Atualmente, a demanda por alimentos com apelo funcional tem se tornado cada vez mais recorrente dentre os consumidores devido a uma crescente busca por hábitos de vida mais saudáveis. Sendo assim, o desenvolvimento de técnicas que possibilitem uma adição mais efetiva de ingredientes funcionais em matrizes alimentícias se torna uma necessidade. Essas técnicas devem possibilitar principalmente (i) a incorporação de mecanismos de liberação sustentada na formulação; (ii) o aumento da bioacessibilidade e biodisponibilidade aos ingredientes, a partir do controle da microestrutura do alimento. Esse projeto visa contemplar essas duas premissas, ao propor a encapsulação de dois bioativos hidrofóbicos, a curcumina e a vitamina D3, conhecidos pelas suas propriedades antioxidantes e nutracêuticas, em carreadores de origem lipídica, os lipossomas, estabilizando-os com diferentes hidrocoloides - goma xantana, goma guar e inulina. Os lipossomas foram produzidos por hidratação de prolipossomas e suas propriedades físico-químicas foram caracterizadas ao longo de 42 dias de armazenagem, a partir de análises de diâmetro médio hidrodinâmico, potencial zeta, colorimetria instrumental e quantificação de bioativos encapsulados. Análises que permitiram a caracterização da microestrutura das dispersões produzidas também foram realizadas, sendo elas: calorimetria diferencial de varredura (DSC), espalhamento de raios-X a baixos ângulos (SAXS) e ensaios reológicos. As análises de SAXS mostraram que lipossomas produzidos na presença de curcumina são mais estáveis que àqueles produzidos na ausência da mesma e que não houve mudança na estrutura da bicamada lipídica das vesículas após a adição de vitamina D3, mesmo quando uma alta concentração foi incorporada ao sistema (80.000 UI). Por fim, verificou-se que a coencapsulação foi possível em lipossomas multilamelares estabilizados apenas com gomas guar e xantana, resultado que pode ser comprovado pelo alto teor de retenção dos bioativos ao longo do tempo de armazenagem. / Currently, the demand for food with functional appeal has become increasingly recurrent among the consumers due to a growing search for healthier living habits. Therefore, the development of techniques that allow a more effective addition of functional ingredients in food matrices becomes a necessity. These techniques should mainly enable to (i) incorporate a sustained release mechanisms into the formulation; (ii) increase the bioaccessibility and bioavailability to these ingredients, from the control of the food microstructure. This project aims to contemplate these two premises by proposing the encapsulation of two hydrophobic bioactives, curcumin and vitamin D3, known for their antioxidant and nutraceutical properties, in liposomes - lipid carriers - stabilizing them with different hydrocolloids - xanthan gum, guar gum and inulin. Liposomes were produced by proliposomes hydration and their physicochemical properties were characterized during 42 days of storage, including analyzes of hydrodynamic average diameter, zeta potential, instrumental colorimetry and quantification of encapsulated bioactives. Analyzes that allowed the microstructure characterization of the produced dispersions were also performed, including: differential scanning calorimetry (DSC), small-angle X-ray scattering and rheological tests. The SAXS analysis showed that liposomes produced in the presence of curcumin were more stable when compared to the empty ones and that there was no change in the lipid bilayer of the vesicles after the addition of vitamin D3, even when a high concentration was incorporated into the system (80,000 IU). Finally, it was concluded that the coencapsulation was possible in multilamellar liposomes stabilized with guar and xanthan gums, a result that can be evidenced by the high content of bioactives retained throughout the storage time.

