Orientador: Jaime Amaya Farfan / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T23:55:38Z (GMT). No. of bitstreams: 1 Lollo_PabloChristianoBarboza_D.pdf: 1703113 bytes, checksum: 420e19e11b5feb4766bf0be0c9076acc (MD5) Previous issue date: 2012 / Resumo: Em situacoes especiais e desejavel estimular o anabolismo e reduzir o catabolismo. As proteinas do soro de leite apresentam excelente balanco de aminoacidos para o anabolismo. A suplementacao com L-leucina vem sendo relacionada a inibicao do catabolismo e ao estimulo do anabolismo, assim como o exercicio fisico. Na busca de estrategias para estimular o anabolismo e reduzir o catabolismo, optou-se por estudar o efeito combinado do consumo de proteinas do soro lacteo e a suplementacao com L-leucina, aliados ao exercicio fisico. Objetivo: verificar o efeito dose-resposta da suplementacao com L-leucina na composicao corporal, ganho de massa corporal, parametros bioquimicos e ativacao da proteina mTOR de ratos Wistar, consumindo proteinas do soro de leite (PSL), quando submetidos ao exercicio. Metodologia: 96 ratos machos Wistar foram divididos em 16 grupos recebendo dieta AIN93 com PSL ou caseina (C) como fonte proteica, com 3, 4.5, ou 6% de suplementacao de L-leucina, exercitados e sedentarios, a saber: a) n=6, exercitados, dieta AIN93 com PSL (PSL E); b) n=6, exercitados dieta AIN93 com PSL + 3% de L-leucina; (PSL L3 E); c) n=6, exercitados, dieta AIN93 com PSL + 4,5% de L-leucina (PSL L4.5 E) e d) n=6, exercitados, dieta AIN93 com PSL + 6% de L-leucina (PSL L6 E). Para cada grupo exercitado houve um grupo controle sedentario e o mesmo desenho experimental foi repetido, com grupos sedentarios e exercitados, alterando somente a fonte proteica da dieta para caseina que foi a proteina controle. Foram analisados: ingestao alimentar; evolucao ponderal; perfil de aminoacidos plasmaticos; composicao corporal dos animais; atividades de CK, LDH, AST ALT e mTOR (muscular, cardiaca e diafragmatica), acido urico, creatinina e massa relativizada do gastrocnemio, coracao e diafragma, tudo em funcao da dieta e da atividade fisica. Para analise estatistica foi utilizado o software SPSS, versao 11.0 for Windows para analise de variancia ¿ ANOVA ¿ utilizando o criterio de significancia de p = 0,05, com teste post-hoc de Tukey. Resultados: A ativacao da via mTOR ocorreu com suplementacao de L-L-leucina em ambas dietas por 30 dias no coracao, diafragma e grastrocnemio dos ratos sedentarios e exercitados. Indicadores gerais de saude, nao pareceram alterados, exceto insulina e glicose, que demosntraram anormalidades nos animais sedentarios com altas doses de Lleucina. O ganho de massa corporal foi significativamente menor nos animais submetidos a suplementacao com 6% de L-L-leucina. As enzimas de prova hepatica (TGO e TGP) mantiveram-se em niveis plasmaticos normais, assim como os indicadores de prova renal (acido urico e creatinina) nao indicando dano renal com a suplementacao descrita. Conclusão: A via mTOR foi ativada no coracao, diafragma e musculo dos animais suplementados com L-L-leucina. A maior dose utilizada (6%) prejudicou o crescimento, niveis de glicose e insulina dos animais / Abstract: In both animals and humans L-L-leucine can activate protein synthesis in skeletal muscle by mTOR thus stimulating body growth. Currently, however, it is not clear if heart tissue is also subject to the same regulatory mechanism of protein synthesis. Objective: The purpose of this study was to assess heart mTOR activation, heart mass, growth and liver function in young Wistar rats fed standard AIN93-G diet supplemented with L-L-leucine at three levels. Methods: Ninety-six weanling male Wistar rats were divided into sixteen groups and fed one of the following diets for 30 days: a) Control (AIN 93-G); b) 3% (AIN93-G +3% L-L-leucine); c) 4,5% (AIN93-G +4,5% L-L-leucine); d) 6% (AIN93- G +6% L-L-leucine). Modified AIN 93-G diets containing whey protein instead of casein performed another 4 groups completing 8 sedentary groups, another set of 8 groups was trained, with the total of 16 groups. The supplemented diets, energy was adjusted at the expense of carbohydrate. mTOR pathway was quantified by Westernblot analysis. Serum insulin, uric acid, glucose, AST, ALT, and cardiac mass, total and body mass-adjusted protein were determined by standard methods. ANOVA and pos-hoc Duncan were applied to compare the means (significance p<0.05). Results: mTOR activation (phosphorylated mTOR/ total mTOR) was reached with L-L-leucine supplementation in heart, diaphragm and gastrocnemius. General health indicators did not show significant modifications except for glucose