A microencapsulação tem sido utilizada promissoramente para melhorar a viabilidade de probióticos. Porém, pouco se sabe sobre o impacto desta na manutenção da funcionalidade do probiótico in vivo. Este trabalho teve como objetivo avaliar duas cepas de lactobacilos, encapsular essas cepas por um sistema envolvendo partículas lipídicas recobertas por interação eletrostática de polímeros e avaliar o efeito da encapsulação na manutenção da capacidade imunomoduladora das cepas. Na primeira etapa do estudo L. rhamnosus 64 e L. paracasei BGP1 foram avaliados quanto à resistência à lisozima e aos fluidos gastrointestinais simulados, perfil de hidrofobicidade da parede celular, susceptibilidade a antibióticos, atividade antagonista contra patógenos e capacidade de utilização de prebióticos. Em etapa seguinte as cepas foram encapsuladas e as microcápsulas avaliadas quanto à morfologia, tamanho e distribuição de partículas, umidade, atividade de água e efeito do pH e temperatura em sua estabilidade. Para avaliar a susceptibilidade dos microrganismos ao processo de encapsulação e estresse tecnológico, investigou-se o impacto do efeito da homogeneização com Ultra-Turrax, tolerância à temperatura, salinidade, diferentes pH e fluidos gastrointestinais simulados na viabilidade das bactérias. A viabilidade dos microrganismos durante a estocagem também foi estudada. Por fim, avaliou-se a manutenção da capacidade imunomoduladora dos microrganismos microencapsulados por meio da dosagem de citocinas pró e anti-inflamatórias e determinação da capacidade protetora contra infecção de Salmonella entérica sorovar Typhimurium em modelo animal. Os microrganismos demonstraram resistência à lisozima, com taxas de sobrevivência superiores a 80%. O perfil de hidrofobicidade da parede celular, foi baixo, entre 8,47 e 19,19%. Demonstraram resistência apenas à vancomicina (35 µg) e eritromicina (15 µg). As duas cepas foram capazes de antagonizar o crescimento de Escherichia coli V517, Salmonella enteritidis OMS-Ca, Staphylococcus aureus 76 e Listeria monocytogenes ATCC 15313. Quanto à capacidade de utilização de prebióticos, os microrganismos apresentaram comportamentos inversos, utilizaram preferencialmente inulina, raftilose 95 e lactulose. Nos testes de resistência aos fluidos gastrointestinais simulados constatou-se declínio significativo de células viáveis, com subtração de até 3,37 log UFC/mL. As cápsulas obtidas apresentaram formato típico e tamanhos médios de 80,12 ± 1,89 e 83,92 ± 1,70 µm. Condições de pH extremos (1,5 e 9,0) e temperatura superior a 50 °C comprometeram a estabilidade das cápsulas. A encapsulação melhorou significativamente a tolerância dos microrganismos à altas concentrações de sal e elevação de temperatura. Além disso, favoreceu a resistência dos microrganismos frente aos fluidos gastrointestinais simulados. A estabilidade dos microrganismos durante o período de estocagem também foi favorecida, após 120 dias de estocagem a 7 e 25 °C a concentração de microrganismos viáveis permaneceu superior a 7,0 log UFC/g. Nos testes in vivo para avaliação da manutenção da capacidade de imunomodulação constatou-se através de dosagem de citocinas (IL-2, IL-6, IL-10 e TNF-α) e IgA secretora, que a encapsulação não alterou a resposta imunológica provocada pelas cepas estudas. Concluiu-se que os microrganismos apresentaram comportamento in vitro de acordo com o desejado para candidatos ao uso de probióticos. A microencapsulação foi bem-sucedida, proporcionando as duas cepas maior resistência frente às condições adversas e de estresse tecnológico. / Microencapsulation has been used successfully to improve the viability of probiotics microorganisms. The aim of this work was to evaluate two strains of lactobacilli, to encapsulate these strains by a system involving lipid particles coated by electrostatic interaction of polymers and to evaluate the effect of encapsulation in the maintenance of immunomodulatory capacity of these strains. In the first stage of the study L. rhamnosus 64 and L. paracasei BGP1 were evaluated for resistance to lysozyme and simulated gastrointestinal fluids, cell wall hydrophobicity profile, susceptibility to antibiotics, antagonist activity against pathogens and prebiotic utilization capacity. In the next step, the strains were encapsulated and the microcapsules evaluated regarding morphology, particle size and distribution, moisture, water activity and pH and temperature. The tolerance to temperature, salinity, different pH and