Duas variações de resíduo agroindustrial foram analisadas como meio de cultura para o bioprocesso de fermentação semissólida pelo fungo Fusarium oxysporum, com o objetivo de obter a melhor produção de peptidases e lipases. Essas enzimas foram microencapsuladas por spray drying, visando garantir sua estabilidade e investigar outros prováveis benefícios obtidos pela técnica. A utilização de planejamento experimental permitiu analisar os efeitos e interações entre as variáveis operacionais do processo (temperatura de secagem, proporção de adjuvantes e relação entre adjuvantes). A caracterização bioquímica e físico-química do extrato enzimático e das micropartículas também foram estudadas. O emprego de farelo de trigo como substrato demonstrou maior produção enzimática que o uso de farelo de algodão. A fermentação produziu uma serinopeptidase e uma lipase, ambas com característica alcalina, com alta estabilidade em ampla faixa de pH e certa estabilidade em diferentes temperaturas. Para ambas as enzimas, observou-se modulação positiva da atividade frente à maioria dos íons estudados e forte inibição pelo surfactante SDS, enquanto a lipase demonstrou superatividade frente a CTAB. A caracterização enzimática permite sugerir a aplicação dessas enzimas na formulação de detergentes enzimáticos, indústria de couro, indústria de papel, agroquímicos, síntese de biopolímeros e biodísel. No processo de microencapsulação, a temperatura foi a variável operacional mais importante para a estabilidade, enquanto a quantidade de adjuvantes em relação à quantidade de extrato enzimático influenciou nas condições de manipulação. O estudo demonstrou que a técnica de microencapsulação por spray drying resultou em grande benefício no armazenamento das enzimas, por aumentar consideravelmente sua estabilidade e melhorar as propriedades físicas do extrato. / Two variations of agroindustrial residue were analyzed as culture medium for the bioprocess of solid-state fermentation by the fungus Fusarium oxysporum, in order to achieve the best production of peptidases and lipases. These enzymes were microencapsulated by spray drying in order to ensure its stability and investigate other potential benefits obtained by the technique. The use of experimental design allowed us to analyze the effects and interactions between the operating variables of the process (drying temperature, proportion of adjuvants and relation among adjuvants). Biochemical and physico-chemical characterization of the enzymatic extract and of the microparticles were also studied. The use of wheat bran as substrate demonstrated a greater enzyme production then the use of cottonseed meal. The fermentation produced serinepeptidases and lipases, both alkaline, with high stability over a wide pH range and some stability at different temperatures. For both enzymes, there was up regulation of activity with most of the ions analyzed and strong inhibition against the surfactant SDS, whereas lipase demonstrated superactivity against CTAB. The enzymatic characterization suggests the application of these enzymes in the formulation of enzymatic detergents, leather industry, paper industry, agrochemicals, synthesis of biopolymers and biodiesel. In the microencapsulation process, the temperature was the most important operating variable for stability, while the amount of adjuvants in relation to the amount of enzymatic extract influenced in terms of handling. The study demonstrated that the technique of microencapsulation by spray drying resulted in benefits for the storage of enzymes, by increasing considerably its stability and improve the physical properties of the extract.

