Pyruvate is a valuable chemical intermediate in the production of fine chemicals used by agrochemical, pharmaceutical, and food industries. Current technology for production of pyruvic acid is based on conversion from tartaric acid and results in environmentally incompatible byproducts. An enzymatic approach to making pyruvate was developed by cloning the glycolate oxidase (GO) gene from spinach into Pichia pastoris (Payne, et al., (1995). High-level production of spinach glycolate oxidase in the methylotrophic yeast Pichia pastoris: Engineering a biocatalyst. Gene, 167(1-2), 215-219). GO is a flavoprotein (FMN dependent) which catalyzes the conversion of lactate to pyruvate with the equimolar production of hydrogen peroxide. Hydrogen peroxide can lower GO activity and make non-catalytic byproducts, so catalase was also cloned into P. pastoris to create a double transformant. Process development work was completed at the University of Iowa's Center for Biocatalysis and Bioprocessing. High-density P. pastoris fermentation (7.2 kg cells/L) was completed at the 100 L scale. Critical fermentation set-points were confirmed at 14 h glycerol feeding followed by methanol induction at 2 - 10 g/L for 30 h. After fermentation, these cells were permeabilized with benzalkonium chloride (BAC) to enable whole-cell biocatalysis and increase enzyme activity, yielding 100 U/g for GO. In 30 L enzyme reactions, permeabilized cells were recycled three times for over 92% conversion of 0.5 M lactate with an "enzyme to product" ratio of approximately 1:2 (Gough, et al., (2005). Production of pyruvate from lactate using recombinant Pichia pastoris cells as catalyst. Process Biochemistry, 40(8), 2597-2601). Though effective, the post-fermentation process for GO recovery involved several unit-operations, including multiple washing steps to remove residual BAC. The present work has focused on minimizing unit-operations by spray-drying the fermentation product to create a powdered biocatalyst. Optimal spray-drying conditions for the Buchi B-190 instrument were 150°C drying air, 15 mL/min liquid feed rate, and 600 mg cells/mL liquid feed. These conditions resulted in P. pastoris biocatalyst with activities of 80 - 100 U/g for GO and 180,000 - 220,000 U/g for catalase. The spray-dried cells retained nearly 100% of the enzyme activity compared to BAC treated cells as reported by Gough et al. Additionally, the spray-dried biocatalyst was stable at room temperature for 30 days, and no measurable enzyme leaching was observed. Then, P. pastoris was spray-dried under optimal conditions and tested for conversion of lactate to pyruvate for an improved "enzyme to product" ratio. Enzyme reaction optimization was done at the one-liter scale in DASGIP reactors. The DASGIP system contained four parallel reactors with control of temperature, pH, and dissolved oxygen. Other key variables included substrate loading, conducting the reaction in buffer or water, minimizing enzyme concentration, and maximizing the number of enzyme recycles. Optimal performance was achieved in water at pH 7.0 with an operating temperature of 25°C and 1.0 M substrate loading. Enzyme loading was at 12 g/L for the first two cycles, and subsequently, 2 - 3 g/L of fresh cells were added every alternate cycle to reach 15 cycles. Under these conditions, 75 - 95% conversion of lactate to pyruvate was accomplished for every 12 - 16 h reaction cycle. Based on these parameters, an "enzyme to product" ratio of 1:41 was achieved.