simulated gastrointestinal fluids in the viability of the bacteria were also evaluated. The probiotics viability during the storage period was also studied. Finally, the maintenance of the immunomodulatory capacity of the encapsulated microorganisms was evaluated by means of the dosage of pro and anti-inflammatory cytokines and IgA. L. rhamnosus 64 and L. paracasei BGP1 demonstrated resistance to lysozyme, with survival rates above 80%. The hydrophobicity profiles of the cell wall were from 8.47 to 19.19%. Susceptibility to antibiotics also corroborated the literature, demonstrating resistance only to vancomycin (35 µg) and erythromycin (15 µg). The two strains were able to antagonize the growth of Escherichia coli V517, Salmonella enteritidis OMS-Ca, Staphylococcus aureus 76 and Listeria monocytogenes ATCC 15313. As far as the capacity of using prebiotics the two strains of lactobacilli presented inverse behaviors, they used preferably inulin, raftilose 95 and lactulose. In the tests of resistance to the simulated gastrointestinal fluids it was verified a significant decline of viable cells, with subtraction of up to 3.37 log CFU / mL, justifying the application of encapsulation technology. In the encapsulation step the capsules were produced with gum arabic, porcine gelatin and vegetable fat. The obtained capsules presented a typical format and average sizes of 80.12 ± 1.89 and 83.92 ± 1.70 µm. Extreme pH conditions (1.5 and 9.0) and temperature above 50 ° C compromised the stability of the capsules. The encapsulation significantly improved the tolerance of microorganisms to high salt concentrations and elevation of temperature. In addition, it favored the resistance of the microorganisms to the simulated gastrointestinal fluids. The stability of the microorganisms during the storage period was also favored, after 120 days of storage at 7 and 25 ° C the concentration of viable microorganisms remained higher than 7.0 log CFU / g. In the in vivo tests for evaluation of the maintenance of the immunomodulation capacity, the cytokines (IL-2, IL-6, IL-10 and TNF-α) and secretory IgA were determined, that encapsulation did not alter the immunological response by the strains of lactobacilli studied. It was concluded that the microorganisms presented in vitro behavior in accordance with the one desired for probiotic candidates. Microencapsulation was successful, giving both strains greater resistance to adverse conditions and technological stress.

Polyelectrolytes of opposite charge in aqueous solution can undergo a liquid-liquid phase separation known as complex coacervation. Complex coacervation of ampholytic proteins with oppositely charged polyelectrolytes is of increasing interest as it results in a protein rich phase that has potential applications in food science, protein therapeutics, protein purification, and biocatalysis. However, many globular proteins do not phase separate when mixed with an oppositely charged polyelectrolyte, and those that do phase separate do so over narrow concentration, pH, and ionic strength ranges. Much of the work that has been done on complex coacervates looks at polymer-polymer systems. While there have been some initial studies showing that proteins can undergo complex coacervation, the major design factor studied to date has been overall protein charge. The tools of genetic engineering, which allow the precise tuning and placement of charge have not been used to more fully understand the design criteria for protein complex coacervation. In this dissertation, we developed a model protein library based on green fluorescent protein (GFP) to study the impact of protein net charge and charge distribution on protein phase separation with polyelectrolytes. We developed a short, ionic polypeptide sequence (6-18 amino acids) that can drive the liquid-liquid phase separation of globular proteins. We characterize the phase behavior of the protein library with a homopolymer and diblock copolymer of similar chemistry to elucidate how protein design impacts macro- and microphase separation. In these phase characterization studies, differences in the nature of phase separation as well as the salt stability of the protein coacervates with the different polymer species are identified. We finally used this model protein library to study the effects of the protein design and phase separation behavior for coacervate-based applications including intracellular protein delivery, purification, and protein stabilization.