Neste trabalho foi investigado o efeito da administração crônica de prolina sobre o conteúdo total de gangliosídios, fosfolipídios e de colesterol, assim como, sobre o perfil de gangliosídios no córtex, no hipocampo, no hipotálamo e no cerebelo de ratos. Também, foi avaliado o conteúdo e o perfil de gangliosídios nas frações solúvel e resistente a detergente obtidas de membranas sinápticas de córtex. Ratos Wistar foram divididos em dois grupos: 1) injetados subcutaneamente com solução 0,9% de NaCl (animais controle) e 2) injetados subcutaneamente com solução de prolina, em concentrações adequadas ao peso corporal (animais hiperprolinêmicos). Tanto a solução de prolina quanto a salina foram administradas do 6° ao 28° dia pós-natal. Doze horas após a última administração, os animais foram sacrificados mediante decapitação sem anestesia. As estruturas cerebrais foram dissecadas e em seguida homogeneizadas em clorofórmio:metanol na proporção 1:1 vpara a extração lipídica. As membranas sinápticas foram obtidas através de centrifugação diferencial e as frações solúvel e resistente a detergente foram isoladas através de tratamento das membranas com Triton X-100 a 4°C para investigação de microdomínios de membrana. Após a realização das análises, os resultados mostraram que os animais submetidos ao tratamento crônico com prolina apresentaram um marcado aumento no conteúdo de gangliosídios no córtex cerebral e no hipocampo, enquanto os conteúdos de fosfolipídios e de colesterol aumentaram somente no hipocampo. Além disso, os conteúdos destes compostos não foram alterados no hipotálamo e no cerebelo de animais hiperprolinêmicos. Por outro lado, o conteúdo de gangliosídios diminuiu nas frações solúvel e resistente a detergente obtidas de membranas sinápticas de córtex de animais hiperprolinêmicos. Embora os perfis de gangliosídios não tenham sido aparentemente modificados, as quantidades absolutas das espécies foram alteradas tanto no extrato total, como nos microdomínios de membrana obtidos do córtex. Estes dados revelam que o tratamento crônico com prolina afeta de forma distinta as diferentes regiões cerebrais quanto à composição lipídica das membranas celulares, refletindo-se sobre a distribuição de lipídios nos microdomínios de membrana do córtex. Entre as conseqüências destes fenômenos poderiam ser sugeridas modulações diferentes nas transmissões sinápticas que contribuiriam para o déficit cognitivo e/ou outras disfunções neurológicas presentes em pacientes com hiperprolinemia tipo II. / In the present work we investigated the effects of chronic proline administration on ganglioside, cholesterol and phospholipid total contents, as well as on ganglioside profile in cerebral cortex, hippocampus, hypothalamus and cerebellum of rats. We also evaluated the ganglioside content and profile in detergent- soluble and resistant fractions isolated from synaptic membranes obtained from cerebral cortex. Wistar rats were divided into two groups: 1) saline (control) and 2) proline injected (hyperprolinemic). Proline solution or saline were administered from 6th to 28th postnatal day, according to body weight. Twelve hours after the last injection, the animals were sacrificed by decapitation without anesthesia. Brain structures were homogenized with chlorophorm:methanol 1:1 for lipid extraction. Synaptic membrane was extracted by differential centrifugation and detergent- soluble and resistant fractions were isolated by cold Triton X-100 treatment. Results showed that rats subjected to chronic proline treatment presented a significant increase of ganglioside content on cortex and hippocampus, while phospholipid and cholesterol contents only increased in hippocampus. However, the content of these components were not altered in hypothalamus and cerebellum of hyperprolinemic rats. On the other hand, ganglioside content decreased in detergent- soluble and resistant fractions isolated from synaptic membrane obtained from hyperprolinemic cortex. Although ganglioside profiles were apparently not modified, the individual absolute quantities were altered in cortex total lipid extract and membrane microdomains obtained from cerebral cortex. Our findings suggest that chronic proline treatment affects, in a distinct manner, different cerebral regions concerning the lipid composition of the cell membranes, reflecting on its distribution in the cortex membrane microdomains. Among these phenomena consequences, different modulations in synaptic transmission may be suggested which may contribute to the impairment in cognition and/or other neurological disfunctions found in hyperprolinemia type II patients.

