A oxidação lipídica é uma das principais reações de deterioração em alimentos, provocando perdas em qualidade e valor nutricional. Para evitar este problema a indústria utiliza antioxidantes, substâncias capazes de retardar essas reações. Os compostos fenólicos constituem-se na principal substância antioxidante utilizada e encontrada em alimentos, sendo que estes podem estar presentes em várias matérias primas vegetais e em resíduos agroindustriais, desprezados pela indústria. O objetivo do presente trabalho foi estudar as condições de extração de compostos fenólicos de frutos de acerola (Malpighia emarginata), borra de café (Coffea arabica) e casca de lichia (Litchi chinensis), bem como avaliar sua atividade antioxidante in vitro e sua aplicação em emulsões. Para extração dos compostos fenólicos foi aplicada a metodologia de superfícies resposta, visando a definição da melhor combinação de variáveis (concentração de etanol, temperatura e tempo) para a máxima recuperação de compostos fenólicos. A avaliação da atividade antioxidante se deu pela estabilização do radical DPPH e estudo da cinética da reação em comparação ao antioxidante sintético TBHQ. Além disso, foi avaliada a atividade antioxidante dos extratos adicionados a emulsões em diferentes concentrações (50 a 200 mg.kg-1), submetidas a teste acelerado de oxidação. O estudo das condições de extração evidenciou que os compostos fenólicos de cada matriz devem ser extraídos em condições distintas. Extratos de frutos de acerola apresentaram o maior teor de compostos fenólicos totais quando foi utilizada apenas água como solvente (175,87 mg GAE.g-1), seguido pelos extratos de borra de café e de casca de lichia extraídos com soluções etanólicas (26,37 e 25,87 mg GAE.g-1 respectivamente). O estudo da cinética de estabilização do radical DPPH demonstrou que o extrato de acerola foi o mais eficiente, seguido pelos extratos de casca de lichia e de borra de café (650,53; 43,09 e 38,74 de TEAC, respectivamente). O estudo de aplicação dos extratos em sistema modelo, com acompanhamento da oxidação por meio da determinação de hidroperóxidos formados (mmol.kg-1), indicou que somente o extrato de acerola, em todas as concentrações utilizadas, apresentou efeito comparável ao TBHQ. Os extratos de casca de lichia e de borra de café, em qualquer concentração utilizada, não apresentaram a mesma proteção à oxidação lipídica. Com os resultados obtidos pode-se concluir que cada matriz se comporta de uma forma na extração de compostos fenólicos, e daí vem a necessidade de se realizar estudo das condições de preparo dos extratos para cada amostra. O extrato de acerola se mostrou o mais eficiente, tanto na cinética de estabilização do radical DPPH quanto na inibição da oxidação no sistema lipídico estudado. No entanto mais trabalhos são necessários para avaliar diferentes sistemas de oxidação, bem como estudos toxicológicos para garantir níveis seguros de consumo. / The oxidation is one of major deterioration in food lipids, causing losses in quality and nutritional value. To avoid this, antioxidants are used and the phenolic compounds are the main component of antioxidant applied and presented in foods, and also may be present in agroindustrial residues, usually discarded by the industry. The objective of this study was to identify ideal conditions to extract phenolic compounds from acerola fruits (Malpighia emarginata), coffee grounds (Coffea arabica) and lychee skins (Litchi chinensis) by means of surface response methodology (RSM), evaluation of antioxidant activity of extracts in in vitro essays, application of selected extracts in emulsions systems during accelerated test. The response surface methodology was applied to each material in order to identify the effects of temperature, ethanol concentration and time in extraction processes. Antioxidant activity by DPPH stabilization and its kinectis evaluation were performed and compared to TBHQ. Furthermore, selected extracts (50 to 200 mg.kg-1) were added in emulsion system and subject to accelerated oxidation test. RSM have shown that phenolic compounds of each material are extractable in different conditions. Acerola fruits aqueous extracts present maximum phenolic content (175.87 mg GAE.g-1) and the higher DPPH antioxidant activity, followed by coffee grounds and lychee skins extracts (26.37 and 25.87 mg GAE.g-1). Acerola extracts also presented the best stabilization of DPPH in kinetic study, with results comparable to the TBHQ effect (650.53 TEAC). Acerola extract added to the emulsion showed a good antioxidant effect in all concentrations during accelerated test, resulting in final oxidation levels (mmol hydroperoxides kg-1) comparable to TBHQ in the same concentration. Coffee grounds and lychee skins extracts were not efficient in avoid hydroperoxides increasing. Results showed the importance of studying extractions conditions for each material. Acerola extract showed the most efficient antioxidant activity both in DPPH kinetics DPPH stabilization and inhibition of lipid oxidation in the system studied. However, further work is needed to evaluate different oxidation systems, as well as toxicological studies to ensure safe levels of consumption.