This thesis describes results of a study aimed at developing and evaluating attrition resistant iron catalysts prepared by spray drying technique. These catalysts are intended for Fischer-Tropsch (F-T) synthesis in a slurry bubble column reactor (SBCR). One of the major challenges associated with the use of SBCR for this purpose is the problem of catalyst/wax separation. If the catalyst particles break up into smaller ones during the F-T synthesis, these small particles (>5-10 m in diameter) will cause problems with the catalyst/wax separation. Several research groups have worked on development of attrition resistant spray-dried iron catalysts, and methodology to measure and predict their attrition behavior. However, these attrition tests were not conducted under conditions representative of those encountered in a SBCR. In this work, the attrition behavior of six spray-dried catalysts and two precipitated catalysts was evaluated under slurry reaction conditions in a stirred tank slurry reactor (STSR). Spray-dried catalysts used in this study were prepared at Texas A&M University (TAMU) and at Hampton University (HU), employing different preparation procedures and silica sources (potassium silicate, tetraethyl orthosilicate or colloidal silica). The attrition properties of F-T catalysts were determined by measuring particle size distribution (PSD) of catalysts before and after F-T synthesis in the STSR. This provides a direct measure of changes in particle size distribution in the STSR, and accounts for both physical and chemical attrition effects. Also, scanning electron microscopy (SEM) was used to investigate the mechanism of attrition - erosion vs. fracture, and to obtain morphological characteristics of catalysts. Spray dried 100Fe/3Cu/5K/16SiO2 catalyst (WCS3516-1), prepared from wet precursors using colloidal silica as the silica source, was the best in terms of its attrition strength. After 337 hours of F-T synthesis in the STSR, the reduction in the average particle size and generation of particles less than 10 m in diameter were found to be very small. This indicates that both particle fracture and erosion were insignificant during testing in the STSR. All other catalysts, except one of the spray dried catalysts synthesized at Hampton University, also had a good attrition resistance and would be suitable for use in slurry reactors for F-T synthesis.

Currently, therapeutic proteins and peptides are delivered subcutaneously, as they are readily denatured in the acidic, protease rich environment of the stomach or gastrointestinal track and low bioavailability results from poor intestinal absorption through the paracellular route. Encapsulation of therapeutic peptides and proteins into polymeric micro- and nano- particle systems has been proposed as a possible strategy to overcome limitations to oral protein administration. Furthermore, it was shown that nanoparticles having diameters less than 5µm are able to be taken up by the M cells of Peyer’s patches found in intestinal mucosa . However, the current methodologies to produce particles within desired range involves organic solvents and several steps. In this study, spray drying was investigated as a microencapsulation alternative, as it offers the potential for single step operation, producing dry particles, with the potential for extending the microparticle size into the nano-range. The particles were produced by spray drying of alginate/protein solutions. The effect of spray drying operational parameters on particle properties such as recovery, residual activity and particle size was studied. Particle recovery depended on the inlet temperature of the drying air, whereas the particle size was affected by the feed rate and the alginate concentration of the feed solution. Increase in alginate:protein ratio increased protein stability during the process and shelf live experiments. Presence of 0.2 g trehalose/g particle increased the residual activity up to 90%. The resulting spherical micro and nanoparticles had smooth surfaces. Stable glycol-chitosan-ca-alginate particles were produced with single step operation. The resulting particles had mean diameter around 3.5μm and released 35% of the initial protein content to the simulated stomach environment within 2 hours. The protein distribution within the particle was studied by confocal laser scanning microscope with florescent labeled protein. The image showed protein deposition toward the surface of the particles. Total drying time and Peclet number was calculated for the particles and found to be 8.5 ms and 240, which indicates that particle formation was governed mainly by convection, which resulted in a hollow central region and protein distribution toward the particle surface. This study shows that stable alginate particles containing proteins can be produced in a single step by spray drying, where the particles had a mean size lower than the critical diameter necessary to be orally absorbed by M cell’s of the Peyer’s patches in the gastrointestinal tract and thus can be considered as a promising technology for oral peptide and protein delivery. / Thesis (Master, Chemical Engineering) -- Queen's University, 2007-10-30 12:20:47.728