and insulin levels, which increased compared to control group. Supplementation did not adversely affect liver function as determined by AST, ALT, but body mass in the 6% group was significantly lower than that of the 4.5% group, showing a negative effect of the highest dose on body mass accretion. Either absolute heart mass or adjusted heart mass showed no difference between any two groups. Conclusion: mTOR activities of heart, diaphragm and gastrocnemius of Wistar rats were increased by supplementing the AIN 93-G diet with L-L-leucine, loss of the body mass and abnormalities in insulin and glucose levels were shown in 6% of L-L-leucine supplementation. Even the 6% supplementation did not alter liver function, but this concentration adversely affected normal growth / Doutorado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Doutor em Alimentos e Nutrição

Les caséines, les protéines les plus abondantes du lait, sont associées sous forme de micelle ayant un rayon moyen de 100nm, pontées par du phosphate de calcium colloïdal (CCP). Lorsque le CCP est retiré par précipitation acide, les micelles se désintègrent formant les caséinates de sodium (NaCas), des particules de 10nm. Les caséines sont très sensibles aux ions calcium et phosphate présents dans certains produits alimentaires qui constituent un levier important dans l’industrie laitière pour contrôler la texture. La viscosité des suspensions de NaCas augmente fortement lorsque la concentration en protéines augmente à cause du remplissage compact par les particules, mais décroît lorsque la température augmente car la fraction volumique efficace des particules diminue. Au-delà d’un ratio critique Ca2+/caséine, les interactions attractives mènent à la formation de microdomaines denses qui diminuent la solubilité et augmententla turbidité. Le phénomène est amplifié lorsque la température augmente et modifie les propriétés viscoélastiques des suspensions de NaCas. En large excès, les ions Na+ entraînent la dissociation des micro-domaines denses par compétition avec les ions Ca2+. Au-dessus de 0.2M de Na+, la viscosité augmente à cause de la déshydratation des protéines (salting-out). L’orthophosphate (Pi) se lie aux protéines en présence de calcium, ce qui conduit à la formation de complexes. En large excès, le Pi se lie directement au calcium et précipite forme de phosphate de calcium ou sous forme de complexes avec les protéines. Lorsque les micelles des caséines sont acidifiées au-dessus de pH 5,2, le phosphateet le calcium solubilisés interagissent avec les protéines lorsque la température augmente, ce qui conduit à une gélification. Cette étude a démontré combien la nature du phosphate et du calcium est fondamentale dans l’organisation des caséines en suspension dense. / In milk, caseins, the main proteins, are associated into a casein micelle (100nm) held together with colloidal calciumphosphate (CCP). When CCP is removed by acid precipitation into sodium caseinate (NaCas), the caseins reassemble into smaller particles, the so-called submicelles (10nm). Caseins are highly sensitive to calcium and phosphate ions,used in most applications. Challenge for dairy industry is to control the texture, mainly determined by protein interactions.The viscosity of dense NaCas suspensions strongly increased with increasing concentration due to jamming of the particles. Increasing the temperature led to a decrease of the viscosity as the effective volume fraction of the particles decreased. When a ratio of Ca2+ is bound by the casein, attractive interactions are introduced and led to the formation of dense micron sized particles that decreased solubility and increased the turbidity. The effect was stronger with increasingtemperature. The visco-elastic properties of NaCas suspensions were also modified. The same behaviour was observed for calcium caseinate suspensions if the ratio of Ca2+ per casein was equal. Large excess of Na+ led to dissociationof the dense protein domains formed by Ca2+ by competiton for specific binding sites of caseins. For increasing the Na+concentration above 0.2M the viscosity increased by decreased hydration of the protein. Orthophosphate (Pi) was bound to the caseins with Ca2+ leading to the formation of complexes. Large excess of Pi competed with Ca2+. The precipitation of the caseins and large calcium phosphate particles were formed. When casein micelles are acidified above pH 5.2, calcium and phosphate are solubilized and interact with casein leading to the formation of a gel. This study showed how calcium and phosphate of different origins are fundamental in the organization of caseins in dense suspensions.