Compartmentalization allows for the spatial organization of cellular components and is crucial for numerous biological functions. One recently uncovered strategy for intracellular compartmentalization is phase separation via the de-mixing of biomacromolecules. Membraneless organelles, also referred to as biomolecular condensates, are compartments formed by phase separation and create distinct environments that are essential to cellular processes ranging from cell signaling to gene expression. Biomolecular condensates offer several advantages – for example, dynamic restructuring of internal constituents and diffusion of cellular components into/out of compartments – that make them suitable for applications in biocatalysis or pharmaceutical production. However, the ability to independently engineer the formation and disassembly of condensates in vivo remains a challenge. Here, concepts from polymer science have been used to understand parameters that govern intracellular phase separation. Many biomolecular condensates exhibit physical properties that are similar to complex coacervates as both are liquid-like phase separated mixtures formed via associative phase separation, frequently with oppositely charged polyelectrolytes. We utilize the physical phenomenon of complex coacervation and principles underlying the formation of liquid-like biological condensates to identify design parameters for engineering synthetic, phase separated organelles in E. coli. In this dissertation, we employed a library of cationic charge variants derived from superfolder green fluorescent protein (sfGFP) to elucidate the effects of overall cationic charge on intracellular phase separation. We first investigated the complex coacervation of engineered proteins with biological polyelectrolytes to determine predictive design rules for protein phase separation and translated these design rules in vivo to engineer bacterial condensates. Characterization of the coacervate-like properties and macromolecular composition revealed that these condensates can undergo dynamic restructuring and exhibit biomolecular specificity. To facilitate the engineering of active supercharged proteins, we also developed short, cationic peptide tags, ranging from 6-27 amino acids in length, that can be appended onto any protein of interest to promote intracellular phase separation. We find that overall charge generally determines protein phase behavior and observe the formation and disassembly of condensates near the physiological phase boundary. Interestingly, we find that small modifications in charge density can tune the interaction strength between associating biomacromolecules and thus tune condensate stability. We demonstrate the use of these protein design parameters and cationic peptide tags to sequester catalytic enzymes and manipulate the intracellular localization of multiple proteins. These studies pave the way to building synthetic, functional organelles.

Flaxseed (Linum usitatissimum L.) gum (FG) is a material with many potential food and non-food applications. Consistent performance is critical for FG utilization and this is possible through selection of genotype, characterization and optimization of constituents, and chemical modification. Physico-chemical and functional properties of FG aqueous solutions from six Canadian flaxseed cultivars were investigated. FG yield, carbohydrate composition, protein content, and zeta potential (ζ) varied among these cultivars. FG solution properties were also affected by temperature, solution pH, NaCl concentration, and sucrose concentration. Detailed studies were conducted on CDC Bethune FG (FGB) proteins that were separated by 2D-gel electrophoresis. Conlinin was identified as the major protein. Protease treatment decreased FGB solution emulsification properties suggesting that conlinin might enhance emulsification. Formation of BSA-FGB coacervates was monitored by turbidimetric analysis as a function of solution pH, biopolymer mixing ratio, NaCl and urea. Coacervates were stabilized primarily by attractive electrostatic forces and secondarily by hydrogen bonds. Further, anionic carboxymethyl ether moieties were introduced to FGB structure through ether forming reactions using monochloroacetic acid (MCA) to produce products with uniform properties. The highest degree of substitution (DS) was obtained at 70 °C, 7.0 M NaOH, and a molar ratio of MCA to FGB of 10:1 over 3 h. Carboxymethylated FGB (CMFG) exhibited both modified surface morphology and thermal behaviour. Solutions of CMFG demonstrated shear-thinning behaviour and apparent viscosity decreased with increased DS. A more liquid-like flow behaviour was observed for CMFG as DS increased. Findings here will introduce and expand FG applications in food or related fields with targeted performance.