Made available in DSpace on 2017-07-21T18:53:04Z (GMT). No. of bitstreams: 1 Suelen Brazo.pdf: 3105120 bytes, checksum: d7c908a74b56c6c9c7dbcc45ef93c32a (MD5) Previous issue date: 2015-02-13 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Anthocyanins are natural colorants that provide attractive and bright colors to most plants, coloring them the bright red to violet. Due to its instability facing the food processing steps and their difficulty of extraction are still little used in the food industry. Flavonoids not anthocyanins, amino acids, nucleosides, organic acids and anthocyanins themselves may act as copigments enabling the anthocyanins as natural colorants. The objective was to evaluate the approximate composition, phenolic compounds, antioxidant activity of blackberries Brazo (Morus nigra L.) and the stability of anthocyanins. The effect of the addition of organic acids and polyphenolic extract of Camellia sinensis raw extract of blackberries anthocyanins as copigments. The copigmentation reaction buffer at pH 3.0, was confirmed by UV-Visible absorption and by high high-performance liquid chromatography analysis. The calculated half-life and the color retention percentage. The response surface methodology was effective to estimate the effect of three independent variables on anthocyanins extraction, the optimal values suggested for the extraction with citric acid were time of 60 min., ratio of 1 g / 30 mL and a temperature of 60 ºC and the extraction with hydrochloric acid have been suggested the same conditions of time and ratio, but at 20 ° C. The increase in relation copigmento / anthocyanins was evidenced by the bathochromic and hyperchromic effects. The interaction of anthocyanins was significantly affected by temperature (5 ± 3 ° C and 25 ± 3 ° C) and the environment (light and dark). The stability of anthocyanins with added copigmento increased half-life and the color retention percentage of sample. Maximum stability was achieved in samples with the addition of caffeic acid at a temperature of 5 ° C ± 3 ° C in the dark. Anthocyanins added caffeic acid and malic showed half-life of 242.40 ± 25.40 and 200.48 ± 12.15 days, respectively, bringing prospects for application of this natural dye. / Antocianinas são corantes naturais que proporcionam cores atrativas e brilhantes à maioria das plantas, colorindo-as do vermelho vivo ao violeta. Devido à sua instabilidade frente às etapas de processamento de alimentos e a sua dificuldade de extração ainda são pouco utilizados na indústria alimentícia. Flavonóides não antociânicos, aminoácidos, nucleosídios, ácidos orgânicos e as próprias antocianinas podem agir como copigmentos viabilizando as antocianinas como corantes naturais. O objetivo do trabalho foi avaliar a composição aproximada, os compostos fenólicos totais, a atividade antioxidante de amoras Brazo (Morus nigra L.) e a estabilidade das suas antocianinas. Foi avaliado o efeito da adição dos ácidos orgânicos e extrato polifenólico de Camellia sinensis ao extrato bruto de antocianinas de amora como copigmentos. A reação de copigmentação em solução tampão pH 3,0, foi confirmada por espectrofotometria de absorção UV-Visível e por análise cromatográfica líquida de alta eficiência. Calculou-se o tempo de meia vida e a percentagem de retenção de cor. A metodologia de superfície de resposta foi eficaz para estimar o efeito de três variáveis independentes sobre a extração de antocianinas, os ótimos valores sugeridos para a extração com ácido cítrico foram tempo de 60 min., razão de 1g / 30 mL e uma temperatura de 60°C e para a extração com ácido clorídrico foram sugeridas as mesmas condições de tempo e razão, porém a uma temperatura de 20°C. O aumento na relação copigmento/antocianinas foi evidenciado pelos efeitos batocrômico e hipercrômico. A interação das antocianinas foi significativamente afetada pela temperatura (5±3ºC e 25±3ºC) e o ambiente (luz e escuro). A estabilidade das antocianinas com adição de copigmento aumentou o tempo de meia vida e a porcentagem de retenção de cor das amostras. A estabilidade máxima foi alcançada nas amostras com adição de ácido cafeico em temperatura de 5ºC±3°C e no escuro. As antocianinas adicionadas de ácido cafeico e málico apresentaram tempo de meia vida de 242,40 ± 25,40 e 200,48 ± 12,15 dias, respectivamente, trazendo perspectivas para aplicação deste corante natural.