La stérilisation déstabilisant les protéines laitières acidifiées, des stabilisants sont généralement ajoutés. L’objectif de cette thèse était de comprendre les mécanismes physico-chimiques induits par l’acidification de formules laitières puis de rechercher des voies de stabilisation clean label pour les caséines acidifiées et traitées UHT. Des analyses décrivant la minéralisation, la charge de surface, l’hydratation et les modifications chimiques des caséines ont été mises en œuvre pour étudier leur stabilité.À l’échelle pilote, les caséines étaient plus stables à pH > 5,8 (entre pH 6,7 et 3,7), à basse température (8 versus 42°C), dans une formule riche en protéines (7 versus 3,2 %) et acidifiées par acide citrique (versus lactique). La meilleure stabilité dans ces conditions (parmi 140 combinaisons) s’expliquerait par des diminutions de répulsions électrostatiques, hydrophobes et de diffusion ainsi que par des augmentations de pouvoir tampon et de calcium chélaté par le citrate.Cependant, un traitement de 120°C / 15 s déstabilisait les micelles de caséines dès pH 6,2. L’ajout de matière grasse n’apportait pas d’amélioration. La lactosylation a ensuite été étudiée pour diminuer le pHi des caséines et donc les stabiliser à pH acide. Un prétraitement thermique (90°C / 60 min) à pH 7,5 avec du lactose leur a été appliqué dans différents environnements physico-chimiques. Les niveaux de lactosylation des caséines en milieu dilué étaient faibles mais augmentés avec une forte concentration en lactose (150 g/L) ou en milieu sec. Ce prétraitement appliqué en présence d’une concentration élevée en caséines (50 g/L) ou en minéraux -teneur en perméat triplée) améliorait davantage la stabilisation thermique des protéines acidifiées que la lactosylation seule. Cela s'exliquerait par un pouvoir tampon supérieur lié à cet environnement plus riche. / As sterilization destabilizes acidified dairy proteins, stabilizers are usually added. The objective of this thesis was to understand the physicochemical mechanisms induced by the acidification of dairy formulas and to look for clean label solutions to stabilize for acidified and heat treated caseins. To study their stability, the mineralization, surface charge, hydration and chemical modifications of caseins were characterized.At the pilot scale, caseins were more stable at pH > 5.8 (between pH 6.7 and 3.7), low temperature (8 versus 42°C), in formulas which were enriched in proteins (7 versus 3.2 %) and acidified with citric acid (versus lactic). This set of conditions (amid 140 combinations) would be the best to preserve the stability of proteins because it would correspond to a decrease of electrostatic and hydrophobic repulsions and of diffusion and to an increase of buffer strength and of citrate-chelated calcium content.However, a heat treatment at 120°C during 15 s destabilized casein micelles from pH 6.2 whatever the set of conditions. Adding fat did not bring any improvement.Next, lactosylation was studied to decrease the pHi of caseins so as to stabilize them at acidic pH. A heat pretreatment (90°C/60 min) at pH 7.5 in the presence of lactose was applied to casein micelles in several physicochemical environments. Lactosylation rates of casein in aqueous conditions were low but they were increased with high lactose content (150 g/L) or in dry conditions. This heat pretreatment, implemented with a high casein (50 g/L) or mineral (triple-concentrated permeate content) concentration improved even more the heat stability of acidified proteins than lactosylation only. This could be explained by higher buffer strength due to this richer environnement

De nos jours, l’entraînement en musculation occupe une part importante dans la programmation d’entraînement des sportifs car il permet d’optimiser deux paramètres essentiels à la performance, la force et la puissance. Les stratégies nutritionnelles qui accompagnent les séances de musculation sont susceptibles d’influencer les réponses à l’entraînement. Parmi ces stratégies, celle qui a surtout retenu l’attention du monde scientifique concerne les caractéristiques des apports protéiques, et notamment les aspects de quantité, de qualité et du moment d’apport optimaux. Alors que la grande majorité des travaux ont été conduit dans le cadre d’un exercice unique, ce travail de thèse se propose d’évaluer l’intérêt d’un apport protéique en récupération d’exercices