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.

Rhizopus oryzae is a filamentous fungi which produces lactic acid and ethanol in fermentations. R. oryzae has numerous advantages for use industrial production of L-(+)-lactic acid but the yield of lactic acid produced on the basis of carbon consumed is low. Metabolic flux analysis of R. oryzae has shown that most of the pyruvate produced at the end of the glycolysis is channelled to ethanol, acetyl-CoA and oxaloacetate production. This study aimed to answer some questions addressed on the regulation of pyruvate branch point in R. oryzae and for this purpose biochemical characterisation of the enzymes acting at this branch point and cloning the genes coding for these enzymes have been done. Pyruvate decarboxylase was purified and characterised for the first time from R. oryzae. The purified enzyme has a Hill coefficient of 1.84 and the Km of the enzyme is 8.6 mM for pyruvate at pH 6.5. The enzyme is inhibited at pyruvate concentrations higher than 30 mM. The optimum pH for enzyme activity shows a broad range from 5.7 and 7.2. The monomer molecular weight was estimated as 59&plusmn / 2 kDa by SDS-PAGE analysis. Pyruvate decarboxylase (pdcA and pdcB) and lactate dehydrogenase (ldhA and ldhB) genes of R. oryzae have been cloned by PCR-cloning approach and the filamentous fungi Aspergillus niger was transformed with these genes. The A. niger transformed with either of the ldh genes of R. oryzae showed enhanced production of lactic acid compared to wild type. Citric acid production was also increased in these transformants while no gluconate production was observed Cloning of hexokinase gene from R. oryzae using degenerate primers was studied by the use of GenomeWalker kit (Clontech). The results of this study were evaluated by using some bioinformatics tools depending on the unassembled clone sequences of R. oryzae genome.

Orientador: Ely Nahas / Banca: Regina Teresa Rosim Monteiro / Banca: Jonas Contiero / Banca: Antônio Carlos Monteiro / Banca: Lúcia Maria Carareto Alves / Resumo: Devido a baixa disponibilidade de P no solo e a alta capacidade do fungo Aspergillus niger F111 em solubilizar fosfatos inorgânicos, este trabalho teve por objetivo geral avaliar a possibilidade de inocular no solo esporos ou pellets imobilizados com vista a prolongar sua habilidade de solubilização e averiguar o mecanismo de solubilização de fosfatos inorgânicos de Ca, Al e Fe por este fungo. Os pellets inoculados em meio de cultura agitado proporcionaram maior solubilização dos fosfatos, principalmente o fosfato de Fe por ser de baixa solubilidade. No solo, os pellets livres e imobilizados promoveram as maiores solubilizações de fosfato de Fe e maior produção de CO2. Avaliando-se o efeito da fonte de N, as seguintes proporções foram obtidas na solubilização dos fosfatos de Ca, glicina > Al, nitrato de amônio > Fe, ácido l-glutâmico. Os açúcares que mais solubilizaram os fosfatos foram manitol, maltose e d-galactose. Dentre os metais somente o FeCl3.6H2O promoveu maior solubilização do fosfato de Fe e os metais FeSO4.7H2O e FeCl3.6H2O promoveram maiores solubilizações do fosfato de Ca. As concentrações de álcoois que mais favoreceram a solubilização do fosfato de Fe foram 3 e 4% de etanol e metanol, para o fosfato de Ca foi 3% de etanol. A combinação dos metais com o metanol, indicou que o metanol foi o principal responsável pela solubilização. Fatores como queda do pH, a maior produção de ácidos e o menor crescimento do fungo influíram neste trabalho, principalmente em relação a solubilização do fosfato de Fe. No solo, os pellets solubilizaram quantidades semelhantes de fosfato de Fe que os esporos imobilizados de A. niger, podendo ser utilizados com vantagem devido a sua facilidade de obtenção. / Abstract: Considering the low P availability in the soil and the high capability of Aspergillus niger F111 in solubilizing inorganic phosphates, this work aimed to evaluate the possibility of inoculating spores or immobilized pellets in the soil to prolong the solubilization capability and study the solubilization mechanism of inorganic calcium phosphate, aluminum phosphate and iron phosphate by this fungus. Pellets inoculated in culture medium under agitation allowed higher phosphate solubilization, especially iron phosphate, which is low soluble. In the soil, free and immobilized pellets allowed the highest solubilization of iron phosphate and CO2 production. Evaluating the effect of N sources, the following proportions were obtained in the solubilization of calcium