O aumento nos rejeitos industriais e a contínua produção de resíduos causam muitas preocupações no âmbito ambiental. Neste contexto, o descarte de pneus usados tem se tornado um grande problema por conta da pequena atenção que se dá à sua destinação final. Assim sendo, essa pesquisa propõe a produção de uma mistura polimérica com polipropileno (PP), a borracha de etileno-propileno-dieno (EPDM) e o pó de pneu (SRT). A Metodologia de Superfície de Resposta (MSR), coleção de técnicas estatísticas e matemáticas úteis para desenvolver, melhorar e optimizar processos, foi aplicada à investigação das misturas ternárias. Após o processamento adequado em extrusora de dupla rosca e a moldagem por injeção, as propriedades mecânicas de resistência à tração e resistência ao impacto foram determinadas e utilizadas como variáveis resposta. Ao mesmo tempo, a microscopia eletrônica de varredura (MEV) foi usada para a investigação da morfologia das diferentes misturas e melhor interpretação dos resultados. Com as ferramentas estatísticas específicas e um número mínimo de experimentos foi possível o desenvolvimento de modelos de superfícies de resposta e a otimização das concentrações dos diferentes componentes da mistura em função do desempenho mecânico e além disso com a modificação da granulometria conseguimos um aumento ainda mais significativo deste desempenho mecânico. / The increase in industrial waste and solid waste production cause many concerns in the environment. In this context, the disposal of used tires has become a major problem because of the little attention given to their final destination. Therefore, this research proposes the production of a polymer blend of polypropylene (PP), rubber of ethylene-propylene-diene (EPDM) and tire dust (SRT). The Response Surface Methodology (RSM), a collection of mathematical and statistical techniques useful for developing, improving and optimizing processes, was applied to the investigation of ternary mixtures. After proper processing on twin screw extrusion and injection molding, the mechanical properties of tensile and impact strength were determined and used as response variables. At the same time, scanning electron microscopy (SEM) was used to investigate the morphology of different blends and better interpretation of results. With specific statistical tools and a minimum number of experiments it was possible to develop models of response surfaces and the optimization of the concentrations of different components of the mixture depending on the mechanical performance and even with the change in particle size could increase even more significant this mechanical performance.