A nutrição é essencial na promoção da saúde da população para estimular o crescimento e desenvolvimento adequado, sendo necessário o planejamento de ações e programas em saúde pública. A falta ou consumo inadequado dos nutrientes pode desencadear distúrbios fisiológicos que levam a doenças. Com o objetivo de prevenir a anemia ferropriva devido à elevada incidência de deficiência de ferro, a Agência Nacional de Vigilância Sanitária do Brasil criou uma resolução para que as farinhas de trigo e milho sejam adicionadas de ferro (Fe) e ácido fólico. Na dieta é possível encontrar dois tipos de ferro, sendo o ferro-heme e o ferro não-heme. O ferro heme é bem absorvido pelo organismo e pouco influenciado pelas reservas de Fe ou outros constituintes da dieta. Já o ferro não-heme apresenta variado grau de absorção, dependente de outros componentes da dieta e das reservas de Fe dos indivíduos. Estudos que testam a bioacessibilidade dos nutrientes têm sido realizados para avaliar a quantidade de nutrientes que é liberada da matriz alimentar no processo de digestão e está disponível para ser absorvida pelo intestino, utilizando a simulação do processo de digestão gastrointestinal aliada as culturas de células Caco-2. Os hábitos saudáveis estimulam o consumo de frutas e vegetais, aumentando também a produção de subprodutos agroindustriais como cascas, sementes, caroço e bagaço que são ricos em vitaminas, minerais, fibras e compostos antioxidantes necessários para funções fisiológicas. O aproveitamento de subprodutos agroindustriais para a obtenção de nutrientes vem sendo aplicado na elaboração de diversos produtos alimentícios, como barras nutritivas e hambúrgueres enriquecidos. O ácido ascórbico pode ser encontrado em subprodutos agroindustriais, mas, por ser instável e muito suscetível a oxidação necessita da aplicação de técnicas como a microencapsulação e liofilização para a preservação do composto. Além disso, o ácido ascórbico quando ingerido juntamente com o ferro não-heme possibilita aumento na absorção deste mineral. O objetivo deste trabalho foi extrair ácido ascórbico de subprodutos agroindustriais, aplicar a tecnologia de microencapsulação (spray drying e emulsão dupla seguida da coacervação complexa) e liofilização a este extrato e adicioná-lo na farinha de trigo comercial, observando a bioacessibilidade do ferro presente na farinha no final deste processo. Primeiramente, foram obtidas farinhas de frutas a partir dos subprodutos de mamão e abacaxi liofilizados. Foi extraído o ácido ascórbico das farinhas das frutas, o qual foi quantificado por cromatografia líquida de alta eficiência (CLAE). O extrato de mamão foi liofilizado e microencapsulado, pelos métodos de spray drying e emulsão dupla seguida da coacervação complexa. Os tratamentos obtidos foram: micropartículas secas por atomização na proporção 1:1 (T1), micropartículas secas por atomização na proporção 1:2 (T2), micropartículas secas por atomização na proporção 1:3 (T3), micropartículas obtidas por emulsão dupla seguida da coacervação complexa com 50% de emulsão extrato líquido + óleo de milho em relação ao polímero (T4), micropartículas obtidas por emulsão dupla seguida da coacervação complexa com 75% de emulsão extrato líquido + óleo de milho em relação ao polímero (T5) e extrato da farinha de subprodutos de mamão liofilizado (T6). Foram realizadas análises de caracterização tratamentos: umidade, atividade de água, solubilidade, higroscopiciade, análises morfológicas, teor de ácido ascórbico, tamanho das partículas. Para avaliar a bioacessibilidade de ferro, realizou-se o processo de digestão das amostras com a farinha de trigo e o cultivo de células Caco-2, no final do processo o material digerido foi fornecido as células. A bioacessibilidade do ferro foi mensurada pela ferritina. Como resultados, o extrato da farinha de subprodutos de mamão apresentou maior concentração de ácido ascórbico (p<0,05) sendo selecionado para as próximas etapas (microencapsulação e teste in vitro). Os resultados para umidade (inferiores a 13%) e atividade de água (inferiores a 0,3) mostraram segurança para todos os tratamentos quanto ao crescimento microbiológico. Os tratamentos obtidos por spray drying mostraram-se mais solúveis quando comparados aos coacervados e ao extrato liofilizado (p<0,05). O extrato liofilizado de mamão apresentou a maior concentração de ácido ascórbico (p<0,05) e maior higroscopicidade (p<0,05) quando comparado aos outros tratamentos. Quanto a morfologia, os tratamentos apresentaram diferenças principalmente devido à técnica aplicada e aos materiais utilizados. Para a bioacessibilidade do mineral presente na farinha de trigo, o teor médio de ferritina formado variou entre 0,02 e 1,19 ng por mg de proteína. Não houve diferenças significativas positivas dos tratamentos realizados em relação a farinha de trigo comercial (p<0,05). Como conclusão, foi possível extrair o ácido ascórbico de subprodutos agroindustriais e aplicar a microencapsulação e liofilização a este extrato sem grandes perdas do composto para alguns tratamentos. Quanto aos ensaios de bioacessibilidade, pode-se concluir que enriquecer a farinha de trigo com ácido ascórbico nas condições e proporções utilizadas neste estudo não foram suficientes para aumentar a bioacessibilidade do ferro presente na farinha de trigo comercial. / Nutrition is essential for the promotion of the health of populations in order to encourage