This thesis describes a novel approach for cell encapsulation in alginate gel microbeads. The main aim of the thesis was to optimise a microfluidic setup and chip to encapsulate cells in monodisperse alginate gel microbeads. A number of cytotoxicity tests were therefore carried out to determine the effect of formulations used for the production, degradation and gelation of calcium alginate gel beads. Results from these tests revealed that the formulations used had little or no significant effect on cell growth, and therefore, alginate was deemed to be a suitable cell encapsulating material for further investigations. Alginate gel microbeads were produced using hydrodynamic focusing techniques. For this purpose two different microfluidic setups were constructed. Fluids (oil, acidified oil and samples) were driven through the microfluidic setup by gravity. However, a number of drawbacks using this setup arose, such as polydispersity and reproducibility. Syringe pumps were introduced into the design of the second microfluidic setup as a means of driving fluids through the setup. In addition three different microfluidic chips were fabricated with the aim of producing the ideal alginate gel microbead. The first microfluidic chip (PMMA MC1) was fabricated from PMMA and involved producing alginate gel microbeads that were internally gelified. This chip suffered from a number of drawbacks such as continuous blockages within the microfluidic channels, which led to the development of the second microfluidic chip. This chip was also fabricated from PMMA (PMMA MC2) but in contrast to PMMA MC1, gelification occurred externally, i.e. gelation took place off chip, and in this case the alginate microdroplets were dropped into a well containing 1 mL of acidified oil. This encapsulating procedure caused immediate cell death, which indicated that the internal gelation of alginate gel microbeads was favoured. These results also indicated that the design of the microfluidic chip needed developing in order to produce the ideal microbeads that can be used for cell encapsulation. This led to the fabrication of a novel microfluidic chip (PC MC3) which was fabricated from polycarbonate (PC) and involved internal gelation of the calcium alginate gel microbeads. The combination of using the optimised microfluidic setup and PC MC3, in addition to alternations in some of the solutions used to make the alginate microbeads, resulted in the production of the desired ideal gel microbeads containing cells. Snap shots of the encapsulated cells obtained using fluorescence microscopy after 24 hours of encapsulation, revealed that the cells showed some characteristics of living cells, yet at the same time they also showed some characteristics of dead cells. These findings demonstrate the potential use of the optimised microfluidic setup and PC MC3 chip for many biological and medical applications.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A incorporaÃÃo de compostos nutracÃuticos como vitaminas, probiÃticos, peptÃdeos bioativos e antioxidantes em sistemas alimentÃcios proporciona uma maneira simples de desenvolver novos alimentos funcionais que podem ter benefÃcios fisiolÃgicos ou reduzir os riscos de doenÃas. Pectinas tÃm sido investigadas por sua capacidade de produzir esferas de gÃis de pectinato de cÃlcio contendo bioativos. Quitosana tambÃm tem sido muito utilizada na preparaÃÃo de esferas por suas propriedades mucoadesivas. Neste estudo, trÃs tipos de esferas de pectina/cÃlcio/quitosana com diferentes graus de reacetilaÃÃo foram produzidas no intuito de encapsular um potente antioxidante natural extraÃdo da manga, a mangiferina. Inicialmente, duas amostras de pectina cÃtrica foram caracterizadas por FTIR, 1H RMN, Reologia e GPC. A pectina cÃtrica com menor grau de metoxilaÃÃo foi escolhida para a preparaÃÃo das esferas por apresentar melhores propriedades geleificantes. As esferas produzidas foram caracterizadas por FTIR, MEV e intumescimento. A formaÃÃo do complexo pectina/cÃlcio/quitosana reacetilada foi observada por FTIR e MEV. Foi realizado