Orientadores: Munir Salomão Skaf, Monica Andrea Pickholz / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-14T17:06:24Z (GMT). No. of bitstreams: 1 Prates_EricaTeixeira_M.pdf: 4627606 bytes, checksum: 4caf846b01f9586ee62d25bee7d6bdc4 (MD5) Previous issue date: 2009 / Resumo: Neste trabalho foi feito o estudo das interações da articaína, um anestésico local de ampla aplicação médico-odontológica, com membranas modelo de POPC (palmitoil-oleil-fosfatidilcolina) em condições próximas às biologicamente relevan- tes empregando-se simulações computacionais de dinâmica molecular. Em uma primeira etapa, empregamos métodos quânticos para modelar a articaína com base no campo de força CHARMM. Das simulações de equilíbrio da articaína em POPC, foi possível obter informações como o seu comportamento conformacional e sua posi- ção transversal na bicamada, assim como suas interações especícas com os lipídios. Os estudos foram realizados para os estados neutro e protonado da articaína, consi- derando também seus isômeros ópticos. Estas análises, em conjunto com resultados experimentais de H-RMN realizados pela Prof. Eneida de Paula (IB-UNICAMP) e pelo Prof. Leonardo F. Fraceto (Dpto. de Eng. Ambiental - UNESP, Sorocaba - SP), demonstram que a articaína, em sua forma neutra, posiciona-se preferencial- mente na interface membrana/água, onde interage frequentemente com os lipídios através de ligações de hidrogênio. Através de ferramentas como perfil de densidade eletrônica do sistema, da parte teórica, e perfil do tempo de relaxação longitudinal para diferentes regiões dos lipídios, da parte experimental, foi discutida a lipossolubilidade da articaína em relação a outros anestésicos. Também foram realizadas simulações de não equilíbrio, utilizando a técnica de Dinâmica Molecular de Caminho Induzido, em que uma molécula de articaína foi removida do interior da membrana para o meio aquoso, através de uma força aplicada em seu centro de massa. Com a aplicação da igualdade de Jarzynski a estas simulações, foi possível estimar a energia livre de partição da ATC neutra (forma mais potente) entre os estados em que encontra-se no seio aquoso e no interior da membrana POPC. / Abstract: We studied the interactions of articaine - a local anesthetic widely used for me- dical and odontological applications - with model membranes of POPC (palmitoyl-oleyl-phosphatidylcholine) at biological relevant conditions. We have employed molecular dynamics technique, which allowed us to investigate the system at molecular level. Firstly, we applied quantum mechanical methods to parametrize articaine molecule based on CHARMM force field. We have done extensive molceular dynamics simulations, taking into account the different ionization states of the drug (neutral and protonated) as well as its optical isomers. From the equilibirum simulations of articaine in POPC membranes, we investigated the conformational behaviour of the drug, its tranversal position and its specific interactions with the lipids and water molecules. Our results show a preferential orientation of the articaine molecule within the membrane. Neutral articaine was mainly found at the lipid head/water interface, in very good agreement with H-RMN experimental results from Prof. Eneida de Paula (IB-UNICAMP) and Prof. Leonardo F. Fraceto (Dpto. de Eng. Ambiental - UNESP, Sorocaba - SP) and from literature (C. Song et al, 2008). By studying properties like electronic density prole and longitudi- nal time relaxation for different regions of the lipid molecules, we discussed the lipossolubility of articaine in comparison to other local anesthetics. We have also performed non-equilibrium simulations, using steered molecular dynamics (SMD) technique. A single articaine molecule was extracted from the membrane to the wa- ter phase, by applying an external force in its mass centre. Coupling the Jarzynski identity to the SMD simulations, we estimated the partition free energy of the neutral drug (the most potent specie) in POPC membranes. / Mestrado / Físico-Química / Mestre em Química

O interesse em criopreservar tecido ovariano, embriões e oócitos, principalmente quando se trata de pacientes oncológicas que irão ser submetidas a tratamentos potencialmente esterilizantes, vem crescendo nas últimas duas décadas. Uma das técnicas propostas para se preservar a fertilidade destas pacientes é o congelamento de oócitos, podendo estes ser obtidos já maturados in vivo após a hiperestimulação ovariana controlada ou na forma de oócitos imaturos na ausência de estimulação, nestes casos procede-se a maturação in vitro (MIV) de oócitos pré congelamento. No entanto sabe-se que a criopreservação causa danos de viabilidade e perda do potencial reprodutivo destes oócitos. Alguns autores têm demonstrado que esses danos podem ser reduzidos por meio de cultivos que modulam o perfil lipídico tanto de oócitos como embriões, fazendo com que estes sejam menos susceptíveis ao congelamento. Uma das técnicas que permite a verificação da composição lipídica de células e outras estruturas é a espectrometria de massas. Os objetivos deste estudo foram comparar o perfil lipídico de oócitos maturados in vitro na presença ou ausência de PL e correlacionar com o perfil lipídico e desenvolvimento embrionário dos embriões produzidos in vitro. Além disso, avaliamos o perfil lipídico dos meios de maturação usando oócitos bovinos como modelo experimental. CCOs foram maturados em meio TCM ou TCM + PL, contendo 10% de soro fetal bovino, 0,5 µg/ml de FSH, 5 ng/ml de LH e 1 mg/mL de 17?