phosphates, glycine > Al, ammonium nitrate > Fe, l-glutamic acid. The sugars that most solubilized phosphates were mannitol, maltose and d-galactose. Among the metals, only FeCl3.6H2O promoted higher iron phosphate solubilization, and FeSO4.7H2O and FeCl3.6H2O promoted higher solubilization of calcium phosphate. The alcohol concentrations that most favored iron phosphate solubilization were 3 and 4% of ethanol and methanol, while the highest solubilization of calcium phosphate was reached with 3% ethanol. The combination of metals with methanol indicated this alcohol was mainly responsible for solubilization. Factors as pH decrease, higher acid production and lower A. niger growth influenced the results, especially in the solubilization of iron phosphate. In the soil, pellets and immobilized spores solubilized similar amounts of iron phosphate. Pellets are thus preferable because they are more easily obtained. / Doutor

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-10-18T15:37:11Z No. of bitstreams: 1 SIUMARA RODRIGUES ALCÂNTARA - DISSERTAÇÃO PPGEA 2008..pdf: 9760309 bytes, checksum: 3c00d833a82841dcdf4d92435feff3e2 (MD5) / Made available in DSpace on 2018-10-18T15:37:11Z (GMT). No. of bitstreams: 1 SIUMARA RODRIGUES ALCÂNTARA - DISSERTAÇÃO PPGEA 2008..pdf: 9760309 bytes, checksum: 3c00d833a82841dcdf4d92435feff3e2 (MD5) Previous issue date: 2008-01 / Capes / Dentre as diversas influências observadas no processo de fermentação semisólida, a atividade de água é um dos fatores mais importantes, pois está diretamente relacionada com a quantidade de água disponível ao microrganismo responsável pela síntese do produto. Assim, objetivou-se a produção de pectinases, usando o pedúnculo de caju seco como meio e o Aspergillus niger CCT 0916 como microrganismo, em um processo fermentativo semi-sólido, verificando a influência da quantidade de água e uma fonte de nitrogénio. Com isso, fez-se a caracterização físico-química do resíduo, o levantamento e ajuste das isotermas de adsorção, o cálculo do calor isostérico total e, verificou-se a influência da umidade inicial e a concentração de sulfato de amónio no meio através da execução de um planejamento fatorial 2 . Observou-se que a caracterização do pedúnculo indicou a necessidade do ajuste da concentração de açúcares redutores e da pectina. O modelo de GAB foi o que melhor ajustou as isotermas de adsorção. A atividade poligalacturonásica máxima foi obtida com atividade de água entre 0,99 e 1,00 e a atividade pectinolítica foi obtida com atividade de água igual a 0,99. Ocorreu inibição pela presença do sulfato de amónio no meio fermentativo com concentrações a partir de 1,5%, tendo apresentado efeito negativo sobre as atividades enzimáticas observadas. Diferentemente da umidade inicial que apresentou efeito positivo sobre as mesmas. Os maiores valores de atividade enzimática foram 11 U/g. Constatou-se também que o bagaço seco do pedúnculo do caju é um substrato competitivo perante outros descritos na literatura. / The water acíivity is one of the most important factor in this process, because it is related with the quantity of available water for the microorganism responsible for the synthesis of the product because the larges quantities of the agro industrial residues. In this context, this work has the objective the production of pectinases, using the cashew peduncle like substratum and the Aspergillus niger CCT 0916 like microorganism, in a solid-state fermentation process, checking the influence of water quantity and nitrogen source. It had done the physiochemical characterization of the residue, the construction and adjusts of the adsorption isotherms, the calculation of the isosteric heat and had checked the influence of the initial moisture and the ammonium sulphate concentration in the médium by factorial design. It observed that the peduncle characterization showed the necessity of adjust of reducing sugar and pectin. The best equation to fit the adsorption isotherms were GAB model. The maximum poligalacturonase activity was obtained with water activity between 0.99 and 1.00. The maximum pectinolytic activity was obtained with 0.99 of water activity. It was observed that occurred inhibition in the médium by presence of ammonium sulphate with concentration equal and over 1.5%. This variable showed negative effect on enzymatic activity. In the other hand, the inicial moisture showed positive effect on the same response. The most values of enzymatic activity were 11 U/g. It had evidenced that the dry bagasse of the cashew peduncle is a competitive substratum.