the adequate growth and development, turning necessary the planning of public health actions and programs. The lack or the inadequate consumption of nutrients can cause physiological disorders that leads to diseases. With the objective of preventing the anemia occurred due to the elevated incidence of iron deficiency, the Brazilian Health Regulatory Agency has created a resolution for the addition of iron and folic acid in wheat and corn flours. In the diet, it is possible to find two types of iron: heme iron and non-heme iron. The heme iron is well absorbed by the organism and is minimally influenced by the iron reserves and other components of the diet. On the other hand, the non-heme iron presents variable degrees of absorption being dependent of the other components of the diet and the iron reserves of the individuals. Studies that test the bioacessibility of nutrients have been conducted in order to evaluate the quantity of nutrients that are released from the food matrix in the digestive process and are available to be absorbed by the intestine, using simulations of gastrointestinal digestion processes allied to cultures of Caco-2 cells. The healthy habits encourage the consumption of fruits and vegetables, also increasing the production of fruit by-products, such as peels, seeds, cores, and bagasses which are rich in vitamins, minerals, fibers, and antioxidant compounds that are necessary for physiological functions. The exploitation of fruit by-products for the obtainment of nutrients have been applied in the elaboration of various food products, such as nutrition bars and enriched burgers. The ascorbic acid can be found in fruit by-products and because it is unstable and very susceptible to oxidation, it requires the application of techniques for the preservation of the compound, such as microencapsulation and lyophilization. Moreover, when the ascorbic acid is ingested together with the non-heme iron it allows an increase in the absorption of this mineral. The objective of this work was to extract ascorbic acid from fruit by-products, apply the microencapsulation (spray drying and double emulsion followed by complex coacervation) and lyophilization techniques to it, and add it to the commercial wheat flour, to observe the bioacessibility of the iron in the end of the process. First, flours were obtained from lyophilized papayas and pineapples by-products. The ascorbic acid was extracted from the flours and it was quantified by high performance liquid chromatography (HPLC). The papaya extract was microencapsulated by the methods of spray drying and complex coacervation, and lyophilized. The treatments obtained were: spray dried microparticles in the ratio 1:1 (T1), spray dried microparticles in the ratio 1:2 (T2), spray-dried microparticles in the ratio 1:3 (T3), microparticles obtained by double emulsion followed by complex coacervation with 50% emulsion liquid extract + corn oil in relation to the polymer (T4), microparticles obtained by double emulsion followed by complex coacervation with 75% emulsion liquid extract + corn oil in relation to the polymer (T5) and lyophilized extract of papaya by-products flour (T6). Characterization analyses of the treatments were performed: humidity, water activity, solubility, hygroscopicity, morphological analyses, ascorbic acid content and particle size. For the evaluation of the bioacessibility, the process of digestion of the treatments together with the wheat flour was performed by the cultivation of Caco-2 cellular tissues; at the end of the process the material digested was given to the cells. The bioacessibility of the iron was measured by the ferritin. As results, the papaya lyophilized extract presented a higher concentration of ascorbic acid (p<0.05), being selected for the next stages (microencapsulation and in vitro test). The results for humidity (less than 13%) and water activity (less tha 0.3) showed microbiological security for all treatments. The treatments obtained by spray drying was more soluble when compared to the coacervated treatments and to the lyophilized extracts (p<0.05). The papaya lyophilized extract present higher concentration of ascorbic acid (p<0.05), and higher hygroscopicity (p<0.05) when compared to the other treatments. Regarding the morphology, the treatments presented differences mainly due to the technique applied, the used materials and the particle size. For the bioacessibility of the mineral, the mean content of ferritin ranged from 0.02 to 1.19 ng per mg of protein There were no positively significant differences in the treatments conducted in relation with the commercial wheat flour (p<0.05). In conclusion, it was possible to extract ascorbic acid from fruit by-products and apply the microencapsulation and lyophilization to this extract, keeping the ascorbic acid levels for some treatments. In terms of the bioacessibility experiments, it can be concluded that enhancing the wheat flour with ascorbic acid in the conditions and proportions used in this study is not enough to increase the bioacessibility of the iron present in the commercial wheat flour.