programmés dans le cadre d’un entraînement en musculation de plusieurs semaines. Il s’agit de 1) préciser l’impact du rapport entre les protéines lentes (caséines) et les protéines rapides (lactosérum) du lait - présentes dans une boisson protéique de récupération - sur les adaptations musculaires à un entraînement en musculation (masse musculaire et force) et, 2) de vérifier l’intérêt et le moment optimal d’un apport en protéines lentes, à distance de l’entraînement en musculation, sur les réponses musculaires à l’entraînement. Les résultats de la première étude montrent une augmentation significative de la concentration plasmatique en leucine lorsque la proportion de protéines rapides est supérieure ou égale à la proportion de protéines lentes, par rapport à la situation inverse (plus de caséine que de lactosérum). Mais, ceci ne se traduit pas par des gains hypertrophiques supérieurs. La seconde étude, 1) confirme l’intérêt de l’ingestion de protéines rapides après chaque séance pour améliorer le gain de masse musculaire en réponse à l’entraînement en musculation, et 2) suggère l’intérêt d’un apport de caséine 3h après la fin de chaque séance d’entraînement pour maximiser les gains de force pendant l’entraînement en musculation. Ces résultats permettent de préciser les recommandations d’apport en protéines en phase de récupération de séances de musculation, tant au plan de la qualité des apports azotés (étude 1), de leur quantité (études 1 et 2), que du moment d’apport (étude 2). / Nowadays, resistance training plays an important part in the training programmes of sportsmen, because it can optimise two essential parameters of physical performance: strength and power. Nutritional strategies following exercise can maximise the muscle responses from resistance training. Quality, quantity and timing of protein intake have particularly engaged the attention of scientific studies. While the majority of relevant research has investigated the intake of protein after a single bout of resistance exercise, this particular research project aims to investigate the effect of post-exercice protein intake during several weeks of resistance training.The objectives of this project are 1) to identify the optimal ratio between slow proteins (casein) and fast proteins (whey) of milk, present in a recovery riched-protein drink, on the muscle adaptations from resistance training (muscle mass and strength) and, 2) to investigate the muscle responses to slow protein intake a few hours after resistance training exercise. Results from the first study show that there is no resulting gain in muscle mass when the ratio of fast protein is equal to or higher than that of slow protein, despite an resulting increase in plasma leucine. The second study 1) confirms that ingestion of fast protein after each resistance training session improves muscle mass and strength gains, and 2) suggests that intake of casein 3 hours after the end of each training session can maximise the gain of strength during resistance training. These results allow specific recommendations to be formulated for protein intake during the recovery period after resistance training, both in planning the quality (study 1), quantity (studies 1 and 2) and timing (study 2) of protein intake during resistance training programmes.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22117号 / 医博第4530号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 松原 和夫, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The objective of the experiments conducted for this dissertation was to determine the standardized ileal digestibility (SID) of amino acids (AA) in a variety of feed ingredients for broiler chickens and pigs. The effects of casein in experimental diets on the SID of AA in corn distillers’ dried grains with solubles (DDGS) fed to pigs were evaluated. The SID of AA in feed ingredients, which include full-fat soybean (FFSB), two soybean meals (SBM), peanut flour (PNF), full-fat canola seeds (FFCS), canola meal (CM), canola expellers (CE), hydrolyzed feather meal (HFM), flash dried poultry protein (FDPP), poultry meal (PM), and meat and bone meal (MBM), were compared in broiler chickens and pigs. One of the studies determined the ileal digestibility of AA in casein by regression analysis and investigated the effects of 60 g/kg casein in experimental diets on the SID of AA in DDGS. The ileal digestibility of AA in