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-02-07T10:26:14Z No. of bitstreams: 1 TeseCF.pdf: 2944475 bytes, checksum: 0b8b7d83e1195e91eb572351858f7b93 (MD5) / Approved for entry into archive by Camila Passos (camilapassos@ufscar.br) on 2017-02-08T12:04:12Z (GMT) No. of bitstreams: 1 TeseCF.pdf: 2944475 bytes, checksum: 0b8b7d83e1195e91eb572351858f7b93 (MD5) / Approved for entry into archive by Camila Passos (camilapassos@ufscar.br) on 2017-02-08T12:08:29Z (GMT) No. of bitstreams: 1 TeseCF.pdf: 2944475 bytes, checksum: 0b8b7d83e1195e91eb572351858f7b93 (MD5) / Made available in DSpace on 2017-02-08T12:10:12Z (GMT). No. of bitstreams: 1 TeseCF.pdf: 2944475 bytes, checksum: 0b8b7d83e1195e91eb572351858f7b93 (MD5) Previous issue date: 2016-04-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Currently, one of the major challenges for second generation ethanol is to reduce the cost of cellulolytic enzymes. Thus, the development of bioprocesses for the enzyme production on-site and strategies to increase the final yield of the enzymatic hydrolysis are required to ensure that biomass conversion to be economically feasible. Therefore, the objective of this work was to study the production and characterization of enzyme cocktails involved in the degradation of plant biomass by filamentous fungi Trichoderma reesei and Aspergillus niger grown in sequential fermentation and evaluate the application of these cocktails in the saccharification process of sugarcane bagasse. Firstly, evaluation and validation of sequential fermentation cultivation methodology to different strains of Trichoderma. Cultivation were made using sugarcane bagasse "in natura" and pretreated by steam explosion, as a carbon source. The result more significantly was observed for T. reesei Rut C30, the endoglucanase production was 4.2-fold higher than the values obtained in conventional submerged fermentation. The enzyme extracts were characterized in terms of optimum pH and temperature and endoglucanase profile. The thermostability was directly influenced by the type of carbon source and type of cultivation method. Subsequently, the proteomic analysis were performed of enzyme cocktails from T. reesei Rut C30 and A. niger A12 produced by submerged and sequential fermentation in the presence of pretreated bagasse. The performance of the enzyme cocktail in saccharification of pretreated bagasse showed that the combination of enzyme cocktails from T. reesei and A. niger produced by sequential fermentation had a yield than 3-fold higher than the enzyme cocktails of submerged fermentation. In order to evaluate the action of the enzyme cocktails produced by T. reesei and A. niger in sugarcane bagasse saccharification, the last step of the work was to study the additives effects during the sugarcane bagasse hydrolysis aiming at reducing non-productive adsorption of enzymes into lignin. The saccharification results in the presence of soybean protein were 2-fold higher than the controls (no additive) to the enzyme cocktails of two fungi studied produced by solid state fermentation, indicating the potential use of soybean protein as an additive to minimize non-productive adsorption of the enzyme into lignin. Overall, this study presents an interesting final contribution in the cellulase production process and the application of the enzyme cocktail in the hydrolysis of sugarcane bagasse. / Atualmente, um dos grandes desafios para a produção de etanol de segunda geração consiste em diminuir o custo das enzimas celulolíticas. Assim, o desenvolvimento de bioprocessos para produção das enzimas on-site e estratégias para aumentar o rendimento final da hidrólise enzimática são necessários para assegurar que a conversão de biomassa seja economicamente viável. Para tanto, o objetivo deste trabalho foi estudar a produção e caracterização de coquetéis enzimáticos envolvidos na degradação da biomassa vegetal pelos fungos filamentosos Trichoderma reesei e Aspegillus niger cultivados por fermentação sequencial, bem como avaliar a aplicação dos mesmos no processo de sacarificação do bagaço de cana-de-açúcar. Primeiramente foi realizada a avaliação e validação da metodologia de cultivo de fermentação sequencial para diferentes linhagens de Trichoderma. Os cultivos foram feitos utilizando o bagaço de cana “in natura” e prétratado por explosão a vapor, como fonte de carbono. O melhor resultado foi observado para T. reesei Rut C30, em que a produção de endoglucanase foi 4,2 vezes maior do que os valores obtidos em cultivo convencional de fermentação submersa. Os extratos enzimáticos