Orientador: Rosiane Lopes da Cunha / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T02:52:06Z (GMT). No. of bitstreams: 1 Takeuchi_KatiuchiaPereira_D.pdf: 2512071 bytes, checksum: bf144af04917964fdd59c0d4a482b84e (MD5) Previous issue date: 2008 / Resumo: A avaliação do processo de gelificação ácida de caseinato de sódio (CS) induzida por glucona-d-lactona (GDL) foi realizada em diferentes taxas e com ampla faixa de concentração de proteína (2-6% p/p). A cinética de acidificação e gelificação foi avaliada desde o pH 6,7 até o ponto isoelétrico das caseínas através da medida de pH e de propriedades mecânicas obtidas em compressão uniaxial (tensão e deformação na ruptura). A formação da rede do gel foi mais influenciada pelas interações eletrostáticas do que pelas diferentes taxas de acidificação, principalmente em pH próximo ao pI das caseínas. Além disso, interações hidrofóbicas e ligações de hidrogênio também estiveram envolvidas na estabilização da estrutura da rede, promovendo géis mais fortes. Também foi avaliado o processo de gelificação de proteínas do leite em pH 6,7 em sistemas contendo carragena. Neste caso, a ?-carragena foi adicionada em concentração de 0,3 a 0,8% (p/p) em misturas contendo caseinato de sódio (2 a 8% p/p), isolado protéico de soro (0,5 a 7% p/p) ou sacarose (5 a 30% p/p). Estes sistemas foram estudados a partir de ensaios reológicos em cisalhamento oscilatório, propriedades mecânicas e microestrutura. A temperatura de início da gelificação ou do desenvolvimento de estrutura (Ts) aumentou com a concentração de carragena em sistemas puros, enquanto que a presença de sacarose ou isolado protéico de soro promoveu um aumento da Ts e a adição de caseinato de sódio não modificou esta temperatura, em relação aos géis puros. Após a fomação do gel, um aumento da concentração de carragena levou a géis mais elásticos, rígidos, deformáveis e firmes, sendo que a adição de sacarose exerceu pouco efeito nas propriedades reológicas destes sistemas. A adição de isolado protéico de soro enfraqueceu a rede do gel em baixas concentrações (até 3%), mas houve formação de uma rede mista em maiores concentrações, sem demonstração de sinergismo entre os biopolímeros. A mistura de carragena e caseinato de sódio mostrou sinergia até 5% (p/p) desta proteína e para maiores concentrações, ocorreu o enfraquecimento da rede, diminuição da rigidez e firmeza do gel, provavelmente relacionada à micro-separação de fases, observada por microscopia confocal. Os espectros mecânicos mostraram que a maioria destes sistemas mistos apresentou comportamento de gel fraco, devido ao efeito simultâneo das interações físicas e incompatibilidade termodinâmica. No entanto, géis fortes foram observados na maior e menor concentração de carragena e caseinato de sódio, respectivamente, indicando a importância da interação eletrostática entre estes dois biopolímeros. O aumento da concentração de carragena e de proteínas promoveu um aumento da heterogeneidade da microestrutura do gel e a intensidade de interações repulsivas entre os biopolímeros e a formação da rede do gel, dominado pela carragena, afetou de maneira complexa as propriedades físicas destes sistemas / Abstract: Evaluation of acid-induced sodium caseinate (CS) gelation promoted by glucono-d- lactone (GDL) was performed at different acidification rates with several protein concentrations (2-6% w/w). The kinetics of acidification and gelation were followed from pH 6.7 to the isoelectric point of casein by evaluation of the pH and mechanical properties using uniaxial compression measurements (stress and strain at rupture). Gel network formation was more influenced by electrostatic interactions than different acidification rates, showing the contribution of rearrangements of bonds to strengthening the network, mainly at steady-state pH close to pI of caseins. Besides the hydrophobic interactions and hydrogen bonds were also important forces involved in structure stabilization, leading to stronger gels. Moreover, evaluation of gelation process of milk proteins at pH 6.7 in systems containing ?-carrageenan. In this study, the ?-carrageenan was added at concentration from 0.3 to 0.8% (w/w) into mixtures containing sodium caseinate (2 a 8% w/w), whey protein isolate (0.5 a 7% w/w) or sucrose (5 a 30% w/w). This systems was analysed using rheological measurements under oscillatory shear, mechanical properties and microstructure. The temperature at which structure development began or initial of gelation (Ts) augmented with increasing carrageenan concentration for pure systems, but sucrose or whey protein isolate addition promoted an increase of Ts and sodium caseinate did not affect this temperature, in relation to pure gel. After gel formation, increasing carrageenan concentration promoted more elastic, stronger, deformable and firmer, as well as sucrose addition showed a little effect to decrease elastic character