The production of free flowing powder by spray drying of sugar-acid rich foods requires an appropriate carrier. High molecular weight materials such as maltodextrins are commercially used as a drying aid because of their high glass transition temperature (Tg). Alternatively, fibre-rich by-products from fruit and vegetable juice processing might provide high molecular weight elements that are suitable as a drying support. This study aimed to understand the variables affecting the spray-dried product of fruit juice so that non-sticky fibre-based juice powder could be obtained. Freeze dried carrot fibre was centrifically-milled to 50-100 µm sizes. Three sugar determination methods; enzymatic, enzyme membrane and HPLC with RID, were compared. The freeze drying performance of fructose, fructose + carrot fibre and fructose + carrot fibre + malic acid had the glass transition temperatures measured by differential scanning calorimetry (DSC) at 0.1 °C min-1. The results from the freeze drying were used as a key for the possibility of spray dried apple juice + carrot fibre. Similar methods were used to study freeze dried fructose + maltodextrin (DE max 9.8) and fructose + maltodextrin + malic acid. Dried sucrose, glucose and fructose were used to study glass transition temperature of melted amorphous sugars and mixtures by the visual experiment and DSC at 0.1°C min-1 of heating and cooling scans. The Gordon-Taylor equation was used to predict the Tg of anhydrous two-sugar mixtures from experimental and literature data. The Coachman and Karaze equation was used to predict Tg of three-sugar mixtures and compared to the experimental data. Spray dried powders of fructose + carrot fibre of 30, 40, 50, 60 and 70% w/w and apple juice concentrate + carrot fibre of 30, 40, 50, 60, 70% w/w at 165/75°C inlet/outlet temperature in a laboratory scale drier were compared to that of fructose + maltodextrin (DE max 9.8) and apple juice concentrate + maltodextrin of 50, 60 and 70% w/w (dry basis). Dielectric analysis in the range 200 Hz -1 MHz between 10-105 °C were applied to find the onset Tg (based on DSC results) from freeze dried mixtures of 14, 21, and 28% w/w (dry basis) carrot fibre+ fructose. The enzymatic method was found to be the most accurate method for sugar determination of fruit juice but the HPLC method was the most practical one. The results of Tg values of sugars and mixtures melted showed that the Tg values from heating and cooling scans of fructose, glucose and sucrose were in good agreement with literature. Fructose acted as a plasticizer; an increase in the fructose fraction decreased the Tg of sugar mixtures. Sucrose increased the Tg of the mixtures while the Tg of the three-sugar mixtures was less variable when there was a moderate to high proportion of glucose. The visual Tg values of sugars and mixtures were 7-28 °C higher than the onset DSC heating and cooling Tg values. This result suggested that more than one method should be used to study the glass transition of substances. The Gordon-Taylor equation did not fit well the Tg values of the dry sugars and their mixtures from this experiment. The variations might have been due to the degradation of sugar samples on the melting process. The Coachman and Karaze equation gave a good prediction of the three-sugar mixtures from this experiment. The carrot fibre was found to be crystalline. Carrot fibre increased the Tg of freeze dried fructose and decreased stickiness of fructose. Increasing malic acid fraction decreased Tg of the mixtures. Freeze dried fructose + maltodextrin showed higher hygroscopicity than freeze dried fructose + carrot fibre. It was not possible to determine Tg of fructose + maltodextrin + malic acid due to the swelling and hygroscopicity of the freeze dried samples. Tg values of freeze dried fructose + carrot fibre and fructose + maltodextrin were found to high enough to allow spray drying of these mixtures. The minimum fraction of carrot fibre to facilitate spray drying of fructose and apple juice concentrate was found to be 30%. Mixtures with maltodextrin at a fraction lower than 50% could not be successfully spray dried. When spray drying fructose + carrot fibre, apple juice + carrot fibre, fructose + maltodextrin and apple juice + maltodextrin at the appropriate ratios most of the powder stuck to the drier walls. The powder swept from the wall was free flowing with moisture content of approximately 2-4%. The Tg values of these powder indicated the wall build-up might be avoided in larger scale drying. Tg values of spray dried powder from the mixtures with fibre and maltodextrin were found to be not very different. The yield from mixtures with carrot fibre was three times higher than those of mixtures with maltodextrin. This cast doubts that Tg alone could be a good indicator for the stickiness of spray dried material. The microscope images and DSC scans of spray dried powders of fructose + carrot fibre and apple juice + carrot fibre showed crystalline material. The particle of spray dried fructose + maltodextrin and apple juice + maltodextrin were mostly amorphous. The crystals are more physically and chemically stable than the amorphous form. Thus carrot fibre is a good additive in spray drying of fruit juice. Dielectric analysis at low frequency was able to possible detect Tg of single and double components. For food polymer with many components it was found that Tg value was not consistently dependent on frequency. In conclusion, carrot fibre was a more effective carrier for spray drying than maltodextrin when compared on a mass basis and spray drying condition. Since edible fibre is an essential element needed by the human body, spray drying of fruit juice using fibre as a carrier showed the great potential of fibre in the application of fruit juice spray drying. In the case of clear juice, after reconstitution, the fibre can be easily separated from the juice as there seemed to be no chemical binding between the juice and the fibre during the spray drying process.