Made available in DSpace on 2014-12-17T15:41:55Z (GMT). No. of bitstreams: 1 IdyllaST_DISSERT.pdf: 1649846 bytes, checksum: b16782ef605bdc7a1d3225b281a6f2b3 (MD5) Previous issue date: 2011-11-08 / Chitosan nanoparticles have been used in several systems for the controlled release of drugs. The aim of this study was to obtain and characterize chitosan nanoparticles prepared by the method of coacervation / precipitation using sodium sulfate at different concentrations as the crosslinking agent. The characterization was done using zeta potential and small angle Xray scattering, SAXS. The dispersions of chitosan were obtained at pH 1 and pH = 3. The results of zeta potential at pH = 1 ranged from +64.8 to +29.27 mV and for pH = 3 they varied from +72.4 to +23.48 mV, indicating that the chain of chitosan is positively charged in acidic pH and the behavior of nanoparticles in terms of surface charge was independent of pH. However, the results indicated a dependence of particle size in relation to pH. This difference in behavior was explained by the influence of enthalpic and entropic components / Nanopart?culas de quitosana t?m sido utilizadas em v?rios sistemas destinados a libera??o controlada de f?rmacos. O objetivo desse trabalho foi a obten??o e caracteriza??o de nanopart?culas de quitosana atrav?s do m?todo de coacerva??o/precipita??o utilizando o sulfato de s?dio em diferentes concentra??es como agente reticulante. A caracteriza??o foi feita utilizando potencial zeta e espalhamento de pequenos ?ngulos, SAXS. As dispers?es de quitosana foram obtidas a pH=1 e pH=3. Os resultados do potencial zeta variaram no pH = 1 de +64,8 a +29,27 mV e no pH = 3 de +72,4 a +23,48 mV, indicando que a cadeia de quitosana fica carregada positivamente em pH ?cido e que comportamento das nanopart?culas em termos de carga superficial foi independente do pH. Entretanto, os resultados indicaram depend?ncia do tamanho de part?cula em rela??o ao pH. Essa diferen?a em termos de comportamento foi explicada pela influ?ncia dos componentes ent?lpicos e entr?picos

L'influence de la concentration totale en biopolymères et de la polydispersité de la protéine sur la formation et la stabilité de coacervats a été étudiée dans un mélange b-lactoglobuline/gomme d'acacia/eau. Les mélanges de b-lactoglobuline (b-lg) native ou agrégée (56% d'agrégats insolubles à pH 4,75) et de gomme d'acacia à des pH compris entre 3,6 et 5,0 sont caractérisés par une séparation de phase par coacervation complexe. Une concentration totale élevée en biopolymères limite l'influence du pH et du ratio de mélange protéine : polysaccharide sur la formation des coacervats dans les deux systèmes. En revanche, une forte polydispersité de la b-lg permet d'étendre l'aire de la zone biphasique des diagrammes de phases ternaires obtenus à pH 4,2. Des coacervats et des précipités sont obtenus en présence d'agrégats de b-lg. Seuls des coacervats sont visibles avec la b-lg native. Leur taille est contrôlée par les agrégats protéiques qui interagissent spécifiquement avec une fraction de la gomme d'acacia. La structure des coacervats est caractérisée par une vacuolisation issue d'une coalescence partielle des plus petits coacervats. Après interaction avec la gomme d'acacia, des mesures de dichroïsme circulaire indiquent une modification de la structure en hélice a de la b-lg, caractérisée par une densité de charge positive. La stabilité et la structuration des coacervats formés résultent d'un équilibre entre des phénomènes de floculation, coalescence et sédimentation des coacervats comme indiqué par des mesure de diffusion de la lumière en milieu turbide et microscopie confocale à balayage laser. Enfin, l'étude de la cinétique de structuration des mélanges b-lg/gomme d'acacia/eau par diffusion de la lumière aux petits angles, révèle des phénomènes de diffusionnels et hydrodynamiques responsables de la croissance des domaines structuraux. Dans certains cas, la cinétique de coacervation complexe peut être décrite par le modèle théorique de décomposition spinodale.

The overall goal of this thesis is to better understand mechanisms governing associative phase separation within admixtures of plant proteins (e.g., pea and canola) and anionic polysaccharides (e.g., gum Arabic, alginate or é-carrageenan). The process involves the electrostatic attraction between two biopolymers of opposing charges, and typically results in the formation of both soluble and insoluble complexes during an acidic pH titration. If successful, polysaccharides could be triggered to coat the proteins surface to give novel, and hopefully improved functionality as ingredients for food and biomaterials.<p> In the first study, the effect of protein enrichment and pH on the formation of soluble and insoluble complexes in admixtures of pea legumin (Lg) and vicilin (Vn) isolates with gum Arabic (GA) was investigated by turbidimetric, surface charge and fluorometric measurements. The solubility of the protein isolates and mixed biopolymer systems was also studied as a function of pH. Enrichment of the crude Lg and Vn isolates by low pressure liquid chromatography led to a shift towards higher pHs at the onset of soluble complex formation in the presence of GA for both protein isolates, whereas the onset of insoluble complex formation was unaffected. Complexation of the Lg (or Vn) isolates with GA resulted in a shift in the pH where neutrality (zeta potential = 0 mV) occurred to lower pH values, relative to the Lg (or Vn) isolates alone. In the case of the enriched Vn isloate, changes to its tertiary structure were observed by fluorometry upon complexation with GA, whereas no changes were found for the enriched Lg isolate. Complexation of Lg and Vn isolates with GA also had little effect on their solubilities relative to protein alone solutions.<p> In the second study, the formation of soluble and insoluble complexes, and the nature of their interactions as determined by optical density analysis, were investigated in admixtures of canola protein isolate (CPI) and anionic polysaccharides (alginate and é-carrageenan) as a function of pH and biopolymer weight mixing ratio. The solubilities of formed complexes were also investigated versus protein alone. In both CPI-polysaccharide systems, critical pH associated with the onset of soluble and insoluble complexes shifted to higher pHs as the mixing ratios increased from 1:1 to 20:1 (CPI:polysaccharide), and then became constant. There complexes formed primarily through electrostatic attractive forces with secondary stabilization by hydrogen bonding. The solubilities of the CPI-alginate complexes were significantly enhanced relative to CPI alone or CPI-é-carrageenan, which were similar.