casein were close to 100%, ranging from 95.5% (SE = 9.10) for Cys to 103.1% (SE = 4.40) for Arg. In addition, the SID of Lys and Phe in DDGS determined by pigs fed the diet containing DDGS and casein were greater (<i>P</i> < 0.05) than the values determined by pigs fed the diet containing DDGS without casein. Based on the results of this experiment, two additional experiments were conducted to determine the effects of graded concentrations of casein from 55 to 165 g/kg in experimental diets on the SID of AA in DDGS and to determine the effects of dietary DDGS concentrations (i.e., 155.6 or 466.8 g/kg) and addition of casein in experimental diets on the SID of AA in DDGS. The SID of indispensable AA, except for Arg and Lys, linearly decreased (<i>P</i> < 0.05) as the concentration of casein in experimental diets increased. Moreover, pigs fed the diets containing 155.6 g/kg DDGS had less (<i>P</i> < 0.05) SID of indispensable AA, except for Trp, in DDGS than those fed the diets containing 466.8 g/kg DDGS regardless of the addition of casein in experimental diets. Therefore, it may be concluded that the addition of casein improves the SID of AA in DDGS, but reduced DDGS concentration in experimental diets decreases the SID of AA in DDGS. In one pair of experiments conducted to compare the SID of AA in FFSB, SBM containing 430 g/kg crude protein, SBM containing 470 g/kg crude protein, and PNF between broiler chickens and pigs, the SID of AA, except for Trp, Ala, and Glu, in test ingredients for pigs were greater (<i>P</i> < 0.05) than the values for broiler chickens. In addition, in both broiler chickens and pigs, the SID of Ile, Leu, and Val in FFSB were less (<i>P</i> < 0.05) than in the other test ingredients. In another pair of experiments conducted to compare the SID of AA in FFCS, CM, and CE between broiler chickens and pigs, interactions (<i>P</i> < 0.05) between experimental diets and species were observed in the SID of AA, except for Lys, Gly, Pro, and Ser. The SID of AA in FFCS for broiler chickens were greater (<i>P</i> < 0.05) than pigs; however, there was no difference in the SID of AA in CM or CE between broiler chickens and pigs. The objective of a third pair of experiments was to compare the SID of AA in HFM, FDPP, PM, and MBM fed to broiler chickens and pigs. There were interactions (<i>P</i> < 0.05) between experimental diets and species in the SID of His, Thr, Trp, and Val. In broiler chickens, the SID of His, Thr, and Trp in FDPP and PM were greater (<i>P</i> < 0.05) than in HFM but were less (<i>P</i> < 0.05) than MBM; however, difference in SID of His, Thr, and Trp among FDPP, PM, and MBM was not observed in pigs. Based on the results of three pairs of studies, it was revealed that differences in SID of AA in common feed ingredients for both broiler chickens and pigs were affected by species. Therefore, it may be concluded that the effects of feed ingredient-specific factors on the SID of AA are different between broiler chickens and pigs.

Inclusion body myositis and Alzheimer's disease are age-related disorders characterized in part by the appearance of intracellular lesions composed of filamentous aggregates of the microtubule-associated protein tau. Abnormal tau phosphorylation accompanies tau aggregation and may be an upstream pathological event in both diseases. Enzymes implicated in tau hyperphosphorylation in Alzheimer's disease include members of the casein kinase 1 family of phosphotransferases, a group of structurally related protein kinases that frequently function in tandem with the ubiquitin modification system. To determine whether casein kinase 1 isoforms associate with degenerating muscle fibers of inclusion body myositis, muscle biopsy sections isolated from sporadic disease cases were subjected to double-label fluorescence immunohistochemistry using selective anti-casein kinase 1 and anti-phospho-tau antibodies. Results showed that the alpha isoform of casein kinase 1, but not the delta or epsilon isoforms, stained degenerating muscle fibers in all eight inclusion body myositis cases examined. Staining was almost exclusively localized to phospho-tau-bearing inclusions. These findings, which extend the molecular similarities between inclusion body myositis muscle and Alzheimer's disease brain, implicate casein kinase 1 alpha as one of the phosphotransferases potentially involved in tau hyperphosphorylation.