Duas variações de resíduo agroindustrial foram analisadas como meio de cultura para o bioprocesso de fermentação semissólida pelo fungo Fusarium oxysporum, com o objetivo de obter a melhor produção de peptidases e lipases. Essas enzimas foram microencapsuladas por spray drying, visando garantir sua estabilidade e investigar outros prováveis benefícios obtidos pela técnica. A utilização de planejamento experimental permitiu analisar os efeitos e interações entre as variáveis operacionais do processo (temperatura de secagem, proporção de adjuvantes e relação entre adjuvantes). A caracterização bioquímica e físico-química do extrato enzimático e das micropartículas também foram estudadas. O emprego de farelo de trigo como substrato demonstrou maior produção enzimática que o uso de farelo de algodão. A fermentação produziu uma serinopeptidase e uma lipase, ambas com característica alcalina, com alta estabilidade em ampla faixa de pH e certa estabilidade em diferentes temperaturas. Para ambas as enzimas, observou-se modulação positiva da atividade frente à maioria dos íons estudados e forte inibição pelo surfactante SDS, enquanto a lipase demonstrou superatividade frente a CTAB. A caracterização enzimática permite sugerir a aplicação dessas enzimas na formulação de detergentes enzimáticos, indústria de couro, indústria de papel, agroquímicos, síntese de biopolímeros e biodísel. No processo de microencapsulação, a temperatura foi a variável operacional mais importante para a estabilidade, enquanto a quantidade de adjuvantes em relação à quantidade de extrato enzimático influenciou nas condições de manipulação. O estudo demonstrou que a técnica de microencapsulação por spray drying resultou em grande benefício no armazenamento das enzimas, por aumentar consideravelmente sua estabilidade e melhorar as propriedades físicas do extrato. / Two variations of agroindustrial residue were analyzed as culture medium for the bioprocess of solid-state fermentation by the fungus Fusarium oxysporum, in order to achieve the best production of peptidases and lipases. These enzymes were microencapsulated by spray drying in order to ensure its stability and investigate other potential benefits obtained by the technique. The use of experimental design allowed us to analyze the effects and interactions between the operating variables of the process (drying temperature, proportion of adjuvants and relation among adjuvants). Biochemical and physico-chemical characterization of the enzymatic extract and of the microparticles were also studied. The use of wheat bran as substrate demonstrated a greater enzyme production then the use of cottonseed meal. The fermentation produced serinepeptidases and lipases, both alkaline, with high stability over a wide pH range and some stability at different temperatures. For both enzymes, there was up regulation of activity with most of the ions analyzed and strong inhibition against the surfactant SDS, whereas lipase demonstrated superactivity against CTAB. The enzymatic characterization suggests the application of these enzymes in the formulation of enzymatic detergents, leather industry, paper industry, agrochemicals, synthesis of biopolymers and biodiesel. In the microencapsulation process, the temperature was the most important operating variable for stability, while the amount of adjuvants in relation to the amount of enzymatic extract influenced in terms of handling. The study demonstrated that the technique of microencapsulation by spray drying resulted in benefits for the storage of enzymes, by increasing considerably its stability and improve the physical properties of the extract.

Foram analisados alguns parâmetros que influenciam os bioprocessos, submerso e sólido, do fungo Eupenicillium javanicum na produção de peptidases. No bioprocesso submerso foram avaliados a influência de diferentes concentrações e tipos de fonte de carbono e concentrações de fonte nitrogênio no meio, pH, temperatura e tempo de incubação. No bioprocesso sólido avaliou-se a influência de dois tipos de resíduos agroindustriais, em diferentes proporções, dois tipos de fonte de nitrogênio, em diferentes concentrações, tempo e temperatura de incubação. As peptidases produzidas em ambos os bioprocessos foram caracterizadas bioquimicamente, avaliando pH e temperatura ótima, estabilidade em diferentes temperaturas e valores de pH e influência da adição de íons e inibidores na atividade da peptidase. A enzima produzida em bioprocesso sólido foi submetida ao processo de purificação utilizando métodos cromatográficos. Utilizando a peptidase pura realizou-se a caracterização bioquímica funcional e a determinação dos parâmetros cinéticos, ambos utilizando o substrato peptídico de supressão intramolecular de fluorescência. Além disso, o extrato enzimático obtido através do bioprocesso foi submetido ao processo de microencapsulação, visando uma maior estabilidade das peptidases e facilidade no armazenamento e transporte, utilizando a técnica de Spray drying, em seguida foi avaliado o rendimento do processo e a estabilidade das peptidases das micropartículas produzidas. O fungo Eupenicillium javanicum mostrou potencial na produção de peptidases em ambos bioprocessos, produzindo peptidases da classe das metalopeptidases com alta estabilidade em diferentes valores de pH e temperatura. O processo de purificação mostrou-se viável e reprodutível. A análise dos parâmetros cinéticos revelou uma grande influência do lado \"linha\" da peptidase na eficiência catalítica. O processo de microencapsulação mostrou-se viável e gerou micropartículas estáveis. A peptidase produzida apresentou características que demonstram seu potencial uso nas diferentes áreas industriais. / Some parameters that influence the submerged and semi solid bioprocesses, by the fungus Eupenicillium javanicum in the peptidases production, were conducted. In submerged bioprocess was evaluated the effect of different concentrations and types of carbon source and nitrogen source in the medium, pH, temperature and incubation time. In semi solid bioprocess semi solid was evaluated the influence of two types of agroindustrial residues, in different proportions, and different concentrations of nitrogen source, temperature and of incubation time. The peptidases produced in both bioprocesses were characterized biochemically, evaluating, the optimum pH and temperature, stability at different temperatures and pH values and the influence of the addition of ions and inhibitors on peptidase activity. The enzyme produced in semi solid bioprocess was subjected to the purification process using chromatographic methods. Using pure peptidase was performed the biochemical characterization and determination of kinetic parameters, both using the fluorescence intramolecular suppression peptide substrate. Furthermore, the enzymatic extract obtained by semi solid bioprocess was subjected to microencapsulation process by using the technique of spray drying, in order to obtain greater stability, ease storage and transport of peptidases, then assessed the yield process and stability of the peptidases of microparticles produced. The fungus Eupenicillium javanicum showed potential production of peptidases in the both bioprocesses, producing peptidases that belong to the class of metallopeptidases, with high stability at different pH and temperature. The purification process was feasible and reproducible. The analysis of kinetic parameters revealed a strong influence of the \"line\" side on the peptidase catalytic efficiency. The microencapsulation process was feasible and generated stable microparticles. The peptidase produced has characteristics that demonstrated it a potential use in different industrial fields.