The overall goal of this thesis is to better understand mechanisms governing associative phase separation within admixtures of plant proteins (e.g., pea and canola) and anionic polysaccharides (e.g., gum Arabic, alginate or é-carrageenan). The process involves the electrostatic attraction between two biopolymers of opposing charges, and typically results in the formation of both soluble and insoluble complexes during an acidic pH titration. If successful, polysaccharides could be triggered to coat the proteins surface to give novel, and hopefully improved functionality as ingredients for food and biomaterials.<p> In the first study, the effect of protein enrichment and pH on the formation of soluble and insoluble complexes in admixtures of pea legumin (Lg) and vicilin (Vn) isolates with gum Arabic (GA) was investigated by turbidimetric, surface charge and fluorometric measurements. The solubility of the protein isolates and mixed biopolymer systems was also studied as a function of pH. Enrichment of the crude Lg and Vn isolates by low pressure liquid chromatography led to a shift towards higher pHs at the onset of soluble complex formation in the presence of GA for both protein isolates, whereas the onset of insoluble complex formation was unaffected. Complexation of the Lg (or Vn) isolates with GA resulted in a shift in the pH where neutrality (zeta potential = 0 mV) occurred to lower pH values, relative to the Lg (or Vn) isolates alone. In the case of the enriched Vn isloate, changes to its tertiary structure were observed by fluorometry upon complexation with GA, whereas no changes were found for the enriched Lg isolate. Complexation of Lg and Vn isolates with GA also had little effect on their solubilities relative to protein alone solutions.<p> In the second study, the formation of soluble and insoluble complexes, and the nature of their interactions as determined by optical density analysis, were investigated in admixtures of canola protein isolate (CPI) and anionic polysaccharides (alginate and é-carrageenan) as a function of pH and biopolymer weight mixing ratio. The solubilities of formed complexes were also investigated versus protein alone. In both CPI-polysaccharide systems, critical pH associated with the onset of soluble and insoluble complexes shifted to higher pHs as the mixing ratios increased from 1:1 to 20:1 (CPI:polysaccharide), and then became constant. There complexes formed primarily through electrostatic attractive forces with secondary stabilization by hydrogen bonding. The solubilities of the CPI-alginate complexes were significantly enhanced relative to CPI alone or CPI-é-carrageenan, which were similar.