foram caracterizados em termos de pH e temperatura ótimos e perfil de endoglucanase. A termo-estabilidade foi diretamente influenciada pelo tipo de fonte de carbono e tipo de cultivo. Posteriormente, foram realizadas as análises proteômicas dos coquetéis enzimáticos do T. reesei Rut C30 e A. niger A12 produzidos por fermentação submersa convencional e fermentação sequencial, na presença de bagaço de cana prétratado. A performance dos coquetéis enzimáticos na sacarificação do bagaço de cana pré-tratado mostraram que a combinação dos coquetéis enzimáticos de T. reesei e A. niger produzidos por fermentação sequencial tiveram um rendimento 3 vezes maior do que os coquetéis da fermentação submersa. A fim de explorar melhor a ação dos coquetéis enzimáticos produzidos por T. reesei e A. niger na sacarificação do bagaço, na última etapa do trabalho foi estudo o efeito de aditivos durante a hidrólise do bagaco de cana visando à redução da adsorção improdutiva de enzimas na lignina. Os resultados de sacarificação na presença da proteína de soja foram 2 vezes maiores do que os controles (sem aditivo) para os coquetéis enzimáticos dos dois fungos estudados produzidos por fermentação em estado sólido, indicando o potencial do uso da proteína de soja como aditivo para minimizar a adsorção improdutiva das enzimas na lignina. De modo geral, o presente trabalho conseguiu uma contribuição final interessante no processo de produção das celulases e a aplicação do coquetel enzimático na hidrólise do bagaço de cana.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-08T14:47:27Z No. of bitstreams: 1 Julio Cezar dos Santos Nascimento.pdf: 1976053 bytes, checksum: 53410347ad1578d486d39f21c31e19aa (MD5) / Made available in DSpace on 2016-06-08T14:47:27Z (GMT). No. of bitstreams: 1 Julio Cezar dos Santos Nascimento.pdf: 1976053 bytes, checksum: 53410347ad1578d486d39f21c31e19aa (MD5) Previous issue date: 2011-02-28 / During ripening, vegetable seeds and grain accumulated substantial amount of phytic acid, representing over 60% of total phosphorus in them. Phytate acts as an energy source for seed germination and is rarely available for non-ruminants, since they do not synthesize the enzyme. Due to the unavailability of biological organic phosphorus in many vegetable foods, research has sought alternative sources of phosphorus in order to better use. Incorporated in this context are the phytases, which form a group of enzymes that catalyze reactions gradual dephosphorylation of phytate. For enzyme purification the aqueous two-phase systems (ATPS) have been widely used for protein separation due to its low cost compared to other purification processes. The aim of this study was to evaluate the variables that influence production and purification of phytase by aqueous two-phase systems, as well as the biochemical characteristics of phytase produced by Aspergillus niger var. phoenicis URM 4924. The culture medium for the production of phytase were studied using factorial design and response surface methodology. The best condition for the production of phytase (8.80 U/mL) was found matching 1.25% of rice bran and 3.0% corn steep liquor. The optimum pH was 5.0, and remains at over 80% of residual activity at pH 5.0 to 9.0 for 15 hours. The phytase showed affinity constant of 0.12 mM and maximum velocity of 7.9 ηmol.s-1. The best conditions of extraction in aqueous two-phase systems were obtained with 26% (w/w) PEG 8000 (g/mol), pH 8.0 and 12% (w/w) citrate thus promoting purification factor of 7.58, partition coefficient of 3.62 and yield of 113.4%. The extraction using ATPS PEG/citrate proved to be promising for purification of phytase produced by A. niger var. phoenicis URM 4924 and may be applied in the composition of animal feed non-ruminants. / Durante o amadurecimento, sementes de legumes e cereais acumulam quantidade substancial de ácido fítico, representando mais de 60% do fósforo total dos mesmos. O fitato serve como fonte de energia para a germinação da semente, sendo pouco disponível para animais não-ruminantes, pois esses não sintetizam a fitase. Devido à indisponibilidade biológica do fósforo fítico em diversos alimentos de origem vegetal, as pesquisas têm buscado por fontes alternativas de fósforo, visando um melhor aproveitamento. Inseridas neste contexto estão as fitases, que formam um grupo de enzimas que catalisam reações graduais de defosforilação do fitato. Para a purificação de enzimas os sistemas de duas fases aquosas (SDFA) têm sido amplamente usados para separação de proteínas por conta do seu baixo custo quando comparado a