The fortification of salt with iron may reduce the prevalence of iron deficiency globally, but fortification is complicated by iron-iodiate interactions. To minimize this interaction, a spray dry microencapsulation system was developed. This study evaluated the creation and use of this system, and produced engineered iron premixes for integration into coarse iodized salt. Bioavailable ferrous fumarate powders were encapsulated to produce small particles (<20μm). Feed systems containing both suspended and dissolved ferrous fumarate were compared to find optimal conditions. The premixes were blended into iodized salt at 1000ppmiron and stored at 40C, ~60%RH. The salt was sampled periodically for 6 months to evaluate iodine stability. All encapsulated samples showed increased stability. The capsules ability to adhere to the salt as well as its colour and apparent bioavailability were evaluated. The evaluated samples indicate that stable double-fortified salt based on inexpensive, coarse, unrefined salt may be obtained economically by this approach.

The fortification of salt with iron may reduce the prevalence of iron deficiency globally, but fortification is complicated by iron-iodiate interactions. To minimize this interaction, a spray dry microencapsulation system was developed. This study evaluated the creation and use of this system, and produced engineered iron premixes for integration into coarse iodized salt. Bioavailable ferrous fumarate powders were encapsulated to produce small particles (<20μm). Feed systems containing both suspended and dissolved ferrous fumarate were compared to find optimal conditions. The premixes were blended into iodized salt at 1000ppmiron and stored at 40C, ~60%RH. The salt was sampled periodically for 6 months to evaluate iodine stability. All encapsulated samples showed increased stability. The capsules ability to adhere to the salt as well as its colour and apparent bioavailability were evaluated. The evaluated samples indicate that stable double-fortified salt based on inexpensive, coarse, unrefined salt may be obtained economically by this approach.

Thesis (Ph.D.)--University of Wollongong, 2003. / Typescript. Includes bibliographical references: leaf 151-160.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

abstract: The behavior of a solid oxide fuel cell (SOFC) cermet (ceramic-metal composite) anode under reaction conditions depends significantly on the structure, morphology and atomic scale interactions between the metal and the ceramic components. In situ environmental transmission electron microscope (ETEM) is an important tool which not only allows us to perform the basic nanoscale characterization of the anode materials, but also to observe in real-time, the dynamic changes in the anode material under near-reaction conditions. The earlier part of this dissertation is focused on the synthesis and characterization of Pr- and Gd-doped cerium oxide anode materials. A novel spray drying set-up was designed and constructed for preparing nanoparticles of these mixed-oxides and nickel oxide for anode fabrication. X-ray powder diffraction was used to investigate the crystal structure and lattice parameters of the synthesized materials. Particle size distribution, morphology and chemical composition were investigated using transmission electron microscope (TEM). The nanoparticles were found to possess pit-like defects of average size 2 nm after subjecting the spray-dried material to heat treatment at 700 &deg;C for 2 h in air. A novel electron energy-loss spectroscopy (EELS) quantification technique for determining the Pr and Gd concentrations in the mixed oxides was developed. Nano-scale compositional heterogeneity was observed in these materials. The later part of the dissertation focuses mainly on in situ investigations of the anode materials under a H2 environment in the ETEM. Nano-scale changes in the stand-alone ceramic components of the cermet anode were first investigated. Particle size and composition of the individual nanoparticles of Pr-doped ceria (PDC) were found to affect their reducibility in H2 gas. Upon reduction, amorphization of the nanoparticles was observed and was linked to the presence of pit-like defects in the spray-dried material. Investigation of metal-ceramic interactions in the Ni-loaded PDC nanoparticles indicated a localized reduction of Ce in the vicinity of the Ni/PDC interface at 420 &deg;C. Formation of a reduction zone around the interface was attributed to H spillover which was observed directly in the ETEM. Preliminary results on the fabrication of model SOFCs and in situ behavior of Ni/Gd-doped ceria anodes have been presented. / Dissertation/Thesis / Ph.D. Materials Science and Engineering 2011

