e5c58f1498940178254f (MD5) Previous issue date: 2007-12-19 / The subclinical mastitis is a mammary gland inflammatory reaction that is characterized by increased levels of somatic cells in the milk. The milk with higher somatic cell counting (SCC) shows alteration in the composition that alters the quality of the milk products. This study aimed to evaluate the effects of the SCC in the milk products in relation to the microbiologic and the physical-chemistry features of the mozzarella cheese. It was chosen cattle with SCC &#61603;200.000 cells/mL; SCC >200 a &#61603;400.000 cells/mL; SCC >400.000 cells/mL to &#61603;750.000 cells/mL and SCC >750.000 cells/mL. The animals did not receive antimicrobial treatment before and on the day of the milk collection. The mozzarella cheese preparation were realized at Planta de Laticínios do Centro de Treinamento da Agência Rural. It was analyzed the milk sub products compositions. The cheese were evaluated after 1, 15 and 30 days of maturation or storage at 7°C for the pH, acidity, humidity, total and soluble protein, fat, total dry extract, defatted, milk acid bacteria count, total and fecal coliforms and psicotrofics microorganism. The experiment was repeated four times in order to demonstrate reproducibility. The results were homogenous for the residues (turkey test) and the variances were compared using a F test, adopting &#61621;=0,05. The milk with high SCC showed low concentration of protein and the higher nitrogen levels. There was loosing of protein and fat to serum. The cheese elaborated from milk with higher SCC (>750.000cells/mL) showed lower protein level, more humidity and less industrial income and presented lower growth of lactic acid bacteria during the maturation time. Mozzarella cheese prepared with milk containing SCC around 400.000 cells/mL presented increased proteolysis. The results showed here demonstrated that in order to get a good quality mozzarella cheese is necessary the utilization of the milk with somatic cell counts bellow 400.000 cells /mL / A mastite sub-clínica é uma reação inflamatória da glândula mamária que cursa com o aumento da contagem de células somáticas (CCS) no leite. Leite com alta CCS apresenta alterações na composição que influenciam a qualidade dos produtos lácteos. A realização do presente estudo teve como objetivo avaliar o efeito do nível de células somáticas (CCS) no leite sobre o rendimento e as características microbiológicas e físico-químicas do queijo mussarela. Foram selecionadas vacas com contagem de células somáticas (CCS) &#61603;200mil células/mL; CCS >200 a &#61603;400mil células/mL; CCS >400mil células/mL a &#61603;750mil células/mL e CCS >750mil células/mL e que não receberam tratamento com antimicrobianos nos dias que antecederam a obtenção do leite utilizado no processamento do queijo mussarela, na Planta de Laticínios do Centro de Treinamento da Agência Rural. Foram avaliadas as composições dos leites, soros e queijos. Os queijos foram avaliados após 1, 15 e 30 dias de armazenamento a 7ºC, por meio das análises de pH, acidez, umidade, proteína total e solúvel, gordura, extrato seco total, desengordurado, contagem de bactérias ácido lácticas, de coliformes totais, fecais e de microrganismos psicrotróficos. O experimento completo foi repetido quatro vezes e o delineamento experimental foi em blocos aleatórios. Os resultados foram inicialmente avaliados para a normalidade dos resíduos, por meio do teste de tukey, e a homogeneidade das variâncias pelo teste F, adotando-se, &#61621;=0,05. O leite com elevada CCS apresentou concentração menor de proteína e maior de nitrogênio não protéico. Observou-se perda de proteína e gordura para o soro. O queijo elaborado a partir do leite com alta CCS, >750 mil céls./mL, apresentou menor teor de proteína, maior umidade e menor rendimento industrial. Observou-se menor crescimento das bactérias ácido lácticas durante o período de armazenamento para o queijo produzido com CCS superior a 750 mil céls./mL. Em relação a proteólise foi constatada o aumento do índice de extensão e profundidade nos queijos mussarela elaborados com leite contendo níveis de CCS superiores a 400 mil céls./mL. Considerando os quatros tratamentos avaliados, pode-se afirmar que para obter queijos mussarela com qualidade satisfatória é necessário a utilização de um leite com CCS inferior a 400 mil céls./mL