-estradiol em atmosfera úmida, com 5% de CO2 durante 24 horas. Após a MIV, os oócitos foram desnudados mecanicamente, lavados em PBS e armazenados a -80 ° C, até a análise de perfil lipídico. Oócitos, meios de maturação e blastocistos foram submetidos à técnica de MALDI-MS (ionização e dessorção a laser assistida por matriz /espectrometria de massas). Diferenças no perfil lipídico foram identificadas por PCA (análise de componentes principais). O perfil lipídico dos meios de maturação determinado por MALDI-MS permitiu a diferenciação entre TCM e TCM + PL. No entanto, a análise dos oócitos maturados in vitro demonstrou que o perfil lipídico dos grupos controle ou suplementado com PL não foram diferentes. Da mesma forma, não foram observadas diferenças no perfil lipídico e na embriogênese dos embriões resultantes. No entanto, diferenças no perfil lipídico entre COC e oócitos desnudos (ODs) maturados in vitro foram detectadas. Oócitos maturados com as células do cumulus contêm íons PC com maiores graus de insaturação dos resíduos de ácidos graxos, enquanto ODs contêm espécies de PC com ácidos graxos insaturados (18:0) ou monoinsaturados (18:1). O MALDI-MS permite a obtenção de perfis lipídicos informativos para meios de cultura e oócitos maturados in vitro. A identificação de mudanças no metabolismo lipídico de oócitos durante a MIV pode contribuir para determinar a suplementação lipídica adequada dos meios de MIV e soluções de vitrificação, contribuindo para otimizar os protocolos de criopreservação de oócitos humanos. / The interest in ovarian tissue, embryos and oocytes cryopreservation has been growing in the last two decades, especially in patients who are faced with potentially sterilizing treatments. One of the techniques proposed to preserve the fertility of these patients is the oocyte cryopreservation. The oocytes can be obtained matured in vivo after controlled ovarian hyperstimulation or as immature oocytes, in the absence of stimulation. In these cases, an option is to proceed the in vitro maturation (IVM) of oocytes before cryopreservation. However, it is known that damage induced by cryopreservation is associated with loss of viability and reproductive potential of oocytes. Some authors have demonstrated that such damage can be reduced by culture media that modulate lipid profile in both oocytes and embryos, making them less susceptible to freezing. One technique that enables the determination of the lipid composition of cells and other structures is mass spectrometry. The objectives of this study were to compare lipid profiles of oocytes matured in vitro in the presence or absence of PL and relate this information to lipid profile and preimplantational development of IVM-derived embryos. Also, we evaluated the lipid profiles of culture media using bovine oocytes as an experimental model. COCs were matured in TCM or TCM + PL, both containing 10% fetal calf serum, 0,5µg/ml FSH, 5 µg/mL LH, and 1 µg/mL 17?-estradiol, with 5% CO2 for 24 h. After IVM, oocytes were mechanically denuded, washed in PBS and stored at -80°C, until lipid analysis. Oocytes, maturation media and blastocysts were submitted to the MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry). Differences in lipid profile were addressed by principal component analysis. Maturation media lipid fingerprint by MALDI-MS allows differentiation among TCM and TCM+PL. However, the MALDI-MS of the in vitro matured oocytes demonstrated that lipid profile of control or PL-supplemented groups were not different. Similarly, no differences were observed in the lipid profile and embryogenesis of resulting embryos. Nevertheless, differences in lipid profiles between COCs and denuded oocytes (DOs) matured in vitro were indicated to occur. The former contain PC ions with higher degrees of unsaturation in the fatty acid residues, while DOs contain PC ions with unsaturated (18:0) or monoenoic (18:1) fatty acids. The MALDI-MS has allowed obtaining informative lipid profiles for culture media and IVM-oocytes. Identification of lipid changes during IVM may contribute to determine appropriate lipid supplementation of IVM/vitrification media and to improve cryopreservation of human oocytes.