A method was developed to produce and incorporate a network of discrete, genipin-crosslinked gelatin microfibers around a pancreatic islet within a barium alginate microcapsule. This technique allows for the encapsulation of a porous fibrous matrix without the geometrical restrictions required for cellular aggregate seeding. Microfibers were produced from a novel vortex-drawn extrusion system with an alginate support matrix. Optimization culminated in a hydrated fiber diameter of 22.3 ± 0.4 μm, a 98% reduction in cross sectional area, while making the process more reliable and less labour intensive. The optimized microfibers were encapsulated at 40 vol% within 294 ± 4 μm 1.6% barium alginate microparticles by an electrostatic-mediated dropwise extrusion system. Pancreatic islets extracted from Sprague Dawley rats were encapsulated within the microparticles, and analyzed over a 21-day preliminary in vitro study. Acridine orange and propidium iodide fluorescent viability staining and light microscopy indicated a significant increase in viability for the fiber-laden particles relative to fiber-free control particles at days 7, 14, and 21. The fiber-laden system also reduced the incidence of disrupted islet cohesion from 31% to 8% at day 21, and showed evidence of islet-fiber adhesion. Preliminary investigations into insulin secretion and metabolic activity showed no significant difference between test and control groups. Further investigation into benefits of islet encapsulation within an extracellular matrix fiber network will be the subject of future studies with this body of work serving as a foundation. The system developed in this investigation could be developed into a modular scaffold system for tissue engineering beyond the field of islet research. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-08-18 15:05:50.917

Foram analisados alguns parâmetros que influenciam os bioprocessos, submerso e sólido, do fungo Eupenicillium javanicum na produção de peptidases. No bioprocesso submerso foram avaliados a influência de diferentes concentrações e tipos de fonte de carbono e concentrações de fonte nitrogênio no meio, pH, temperatura e tempo de incubação. No bioprocesso sólido avaliou-se a influência de dois tipos de resíduos agroindustriais, em diferentes proporções, dois tipos de fonte de nitrogênio, em diferentes concentrações, tempo e temperatura de incubação. As peptidases produzidas em ambos os bioprocessos foram caracterizadas bioquimicamente, avaliando pH e temperatura ótima, estabilidade em diferentes temperaturas e valores de pH e influência da adição de íons e inibidores na atividade da peptidase. A enzima produzida em bioprocesso sólido foi submetida ao processo de purificação utilizando métodos cromatográficos. Utilizando a peptidase pura realizou-se a caracterização bioquímica funcional e a determinação dos parâmetros cinéticos, ambos utilizando o substrato peptídico de supressão intramolecular de fluorescência. Além disso, o extrato enzimático obtido através do bioprocesso foi submetido ao processo de microencapsulação, visando uma maior estabilidade das peptidases e facilidade no armazenamento e transporte, utilizando a técnica de Spray drying, em seguida foi avaliado o rendimento do processo e a estabilidade das peptidases das micropartículas produzidas. O fungo Eupenicillium javanicum mostrou potencial na produção de peptidases em ambos bioprocessos, produzindo peptidases da classe das metalopeptidases com alta estabilidade em diferentes valores de pH e temperatura. O processo de purificação mostrou-se viável e reprodutível. A análise dos parâmetros cinéticos revelou uma grande influência do lado \"linha\" da peptidase na eficiência catalítica. O processo de microencapsulação mostrou-se viável e gerou micropartículas estáveis. A peptidase produzida apresentou características que demonstram seu potencial uso nas diferentes áreas industriais. / Some parameters that influence the submerged and semi solid bioprocesses, by the fungus Eupenicillium javanicum in the peptidases production, were conducted. In submerged bioprocess was evaluated