of gel. Whey protein addition weakened gel network at lower concentrations (up to 3% w/w), but at higher concentration was observed a mixed gel network entanglement of carrageenan and whey protein, without sinergism between this biopolymers. Mixed gels of carrageenan and sodium caseinate showed synergism up to 5% (w/w/) of this protein and increasing concentration led to weaken the gel network, decreasing the rigidity and the firmness of gel, probably related to micro-phase separation, observed by confocal microscopy. Mechanical spectra showed that most of mixed systems presented weak gel behaviour, due to simultaneous effect of physical interactions and thermodynamic interactions. However, stronger gels were formed at higher and lower concentration of carrageenan and sodium caseinate, respectively, which indicate the relevance of electrostatic interactions between these biopolymers. Increasing carrageenan and proteins concentrations led to greater microstructure heterogeneity and increased repulsive interactions between biopolymers and thus gel formation, dominated by carrageenan, affected in a complex way the physical properties of these systems / Doutorado / Doutor em Engenharia de Alimentos

L’ostéoporose touche principalement les femmes après la ménopause, c’est une maladie caractérisée par une détérioration de la minéralisation et de la micro-architecture de l’os. L’objectif du travail de thèse présenté ici est d’identifier une fraction protéique laitière ayant un effet stimulant sur la formation osseuse. Une telle fraction, ajoutée dans un produit ou un complément alimentaire, pourrait contribuer à réduire la perte osseuse. La première étape du projet consiste à produire les fractions laitières. Des protéines laitières (caséine ou protéines sériques) ont été digérées par des enzymes puis filtrées pour les fractionner selon leur poids moléculaire. Les fractions obtenues ont ensuite été testées sur des cultures primaires de cellules osseuses. Certaines fractions protéiques laitières ont augmenté la prolifération et la différenciation des ostéoblastes. Parmi ces fractions actives, la fraction correspondant au rétentat d’une filtration sur un filtre à 10kDa d’un hydrolysat de caséine par de la chymotrypsine a été sélectionnée pour être testée sur animaux. Cette fraction a été nommée fraction CR10. Pour étudier l’activité du CR10 in vivo sur le métabolisme osseux, un modèle de souris sous restriction protéique est mis au point. Nos études démontrent que, lorsque le régime est basé sur des protéines de soja, le passage d’un régime contenant 20% de protéines à un régime contenant 6% de protéines induit une réduction de la formation osseuse. Le traitement des souris sous restriction protéique avec du CR10 n’a eu aucun effet, ce qui signifie que le CR10 n’arrive pas à exercer son activité anabolique in vivo. En revanche, si de la caséine est donnée à la place du soja ou si de la PTH est injectée aux souris, la formation osseuse est augmentée. Ces résultats suggèrent que la fraction CR10 n’est pas un bon candidat comme fraction anabolique. En revanche, l’effet positif de la caséine par rapport au soja pourrait être exploité lors de futures études visant à mettre au point une fraction caséique ostéoanabolique. / Osteoporosis is a disease mainly affecting women after menopause, characterized by a reduced bone mineralization and a deterioration of bone micro-architecture. The aim of this thesis is to identify a milk protein fraction able to stimulate bone formation. When added to a food product, this fraction could reduce bone loss. The first task of this project was to produce the milk protein fractions. Milk proteins (casein or whey proteins) were digested by enzymes and fractionated by filtration according to their molecular weight. The fractions obtained were then tested on primary cultures of bone cells. Some of the milk protein fractions tested were able to increase proliferation and differentiation of osteoblasts. Among these active fractions, the one obtained by digestion of casein by chymotrypsin followed by filtration through a 10 kDa filter have been selected to be tested on animals. This fraction is named CR10. To study the activity of CR10 in vivo, a protein-restricted mouse model has been developed. Our studies showed that a reduction of protein in the diet from 20% to 6% impaired bone formation when the diet was based on soy protein. When these protein-restricted mice ingested the CR10 fraction, no improvement of the BMD was reported, which means that the CR10 cannot exert its anabolic activity in vivo. However, if casein is given instead of soy or if PTH is injected to the mice, bone formation is increased. These results suggest that the CR10 is not a good candidate as an anabolic fraction. However, the positive effect of casein compared to soy could be exploited in future studies aimed at finding an osteoanabolic casein fraction.