O chá verde (Camellia sinensis) é reconhecido mundialmente por seu alto teor de polifenóis, em especial as catequinas. As catequinas estão relacionadas à prevenção de várias doenças degenerativas, como o câncer e diabetes, devido ao grande potencial antioxidante. Contudo, vários incovenientes precisam ser superados para aprimorar o uso destes produtos, principalmente em relação à sua biodisponibilidade. O desenvolvimento de carreadores lipídicos na encapsulação de compostos bioativos é uma tecnologia recente capaz de resolver vários problemas de biodisponibilidade apresentadas pelos produtos naturais, produzindo uma estrutura capaz de proteger os compostos ativos. O objetivo deste trabalho foi o desenvolvimento de uma formulação utilizando carreador lipídico, empregando métodos de secagem para aumentar a estabilidade dos compostos bioativos, enfatizando-se processos de preparação, tipo de excipientes e procedimentos para a caracterização físico-química, estabilidade e avaliação da atividade antioxidante in vitro do produto final. Os compostos bioativos foram extraídos a partir das folhas secas e moídas de chá-verde através do processo de maceração dinâmica, sendo filtrado, concentrado e liofilizado. A formulação foi desenvolvida utilizando o sistema de balanço hidrófilo-lipófilo (EHL) com tensoativos nãoiônicos e co-solvente, e caracterizados pelas análises organolépticas, centrifugação, reologia, microscopia óptica, distribuição de tamanho e potencial zeta. Para a etapa de secagem, selecionou-se a formulação mais estável. As formulações foram secas em spray dryer de escala laboratorial a uma vazão de 4,0 g/min, e temperaturas de 100, 120 e 150 °C. O desempenho de secagem foi avaliado pela recuperação do produto. Os pós obtidos por spray drying foram caracterizados quanto ao teor de umidade, atividade da água, densidade, distribuição granulométrica, propriedades de fluxo, cristalinidade, morfologia e redispersibilidade. Após, se avaliou a atividade antioxidante do produto em óleo vegetal (óleo de soja) utilizando o teste acelerado de estabilidade oxidativa em Rancimat®. O EHL, o tipo de tensoativo e a técnica de preparo da formulação influenciaram diretamente na estabilidade do sistema. Para as formulações estudadas, o tensoativo que conferiu uma maior estabilidade foi o Gelucire® 44/14, sendo selecionado para prosseguimento com o processo de secagem das composições. O rendimento do processo atingiu em média 51,3 ±3,5 %, típico para secadores do tipo spray dryer em escala laboratorial. A maioria das partículas apresentou aparência arredondada, sem presença visível de porosidade, independente do adjuvante (lactose ou trealose). O produto apresentou baixa atividade de água (<= 0,22) e teor de umidade (<= 1,79). O aumento da temperatura de secagem provocou um ligeiro aumento no diâmetro médio de partícula quando a lactose foi utilizada como adjuvante de secagem (9,8 ± 5,9 ?m para 13,65 ± 8,4 ?m). Os pós obtidos tiveram baixa densidade, sendo menor em temperaturas mais altas. O índice de Carr e Fator de Hausner (15 % e < 1,25, respectivamente) indicaram boas propriedades de compressão e fluidez. O produto foi prontamente redispersível, recuperando facilmente sua consistência e características originais. A avaliação da estabilidade oxidativa acelerada utilizando Rancimat demonstrou que o processo de encapsulação conferiu uma maior solubilidade e proteção dos compostos bioativos, levando ao aumento da atividade antioxidante. Os resultados aqui relatados confirmam a viabilidade da encapsulação dos compostos bioativos de Camellia sinensis em carreadores lipídios empregando o processo de spray drying. O produto apresenta potencial de aplicação como matéria-prima para a produção de formas orais, inclusão em produtos nutracêuticos e cosméticos / Green tea, a product made from Camellia sinensis leaves, is recognized worldwide by its high polyphenol content, in special the catechins. Tea catechins are linked to the prevention of several degenerative diseases as cancer and diabetes. However, several drawbacks need to be overcome in order to increase the use of this products, being their bioavailability one of the upmost. The development of carrier lipid based containing bioproducts is a recent technology, which can solve several bioavailability problems presented by natural products, producing a structure which confer protection to active compounds. The aim of this work was the development of carrier lipid based compositions containing bioactive compounds of Camellia sinensis (green tea) by spray drying evaluating processes of preparation, type of excipients and characterization of physicochemical properties and evaluation the antioxidant activity in vitro of the product. Bioactive compounds from dried and milled green tea leaves was extracted by dynamic maceration, filtered, concentrated and freeze-dried. The formulation was developed through the utilization of Hydrophilic Lipophilic Balance System (HLB) using the