outros processos de purificação. O objetivo deste trabalho foi a avaliação das variáveis que influenciam a produção e purificação da enzima por sistemas de duas fases aquosas, bem como a determinação das características bioquímicas de fitase produzidas por Aspergillus niger var. phoenicis URM 4924. Os meios de cultivo para a produção de fitases foram estudados utilizando planejamento fatorial e metodologia de superfície de resposta. A melhor condição para a produção de fitase (8,80 U/mL) foi encontrada combinando 1,25% de farelo de arroz e 3,0% de milhocina. A fitase apresentou temperatura ótima a 60°C e manteve 38,4% da atividade residual a 90°C por 120 minutos. O pH ótimo foi 5,0, e permaneceu com mais de 80% da atividade residual na faixa de pH 5,0 a 9,0 por 15 horas. A fitase apresentou constante de afinidade de 0,12 μM e velocidade máxima de 7,9 ηmol.s-1. As melhores condições de extração em sistemas de duas fases aquosas foram obtidas com 26% (m/m) de PEG 8000 (g/mol), pH 8,0 e 12% (m/m) de citrato promovendo assim, aumento de pureza de 7,58, coeficiente de partição de 3,62 e recuperação de 113,4%. A extração utilizando SDFA PEG/citrato demonstrou ser promissora para purificação de fitase produzida por A. niger var. phoenicis URM 4924, podendo ser aplicada na composição de rações de animais não-ruminantes.

Orientadores: Marcelo Cristianini, Pilar Rodriguez de Massaguer / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-12T13:04:24Z (GMT). No. of bitstreams: 1 Tribst_AllineArtigianiLima_M.pdf: 3194893 bytes, checksum: 2ef1a8dcf449b5ddd301d699b525063b (MD5) Previous issue date: 2008 / Resumo: O néctar de manga é apreciado por sua cor e aroma. Este produto, pela sua alta acidez, pode ser contaminado por bolores, dentre os quais são de grande relevância aqueles capazes de sobreviver ao processamento térmico. O processamento a alta pressão dinâmica (APD) é um tratamento ¿a frio¿ realizado para inativação de microrganismos visando uma melhor qualidade sensorial e nutricional do produto obtido. Neste estudo, avaliou-se o efeito do processamento a APD isoladamente ou combinado com tratamento térmico (TT) brando na inativação de Aspergillus niger em néctar de manga e, posteriormente, foi realizada a avaliação do efeito dos processos sobre a cor e a concentração de vitamina C do néctar. Paralelamente foi desenvolvida uma metodologia para limpeza e sanificação do equipamento e um processo para a redução de viscosidade do néctar de manga. Foram estudadas pressões entre 100 e 300 MPa para a inativação do Aspergillus niger. A pressão de 300 MPa inativou toda a carga inicial (> 6,24 reduções decimais), 200 MPa provocou uma inativação parcial (aproximadamente 2 ciclos) e, pressões inferiores a 150 MPa não resultaram em inativação significativa do A. niger. Quando o processo de APD foi associado à TT brando (80ºC/15minutos) foi observado um efeito sinérgico entre pré tratamento a APD e posterior TT (aproximadamente 1 ciclo logaritmo); já quando utilizado pré TT seguido de tratamento a APD, foi observado apenas um efeito aditivo. Utilizando-se aplicação de APD seguida de TT, foi realizado um planejamento experimental fixando-se a pressão em 200 MPa e variando-se temperatura e tempo do TT e ratio do néctar de manga (brix/acidez), de forma a obter um modelo que descrevesse o processo. Os resultados indicaram que tempo e temperatura afetaram positivamente a inativação e o ratio do néctar afetou negativamente. A partir do modelo foram estabelecidos que processos a 200 MPa seguido de TT a 73,5ºC/10 minutos ou 61,5ºC/20 minutos eram suficientes para obter 5 reduções decimais do bolor. A avaliação desses processos combinados, do processo térmico tradicional (100ºC/10 minutos) e do processo utilizando apenas pressão (300 MPa) sobre o conteúdo de vitamina C e cor do néctar demonstrou uma inativação de vitamina C muito similar para todos os processos (perdas de aproximadamente 45%) e uma melhor retenção de cor para os produtos processados à APD. O desenho do processo de limpeza e sanificação para o equipamento foi considerado eficaz uma vez que reduziu para < 1 UFC.mL-1 a contaminação de A.niger inicial de aproximadamente 106 UFC.mL-1 na água de enxágüe. Foi utilizada concentração de 0,05% de ácido peracético e 2,5% de detergente comercial cujos princípios ativos eram o toluenosulfonato de sódio e cloreto de didecilmetilamônio. O processo de redução de viscosidade do néctar também foi efetivo através de seu pré tratamento com enzimas pectinolíticas e celulases, que reduziram a viscosidade inicial do néctar em 50%. Pelos resultados obtidos