In this research, beta-lactoglobulin was chemically modified by attaching different levels of stearic acid to the protein. The effect of this modification on hydrophobic!ty, emulsifying and foam properties, digestibility and allergenicity of the protein was investigated. It was found that the effect of fatty acid attachment or lipophilization depended on the amount of fatty acids attached to the protein. Incorporation of the hydrophobic ligands led to increased hydrophobic interactions, resulting in a decreasing solubility with extent of incorporation. Furthermore, the surface hydrophobicity measurements showed that the two fluorescence probes 8-anilinonaphthalene-l-sulfonate (ANS) and cis-parinaric acid (CPA) used for the surface hydrophobicity measurements were not equivalent This may support the. observation by earlier workers that ANS measures aromatic hydrophobicity and CPA aliphatic hydrophobicity. The studies on surface functional properties i.e. emulsifying and foaming properties, indicated that there was some improvement in these functional properties at low and medium levels of incorporation which decreased as the extent of fatty acid attachment further increased. The improvement, of these functional properties could be attributed to improved amphiphilicity of the proteins at these levels of incorporation. This research also showed that both high solubility and high ANS surface hydrophobicity is needed for the best emulsifying properties. In vitro digestibility studies showed a decrease in digestibility of the modified proteins with increased lipophilization. From the passive cutaneous anaphylaxis experiments, it was found that the level of fatty acid attachment to the protein had a significant effect on its ability to elicit IgE antibodies. Increased ability to elicit IgE antibodies was observed at a low level of fatty acid. When a medium level of fatty acid was attached the ability to elicit antibodies was reduced and almost completely destroyed when a higher level of fatty acid was incorporated. The above observations could be explained by the fact that the low level incorporation of fatty acid led to changes in the protein structure which exposed more allergenic sites. The almost complete destruction of the allergenicity could be attributed to denaturation of the protein which reduced or destroyed available allergenic sites. The antigenicity or binding of the modified proteins to the IgG antibodies raised against the native protein was studied by both direct and competitive enzyme linked immunosorbent assay. It was found that at low and medium levels of incorporation, the proteins demonstrated increased binding ability compared to the native protein. This was attributed to the increased exposure of antigenic sites on the protein with fatty acid incorporation. However, the protein with high level of incorporated fatty acid showed decreased binding ability. / Land and Food Systems, Faculty of / Graduate

Mammary cells from lactating rats and dairy cows were cultured in Eagle's minimal essential medium (MEM) with added amino acids. Changes in free intracellular amino acid pools and milk protein synthesis in response to amino acid additions to the medium were measured. Increases in free intracellular amino acid pools are associated with increased protein synthesis and the rate of their change in response to extracellular amino acids would partially reflect the cell's amino acid requirement. The intracellular pools from medium amino acids (except methionine, tryptophan and glutamine) increased with extracellular amino acids but at their own characteristic rate. The ratio of medium amino acids to nonmedium amino acids inside the mammary cell increased with the concentration of amino acids in the medium. Methionine and tryptophan did not have measurable pools in the rat mammary cell and only very small pools in the bovine mammary cell which did not increase with extracellular amino acids. Culturing rat mammary cells with labeled methionine showed only 42% of the intracellular radioactivity was still associated with labeled methionine indicating significant conversion of this amino acid after it entered the cell. A small linear increase in intracellular cystine was observed with elevated cystine in the medium. The responses in cystine, tryptophan and methionine intracellular pools to extracellular amino acids suggest the concentration of these amino acids in the medium are insufficient to meet the bovine mammary cells requirement. Increasing the concentration of amino acids in MEM 1-, 3-, 5- and 7 fold significantly (P < .05) increased β-casein and to a lesser degree β-lactoglobulin synthesis by bovine mammary cells in culture. Individually increasing each of the 13 amino acids in MEM 3-fold showed cystine followed by threonine and then methionine significantly (P < .05) increased the combined synthesis of β-casein and β-lactoglobulin. The correlation between intracellular pool size change and the response in milk protein synthesis to increased individual amino acids was -.51 which was not significant at the .05 level. / Ph. D.