Orientadores: Carlos Raimundo Ferreira Grosso, Maria Ines Re / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-06T03:05:42Z (GMT). No. of bitstreams: 1 Prata_AnaSilvia_D.pdf: 8487208 bytes, checksum: c134b0c8ddb26e852727eca6b6bef640 (MD5) Previous issue date: 2006 / Resumo: Microencapsulação é uma tecnologia usada como ferramenta potencial para proteção de um composto e modulação de sua liberação. Atualmente, estudos na área de micropartículas e liberação controlada estão bastante avançados e uma variedade de técnicas estão disponíveis entre as quais destacam-se processos por atomização (spray drying, spray cooling e spray chilling), coacervação e recobrimento por leito fluidizado e por complexação. A coacervação complexa envolve a associação reversível de dois polímeros e apresenta algumas vantagens frente a outras técnicas como a possibilidade de se trabalhar com biopolímeros, a ausência de solvente orgânico e condições brandas de temperatura no processamento. Porém a complexidade do processo dificulta a aplicação da técnica a biopolímeros com diferentes características físico-químicas. Assim, neste trabalho foram estudadas as principais condições que podem interferir na formação das partículas por coacervação complexa do sistema clássico goma-arábica:gelatina e pectina:gelatina, na tentativa de elucidar os mecanismos envolvidos. Além disso, as propriedades de barreira das partículas encapsuladas com óleo essencial de vetiver foram alteradas sob duas condições de reticulação (glutaraldeído e transglutaminase) como forma de prever o comportamento da membrana polimérica em diferentes meios. O ensaio de liberação foi mensurado a partir de um composto apolar fluorescente (dansilato de kusimila - KD) em meios aquosos com polaridade modificada pela adição de tensoativos de natureza iônica (dodecilsulfato de sódio) e não-iônica (Tween 80). Os resultados obtidos confirmaram que existe uma faixa ótima de formação de partículas dependente do pH. Acredita-se que as partículas são formadas pela coacervação dos polímeros no sistema líquido-líquido sobre a superfície hidrofóbica do agente de núcleo. O planejamento experimental possibilitou a otimização do processo clássico de formação de partículas gelatina-goma arábica resultando em uma proporção ótima de 2:1. As condições estudadas para a pectina não possibilitaram otimizar o processo nem produzir cápsulas esféricas, sendo que uma massa coacervada foi formada arrastando a porção hidrofóbica e conseqüentemente encapsulando-a. Nas melhores condições experimentais encontradas foram testados agentes reticulantes (glutaraldeído e transglutaminase) que apresentaram diferenças quanto à retenção do composto fluorescente encapsulado e diferenças morfológicas nos meios estudados. Para as partículas não reticuladas úmidas, KD foi completamente liberado após 5 horas enquanto para partículas secas esta quantidade foi de 80%. Nestes sistemas liberação do óleo de vetiver foi avaliada por quantificação cromatográfica através da medida de liberação de alguns compostos presentes no óleo. Em geral, maior porcentagem de liberação foi observada para álcoois e cetonas e menor porcentagem de liberação observada para os hidrocarbonetos. Partículas reticuladas com glutaraldeído apresentaram menor liberação de KD em relação às reticuladas com transglutaminase, que por sua vez apresentaram liberação ligeiramente inferior às partículas não-reticuladas, independentemente dos meios de liberação estudados. Considerando os meios, a ordem de liberação de KD observada foi : etanol anidro>SDS>tween80 / Abstract: Microencapsulation is a technology used as a potential tool in the protection of a product and the modulation of its core release. Currently, studies in the area of microparticles and controlled release are considerably advanced and a variety of techniques and products are available, among them differents processes for atomization (spray drying, spray cooling and spray chilling), coacervation and fluidized bed and complexation. The complex coacervation involves the reversible association (ionic) of two polymers and presents some advantages in comparison to other techniques such as the possibility to work with biopolymers, the absence of organic solvent and mild temperatures. However the complexity of the process turns the application of the technique difficult to biopolymers due to different physical and chemical characteristics. Thus, this work considered a study of the main conditions that can influence the formation of particles by complex coacervation in an attempt to elucidate the mechanisms involved. Moreover, the barrier properties of these particles were modified under two conditions of crosslinking. The release assay was measured with a hydrophobic fluorescent (khusimyl dansylate - KD), substance in solvents with their polarity modified by the addition of ionic and non-ionic surfactants (SDS and Tween 80). The results confirmed that an optimum band of particle formation exists and depends on the pH. The particles are formed by the coacervation of polymers in the liquid-liquid system over the hydrophobic surface of the core agent. The experimental design made possible the optimization of the classic process of particle formation when the arabic gum-gelatin ratio was 1:2. For the pectin, the range of conditions studied did not allow to optimize the process or to produce spherical capsules, but a coacervated mass was formed dragging the hydrophobic portion and consequently encapsulating it. In the best conditions found, the crosslinking agents (glutaraldehyde and transglutaminase) presented different retention capacities for the encapsulated fluorescent probe and morphological differences in the solvents studied. The KD in the moist capsules without reticulation was completely released after 5 hours, whilst for the freeze-dried particles, this amount was 80%. Release of vetiver oil was evaluated indirectly by chromatographic quantification following the release of some of the compounds present in the oil. In general, a greater percent of release occurred for the alcohols and ketones and smaller percent release was observed for the hydrocarbon compounds. Crosslinking using glutaraldehyde showed smaller release of the KD than particles crosslinked with transglutaminase. The release KD profile of the particles crosslinked with transglutaminase was almost the same compared with particles without crosslinking, in all release medium used. The release order observed for KD was: anhydrous ethanol>SDS>tween80 / Doutorado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Doutor em Alimentos e Nutrição