um estudo de liberaÃÃo controlada de mangiferina a partir de trÃs tipos de esferas obtidas por duas metodologias diferentes. Os valores de percentual de liberaÃÃo foram bastante significativos, chegando atà 75%. O comportamento dos mecanismos de liberaÃÃo de mangiferina das esferas de pectina/cÃlcio/quitosana mostrou que o bioativo foi liberado a partir da matriz principalmente por meio de relaxaÃÃo das cadeias polimÃricas. / Incorporation of nutraceuticals compounds such as vitamins, probiotics, bioactive peptides and antioxidants in food systems provides a simple way to develop new functional foods that can have physiological benefits or reduce risks of disease. Pectins have been investigated for their ability to produce spheres of calcium pectinate gels containing bioactive. Chitosan has also been widely used in the preparation of beads due to its mucoadhesive properties. In this study, three types of spheres of pectin/calcium/chitosan with different degrees of reacetylation were produced in order to encapsulate a powerful natural antioxidant extracted from mango, mangiferin. Initially, two samples of citrus pectin were characterized by FTIR, 1H NMR, Rheology and GPC. The citrus pectin with the lowest degree of methoxylation was chosen for the preparation of beads due to its better gelling properties. The beads produced were characterized by FTIR, SEM and swelling. The formation of the reacetylated complex pectin/calcium/chitosan was observed by FTIR and SEM. A study of controlled release of mangiferin was conducted from three types of beads obtained by two different methodologies. The values of percentage of release were quite significant, reaching up to 75%. The mechanisms behaviour for the release of mangiferin from spheres of pectin/calcium/chitosan showed that the bioactive was released from the matrix mainly through relaxation of the polymer chains.

Heavy metals and hydrocarbons are persistent pollutants in the environment. Problems associated with the cleanup of sites contaminated by metals and hydrocarbons have demonstrated the need to develop remediation technologies that are feasible, quick, and effective in a wide range of physical settings. Experiments were conducted to investigate the efficiency of silica encapsulation and the factors that influence its performance. Analysis was done by ICP-OES and GC-FID for metals and hydrocarbons respectively. This technology was tested using sodium silicate and ChemcapTM. Soils and water contaminated with hydrocarbons and heavy metals were successfully remediated by silica encapsulation. The silica coating was stable under both acidic and alkaline conditions. A new product that is based on sodium silicate formulation was developed and was more effective at encapsulating hydrocarbons and heavy metals. Laboratory tests indicated that it is more effective in an acidic medium and it continues to strengthen with time. Metal encapsulation was affected by the sizes of metal ions and the presence of hydrocarbons.

The use of probiotics and prebiotics has become a popular trend in the food industry. The main goal of this study was to produce a synbiot by encapsulating a probiotic and a prebiotic within a matrix that would provide sufficient protection to the probiotic against simulated gastric juice (SGJ). The ability of the probiotic, Bifidbacterium adolescentis, to grow on short chain fructooligosaccharides (FOS; DP 2-8, P95), inulin (DP 2-60, ST), and FOS/inulin mixture (DP 2-60, Syn), as well as glucose and a glucose-free maltooligosaccharide (MOS), were evaluated. Bifidobacterium adolescentis had a significantly higher specific growth rate on P95 (0.47 h-1), than glucose (0.40 h-1). Examination of the growth medium containing P95 and MOS by high performance anion exchange with pulsed amperometric detection (HPAE-PAD) revealed that B. adolescentis utilised the oligosaccharides to the same extent as the monosaccharides.