the effect of different concentrations and types of carbon source and nitrogen source in the medium, pH, temperature and incubation time. In semi solid bioprocess semi solid was evaluated the influence of two types of agroindustrial residues, in different proportions, and different concentrations of nitrogen source, temperature and of incubation time. The peptidases produced in both bioprocesses were characterized biochemically, evaluating, the optimum pH and temperature, stability at different temperatures and pH values and the influence of the addition of ions and inhibitors on peptidase activity. The enzyme produced in semi solid bioprocess was subjected to the purification process using chromatographic methods. Using pure peptidase was performed the biochemical characterization and determination of kinetic parameters, both using the fluorescence intramolecular suppression peptide substrate. Furthermore, the enzymatic extract obtained by semi solid bioprocess was subjected to microencapsulation process by using the technique of spray drying, in order to obtain greater stability, ease storage and transport of peptidases, then assessed the yield process and stability of the peptidases of microparticles produced. The fungus Eupenicillium javanicum showed potential production of peptidases in the both bioprocesses, producing peptidases that belong to the class of metallopeptidases, with high stability at different pH and temperature. The purification process was feasible and reproducible. The analysis of kinetic parameters revealed a strong influence of the \"line\" side on the peptidase catalytic efficiency. The microencapsulation process was feasible and generated stable microparticles. The peptidase produced has characteristics that demonstrated it a potential use in different industrial fields.

The aim of our experiment was proving the suitability and effectivity of artemia nauplii enrichment in pikeperch fry rearing. 24 hour enrichment took place in 3 variants: 1. SELCO, 2. 18 (n-3) (alfa-linolenic acid) and 3. 18 (n-3) + vitamin C. Original intention was initializing fry feeding with different types of enriched artemia metanauplii. Due to excessive size of artemia after 1 day enrichment (Artemia franciscana) (in comparison with size of fry oral cavity) the intake of such food was not possible, thus the metodics had to be modified. From the given reason, fish were initially fed with unenriched nauplii ; firstly with dose 100 ml (4 - 8 day post hatch - dph) after that with dose 50 ml per tank (9. dph 25.4.). As soon as fry reached suitable size and was able to intake bigger size of nauplii, was in the second part feeding diferenciated (use of different types of enrichment and control group was fed with unenriched artemia). First part of experiment lasted 22 days (5.4. - 26.4.), fish must have been netted and again placed in tanks for experiment with enriched artemia. Second part of rearing varied in use of differently enriched metanauplii and lasted 8 days (27.4. - 4.5.). At the end of second part of feeding (from 1.5.) was involved co-feeding, which means combined feeding (living food and starter feed). 5.5. was experiment terminated. The highest survival was reached in group fed with artemia enriched in comercial preparate SELCO (28,67 %), second highest survival was reached in group fed metanauplii enriched in alfa-linolenic acid (27,88 %) and third highest survival was reached in group fed with alfa-linolenic acid and vitamin C enriched metanauplii (19,00 %). The highest individual fry weight at the end of experiment was reached in group fed with artemia enriched in alfa-linolenic acid and vitamin C : 306,16 +- 64,27 mg, the lowest individual weight was reached in control group : 216,9 +- 39,96 mg. The highest average total lenght (l.t.) was measured in SELCO group 30,13 +- 2,47 mm, the lowest total lenght reached fish in control group 27,37 +- 1,32 mm. In group fed with artemia enriched in vitamin C was noticed the highest percentage of starter feed intake (8 %), but simultaneously in the same group was reached the highest rate of cannibalism (6 %).

