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.

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.

&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 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.

The thesis focuses on the development of statistical theory in experimental design with applications in global optimization. It consists of four parts. In the first part, a criterion of design efficiency, under model uncertainty, is studied with reference to possibly nonregular fractions of general factorials. The results are followed by a numerical study and the findings are compared with those based on other design criteria. In the second part, optimal designs are dentified using Bayesian methods. This work is linked with response surface methodology where the first step is to perform factor screening, followed by response surface exploration using different experiment plans. A Bayesian analysis approach is used that aims to achieve both goals using one experiment design. In addition we use a Bayesian design criterion, based on the priors for the analysis approach. This creates an integrated design and analysis framework. To distinguish between competing models, the HD criterion is used, which is based on the pairwise Hellinger distance between predictive densities. Mixed-level fractional factorial designs are commonly used in practice but its aliasing relations have not been studied in full rigor. These designs take the form of a product array. Aliasing patterns of mixed level factorial designs are discussed in the third part. In the fourth part, design of experiment ideas are used to introduce a new global optimization technique called SELC (Sequential Elimination of Level Combinations), which is motivated by genetic algorithms but finds the optimum faster. The two key features of the SELC algorithm, namely, forbidden array and weighted mutation, enhance the performance of the search procedure. Illustration is given with the optimization of three functions, one of which is from Shekel's family. A real example on compound optimization is also given.

The first aim of this study is the development of extracellular recombinant therapeutic protease streptokinase producing Bacillus sp., and the second aim is to determine fermentation characteristics for streptokinase production. In this context, the signal (pre-) DNA sequence of B.licheniformis (DSM1969) extracellular serine alkaline protease enzyme gene (subC: Acc. No. X03341) was ligated to 5&rsquo / end of the streptokinase gene (skc: Acc. No. S46536) by SOE (Gene Splicing by Overlap Extension) method through PCR. The resulting hybrid gene pre(subC)::skc was cloned into the pUC19 plasmid. Then, the hybrid gene was sub-cloned to pMK4 plasmid which is an E. coli-Bacillus shuttle vector with high copy number and high stability. Recombinant plasmid pMK4::pre(subC)::skc was finally transferred into B. subtilis (npr- apr-) and B. licheniformis 749/C (ATCC 25972) species. Streptokinase production capacities of these two recombinant Bacillus species were compared. The highest production was observed in recombinant B. lichenifomis 749/C (ATCC 25972) strain in a defined medium which was optimized in terms of carbon and nitrogen sources by a statistical approach, namely Response Surface Methodology (RSM). RSM evaluated the streptokinase concentration as the response and the medium components as the independent variables. The highest recombinant streptokinase concentration was found as 0.0237 kgm-3 at glucose and (NH4)2HPO4 concentrations of 4.530 and 4.838 kgm-3 respectively. The fermentation and oxygen transfer characteristics of the streptokinase production were investigated in a 3 dm3 pilot scale batch bioreactor (Braun CT2-2) equipped with temperature, pH, foam, air inlet and agitation rate controls having a working volume of VR=1.65 dm3 using the production medium optimized for the recombinant B. lichenifomis 749/C (ATCC 25972) strain. Streptokinase and &amp / #946 / -lactamase activities, cell, glucose and organic acid concentrations, dissolved oxygen, pH, oxygen uptake rate, overall liquid phase mass transfer coefficient for oxygen, maintenance coefficient for oxygen, specific cell growth rate and yield coefficients were determined through the bioprocess. The bioprocess of recombinant streptokinase production was performed at uncontrolled pH of these bioreactor operation conditions: air inlet rate of Q0/VR=0.5 vvm, and the agitation rate of N=400min-1. The resulting streptokinase volumetric activity reached its maximum as 1.16 PUml-1 (0.0026 g/l streptokinase) at t=20 h.