<p> Bifidobacterium adolescentis was successfully encapsulated with and without P95 using extrusion and emulsion methods, at cell concentrations of 8-9 log colony forming units (CFU) mL-1. Capsules formed by the extrusion method with 1.0% alginate (AL), 4.0% pea protein isolate (PPI) + 0.5% AL, and 4.0% whey protein isolate (WPI) + 0.5% AL ranged in geometric mean diameter from 2.0 to 2.2 mm. Capsules formed by emulsion with 4.0% WPI + 0.5% AL had geometric mean diameter of 53 ìm. Extrusionbased encapsulated probiotics in either PPI + AL or WPI + AL showed improved survival in SGJ at pH 2.0 for 2.0 h with log CFU mL-1 reductions of 3.6 and 1.1, respectively. Free cells, AL extrusion-based and WPI + AL emulsion-based encapsulated probiotics showed no survival after 30 min in SGJ at pH 2.0. The addition of 1.0% (w/w) P95 to the PPI + AL capsules improved probiotic survival such that 1.0 log CFU mL-1 reduction was observed. The amount of P95 encapsulated ranged from 4.0 to 4.4 mg per gram of capsules.<p> The external surface of the PPI + AL capsules as examined by cold stage scanning electron microscopy (cryo-SEM) and atomic force microscopy (AFM) was smooth with the presence of pores ranging in diameter from 0.25 to 1.00 ìm. The addition of P95 to the capsules had no significant effect on surface roughness as measured by AFM, but significantly increased the external capsule thickness. The internal structure of the PPI + AL capsules examined by cryo-SEM revealed a porous honeycomb-like structure, with inner pore diameters ranging between 13.0 and 21.9 ìm. Probiotic cells were found to be randomly dispersed on the surface and in the interior of the honeycomb pores. In contrast, the prebiotic was found to be distributed throughout the capsule as observed by confocal laser scanning microscopy (CLSM), indicating that it would be readily available to the probiotic as a carbon source

The focus of this research was to develop a plant-based microcapsule for flax oil by complex coacervation. Complex coacervation involves the electrostatic attraction between two polymers of opposing charges. Specifically, the research aimed to: a) identify the ideal biopolymer and solvent conditions required for complex coacervation involving pea protein isolate (PPI) and gum Arabic (GA); b) understand the functional behaviour of PPI-GA complexes as food and biomaterial ingredients; and c) develop methodologies for encapsulating flax oil within PPI-polysaccharide capsules. Complex coacervation between PPI-GA was found to be optimized at a biopolymer weight mixing ratio of 2:1 in the absence of salt. The functional behaviours of the optimized biopolymer mixture were then investigated as a function of pH (4.30-2.40) within a region dominated by complex coacervation. Emulsion stability was found to be greater for PPI-GA mixture systems relative to PPI alone at pH values between 3.10 and 4.00; emulsions produced under one-step emulsification exhibited higher stability compared to those of two-step emulsification at all pH values. Foam expansion was independent of both biopolymer content and pH, whereas foam stability improved for the mixed system between pH 3.10 and 4.00. The solubility minimum was broadened relative to PPI at more acidic pH values. These findings suggested that the admixture of PPI and GA under complexing conditions could represent a new food and/or biomaterial ingredient, and has potential as an encapsulating agent. Two encapsulation processes were employed in this research: high speed mixing (two-step emulsification) and low speed mixing (one-step emulsification). Flax oil capsules formed using the gelatin-GA mixture (as control) under high speed mixing exhibited low moisture content, water activity and surface oil, and afforded adequate protection against oxidation relative to free oil over a 25 d storage period. The PPI-GA mixture failed to produce acceptable capsules using either high or low speed mixing. In contrast, PPI-alginate capsules were produced and exhibited similar chemical properties as gelatin-GA capsules, except with lower encapsulated flax oil content (30% vs. 50% w/w). However, oxidative stability may adversely affected by the low speed mixing condition during encapsulation.