This dissertation used a person-environment fit theoretical framework to examine the influence of person-job misfit as an organizational stressor on strain and well-being outcomes for emergency responders. Independent variables consisted of job attributes such as skill variety, task identity, task significance, autonomy and job-based feedback. These job characteristics are often used in work redesign efforts as they are amendable to organizational change initiatives. Dependent variables included strain outcomes relevant to those working in emergency services: physical symptoms, burnout, and secondary traumatic stress. Also, to include a positive aspect of emergency services work, the well-being outcome of compassion satisfaction was examined. Data were collected from 358 emergency responders across the United States via online survey, including law enforcement, firefighters, police/fire/medical dispatch, emergency medical technicians, and paramedics. Methodology utilized polynomial regression analysis in which joint linear and curvilinear effects from two predictors upon one outcome correspond to a three-dimensional response surface reflecting the fit-outcome relationship. This approach allowed a detailed examination of the nature of fit and the nature of misfit for each job attribute in relation to strain and well-being. Maximum likelihood with bootstrapping was used to estimate model parameters and test response surface features. Findings identified several influential fit-outcome relationships including skill variety fit-compassion satisfaction (a1 = 0.366), task identity fit-burnout (a2 = -0.083), task significance fit-burnout (a1 = -0.241) task significance fit-compassion satisfaction (a1 = 0.496,), job-based feedback fit-physical symptoms (a1 = -3.807), job-based feedback fit-burnout (a1 = -0.323), and job-based feedback fit-compassion satisfaction (a1 = 0.391). In terms of misfit, task identity misfit was related to secondary traumatic stress (a3 = -0.209) and job-based feedback misfit was related to burnout (a3 = -0.234). Conclusions regarding identified fit-outcome relationships suggested a potential to reduce frequency of physical symptoms, burnout, and secondary traumatic stress and increase employees’ experience of compassion satisfaction by considering employees’ preference for these job characteristics. On the basis of these findings, opportunities for Emergency Services Management agencies to facilitate wellness for personnel, as well as future research directions are discussed.

La banane plantain est un fruit couramment consommé au Congo-Brazzaville sous la forme cuite en tant qu’accompagnement. Le procédé de friture profonde de la banane plantain est une pratique très répandue, souvent mal maitrisé, compte tenu de l’utilisation prolongée des huiles de friture entrainant la formation de composés néfastes à la santé. En effet, depuis un certain nombre d’années, la consommation d’huiles locales (soja, palme) et leurs dégradations après chauffage constituent une des préoccupations principales pour la sécurité sanitaire du consommateur. Les objectifs de cette thèse consistaient à étudier l’influence du procédé de la friture profonde appliqué à la banane plantain Musa AAB, variété « Harton » au stade de maturité 7, consommée sous la forme de disque. Nous avons dans un premier temps appliqué la méthodologie des surfaces de réponse au procédé de friture profonde, afin de déterminer l’influence de trois paramètres (temps / température / quantité) sur différentes réponses (couleur, dureté, teneurs en huile et en eau des produits frits) en fonction de la nature de l’huile utilisée (soja, palme, huile Frial). Une fois les conditions optimales obtenues sur la banane plantain en condition domestique, l’étude s’est focalisée sur l’évolution de la stabilité des huiles de friture par le suivi des paramètres physico-chimiques de dégradation de ces dernières (composés polaires totaux, acides gras libres, composés primaires et secondaires d’oxydation). Des analyses de la couleur, du point de cristallisation et de la viscosité sont venues compléter l’étude et ont permis d’établir la durée d’utilisation optimale de ces huiles en fonction de la quantité de banane plantain à frire. Enfin, les mécanismes de transfert d’eau et d’absorption d’huile dans la matrice ont été étudiés et modélisés en fonction de la température, de l’épaisseur et du temps d’immersion. / Plantain is a fruit commonly consumed in Congo - Brazzaville in its cooked form, as an accompaniment. The process of deep frying plantain is a widespread practice, often poorly mastered, given the prolonged use of frying oils, leading to the formation of compounds harmful to health. Indeed, for a number of years, the consumption of local oils (soybean, palm) and their degradation after heating are cause of major concern for consumer safety. The objectives of this study were to investigate the influences of the deep frying process applied on the Musa AAB plantain of "harton" variety, maturity level 7, as consumed in sliced form. We initially applied response surface methodology (RSM) to the deep frying process to determine the influence of three parameters (time / temperature / quantity) on different responses (color, hardness, oil uptake and water content) according to the type of oil used (soybean, palm, Frial oils). Once optimal conditions were obtained from the plantain in domestic conditions, the study focused on the evolution of frying oil stability, by monitoring physicochemical parameters of the degradation of the aforementioned (total polar compounds, free fatty acids, primary and secondary oxidation products). Analyses of color, crystallization point and viscosity, were added to the study and helped establish the optimal duration of use for these oils depending on the quantity of plantains fried. Lastly, the mechanisms of water transfer and oil uptake in the matrix has been studied and modeled according to temperature, thickness and immersion time.