Introdução e objetivos: As nanocápsulas, uma vez que são produzidas com polímeros, representam sistemas mucoadesivos promissores. O uso desse tipo de sistema é importante no delineamento de medicamentos que vislumbrem a membrana sublingual como via de administração, devido ao constante fluxo de saliva. Em vista disso, esse trabalho tem como objetivos: estudar o efeito da nanoestruturação em nanocápsulas de polímeros de diferentes características iônicas, quanto as suas propriedades mucoadesivas, quando veiculadas em suspensão, hidrogel ou pós, e frente a distintas superfícies mucoadesivas (discos de mucina, mucosa vaginal ou mucosa bucal); desenvolver nanocápsulas contendo carvedilol, avaliando as suas propriedades mucoadesivas e perfil de permeação do fármaco em diferentes modelos de membrana sublingual; e produzir, a partir das nanocápsulas secas, comprimidos sublinguais contendo carvedilol nanoencapsulado. Metodologia: Nanocápsulas formadas por Eudragit® RS100, Eudragit® S100 ou poly(ε-caprolactona) [PCL] foram produzidas pelo método de deposição interfacial do polímero. Suas propriedades mucoadesivas foram avaliadas empregando analisador de textura. As nanocápsulas contendo carvedilol foram produzidas pelo mesmo método citado acima, utilizando Eudragit® RS100 e a PCL. A mucoadesão dessas nanocápsulas foi avaliada quanto a sua interação com moléculas de mucina, além do efeito da sua interação com a mucosa sublingual de porco na permanência do fármaco sobre a mucosa e na sua permeação, em presença de um fluxo salivar mimetizado. O transporte de carvedilol através de uma monocamada celular de células de epitélio oral (SCC4) também foi estudado. As suspensões de nanocápsulas foram, então, secas por aspersão e as propriedades das nanocápsulas redispersas foram reavaliadas. Na última etapa, foram produzidos comprimidos sublinguais pelo método de compressão direta, a partir dos pós desenvolvidos. Resultados: A mucoadesividade dos polímeros Eudragit® RS100, Eudragit® S100 e PCL foi potencializada pela sua estruturação em nanocápsulas. Dentre as formulações analisadas, as nanocápsulas catiônicas, formadas por Eudragit® RS100, veiculadas em gel, foram as que apresentaram melhores propriedades adesivas. Além disso, o processo de secagem não interferiu na adesividade das nanocápsulas originais. Em relação a superfície utilizada, a mucina se mostrou uma superfície mais adesiva comparada as mucosas suínas. Entretanto, a mucina reproduziu as diferenças observadas entre as formulações. As nanocápsulas contendo carvedilol interagiram bem com moléculas de mucina, sendo essa interação mais intensa para as nanocápsulas catiônicas [Eudragit® RS100], que para as aniônicas [PCL]. No entanto, ambas as nanocápsulas melhoraram o contato do carvedilol com a mucosa sublingual suína, o que fez com que mais fármaco permeasse através da mucosa, na presença de um fluxo salivar mimetizado, em comparação com uma solução do fármaco. Além disso, as nanocápsulas controlaram a permeação do fármaco através de mucosa sublingual de porco, bem como através de monocamadas de células SCC4. A partir destes resultados, as suspensões de nanocápsulas foram secas por aspersão. As nanopartículas foram recuperadas após redispersão aquosa dos pós e mantiveram suas propriedades mucoadesivas e biofarmacêuticas. Na sequência, os comprimidos foram produzidos como forma farmacêutica final. A presença de nanoestruturas foi observada nos comprimidos, as quais foram liberadas após total desintegração destes em saliva artificial. Além disso, a liberação do fármaco partir dos comprimidos contendo as nanocápsulas apresentou um perfil controlado comparado aos comprimidos contendo o fármaco livre, reforçando a manutenção da estrutura supramolecular das nanocápsulas nos comprimidos. Conclusão: As nanocápsulas produzidas com Eudragit® RS100, Eudragit® S100 ou PCL apresentaram boas características mucoadesivas. As, nanocápsulas de Eudragit® RS100 e PCL também melhoraram a interação do carvedilol com a membrana sublingual de porco. Em ambos os estudos, um melhor desempenho mucoadesivo foi observado para as nanocápsulas catiônicas. Além disso, o carvedilol apresentou boa permeação através de mucosa sublingual suína e através de monocama celular de células de epitélio oral. Ainda, a secagem por aspersão das suspensões de