non-ionic surfactants and a co-solvent and characterized by organoleptic analysis, centrifugation, rheology, optical microscopy, size distribution and zeta potential. The most stable compositions were submitted to spray drying. The compositions were dried in a labscale spray dryer at flow rate of 4.0 g/min at temperatures of 100, 120 and 150 °C. The spray drying performance was characterized by determination of the powder production yield. Spray dried powders were characterized by moisture content, water activity, density, size distribution, flow properties, crystallinity, morphology, and redispersibility. After, it was evaluated the antioxidant activity of the product in vegetable oil (soybean oil) using the accelerated oxidative stability test Rancimat®. The HLB, the type of surfactant and the preparation method of the formulation influenced the system stability. For the formulations studied, the surfactant which confers the greater stability was Gelucire® 44/14, which was selected to prepare composition for spray drying. The powder production yield falls around 51.3 ±3.5 %, typical for lab scale spray dryers. Wrinkled and rounded particles, without visible presence porosities were mostly generated, independent of the adjuvant (trehalose or lactose). The product presented low water activity (<= 0.20) and low moisture content (<= 1.79). Increasing in drying temperature caused a slight increase in mean particle diameter, when lactose was used as drying carrier (9.8 ±5.9 ?m to 13.65 ±8.4 ?m). Low density powders were generated, but density tends to be lower at high drying temperatures composition were submitted to spray drying. Carr index and Hausner ratio of the product (< 15% and < 1.25, respectively) were indicative of good compressive and flow properties. The product was promptly redispersible, regaining its original consistency straight forwardly. The accelerated oxidative stability using the Rancimat demonstrated that the encapsulation increased the solubility and protection of bioactive compounds, resulting to increased antioxidant activity of the product. The results here reported confirm the feasibility of entrapment of herbal bioactive compounds of Camellia sinensis in lipid carrier by spray drying. The product has potential to be used as raw material for production of oral dosage forms, inclusion in nutraceutical and cosmetic products.

Master of Science / Food Science Institute / Jayendra K. Amamcharla / In recent years, the interest in functional components such as phospholipids (PLs) is increasing as a result of growing awareness of their health benefits. PLs affect several cell functions, such as growth, molecular transport system, memory processing, stress responses, and central nervous system myelination. Many studies have shown that the neutral lipids can be successfully extracted using supercritical carbon dioxide (SCO₂) from different types of foods such as egg, canola, pumpkin seed, fish and dairy powders. It is an alternative method to avoid the use of large quantities organic solvents. The SCO₂ is a safe, environmentally friendly and economical process to extract edible lipids from a variety of matrices. However, a modifier such as ethanol is needed to fractionate PLs due to limited solubility of PLs in SCO₂. The objectives of this study were to optimize the SFE process parameters and to determine the effect of pressure, temperature, and ethanol concentration on the extraction efficiency of PLs from whey protein phospholipid concentrate (WPPC) and buttermilk powder (BMP). Three different batches of WPPC and BMP were obtained from a commercial manufacturer and followed a unique two-step extraction process to isolate PLs from WPPC and BMP. In Step-1, neat supercritical CO₂ was used to remove all the neutral lipids at 414 bar pressure, 60 °C sample temperature, and 5 L/min CO₂ flow rate. The spent solids, the powder left after the first step extraction, were used to extract PLs in the second step. The Step-2 (SCO₂-Ethanol) process was optimized in terms of pressure (350, 414 and 550 bar), temperature (40 °C and 60 °C) and concentration of ethanol (10%, 15% and 20%) as independent factors. All the lipid fractions were analyzed by high performance lipid chromatography (HPLC) and thin layer chromatography (TLC). For WPPC, only ethanol concentration had significant effect (P < 0.05) on the amount of PLs extracted after the Step-2. On the other hand, temperature and ethanol concentration were significantly (P < 0.05) affected the efficiency of SFE for BMP. The optimal processing conditions for WPPC and BMP were 350 bar pressure, 60 °C sample temperature and 15% concentration of ethanol, and 550 bar of pressure, 60 °C sample temperature and 15% concentration of ethanol, respectively. This study allowed obtaining PLs from dairy co-products such as WPPC and BMP as a separate ingredient and this could be useful in nutraceutical and infant formulations as well as different food products formulations.