In many instances arising prominently, but not exclusively, in imaging problems, it is important to condense the salient information so as to obtain a low-dimensional approximant of the data. This thesis is concerned with two basic situations which call for such a dimension reduction. The first of these is the statistical recovery of smooth edges in regression and density surfaces. The edges are understood to be contiguous curves, although they are allowed to meander almost arbitrarily through the plane, and may even split at a finite number of points to yield an edge graph. A novel locally-parametric nonparametric method is proposed which enjoys the benefit of being relatively easy to implement via a `tracking' approach. These topics are discussed in Chapters 2 and 3, with pertaining background material being given in the Appendix. In Chapter 4 we construct concomitant confidence bands for this estimator, which have asymptotically correct coverage probability. The construction can be likened to only a few existing approaches, and may thus be considered as our main contribution. ¶ Chapter 5 discusses numerical issues pertaining to the edge and confidence band estimators of Chapters 2-4. Connections are drawn to popular topics which originated in the fields of computer vision and signal processing, and which surround edge detection. These connections are exploited so as to obtain greater robustness of the likelihood estimator, such as with the presence of sharp corners. ¶ Chapter 6 addresses a dimension reduction problem for spatial data where the ultimate objective of the analysis is the discrimination of these data into one of a few pre-specified groups. In the dimension reduction step, an instrumental role is played by the recently developed methodology of functional data analysis. Relatively standar non-linear image processing techniques, as well as wavelet shrinkage, are used prior to this step. A case study for remotely-sensed navigation radar data exemplifies the methodology of Chapter 6.

&alpha / -Galactosidase is an exo-glycosidase that hydrolyses non-reducing, &alpha / -1,6-linked &alpha / -galactose units from oligosaccharides, galactomannans, and galactolipids. &alpha / -Galactosidase activity has biotechnological, industrial, and medical importance. &alpha / -Galactosidase from A. fumigatus IMI 385708, in particular, can catalyse unique hydrolysis and transgalactosylation reactions on polymeric substrates. In this study, &alpha / -galactosidase of the human pathogen A. fumigatus IMI 385708 was first produced in a GRAS organism, Aspergillus sojae. For this aim, &alpha / -galactosidase gene (aglB) of A. fumigatus IMI 385708 was ligated onto pAN52-4 vector (Acc. No: Z32699) and transformed into Aspergillus sojae ATCC11906, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA) of A. nidulans and the signal sequence of glucoamylase gene (glaA) of A. niger. This allowed high level of &alpha / -galactosidase production on glucose instead of locust bean gum (2.45 U/mL), corresponding to a 3-fold increase in volumetric production. Next, using response surface methodology, carbon and nitrogen sources and agitation speed were optimized (10.5% molasses (w/v) / 1.3% NH4NO3 (w/v) / 276 rpm). Compared to non-optimized cultivation, a further 4-fold increase in &alpha / -galactosidase production (10.4 U/mL) was achieved. Recombinant &alpha / -galactosidase was purified 18.7-fold using Anion Exchange and Hydrophobic Interaction Chromatography with an overall yield of 56% and 64.7 U/mg protein. The Vmax and Km values for the hydrolysis of p-nitrophenyl &alpha / -D-galactopyranoside were 78 U/mg protein and 0.45 mM, respectively. Optimum pH and temperature for &alpha / -galactosidase activity were between pH 4&ndash / 6 and 50&ndash / 60 &deg / C, respectively. Among the tested chemical agents, Ag+, Hg2+, and Fe2+ drastically decreased the activity, while biotin, I+1, Mn+2, Pb+2, Li+1, and Mg+2 enhanced between 12&ndash / 29%. To analyse the influence of osmotic stress as a means of further inducing &alpha / -galactosidase production, salt was added into the complete growth medium. In addition to enzyme production, fungal growth and morphology were analysed for both &lsquo / salt-adapted&rsquo / and &lsquo / salt non-adapted&rsquo / A. sojae Ta1 cells in the presence of KCl, MgCl2, MgSO4, NaCl, and Na2SO4 at 1 M and 2 M. Accordingly, 3-fold increase in &alpha / -galactosidase production was achieved by non-adapted cells in the presence of 1 M NaCl. Exposure of A. sojae Ta1 cells to salt resulted in predominantly mycelial form, rather than the pellet form observed under normal conditions. Finally, the transgalactosylation ability of &alpha / -galactosidase was studied. &alpha / -Galactosidase efficiently catalysed galactose transfer to different monosaccharides and disaccharides in the presence of pNP&alpha / Gal as monitored by TLC, ESI-MS, and HPLC.