Methionine- and lysine-containing di- to octapeptides were evaluated for their ability to serve as methionine and lysine sources respectively for the synthesis of secreted proteins. Mammary tissue explants from lactating (10 to 11 d) CD-1 mice and cultured bovine mammary epithelial cells (MAC-T) were used as experimental models. Explants and cultured cells were incubated at 37°C in a humidified atmosphere of 90% air/10% CO₂ and 95% air/5% CO₂, respectively, for 1 to 24 h in Dulbecco's modified Eagle's medium containing hormones, ³H-leucine, and methionine or lysine substrate in either free or peptide-bound form. The ability of methionine and lysine substrates to promote incorporation of ³H-leucine into secreted proteins was quantified. Mouse mammary explants were able to utilize methionine and lysine from all peptides tested except the lysyl octapeptide. All the methionyl peptides were at least as effective as free methionine in promoting ³H-leucine incorporation into secreted proteins. Most methionyl di- and tri-peptides promoted 15 to 76% greater (P < .05) ³H-leucine incorporation than did free methionine. A negative correlation (r = -.89, P < .01) was detected between the rate of ³H-leucine incorporation and the number of amino acid residues in the peptides. The incorporation of ³H-leucine promoted by some methiony] dipeptides was reduced (P < .05) in the presence of a 200-fold higher concentration of glycylsarcosine or carnosine. Incorporation of ³H-leucine promoted by lysyl peptides ranged from 91 to 117% of the incorporation promoted by free lysine. MAC-T cells were also able to utilize methionine from all di- and tri-peptides studied. The ability of the peptides to promote ³H-leucine incorporation varied with experimental conditions. For cells allowed to grow/differentiate for 3 or 8 d, incorporation of ³H-leucine promoted by peptides ranged from 67 to 85% and 86 to 110% of the incorporation promoted by free methionine, respectively. The effect of extracellular matrix on the utilization of peptide-bound methionine by MAC-T cells was also examined. Generally, there was no difference in ³H-leucine incorporation/DNA promoted by methionyl dipeptides in MAC-T cells cultured on matrigel, collagen, laminin, or fibronectin coated or uncoated plates. These results suggest that peptides can serve as sources of amino acids for the synthesis of secreted proteins by both lactating mammary explants and cultured mammary epithelial cells. Mouse mammary explants appear to have a greater ability to utilize peptide-bound methionine than to utilize peptide-bound lysine. Mediated transport of some methiony] peptides may be involved in the peptide utilization by the mammary explants. More extensive utilization of peptides by MAC-T cells following a longer (3 vs 8 d) growth/differentiation period may indicate that some maturation process(s) in the cells was necessary for the most effective utilization of peptides. / Ph. D.

Le fromage fondu est un produit alimentaire de seconde transformation obtenu après mélange et cuisson de fromages, additionnés éventuellement d'autres ingrédients laitiers. Ce produit, dont la consommation croit dans de nombreuses régions du monde, présente une grande variété d'applications et peut être conservé plusieurs mois à température ambiante. Cependant, les mécanismes moléculaires sous-jacents à sa structuration biochimique demeurent mal connus et sa fabrication industrielle reste souvent empirique.L'objectif général de ce travail est d'améliorer la compréhension des étapes de structuration biochimique du fromage fondu de type « portion triangulaire tartinable », au cours des étapes de sa fabrication. Dans un premier temps, les propriétés physico-chimiques des sels de fonte, additifs ajoutés au fromage fondu, ont été étudiées dans des milieux modèles de complexités croissantes, de la solution aqueuse jusqu'au lait écrémé. La composition de ces sels de fonte, leur hydrolyse après traitement thermique ainsi que leur interaction avec certains constituants laitiers ont été évaluées par deux méthodes complémentaires : la chromatographie ionique et la RMN du phosphore. Par ailleurs, il a été montré que ces sels, via la chélation du calcium, induisaient la dissociation des caséines, modifiant les propriétés d'hydratation et d'émulsification de ces dernières.Dans un deuxième temps, le rôle des sels de fonte quant à la structuration des protéines, de l'eau, des minéraux et de la matière grasse a été analysé dans des matrices plus complexes : les portions triangulaires tartinables, aux étapes clés de leur fabrication. Un mécanisme d'interaction des constituants biochimiques majoritaires a été proposé, prenant en compte l'évolution des molécules sous l'effet des contraintes physiques et chimiques appliquées au cours du procédé. / Processed cheese is manufactured by the secondary processing food industry, by mixing and heating cheese, along with other dairy ingredients. This product, whose consumption grows in many parts of the world, has a wide variety of applications and can be stored for several months at room temperature. However, the molecular mechanisms underlying its structure remain poorly understood and industrial production is often empirical. The general objective of this work is to improve the understanding of biochemical steps structuring spreadable processed cheese during its manufacture. At first, the physico-chemical properties of additives used in processed cheese, i.e. emulsifying salts, have been studied in simplified environments of increasing complexity, from aqueous solutions to skimmed milk. The composition of theses salts, their hydrolysis after heat treatment and their interaction with some dairy constituents were assessed by two complementary methods: ion chromatography and phosphorus NMR. Moreover, it has been shown that these salts, through the calcium chelation, induced casein dissociation and modified their hydration and emulsifying properties. Then, the role of these salts on the structure and interactions between proteins, water, minerals and fat were analyzed in spreadable processed cheese, at different steps of its manufacture. A mechanism of interaction between the major biochemical constituents has been proposed, taking into account the evolution of molecules under the influence of physical and chemical constraints applied during the process.