The focus of this research was to develop a plant-based microcapsule for flax oil by complex coacervation. Complex coacervation involves the electrostatic attraction between two polymers of opposing charges. Specifically, the research aimed to: a) identify the ideal biopolymer and solvent conditions required for complex coacervation involving pea protein isolate (PPI) and gum Arabic (GA); b) understand the functional behaviour of PPI-GA complexes as food and biomaterial ingredients; and c) develop methodologies for encapsulating flax oil within PPI-polysaccharide capsules. Complex coacervation between PPI-GA was found to be optimized at a biopolymer weight mixing ratio of 2:1 in the absence of salt. The functional behaviours of the optimized biopolymer mixture were then investigated as a function of pH (4.30-2.40) within a region dominated by complex coacervation. Emulsion stability was found to be greater for PPI-GA mixture systems relative to PPI alone at pH values between 3.10 and 4.00; emulsions produced under one-step emulsification exhibited higher stability compared to those of two-step emulsification at all pH values. Foam expansion was independent of both biopolymer content and pH, whereas foam stability improved for the mixed system between pH 3.10 and 4.00. The solubility minimum was broadened relative to PPI at more acidic pH values. These findings suggested that the admixture of PPI and GA under complexing conditions could represent a new food and/or biomaterial ingredient, and has potential as an encapsulating agent. Two encapsulation processes were employed in this research: high speed mixing (two-step emulsification) and low speed mixing (one-step emulsification). Flax oil capsules formed using the gelatin-GA mixture (as control) under high speed mixing exhibited low moisture content, water activity and surface oil, and afforded adequate protection against oxidation relative to free oil over a 25 d storage period. The PPI-GA mixture failed to produce acceptable capsules using either high or low speed mixing. In contrast, PPI-alginate capsules were produced and exhibited similar chemical properties as gelatin-GA capsules, except with lower encapsulated flax oil content (30% vs. 50% w/w). However, oxidative stability may adversely affected by the low speed mixing condition during encapsulation.

The use of probiotics and prebiotics has become a popular trend in the food industry. The main goal of this study was to produce a synbiot by encapsulating a probiotic and a prebiotic within a matrix that would provide sufficient protection to the probiotic against simulated gastric juice (SGJ). The ability of the probiotic, Bifidbacterium adolescentis, to grow on short chain fructooligosaccharides (FOS; DP 2-8, P95), inulin (DP 2-60, ST), and FOS/inulin mixture (DP 2-60, Syn), as well as glucose and a glucose-free maltooligosaccharide (MOS), were evaluated. Bifidobacterium adolescentis had a significantly higher specific growth rate on P95 (0.47 h-1), than glucose (0.40 h-1). Examination of the growth medium containing P95 and MOS by high performance anion exchange with pulsed amperometric detection (HPAE-PAD) revealed that B. adolescentis utilised the oligosaccharides to the same extent as the monosaccharides.<p> Bifidobacterium adolescentis was successfully encapsulated with and without P95 using extrusion and emulsion methods, at cell concentrations of 8-9 log colony forming units (CFU) mL-1. Capsules formed by the extrusion method with 1.0% alginate (AL), 4.0% pea protein isolate (PPI) + 0.5% AL, and 4.0% whey protein isolate (WPI) + 0.5% AL ranged in geometric mean diameter from 2.0 to 2.2 mm. Capsules formed by emulsion with 4.0% WPI + 0.5% AL had geometric mean diameter of 53 ìm. Extrusionbased encapsulated probiotics in either PPI + AL or WPI + AL showed improved survival in SGJ at pH 2.0 for 2.0 h with log CFU mL-1 reductions of 3.6 and 1.1, respectively. Free cells, AL extrusion-based and WPI + AL emulsion-based encapsulated probiotics showed no survival after 30 min in SGJ at pH 2.0. The addition of 1.0% (w/w) P95 to the PPI + AL capsules improved probiotic survival such that 1.0 log CFU mL-1 reduction was observed. The amount of P95 encapsulated ranged from 4.0 to 4.4 mg per gram of capsules.<p> The external surface of the PPI + AL capsules as examined by cold stage scanning electron microscopy (cryo-SEM) and atomic force microscopy (AFM) was smooth with the presence of pores ranging in diameter from 0.25 to 1.00 ìm. The addition of P95 to the capsules had no significant effect on surface roughness as measured by AFM, but significantly increased the external capsule thickness. The internal structure of the PPI + AL capsules examined by cryo-SEM revealed a porous honeycomb-like structure, with inner pore diameters ranging between 13.0 and 21.9 ìm. Probiotic cells were found to be randomly dispersed on the surface and in the interior of the honeycomb pores. In contrast, the prebiotic was found to be distributed throughout the capsule as observed by confocal laser scanning microscopy (CLSM), indicating that it would be readily available to the probiotic as a carbon source

Thèse de doctorat : Sciences physiques et de l'Ingénieur. Microélectronique : Bordeaux 1 : 2008. / Titre provenant de l'écran-titre.