no presente trabalho, observa-se que a APD isoladamente ou aplicada junto com TT brando é uma alternativa viável para a inativação de A. niger termorresistente em néctar de manga. O processo teve efeito negativo sobre a vitamina C do néctar de manga, o que pode ser atribuído tanto à presença de oxigênio na amostra quanto à presença de selos metálicos no equipamento (selos de berílio-cobre) que promovem a passagem de íons metálicos para o suco, os quais atuam como pró-oxidantes de vitamina C, agravando a oxidação da vitamina / Abstract: Mango nectar is appreciated by its color and flavor. Due to its high acidity, this nectar can be contaminated by molds and the most important molds are those able to survive to the heating process. The dynamic high pressure (DHP) is a cold treatment used to inactivate microorganisms with better nutritional and sensory retention in food. In this study, the DHP process isolated or combined with mild thermal treatment (TT) was used to inactivate Aspergillus niger in mango nectar. The effects of these processes were evaluated on color and vitamin C retention. In parallel, a methodology was developed to clean in place (CIP) the DHP equipment. A process to reduce the viscosity of mango nectar was also developed. Pressures between 100 and 300 MPa were studied to Aspergillus niger inactivation. Pressure of 300 MPa completely inactivated the initial load (> 6.24 decimal reduction), 200 MPa caused a partial inactivation (2 log cycles) and pressures up to 150 MPa did not inactivate the mold. A synergistic effect was observed when pre treatment of DHP was associated to mild TT (80ºC/ 15 minutes) with increase of 1 log cycle on mold inactivation. However, only an additive effect was observed when pre TT was associated to DHP. An experimental design was carried out using fixed pressure of 200 Mpa followed by mild TT. Temperature and time of thermal treatment and ratio of mango nectar (brix/acidity) was select as experimental design variables. The results indicated that time and temperature affected positively and nectar ratio affected negatively the mold inactivation. By the mathematical model, process of 200 MPa followed by 73.5ºC/10 minutes or by 61.5ºC/20 minutes was able to promote 5 decimal reduction of A. niger. The effect of these processes, the conventional heat and DHP at 300 MPa on vitamin C concentration and nectar color retention was evaluated. The vitamin C losses were around 45% for all processes studied but better color retention was observed on the nectar processed by DHP. The CIP design was considered efficient once it was able to reduce the initial load of A. niger from ~106 CFU.mL-1 to <1 CFU.mL-1. 2.5% of a commercial detergent (active agents: sodium toluenosulfonate and didecylmethilamonium chlorine) and peracetic acid at 0.05% were used in this process. The nectar viscosity reduction was effective by using pectic enzymes and celullase, being able to reduce natural mango nectar viscosity by 50%. The results obtained indicated that DHP isolated or combined with mild thermal treatment are a viable alternative to inactivate A. niger in mango nectar. The process has a negative effect on vitamin C retention. It can be attributed to the oxygen dissolved in mango nectar and also to metals seals of equipment (Be-Cu) which can had promoted an increase in metallic ions concentration in nectar. These ions are pro oxidant of vitamin C, resulting in intense vitamin oxidation / Mestrado / Mestre em Tecnologia de Alimentos

M. Tech (Department of Biotechnology, Faculty of Applied and Computer Sciences) Vaal University of Technology. / Water shortages, water pollution and climate changes are highly interrelated global issues. These have raised immense concerns about serious adverse effects on the quality, treatment and re-use of wastewater. A major role of water is for vitality of life on earth. Water is recognized as source of evolution from origin to degree of civilization, since it is an essential resource its treatment becomes a necessity for day to day for life. Nanoparticles and their application in treatment of wastewater is becoming a major area of research. It is mainly applicable to the removal of major contaminants like microorganisms. This study was carried out with an objective to investigate the antibacterial and antifungal potentials of nanoparticles. Cobalt and cobalt complexes of urea and thiourea were synthesized and characterized using UV-Vs, PL, FTIR, TEM, SEM, XRD and TGA techniques. The Co particles are in a mixture of rod, agglomerates with irregular shape around 50 – 100 