Para produção de biofármacos protéicos, como a eritropoietina recombinante humana (EPOrh), são necessárias alterações pós-traducionais adequadas que garantam a sua especificidade e atividade biológica. Essas características são obtidas apenas em biorretores baseados em células eucarióticas, como as da glândula mamária. Sistemas baseados nesse tipo celular, tanto in vivo quanto in vitro, já são utilizados para produção estratégica e viável de proteínas recombinantes biologicamente ativas. Assim, tanto o estabelecimento de novas linhagens de células mamárias que apresentem boa expressão protéica quanto o desenvolvimento de sistemas in vivo que utilizem a estrutura da glândula mamária para essa produção de proteínas recombinantes são de grande valia. O presente trabalho teve como objetivo comparar dois métodos de estabelecimento de uma cultura de células de glândula mamárias ovinas, enzimático e não enzimático, e verificar sua capacidade de expressão das proteínas do leite &beta;-lactoglobulina, &alpha;-caseína, &beta;-caseína e &kappa;-caseína mediante o tratamento com SFB (soro fetal bovino) ou SOL (soro de ovelha lactante), na presença ou não de Matrigel. Para isso, foi realizado um experimento in vitro, no qual foi estabelecido o cultivo celular até a passagem 12 (P12) de duas linhagens celulares: digerida (LD) e não digerida (LND). Para a LD na P12 foi observado apenas um tipo celular, o qual era positivo para a marcação com vimentina. Essa linhagem apresentou expressão gênica de &beta;-caseína e &beta;-lactoglobulina apenas quando tratada com meio de cultivo acrescido de SFB, sendo a expressão inferior (P=0,001) ao grupo da LND submetido ao mesmo tratamento. Já a LND, quando tratada com meio adicionado com SFB expressou &kappa;-caseína além da &beta;-caseína e &beta;-lactoglobulina. A troca do SFB do meio de cultivo por SOL aumentou a expressão gênica de &beta;-lactoglobulina (P=0,001) para ambas linhagens. Foi realizada a curva de crescimento para LD e LND na P12 com o meio de cultivo acrescido com SFB ou SOL. Para a LND observou-se o efeito do meio na velocidade de crescimento celular, sendo que foi maior para o grupo tratado com SFB (P&lt;0,05). Para a LD, não ocorreu o efeito do meio na velocidade de crescimento celular (P&gt;0,05), não sendo observada diferença com a LND tratada com SOL (P&gt;0,05). A LND apresentou marcação positiva para a presença de vimentina e citoqueratina. Este trabalho visou, ainda, estabelecer um sistema de produção da EPOrh no leite de ovelhas não transgênicas pela técnica de infusão intra-mamária in vivo de dois plasmídeos diferentes e verificar a secreção qualitativa desta proteína por Western-blotting. Assim, foi feito um experimento in vivo no qual glândulas mamárias de ovelhas foram transfectadas com dois plasmídeos diferentes: ALAC (n=2), BGL (n=2) e controle negativo (n=2). Após a infusão dos plasmídeos, foi realizada a eletroporação de cada teto (3 choques de 500 volts com a duração de 15ms cada, sendo realizada a inversão da polaridade). Os animais foram ordenhados durante 20 dias após a transfecção, porém não foi possível detectar a presença de EPOrh nas amostras de leite analisadas. O limiar de detecção do teste utilizado foi de 67,5pg de EPOrh (Eritromax&reg;) em leite controle negativo de ovelha. Concluindo, foi possível estabelecer o cultivo in vitro das LD e LND com capacidade de expressar proteínas do leite, sendo a expressão da &beta;-lactoglobulina aumentada pelo tratamento com SOL. Ambas as linhagens apresentaram marcação positiva para vimentina, mas apenas LND para citoqueratina. Ainda, para o experimento in vivo, não foi possível detectar a expressão de EPOrh no leite das ovelhas transfectadas com os plasmídeos ALAC e BGL. / Some post-translational modifications are necessary for the production of biopharmaceutical proteins, such as recombinant human erythropoietin (rhEPO), with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors, in vivo or in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression and the development of systems for production of recombinant proteins by the mammary gland in vivo are essential studies. One of the main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as &beta;-lactoglobulin, &alpha;-casein, &beta;-casein and &kappa;-casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel&reg;. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed &beta;-lactoglobulin and &beta;-casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0,001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed &beta;-lactoglobulin, &beta;-casein and &kappa;-casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of &beta;-lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P&lt;0,05). However, the medium did not have effect on growth speed of LD (P&gt;0,05) and no difference was observed at the NDL treated with LOS (P&gt;0,05). The NDL was positive for staining with vimentin and cytokeratin. The second main objective of this study was to establish an in vivo system for the production of rhEPO in milk of non-transgenic ewes by the intra-mammary infusion of two different plasmids and verify the qualitative milk secretion of this protein by western-blotting. In this way, in the in vivo experiment ovine mammary glands were transfected with two different plasmids: ALAC (n=2), BGL (n=2) and negative control (n=2). Each half udder was filled with plasmid solution and three 3 electric pulses of 500 volts were applied for 15ms each, followed by another three pulses with reversed polarity. The three animals were milked for 20 days after transfection, nevertheless it was not possible to identify rhEPO in any milk sample. The test threshold to identify rhEPO (Eritromax&reg;) in milk from a negative control animal was 67,5pg. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of &beta;-lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. In the in vivo experiment, rhEPO secretion was not detected in the milk from ewes transfected with ALAC and BGL plasmids.

Para produção de biofármacos protéicos, como a eritropoietina recombinante humana (EPOrh), são necessárias alterações pós-traducionais adequadas que garantam a sua especificidade e atividade biológica. Essas características são obtidas apenas em biorretores baseados em células eucarióticas, como as da glândula mamária. Sistemas baseados nesse tipo celular, tanto in vivo quanto in vitro, já são utilizados para produção estratégica e viável de proteínas recombinantes biologicamente ativas. Assim, tanto o estabelecimento de novas linhagens de células mamárias que apresentem boa expressão protéica quanto o desenvolvimento de sistemas in vivo que utilizem a estrutura da glândula mamária para essa produção de proteínas recombinantes são de grande valia. O presente trabalho teve como objetivo comparar dois métodos de estabelecimento de uma cultura de células de glândula mamárias ovinas, enzimático e não enzimático, e verificar sua capacidade de expressão das proteínas do leite &beta;-lactoglobulina, &alpha;-caseína, &beta;-caseína e &kappa;-caseína mediante o tratamento com SFB (soro fetal bovino) ou SOL (soro de ovelha lactante), na presença ou não de Matrigel. Para isso, foi realizado um experimento in vitro, no qual foi estabelecido o cultivo celular até a passagem 12 (P12) de duas linhagens celulares: digerida (LD) e não digerida (LND). Para a LD na P12 foi observado apenas um tipo celular, o qual era positivo para a marcação com vimentina. Essa linhagem apresentou expressão gênica de &beta;-caseína e &beta;-lactoglobulina apenas quando tratada com meio de cultivo acrescido de SFB, sendo a expressão inferior (P=0,001) ao grupo da LND submetido ao mesmo tratamento. Já a LND, quando tratada com meio adicionado com SFB expressou &kappa;-caseína além da &beta;-caseína e &beta;-lactoglobulina. A troca do SFB do meio de cultivo por SOL aumentou a expressão gênica de &beta;-lactoglobulina (P=0,001) para ambas linhagens. Foi realizada a curva de crescimento para LD e LND na P12 com o meio de cultivo acrescido com SFB ou SOL. Para a LND observou-se o efeito do meio na velocidade de crescimento celular, sendo que foi maior para o grupo tratado com SFB (P&lt;0,05). Para a LD, não ocorreu o efeito do meio na velocidade de crescimento celular (P&gt;0,05), não sendo observada diferença com a LND tratada com SOL (P&gt;0,05). A LND apresentou marcação positiva para a presença de vimentina e citoqueratina. Este trabalho visou, ainda, estabelecer um sistema de produção da EPOrh no leite de ovelhas não transgênicas pela técnica de infusão intra-mamária in vivo de dois plasmídeos diferentes e verificar a secreção qualitativa desta proteína por Western-blotting. Assim, foi feito um experimento in vivo no qual glândulas mamárias de ovelhas foram transfectadas com dois plasmídeos diferentes: ALAC (n=2), BGL (n=2) e controle negativo (n=2). Após a infusão dos plasmídeos, foi realizada a eletroporação de cada teto (3 choques de 500 volts com a duração de 15ms cada, sendo realizada a inversão da polaridade). Os animais foram ordenhados durante 20 dias após a transfecção, porém não foi possível detectar a presença de EPOrh nas amostras de leite analisadas. O limiar de detecção do teste utilizado foi de 67,5pg de EPOrh (Eritromax&reg;) em leite controle negativo de ovelha. Concluindo, foi possível estabelecer o cultivo in vitro das LD e LND com capacidade de expressar proteínas do leite, sendo a expressão da &beta;-lactoglobulina aumentada pelo tratamento com SOL. Ambas as linhagens apresentaram marcação positiva para vimentina, mas apenas LND para citoqueratina. Ainda, para o experimento in vivo, não foi possível detectar a expressão de EPOrh no leite das ovelhas transfectadas com os plasmídeos ALAC e BGL. / Some post-translational modifications are necessary for the production of biopharmaceutical proteins, such as recombinant human erythropoietin (rhEPO), with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors, in vivo or in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression and the development of systems for production of recombinant proteins by the mammary gland in vivo are essential studies. One of the main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as &beta;-lactoglobulin, &alpha;-casein, &beta;-casein and &kappa;-casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel&reg;. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed &beta;-lactoglobulin and &beta;-casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0,001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed &beta;-lactoglobulin, &beta;-casein and &kappa;-casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of &beta;-lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P&lt;0,05). However, the medium did not have effect on growth speed of LD (P&gt;0,05) and no difference was observed at the NDL treated with LOS (P&gt;0,05). The NDL was positive for staining with vimentin and cytokeratin. The second main objective of this study was to establish an in vivo system for the production of rhEPO in milk of non-transgenic ewes by the intra-mammary infusion of two different plasmids and verify the qualitative milk secretion of this protein by western-blotting. In this way, in the in vivo experiment ovine mammary glands were transfected with two different plasmids: ALAC (n=2), BGL (n=2) and negative control (n=2). Each half udder was filled with plasmid solution and three 3 electric pulses of 500 volts were applied for 15ms each, followed by another three pulses with reversed polarity. The three animals were milked for 20 days after transfection, nevertheless it was not possible to identify rhEPO in any milk sample. The test threshold to identify rhEPO (Eritromax&reg;) in milk from a negative control animal was 67,5pg. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of &beta;-lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. In the in vivo experiment, rhEPO secretion was not detected in the milk from ewes transfected with ALAC and BGL plasmids.

L'appellation ‘desserts laitiers neutres' regroupe de nombreux produits de composition similaire mais de structure et de texture différente. Trois de leurs composants et leurs interactions définissent leur structure : l'amidon, les carraghénanes et les protéines de lait. L'objectif de cette thèse était de mieux appréhender les mécanismes physico-chimiques impliqués dans la mise en place de la structure de ces produits et leur impact sur la perception sensorielle.Ce travail s'est focalisé sur les interactions entre l'amidon et les carraghénanes en présence ou non de protéines de lait. De l'amidon modifié de maïs cireux, du lait écrémé reconstitué et des kappa-carraghénanes ont été utilisés. Différentes interactions ont été mises en avant par l'utilisation d'outils rhéologiques et microscopiques. En mélange binaire le carraghénane s'adsorbe sur le granule d'amidon gonflé. Cette interaction dépend de la densité de charge, et de la masse moléculaire du carraghénane utilisé. En mélange ternaire le carraghénane interagit préférentiellement avec les micelles de caséines et ce quel que soit le moment ou le lait est ajouté (avant ou après empesage de l'amidon). Cette modification de l'ordre d'incorporation des ingrédients permet d'obtenir des produits de microstructures différentes. Dans les deux cas les granules d'amidon sont inclus dans un réseau carraghénanes / micelles de caséines mais les caractéristiques de ces deux phases (amidon et carraghénanes / micelles de caséines), responsables de la structuration des produits, sont modifiées. Afin d'en évaluer l'impact sur la perception sensorielle de crèmes, neuf produits de formules identiques mais assemblés différemment ont été fabriqués. Un tri libre, suivi d'un classement des groupes sur des termes discriminants à consonance personnelle, ont été mis en place. Des différences entre les produits ont été perçues. Ces différences ont été décrites par des termes de texture corrélés à des mesures instrumentales de texture.Ce travail a permis de mettre en avant les mécanismes physico chimiques déterminants dans la mise en place de la structure ainsi que leur potentiel impact sur les propriétés sensorielles de produits de type crèmes desserts. Ces connaissances pourraient être mises à profit dans des démarches de conception raisonnée ou de rétro ingénierie pour formuler de nouveaux produits. / Neutral dairy desserts are composed of a large diversity of products of similar composition but different structures and textures. Their structure set up is defined by the interactions between three of their quantitatively minor components: starch, milk proteins and carrageenan. The objective of this PhD project was to better understand the chemical-physics mechanisms beyond the neutral dairy desserts structure set up and their impact on the sensory perception of the product.The