Le travail décrit dans cette thèse a été mené à l'interface entre trois disciplines: l'électrochimie, la science des matériaux et la microbiologie. L'objectif de cette recherche était tout d'abord d'étudier l'activité de bactéries immobilisées dans un film de silice déposé par le procédé sol-gel à la surface d'électrodes. Les applications potentielles de ce travail fondamental sont les biocapteurs, les bioréacteurs ou biopiles. L'encapsulation bactérienne assistée par électrochimie a été développée en utilisant l'électrolyse du sol de départ pour immobiliser la bactérie Escherichia Coli dans une couche mince sol-gel hybride. La combinaison de précurseurs de silice, de chitosan, de poly(ehtylène glycol) et de tréhalose permet de préserver l'intégrité membranaire et l'activité métabolique. L'électrochimie a ensuite été utilisée comme moyen analytique. Shewanella putrefaciens et Pseudomonas fluorescens ont été encapsulées dans un film à base de silice et les réactions de transfert d'électron de la bactérie à différent médiateurs rédox ont été analysées. Des nanotubes de carbone fonctionnalisés par des espèces ferrocène et la protéine rédox cytochrome c ont été utilisés pour faciliter ce transfert électronique au sein de cette matrice de silice isolante, permettant l'obtention d'un biofilm artificiel. Ces deux types de médiateurs, chimique ou biologique, ont conduit à des sensibilités différentes de la bioélectrode à l'ajout du substrat pourvoyeur d'électron en raison des mécanismes différents impliqués pour transférer ces électrons. L'immobilisation de protéines rédox membranaires a également été considérée dans ces couches minces inorganiques pour favoriser la stabilité de la réponse électrocatalytique. Les protéines considérées impliquent des mécanismes de transfert électronique différents, soit direct pour le cytochrome P450 (CYP1A2), soit médié pour la mandélate déshydrogénase. Finalement, l'influence de l'encapsulation dans une matrice sol-gel hybride sur l'infectivité du bactériophage [phi]X174 a été étudiée, montrant l'effet protecteur de la polyéthylènenimine ou du glycérol / The work reported in this thesis has been developed at the interface between three disciplines, i.e., electrochemistry, material science and microbiology. The purpose of this research was first to study the activity of bacteria immobilized in silica-based films prepared by the sol-gel process on electrode surfaces. Potential applications concern biosensors, bioreactors and biofuel cells. Electrochemically assisted bacterial encapsulation has been developed, using sol electrolysis to immobilize Escherichia coli in a hybrid sol-gel layer. The combination of silica precursors, chitosan, poly(ethylene glycol) and trehalose allowed preservation of cell membrane integrity and metabolic activity. Electrochemistry was then considered as an analytical method. Shewanella putrefaciens and Pseudomonas fluorescens have been encapsulated in silica-based films and the electron transfer reactions from bacteria to different redox mediators have been monitored. Single-walled carbon nanotubes functionalized with ferrocene moieties and bovine heart cytochrome c have been considered as redox shuttles to facilitate the electron transfer in the non-conducting silica matrix, leading to the elaboration of artificial biofilms. Interestingly, these two classes of mediator, i.e. chemical and biological, led to different substrate sensitivity because of their different mechanism of interaction with the bacteria. Immobilization of membrane associated redox proteins in sol-gel films have been then considered and applied for electrocatalysis. Direct and mediated electrochemical communication has been investigated between the electrode surface and cytochrome P450 (CYP1A2) or mandelate dehydrogenase, respectively, showing the interest of sol-gel to stabilize the bioelectrocatalytic reaction. Finally, the influence of encapsulation in a hybrid sol-gel matrix on the infectivity of bacteriophage [phi]X174 has been studied and the protective effect of polyethyleneimine or glycerol was shown

One of the pathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque deposition in aging brains by aggregation of amyloid-β (Aβ) peptides. In this study, the effect of chloroplast derived myelin basic protein (MBP) fused with cholera toxin subunit B (CTB) was investigated in advanced diseased stage of human and mouse AD brains. The CTB-fusion protein in chloroplasts facilitates transmucosal delivery in the gut by the natural binding ability of CTB pentameric form with GM1 receptors on the intestinal epithelium. Further, bioencapsulation of the MBP within plant cells confers protection from enzymes and acids in the digestive system. Here, 12-14 months old triple transgenic AD mice were fed with CTB-MBP bioencapsulated in the plant cells for 3 months. A reduction of 67.3% and 33.3% amyloid levels in hippocampal and cortical regions, respectively were observed by immunostaining of brain sections with anti- Aβ antibody. Similarly, 70% decrease in plaque number and 40% reduction of plaque intensity was observed through thioflavin S (ThS) staining that specifically stains amyloid in the AD brain. Furthermore, ex vivo 3xTg AD mice brain sections showed up to 45% reduction of ThS stained amyloid levels when incubated with enriched CTB-MBP in a concentration dependent manner. Similarly, incubation of enriched CTB-MBP with ex vivo postmortem human brain tissue sections with advanced stage of AD resulted up to 47% decrease of ThS stained amyloid plaque intensity. Lastly, lyophilization of plant material facilitates dehydration and long term storage of capsules at room temperature, in addition to increasing CTB-MBP concentration by 17 fold. These observations offer a low cost solution for treatment of even advanced stages of the AD by facilitating delivery of therapeutic proteins to central nervous system to address other neurodegenerative disease.