The physiological benefits of flaxseed against pathological disturbances, such as cancers and heart diseases, are mainly attributed to its high lignan content. This study (Experiment 1) examined the application of pressurized low polarity water (PLPW) for extraction of lignans, proteins and carbohydrates from defatted flaxseed meal. Key processing conditions included temperature (130, 160, 190°C), solvent pH (4, 6.5 and 9), solvent to solid ratio (S/S) (90, 150 and 210 mL/g) and introduction of co-packing material (0 and 3 g glass beads). The addition of 3 g glass beads as co-packing material facilitated extraction by enhancing surface contact between the liquid and solid thus shortening extraction time. Elevated temperature accelerated the extraction rate by increasing the solid diffusion coefficient thereby reducing the extraction time. The maximum yield of lignans (99 %) was obtained at temperatures ranging from 160°C to 190°C, with solvent volume of 180 mL (90 mL/g meal) at pH 9. Optimal conditions for protein extraction (70 %) were pH 9, extraction volume of 420 mL (210 mL/g meal) and 160°C. Total carbohydrates yield was maximized at 50% recovery at pH 4 and 160°C with 420 mL solvent (210 mL/g meal). Increased temperature accelerated extraction, thus reducing solvent volume and time to reach equilibrium. For the extraction of proteins, however, a temperature of 130-160°C is recommended, as proteins are vulnerable to thermal degradation due to heat decomposition. The effects of flow rate and geometric dimensions for extraction of lignans and other flaxseed meal bioactives were further investigated in Experiment 2, based on the variables optimized in the previous experiment. Defatted flaxseed meal was extracted with pH 9 buffered water with meal to co-packing glass beads ratio of 1:1.5 at 5.2 MPa (750 psi) and 180°C. The aqueous extracts were analyzed for lignan, protein and carbohydrate using HPLC and colorimetric methods. The optimal extraction yields for lignan, protein and carbohydrate were found at flow rates of 1 to 2 mL/min with bed depth between 20 and 26 cm and a S/S ratio of 40 to 100 mL/g. The combination of low flow rate and high bed depth allowed the use of lower S/S ratio with reduced total solvent volume consumption. This study also evaluated the mass transfer kinetics governing the process of lignan extraction from flaxseed meal in a fixed bed extraction cell. Diffusion of solute into the continuously flowing solvent was mainly responsible for the mass transfer mechanism as flow rate did not increase proportionally with the yield and rate of extraction. The extraction kinetics were studied on the basis of two approaches: Fick’s diffusion equation and a two-site exponential kinetic model. The proposed two-site exponential kinetic model corresponding to the two-stage extraction (rapid and slow phases) successfully described the experimental data. Diffusivities attained from Fick’s diffusion model ranged from 2 x 10-13 to 9 x 10-13 m2s-1 while mass transfer coefficients were between 4.5 x 10-8 and 2.3 x 10-7 ms-1 for extraction of lignans at 180°C, pH 9 with 1:1.5 meal to co-packing material ratio. / February 2007

The natural ability of filamentous fungi to synthesize, glycosylate, and secrete high levels of protein products has made them potentially attractive hosts for heterologous protein production. Advances in fungal genetics enabled the expression of several high value proteins in filamentous fungi. Particularly the genus, Aspergillus has proven to be potentially useful for the expression of eukaryotic gene products. This thesis pertains to the optimization of recombinant protein production by the fungal host, Aspergillus niger. The target recombinant protein of interest is hen egg white lysozyme (HEWL). This protein encoded in the genome resulting in relatively stable gene construct; however, it is subject to extracellular protease attack. The objective of the proposed research is the development and application of engineering methodology for the analysis and optimization of a fungal bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. To accomplish this, during the first phase of this study a statistically based experimental method was used to systematically elucidate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl₂.2H₂O) on hen egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2⁵⁻¹ fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other