study focused on single, binary and ternary mixtures containing starch. It was performed with a modified waxy maize starch, reconstituted skim milk and kappa carrageenan mostly. Using rheological and microscopic tools, different interactions were highlighted. In binary mixtures, carrageenan was found to adsorb on the starch granules. A specific study on those interactions pointed out that they depended on carrageenans' charge density and molecular weights. In ternary mixtures, preferential carrageenan / casein micelles interactions in comparison to starch / carrageenan ones were pointed out even when milk proteins were added after starch pasting in a carrageenan solution. The addition of milk before or after starch pasting led to products of same formula but different microstructures. Starch granules were in both cases embedded in a carrageenan / casein micelles network still, the starch granules states or characteristics and the network formed differed. Dispersed starch and carrageenan / milk phases were defined as the key structure parameters of neutral dairy desserts. To assess their impact on sensorial perception, nine products of same formula but built differently were produced. The evaluation was done by subjects by means of a free sorting task followed by a ranking task on free discriminating terms. Sensory differences were found between the products. These differences were characterized and correlated with instrumental attributes.This work permitted to collect numerous data on chemical-physics properties, link them to the structure set up and to their potential impact on sensory properties of dairy creams. All this knowledge would be easily used to improve tailor food development and particularly to formulate new dairy cream texture through a reverse engineering approach.

Le séchage par atomisation est un procédé relativement bien maîtrisé, certains aspects de la transition goutte-particule n’étant pas encore totalement compris. Ainsi, comprendre précisément comment la particule est formée et comment ce phénomène peut être contrôlé reste aujourd’hui un défi majeur. Ce projet visait à découpler la complexité du phénomène de séchage via une approche multi-échelles. La formation de particules à partir de protéines laitières (protéines de lactosérum et micelles de caséines) a été étudiée avec différents systèmes expérimentaux (gouttes suspendue, confinée, mono-dispersées et enfin pulvérisées) dans des environnements de séchage contrôlés (température de séchage: 20°C à 190°C et l'humidité relative: 40% à 2%).Les résultats obtenus montrent que le séchage d'une goutte de protéine comprend trois étapes distinctes, mises en évidence par l’apparition d'événements morphologiques spécifiques (rétrécissement à vitesse établie, flambage, formation de vacuole). Selon le type de protéines, ces étapes diffèrent en termes de cinétique de séchage et de dynamique structurale, conduisant à des formes de grains caractéristiques. Ces différents comportements peuvent être rattachés aux conditions particulières de la formation d’une peau en surface et aux modes de dissipation des contraintes internes par les matériaux protéiques. De manière générale, l’approche multi-échelles de ce travail a permis de mettre en évidence la signature particulière de protéines laitières dans un état concentré et l'impact de la matière dans le processus de séchage. / Spray drying is a well-established process but certain aspects of droplet-particle transition are not yet fully understood, resulting in variability in terms of powder quality and performance. Therefore, understanding precisely how the particle is formed and how it can be controlled still remain a major challenge. This PhD project aims to break down the complexity of the drying phenomenon using an exploratory multi-scale approach. Particle formation of milk proteins (whey proteins and casein micelles) was investigated using different experimental systems (single pendant droplet, confined droplet, mono-dispersed droplets and spraying cone droplets) in controlled drying environments (drying temperature: 20°C to 190°C and relative humidity: 40% to 2%).The results showed that the drying of a single protein droplet included three distinct stages highlighted with the occurrence of specific morphological events (constant rate shrinkage, buckling instability, vacuole nucleation). According to the type of proteins, these drying stages differed in drying kinetics and droplet dynamics, leading to characteristic and reproducible particle shapes whatever the droplet configuration and the drying conditions. These different kinds of drying behaviour were related to specific skin formation conditions and different responses of the protein material to internal stress. Finally, by means of this multi-scale approach, this work highlighted the particular signature of milk proteins in a concentrated state and in general the impact of the matter in the droplet drying process.