Enzymic milk coagulation was monitored by measuring changes in curd firmness and apparent absorbance of undiluted milk. Detection of coagulation, visually or rheologically, occurred after the milk changes from a system of aggregating particles to an extended space network. This change was observed as a shoulder in apparent absorbance plots and coagulation time was defined as the critical point in the aggregation process analogously to non-linear condensation polymerization reactions. It corresponds to the inflexion point during the period when apparent absorbance was rapidly increasing and can be calculated by fitting curd firmness data to an exponential equation. Addition of calcium chloride to milk reduced coagulation time with a minimum occurring at .05M calcium. Also, curd firmness increased with a maximum at .02M calcium. It appears that calcium affects all stages of coagulation: proteolysis, micelle aggregation, and gelation. When bulk culture media was added to milk, the pH of the media had a greater effect on coagulation time than did presence of phosphate in the media. Non-specific proteolytic activity of milk coagulants affects the initial rate of curd firming but not the maximum firmness. The more proteolytic the enzyme the slower the curd firming rate. This can be used to rapidly assay for pepsin content of bovine rennets.

The Western Dairy Center at Utah State University funded this project to investigate cheese research using concentrated milks. Concentrated milk was provided by the South Dakota State University and starter culture for this study was prepared and donated by Vivolac Cultures Corporation, Greenfield, Indiana. The project initiated as a continuation of a previous study on effects of protein concentration, coagulum cut size and set temperature on curd moisture loss kinetics while stirring during cheesemaking. It was aimed at determining the extent to which pH drop prior to draining and final cheese moisture when using microfiltered concentrated milk. We performed twelve cheesemaking trials using recombined milk from micellar casein concentrate, cream and skim milk according to a modified cheddar cheese-make procedure. Four different levels of starter cultures were used to achieve different acidification rates for pH change during cheesemaking. The amount of starter culture added had significant effect on moisture of cheese at whey drainage, moisture and pH of cheese. Thus, it can be said that the pH drop that occurs during the cheesemaking increases rate and extent of whey expulsion.

Mastitis, an infection of the mammary gland, is the most common and expensive animal health problem for the dairy industry and affects every dairy farm to some degree. This disease complex is painful for dairy cows, increases the on-farm use of antibiotics, presents a threat to milk quality and is a waste of time, money and milk production. Each year, the dairy industry loses as much as a billion dollars to mastitis. Many cows will experience mastitis at least once during a lactation cycle and some animals will develop recurring mastitis episodes in a single mammary quarter. These mastitic quarters can be difficult to manage during the lactation cycle. Cessation of production in the quarter while continuing to milk the other three can be a beneficial management decision in this scenario. However, the current methods available for cessation of lactation in a single quarter are limited. This study investigated the use of casein hydrolysate as a non-antibiotic option for causing cessation of lactation in a quarter. From this preliminary study we were able to apply our results to another aspect of mastitis prevention, which is the routine use of intramammary antibiotics at the end of the lactation cycle. This management practice is known as dry treatment and is a standard practice in the dairy industry with many years of proven efficacy against clearing infections present at the end of the lactation cycle. Increasing pressure from consumers to decrease antibiotic use in food production animals has caused this practice to come under scrutiny. This secondary study investigated the use of casein hydrolysates as a nonantibiotic alternative to standard antibiotic dry cow treatment. Overall, these studies demonstrated that casein hydrolysate has some efficacy in inducing mammary involution of a single quarter mid-lactation and also potentially as an alternative dry cow treatment. None of the animals treated in these studies displayed any symptoms of pain or discomfort, and all treated quarters resumed milk production after the next calving. Additionally, all antimicrobial milk tests on treated animals were negative. Casein hydrolysates may be a useful management tool for milk quality and animal health within the dairy industry.