medium components were not important within the levels tested. Then, the method of steepest ascent was employed to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g/L, peptone 34 g/L, ammonium sulfate 11.9 g/L, yeast extract 0.5 g/L, and CaCl₂.2H₂O 0.5 g/L. This medium was projected to produce theoretically 212 mg/L lysozyme. Using this optimized medium, an experimentally observed maximum lysozyme concentration of 209±18 mg/L verified the applied methodology. A second optimization approach was based on metabolic flux analysis (MFA). A comprehensive metabolic network comprising three intracellular compartments (cytoplasm, mitochondrion and peroxisome) was developed for Aspergillus niger. The metabolic flux network included carbohydrate and amino acid metabolism in both anabolic and catabolic reactions. According to experimental observations, the time course of fermentation was divided into five phases, each with unique physiological properties. The network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consists of 137 metabolites and 287 processes, of which 181 represent biochemical conversions and 106 represent transport processes between the different compartments and the extracellular environment. In addition, due to the physiological evidence some biochemical reactions considered to be active only in one direction. Linear programming was used for optimizing of the specific growth rate as the objective function in combination with 37 measured input and output fluxes of the key metabolites to evaluate corresponding intracellular flux distributions throughout the batch fermentations. The general applicability of the methodology was evaluated by establishing commonality to optimize recombinant HEWL production. The proposed model was able to predict correctly the specific growth rate, oxygen uptake rate, and carbon dioxide evolution rate with good precision. The results of the metabolic flux and sensitivity analysis were employed for medium design. Growth was biphasic; glucose was utilized initially as the carbon source and was followed by its oxidation product, gluconate, later. Logarithmic sensitivity analysis revealed that the addition of proline, alanine and glutamate benefited growth in defined media. The experimental observations and flux analysis showed that tyrosine was a potential candidate for biomass production improvement. The two amino acids, namely proline and tyrosine benefited biomass production during the initial growth phases. Glutamate and alanine were particularly important during the latter stages of the batch process. A series of growth studies were conducted with the identified amino acids added in the medium. In these preliminary nutritional experiments the contribution to growth enhancement was 46% for proline, 23% for glutamate, and 22% for tyrosine. Model predictions were further verified by conducting batch and fed-batch fermentations in a 7- liter bioreactor. The programmed addition of four amino acids (proline, glutamate, alanine, and tyrosine) according to a predetermined schedule resulted in a 44% improvement in biomass and 41% improvement in recombinant protein production. The experiments also confirmed the model prediction that extra amount of amino acids besides the identified ones would not significantly enhance biomass and the recombinant protein production. A computer-based control system was developed for the on-line monitoring and control of the major state variables (e.g., temperature, pH, and DO) during the time course of fermentation. The graphical programming environment, LabVIEW was used to acquire and integrate these variables in a supervisor computer. The temperature of the bioreactor during sterilization and fermentation was controlled using a cascade methodology. The controller parameters of the master and slave loops were determined experimentally to yield a smooth response with minimum overshoot of both the bioreactor and jacket temperatures. The program scheduled various required steps in an established order during the fermentation. This feature of the software guarantees that every necessary operation will be met. The graphical representation of the process is displayed on the screen and helps the user to follow the process and perform the required adjustments. Furthermore, different variables can be observed simultaneously and saved in text or spreadsheet files for further analysis.