The aim of this study was to prepare biologically active insulin-loaded alginate microparticles by spray drying. Particles were produced from three alginate feed concentrations of 1, 1.5 and 2% w/v, with respective insulin loadings of 11.8, 7.8 and 5.8 mg/g of alginate and investigated in terms of mass yield, moisture content, particle size, morphology and encapsulation efficiency. The mass yield of the system was determined to be between 15 and 30%, with approximately 3% of the initial dry mass ending up in the exhaust filter. The moisture content of the particles was found to be between 4.9 and 11.1% and the mean size ranged between 1.2 and 1.6 μm. Particulate morphologies were observed to be mostly spherical with some ‘divots’ present on the surface. Lastly, the encapsulation efficiency determined by absorbance assay was approximately 40%. Particles produced from a 2% alginate feed were further assayed by determining the release of insulin in simulated gastrointestinal conditions and looking at the insulin and alginate distribution within spray dried particles. A steep release profile was observed in the first 120 min of the simulation in a gastric pH of 1.2 and a longer, more sustained release is observed in intestinal conditions, where an additional 20% of the total insulin in the particles is released over 600 min. Fluorescent labels revealed that insulin and alginate are concentrated towards the periphery of the particles. The residual bioactivity of insulin was assessed by an in vitro bioactivity assay, which was developed using Fast Activated Cell Based ELISA (FACE™) AKT kits specific for phosphylated AKT. The bioactivity of insulin in the particles after spray drying was determined to be 87.9 ± 15.3%. / Thesis (Master, Chemical Engineering) -- Queen's University, 2009-09-20 20:32:29.103

Ce travail de thèse avait pour objectif de mieux comprendre l'influence de l'incorporation d'une molécule active (arôme) et/ou de particules lipides sur la structure et les propriétés physico-chimiques de matrices filmogènes à base d'iota-carraghènane ou d'alginate de sodium. La microstructure, la granulométrie de l'émulsion filmogène, les caractéristiques thermiques et mécaniques des films ont été étudiées en présence ou non de n-hexanal. Les propriétés de transfert de ces films ont été appréhendées grâce aux mesures de perméabilité à l'oxygène, à la vapeur d'eau et à l'eau liquide, au n-hexanal et au D limonène à l'état vapeur et liquide et à 6 autres composés d'aromes (esters d'éthyle, cétone et alcools). L'ajout de n-hexanal dans la matrice d'iota-carraghènane modifie le processus de gélification ce qui induit la formation d'une microstructure plus homogène mais ayant une résistance mécanique moindre, augmente la perméabilité à la vapeur d'eau mais diminue légèrement celle de l'hexanal. Quant aux films d'alginate, ils sont peu sensibles à la présence de n-hexanal. Cette différence de comportement est attribuée aux interactions entre la fonction aldéhyde de l'arôme et les groupes OH et sulfate du carraghènane qui perturbent la formation des hélices lors de la gélification. L'organisation du gel d'alginate en boite à œuf lui confère une plus grande stabilité. L'ajout de lipide permet de diminuer la sorption et la perméabilité à la vapeur d'eau des matrices filmogène. En présence de la matière grasse, l'hexanal se comporte comme un émulsifiant et semble être localisé en surface des globules en augmentant la finesse et la stabilité des émulsions filmogènes. Les paramètres cinétique (diffusivité) et thermodynamique (sorption) ont été mesurés pour mieux caractériser les mécanismes impliqués dans le transfert Enfin, le paradoxe de Schroeder, correspondant à la différence de flux selon l'état physique de la molécule diffusante, a été observé et expliqué pour l'eau dans les films d'alginate et pour l'hexanal dans les films de carraghènanes.