Abstract The Eustachian tube (ET) connects the upper respiratory tract and the middle ear. It equilibrates the pressure in the middle ear and prevents the harmful impact of the airways. Middle ear infection, or otitis media, is one of the most common illnesses in childhood and affects nearly all children at least once. ET dysfunction is considered to be a major pathogenic factor in the development of otitis media. Surfactant proteins A (SP-A) and D (SP-D) have an important role in the pulmonary host defence system, and interactions with bacteria that also cause middle ear infections raised a question about the expression of surfactant proteins in ET. The local defence system could be significant in preventing ear infections. The purpose of the study was to increase our basic knowledge of the putative ET surfactant system. ET and lung epithelia are both of endodermal origin, and ET epithelium with its mucociliary system resembles that of the lower airways. The aim was to determine whether SP-A, SP-B and SP-D are expressed in ET. The expressions were characterized with reverse transcriptase polymerase chain reaction (RT-PCR), Northern hybridizations and in situ hybridizations. The surfactant proteins were localized using immunohistochemistry and immunoelectron microscopy. The proteins were characterized with Western analyses, and the properties of the ET surfactant were evaluated using electrospray ionization mass spectrometry and a pulsating bubble surfactometer. SP-A, SP-B and SP-D were found to be expressed in porcine ET epithelium, and the cDNA sequences were homologous to those detected in lung tissue. The proteins were localized to specific ET epithelial cells, and their sizes were characteristic of lung SP-A, SP-B and SP-D. ET lavage fluid contained the surfactant proteins and phospholipid. However, the phospholipid molecular species and surface tension measurements showed the structure and function of the surfactant in ET to be different from lung surfactant, suggesting that a very low surface tension is not a critical determinant of ET surfactant function. As a conclusion, surfactant proteins in ET are likely to be involved in the local host defence system and may contribute to the mucociliary function of the tube.

No description available.

Les corps lipidiques (CLs) sont des organites de stockage de lipides neutres rencontrés dans des organismes très variés, depuis les procaryotes jusqu’aux organismes complexes (animaux, végétaux). La surface des CLs est constituée d’une monocouche de phospholipides (PLs) entourant un coeur hydrophobe dans lequel sont stockés les lipides neutres. La monocouche de PLs est associée plus ou moins étroitement avec des protéines structurales, capables de stabiliser les CLs et d’accompagner certaines de leurs modifications morphologiques. Dans les graines de plantes oléagineuses, les CLs sont stabilisés par les oléosines. Ces protéines contiennent le plus long domaine hydrophobe connu (70 résidus) situé entre deux extrémités N et C-terminales hydrophiles. Leur mode d’association avec les CLs n’est pas connu et la littérature fait état de résultats contradictoires concernant leur structure secondaire. Nous avons montré que les oléosines de graines d’Arabidopsis thaliana sont maintenues en solution par différentes catégories de surfactants, comme les détergents anioniques ou des polymères amphiphiles appelés amphipols (Apols). La détermination de la structure secondaire des oléosines maintenues en solution dans ces différents surfactants, par dichroïsme circulaire utilisant le rayonnement synchrotron, a mis en évidence des profils contrastés. Les détergents chargés augmentent le contenu en hélices α des oléosines alors que des proportions plus importantes de feuillets β sont observées avec le détergent zwitterionique (Foscholine-12) ou les Apols. Afin d’obtenir un profil structural modèle dans un système proche du naturel, nous avons réalisé une expression hétérologue d’une isoforme d’oléosine pour la cibler dans les CLs de Saccharomyces cerevisiae. Les CLs purifiés de levures restent intacts et contiennent une forte majorité de cette isoforme d’oléosine à leur surface. Nous avons été les premiers à montrer que les oléosines étaient repliées dans un tel environnement, avec un profil structural majoritairement β. Celui-ci se rapproche du profil observé en Foscholine-12. Ce détergent est par conséquent un outil de choix pour envisager des études structurales plus résolutives (structures tridimensionnelles). / Lipid Bodies (LBs) are neutral lipid storage organelles found in various organisms from procaryotic cells to complex organisms. These neutral lipids are