The natural ability of filamentous fungi to synthesize, glycosylate, and secrete high levels of protein products has made them potentially attractive hosts for heterologous protein production. Advances in fungal genetics enabled the expression of several high value proteins in filamentous fungi. Particularly the genus, Aspergillus has proven to be potentially useful for the expression of eukaryotic gene products. This thesis pertains to the optimization of recombinant protein production by the fungal host, Aspergillus niger. The target recombinant protein of interest is hen egg white lysozyme (HEWL). This protein encoded in the genome resulting in relatively stable gene construct; however, it is subject to extracellular protease attack. The objective of the proposed research is the development and application of engineering methodology for the analysis and optimization of a fungal bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. To accomplish this, during the first phase of this study a statistically based experimental method was used to systematically elucidate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl₂.2H₂O) on hen egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2⁵⁻¹ fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other medium components were not important within the levels tested. Then, the method of steepest ascent was employed to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g/L, peptone 34 g/L, ammonium sulfate 11.9 g/L, yeast extract 0.5 g/L, and CaCl₂.2H₂O 0.5 g/L. This medium was projected to produce theoretically 212 mg/L lysozyme. Using this optimized medium, an experimentally observed maximum lysozyme concentration of 209±18 mg/L verified the applied methodology. A second optimization approach was based on metabolic flux analysis (MFA). A comprehensive metabolic network comprising three intracellular compartments (cytoplasm, mitochondrion and peroxisome) was developed for Aspergillus niger. The metabolic flux network included carbohydrate and amino acid metabolism in both anabolic and catabolic reactions. According to experimental observations, the time course of fermentation was divided into five phases, each with unique physiological properties. The network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consists of 137 metabolites and 287 processes, of which 181 represent biochemical conversions and 106 represent transport processes between the different compartments and the extracellular environment. In addition, due to the physiological evidence some biochemical reactions considered to be active only in one direction. Linear programming was used for optimizing of the specific growth rate as the objective function in combination with 37 measured input and output fluxes of the key metabolites to evaluate corresponding intracellular flux distributions throughout the batch fermentations. The general applicability of the methodology was evaluated by establishing commonality to optimize recombinant HEWL production. The proposed model was able to predict correctly the specific growth rate, oxygen uptake rate, and carbon dioxide evolution rate with good precision. The results of the metabolic flux and sensitivity analysis were employed for medium design. Growth was biphasic; glucose was utilized initially as the carbon source and was followed by its oxidation product, gluconate, later. Logarithmic sensitivity analysis revealed that the addition of proline, alanine and glutamate benefited growth in defined media. The experimental observations and flux analysis showed that tyrosine was a potential candidate for biomass production improvement. The two amino acids, namely proline and tyrosine benefited biomass production during the initial growth phases. Glutamate and alanine were particularly important during the latter stages of the batch process. A series of growth studies were conducted with the identified amino acids added in the medium. In these preliminary nutritional experiments the contribution to growth enhancement was 46% for proline, 23% for glutamate, and 22% for tyrosine. Model predictions were further verified by conducting batch and fed-batch fermentations in a 7- liter bioreactor. The programmed addition of four amino acids (proline, glutamate, alanine, and tyrosine) according to a predetermined schedule resulted in a 44% improvement in biomass and 41% improvement in recombinant protein production. The experiments also confirmed the model prediction that extra amount of amino acids besides the identified ones would not significantly enhance biomass and the recombinant protein production. A computer-based control system was developed for the on-line monitoring and control of the major state variables (e.g., temperature, pH, and DO) during the time course of fermentation. The graphical programming environment, LabVIEW was used to acquire and integrate these variables in a supervisor computer. The temperature of the bioreactor during sterilization and fermentation was controlled using a cascade methodology. The controller parameters of the master and slave loops were determined experimentally to yield a smooth response with minimum overshoot of both the bioreactor and jacket temperatures. The program scheduled various required steps in an established order during the fermentation. This feature of the software guarantees that every necessary operation will be met. The graphical representation of the process is displayed on the screen and helps the user to follow the process and perform the required adjustments. Furthermore, different variables can be observed simultaneously and saved in text or spreadsheet files for further analysis.