nanocápsulas não alterou significativamente as suas propriedades. A compressão direta dos pós secos por aspersão produziu comprimidos inovadores contendo um sistema nanotecnológico mucoadesivo para administração sublingual de carvedilol, como um nanomedicamento. / Introduction and objectives: Nanocapsules may represent promissing mucoadhesive systems since they are produced with polymers. The use of these systems is very important for drug administration by the sublingual route due to the constantly salivary flux in the oral cavity. In view of this, the objectives of this study were: to study the effect of the nanostructuration in nanocapsules on the mucoadhesiveness of polymers with different charge surface and the effect of the vehicle (suspension, hydrogel, and powder) on the mucoadhesiveness of nanocapsules as well as the effect of different mucosal surfaces (mucin, vaginal mucosa, and buccal mucosa); to develop carvedilol-loaded nanocapsules and to evaluate their mucoadhesive properties and drug permeation profiles using different models of sublingual membrane; and to produce sublingual tablets using spray-dried carvedilol-loaded nanocapsules. Methods: Eudragit®RS100, Eudragit®S100 or poly(ε-caprolactone) [PCL] nanocapsules were produced by interfacial deposition of the polymer method. Their mucoadhesiveness were evaluated by tensile stress tester. Carvedilol-loaded nanocapsules were produced by the method cited above and using Eudragit® RS100 or PCL as polymers. Mucoadhesiveness of nanocapsules were studied analyzing their interaction with mucin molecules and analyzing the effect of their interaction with porcine sublingual mucosa on drug retention as well on the amount of drug permeated to the receptor fluid in the presence of simulated salivary flux. The transport of carvedilol across monolayers of oral epithelial cells (SCC4) was also evaluated. In the next step, nanocapsules suspensions were spray-dried and the properties of redispersed nanocapsules were evaluated. In the last step, sublingual tablets were produced by direct compression using the spray-dried nanocapsules. Results: Mucoadhesiveness of Eudragit® RS100, Eudragit® S100 and PCL were improved by their structuration in nanocapsules. Among the tested formulations, the cationic Eudragit® RS100 nanocapsules formulated as a hydrogel showed the best behavior. Moreover, the drying process did not interfer in the adhesiveness of original nanocapsules. Regarding the surface substrate, mucin discs were more adhesive than porcine mucosas. However, mucin was able to reproduce the differences observed between the formulations. Carvedilol-loaded nanocapsules interacted with mucin molecules and this interaction was more intense for cationic Eudragit® RS100 nanocapsules than for anionic PCL nanocapsules. However, both nanocapsules increased the amount of drug retained on porcine sublingual mucosa and improved the amount of drug permeated through mucosa, in comparison to the drug solution, in presence of a mimetic salivary flux was present. Furthermore, nanocapsules were able to control the drug permeation across porcine sublingual and through SCC4 monolayer. Subsequently, suitable powders were obtained by spray-drying. The original nanoparticles were recovered after aqueous redispersion of powders and the maintenance of their mucoadhesiveness and biopharmaceutics properties was observed. Moreover, sublingual tablets were produced as a final pharmaceutical form. The presence of nanometric particles in the tablets was observed and they were released after tablet disintegration in artififcial saliva. The drug was released by a controlled way from tablets containing nanocapsules when compared to tablets containing the non-encapsulated drug, reinforcing the maintenance of supramolecular structure of nanocapsules in the tablets. Conclusion: The Eudragit® RS100, Eudragit® S100 and PCL nanocapsules showed good mucoadhesive characteristics. Moreover, Eudragit® RS100 and PCL nanocapsules improved the carvedilol interaction with porcine sublingual mucosa. In both studies, cationic nanocapsules showed the best mucoadhesive performance. Additionally, carvedilol showed a good permeation across porcine sublingual mucosa and through oral epithelial cells monolayer. The spray-drying process did not change the properties of the original aqueous nanocapsules. Furthermore, their direct compression produced innovative tablets containing a mucoadhesive nanotechnological system for sublingual administration of carvedilol as a nanomedicine.