packed into the core of the particle which is surrounded by a phospholipid monolayer. The surface of LBs is more or less tightly associated with structural proteins involved in their stabilization and able to assist modifications of their shape or size. In oleaginous seeds, LBs are stabilized by oleosins. These proteins contain the longest known hydrophobic domain (70 residues) flanked by hydrophilic N and C-termini. The way of association of these proteins with LBs is poorly known and secondary structure descriptions in the literature are contradictory. We have shown that Arabidopsis thaliana seed oleosins could be solubilized by various surfactants such as detergents or amphiphatic polymers called amphipols (Apols). Secondary structure determination of solubilized oleosins using synchrotron radiation circular dichroism gave contrasted profiles. Negatively charged detergents increase the α-helix content of oleosins whereas the zwitterionic detergent (Foscholine-12) or Apols allow higher proportions of β-sheets. In order to get closer to the natural environment of olesins, we have opted for the heterologous expression of one oleosin isoform in the yeast Saccharomyces cerevisiae. This approach allows the biological targeting and insertion of oleosins into cytosolic LBs. Purified yeast LBs remain intact and contain a large majority of oleosins at their surface. In this natural like environment, oleosins are folded and contain a majority of β-sheets. This secondary structure profile is close to that of oleosins solubilized by Foscholin-12, making it a suitable detergent for more resolutive structural studies (three-dimensional structures).

Ce travail est basé sur l'intuition que seul de nouveaux systèmes biomimétiques permettant un contrôle de la localisation spatiale des phénomènes d’oxydation peuvent conduire à une compréhension profonde de l’oxydation des lipides dans les cellules eucaryotes. Nous avons donc développé un nouveau type de molécule photosensible pouvant être ancré dans des Vésicules Géantes Unilamellaires (GUVs). Nous montrons dans cette étude que, pour le cas particulier de l’hydroperoxydation, contrôler la distribution spatiale permet une sélection des réactions d’oxydation, ce qui nécessite également une adaptation des stratégies de traitement antioxydant. En association avec de nouvelles techniques pour la quantification des événements d’oxydation, ces nouveaux modèles fournissent un scénario complet des mécanismes d’hydroperoxydation, de la production des espèces réactives (1O2) aux modifications physiques et chimiques induites dans les bicouches auto-assemblées. Nous montrons que les GUVs sont capables de survivre lorsque tous les lipides sont hydroperoxydés, confirmant que l’intégrité de la membrane est conservée dans ces conditions d’oxydation. Notre expérience permet de mesurer avec une bonne précision : l’augmentation d’aire produite sous hydroperoxydation, les modifications des propriétés mécaniques de la membrane, ainsi que l’efficacité d’hydroperoxydation. Pour une compréhension approfondie, les modifications moléculaires sous oxydation ont été étudiées à l’interface eau-air en utilisant des monocouches de lipides. / Our contribution to research in the area of lipid oxidation in eukariotic cells is based on the central intuition that progress can only be achieved in new biomimetic membrane systems where the spatial localization of the oxidation events might be controlled and monitored. Accordingly, we have developed new photosensitizer agents that can be anchored in Giant Unilamelar Vesicles (GUVs). It is important to stress that progress in the control of the spatial distribution of oxidation allows for a selection of the oxidation pathways, as we show in this study for the particular case of hydroperoxidation, and therefore constrains anti-oxidant strategies. In association with new tools for the quantification of the oxidation events, these new models have provided a complete scenario for the hydroperoxidation mechanisms, from the production of the oxidant species (1O2) to the final chemical and physical modifications induced on the self-assembled bilayers. We report that GUVsare able to survive full hydroperoxidation, showing that membrane integrity can be preserved under these oxidation conditions. Our experimental setup allows to measure the relative area increase produced upon peroxidation, the associated change in mechanical properties of the membrane and also the hydroperoxidation efficiency, all of them with good precisions. Further insights into the molecular modifications under oxidation have been studied at the air –water interface, using lipid monolayers.