Aspergillus fumigatus produces enzymes required for the hydrolysis of galactomannans like locust bean gum. Among these enzymes endo-beta-1,4 mannanase is also produced at high levels. However, the fungus is not safe for use in the food industry. Therefore, the gene encoding endo-beta-1,4-mannanase of A. fumigatus IMI 385708 was previously cloned in our laboratory into Aspergillus sojae ATCC11906 which is a safe microorganism for use in food applications. Altogether eight transformants were obtained. It was shown that some of these transformants overproduce the enzyme because of expression under the control of glyceraldehyde-3-phosphate dehydrogenase promoter and fusion to the glucoamylase signal and pro-peptide coding region of Aspergillus niger. In this study, mannanase production of these transformants was compared with A. fumigatus and A. sojae transformant AsT1 showed c. 12 fold increase with the maximum activity of 352 U/ml. The effects of initial medium pH and number of spores on activity were investigated and maximum activity was achieved at pH 7.0 and the number of spores was found as 3.6 &times / 106. Optimization of the growth conditions for maximum mannanase production in shake flasks by using the best mannanase producing transformant AsT1 was carried out by using Box-Behnken design under Response Surface Methodology. The highest beta-mannanase activity on the fourth day of cultivation at 30 &ordm / C was obtained as 363 U/ml in the optimized medium containing 7% sugar beet molasses, 0.43% NH4NO3, 0.1% K2HPO4, 0.05% MgSO4 as the weight/volume percentage at 207 rpm. On sixth day of cultivation under the optimized conditions, the highest mannanase activity was achieved as 482 U/ml which is 1.4 fold of 352 U/ml activity found on glucose medium previously. After 48 h of LBG hydrolysis by 40 U of mannanase, mannotriose, 61-galactosyl-beta-D-mannotriose and 63,64-di-alpha-galactosyl-beta-1,4-mannopentaose were found as the main products via HPLC analysis.

Kitchen waste, which is collected in large amounts from cafeterias, restaurants, dining halls, food processing plants, and household kitchens, have become a valuable material for bioprocess engineering. Due to the high carbohydrate fraction, kitchen waste has great potential to be used as a potential substrate for ethanol production. Utilization of it as a raw material in ethanol fermentation would also contribute to reduction of costs. In the first part of this study, the effect of pretreatment method and enzymatic hydrolysis on glucose production was evaluated. Dry baker&rsquo / s yeast, Saccharomyces cerevisiae, was used in fermentation experiments conducted with and without fermentation medium at pH 4.5 and 30oC for 48 hours. Close values of glucose concentration were obtained from no pretreated and hot water treated samples. The fermentation results indicated that ethanol can be produced at similar concentrations in bioreactors with and without fermentation medium addition (p &gt / 0.05). Thus, it is concluded that use of kitchen wastes as is disposed and without fermentation medium in ethanol fermentation could lower the cost to a large extent. In the second part of this study, the effects of solid load, which is proportional to the glucose concentration (10% to 20% (w/w)), inoculum level of Saccharomyces cerevisiae (5% to 15% (v/v)), and fermentation time (48 to 96 h) on production of bioethanol from kitchen waste were studied using Response Surface Methodology (RSM). A three-factor Box Behnken design was used. Ethanol concentration was used as a response in the resulting experimental design. High Pressure Liquid Chromatography (HPLC) method was used to determine ethanol and glucose concentrations. The statistical analysis of the constructed model developed by RSM suggested that linear effects of solid load, inoculum level, and fermentation time and quadratic effects of inoculum level and fermentation time were all significant (p &lt / 0.05) on bioethanol production. The model was verified by additional runs, which were not present in the design matrix. It was found that the constructed model could be used to determine successfully the bioethanol concentration with &gt / 90% precision. An optimum ethanol concentration of 32.16 g/L was suggested by the model with 20% (w/w) solid load, 8.85% (v/v) inoculum level and 58.8 hours of fermentation. Further study is needed to evaluate the optimal fermentation conditions in a large scale fermentation