nm in diameter. The Co/Thiourea particles appear to be around 10 – 30nm in size. The Co complexed with urea images showed spherical to hexagonal shape with 50 nm size in diameter. The antimicrobial activity was determined using Minimum Inhibitory and bactericidal concentration and the well diffusion method. The antibacterial and antifungal activities of ratios (1:1, 1:2, 1:3, 2:1 μg/mL) of doped cobalt nanoparticles were tested against a panel of five Gramnegative bacteria - (Escherichia coli, Pseudomonas aeruginosa, Shigella enterica, Salmonella typhi and Salmonella sonnei) human pathogenic bacteria; and two fungal strains - Aspergillus niger and Candida albicans. Zones of inhibition as a consequence of nanoparticles were compared with that of different standards like Neomycin for antibacterial activity and Amphotericin B for antifungal activity. The results showed a remarkable inhibition of the bacterial growth against the tested organisms. The most striking feature of this study is that Cobalt, Urea and Thiourea nanoparticles have antifungal activity comparable or more effective (as in case of Thiourea on A. niger) than Amphotericin B and nearly promising antibacterial activity although not comparable to Neomycin.

The overall goal of the presented dissertation thesis was to study the sterilization efficiency of dielectric barrier discharge operated at atmospheric pressure. The fungi Aspergillus niger, gram-positive bacteria Bacillus subtilis and in some experiments also gram-negative bacteria Escherichia coli were used as a bio-indicator enabling to evaluate the effect of plasma assisted microbial inactivation. The samples of microorganism were placed on paper Whatman 1 or PET foil and exposed to plasma. The plasma was generated in argon, nitrogen, synthetic dry/humid air with frequency up to 10 kHz and plasma power density in the range of 1,2-2,9 W/cm3 (according to the process gas). The influence of process gas, plasma power density, plasma exposition time, type of microorganism and material of the substrate on the sterilization effect of dielectric barrier discharge was evaluated. Furthermore the contribution of each single mechanism (UV radiation, temperature and reactive species) to the sterilization effect of plasma and influence of gas humidity was evaluated. The DBD was analysed by means of optical emission spectroscopy, thermocouple was used to measure temperature during a sterilization process. In order to verify the mechanical damage of the microbial cell or the substrates during the plasma process the samples were studied by scanning electron microscopy. Generally, on the basis of experimental results, at increasing treatment times, the remaining number of spores (CFU) decreased. Similarly at increasing the plasma power input, the sterilization rate increased. When sterilising the spores of A. niger in plasma using different process gasses, the efficiency of plasma sterilization decreased as follows: argon, humid synthetic air, nitrogen and dry synthetic air. The results observed in argon plasma using different microorganism demonstrated that the sensitivity of vegetative cells resp. spores to DBD decreased as follows: A. niger spores, B. subtilis vegetative cells, E. coli vegetative cells and B. subtilis spores. Simultaneously results observed for sterilization of spores and vegetative cells of B. subtilis and A. niger demonstrated that the spores are generally more resistant to plasma than are the corresponding vegetative cells. Combining the results of contribution of each single mechanism, optical emission spectroscopy and inactivation characteristic it was found out that the reactive species significantly contribute to the plasma sterilization in all process gasses. Furthermore the inactivation process can be partly assisted by UV radiation and also the temperature can contribute in limited extent to inactivation process in some gasses. The contribution of UV radiation to the plasma sterilization decreased as follows: nitrogen, argon, dry syntetic air and humid syntetic air. Moreover it was found out that the contribution of each single mechanism can be species dependent, this is due to the different response of microorganism to the unfavorable external conditions. SEM analysis of the substrates prooved the etching actions of the plasma generated in all process gasses on the surface of the PET foil. The several minute plasma exposition of the PET foil resulted in the occurence of the „hole corrosion“ on the PET surface. Contrary to these there were no visible changes observed in the paper structure.