Fructooligosaccharides (FOS) are short-chain fructans with a terminal glucose moiety and are found naturally in many plant species. Besides their wide use as an alternative sweetener in food and beverage industry, FOS have shown great potential as neutraceuticals against diabetes, colon cancer and bowel disease. The uses of FOS are dependent on the degree of polymerisation that they exhibit. β-fructofuranosidase (FFase) and fructosyltransferase (FTase) enzymes are capable of synthesing FOS from carbohydrate raw materials such as chicory and sugar beet. The aim of this study was to investigate the synthesis of FOS of a pre-defined chain length, from sucrose, by the enzyme FopAp; a β-fructofuranosidase from Aspergillus niger. ATCC 20611. The crude enzyme FopAp was successfully purified, with a yield of 78.20 %, by ammonium sulphate precipitation and anion exchange chromatography. Two protein fractions, named FA and FB were shown to exhibit FFase activity. SDS PAGE analysis revealed two proteins with molecular weights of 112 kDa and 78 kDa, which were identified as a FFase and a hydrolase. Temperature and pH optima of 20 ºC and 9, respectively, were observed for the transfructosylation activity in the FFase. The purified FFase exhibited a half life of 1.5 hrs under optimal conditions. Substrate kinetic studies indicated a high hydrolytic activity at low sucrose concentrations, with Vmax and Km of 1.25 μmol/ml/min and 3.28 mM, respectively. Analysis by response surface methodology identified temperature and pH to be significant factors for the production of kestose and nystose, at a 95 % level of confidence. These findings were confirmed by neural networks constructed to identify optimal conditions of FOS synthesis.FOS synthesis was found to be optimal between pH 6 and pH 9 at 25 ºC. The factor of reaction time was found to be insignificant within the selected experimental constraints, for both FOS species. The findings of this investigation are very important as the foundations of a commercially viable synthetic process for the production of FOS.

Optimizar un medio de cultivo para un linaje nativo Aspergillus niger Para-3 para demostrar la actividad β-fructofuranosidasa. Métodos: Investigación exploratoria- correlativa, de tipo básica y de diseño experimental de post prueba y factorial simple. El método específico estuvo basado en el análisis observacional. Se analizaron 167 especies nativas Aspergillus spp. la recolección de datos fue a partir de la colectas de especies en muestras de suelo por la técnica de aislamiento, identificación morfológica y aplicando protocolos de microbiología. Resultados: La identificación de dos cepas Aspergillus niger y phoenicis que demostraron tener actividad β-fructofuranosidasa intracelularmente con un aproximado de 613,31 y 390,16 U/g respectivamente. Mejorando la concentración de la sacarosa inicial y las condiciones para la activación de la enzima del linaje que mostró el mayor rendimiento entres las especies nativas Aspergillus spp. se obtuvo una actividad β-fructofuranosidasa de 1146,76 U/g. Conclusiones: Se identificó que la cepa nativa Aspergillus niger Para-3 es un potencial productor β-fructofuranosidasa y por ende de síntesis de fructooligosacáridos, así mismo que la activación de esta enzima se ve relacionada directamente al pH, temperatura e inducción de su fuente de carbono / Tesis

Recent data on the effect of trace elements on the production of citric acid by Aspergillus niger 72-4 suggested that at last a firm basis had been established for studies on the mechanism of production. Citric acid production is an important commercial process and most research had been directed toward obtaining high yields of the acid. The small amount of information on mechanisms is invalidated by the new knowledge of the importance of trace minerals in citric acid synthesis. The attempt at elucidating a system was approached by studying the distribution of organic phosphates in the mats and by manometric experiments. By using the Umbreit fractionation method combined with chromatographic analysis none of the phosphorylated intermediates present in the Embden-Meyerhof system could be identified. Evidence for a pentose and a ketose phosphate is presented. The manometric studies on still cultures were unsatisfactory due to a high endogenous rate and to difficulties in handling the mat. Shake cultures grown for four days and then depleted for 24 hours in the medium minus sucrose and manganese were shown to be a workable method for manometric studies. Using this method evidence for the presence of most of the enzymes required for the oxidation of the Krebs cycle intermediates is presented. A survey of the literature on cell preparations was made. Attempts to prepare active cell preparations failed since enzyme activity seems to be associated with the structural integrity of the mycelium. / Land and Food Systems, Faculty of / Graduate

Orientador : Renato Bonatelli Junior / Dissertação (mestrado) - Universidade Estadual de Campinas. Instituto de Biologia / Made available in DSpace on 2018-07-17T01:59:07Z (GMT). No. of bitstreams: 1 Vialta_Airton_M.pdf: 5894822 bytes, checksum: 4493787d6f88a9c0cd04c4bbaac32710 (MD5) Previous issue date: 1987 / Resumo: O presente trabalho teve como objetivos principais, verificar a ocorrência do ciclo parassexual em Aspergillus awamori, testar a produção de amiloglicosidase dos derivativos, mutantes e diplóides obtidos e realizar o cruzamento interespecífico com A. niger. Além desses, estudos foram desenvolvidos com a instabilidade apresentada por A. awamori.. A linhagem NRRL 3112 segrega conídios pro e forma setores espontaneamente. Este comportamento não seria o esperado para um clone e sugere a existência de heterozigose para as características estudadas, a qual poderia estar contida numa duplicação parcial ou total de genoma. Mutantes auxotróficos e morfológicos de Á. awamori foram conseguidos utilizando-se os métodos de isolamento total e de enriquecimento por filtração. Este último mostrou freqüências de isolamento 12 vezes maiores. Mutantes resistentes ao Brometo de Etídio também foram isolados, mas somente após indução com luz ultravioleta. Os mutantes foram utilizados em cruzamentos que permitiram verificar a ocorrência do ciclo parassexual. Através da análise dos segregantes, pode ser evidenciada ligação entre os genes etbl, grel bwnl; morl, arg2 e leul, mor2. O gene pabl segregou independentemente e, assim, foi possível sugerir 4 como o numero mínimo de grupos de ligação nessa espécie. Entre os critérios não geneticos utilizados na caracterização, o diâmetro de conídios e o tratamento com Benlate mostraram-se eficientes para separar haplóides de diplóides. O método de extração e quantificação de DNA por núcleo também foi adequado para esse fim. Com relação ã enzima, o primeiro passo foi averiguar se o método empregado estava medindo a atividade da amiloglicosidase, fato que foi confirmado fazendo o teste com o inibidor e a dextrina limite. Foi constatada uma relação inversamente proporcional entre a porcentagem de segregação de conídios pro e a produção de amiloglicosidase. Só foi possível obter o cruzamento entre A. awamori e Á. niger através de fu são de protoplastos. A freqüência de formação de colônias prototróficas foi relativamente baixa, situando-se na faixa de 0,6%, possivelmente devido ao pequeno número de protoplastos utilizados para a fusão e a um provável efeito tóxico diferencial do agente fusogênico utilizado. As colônias prototróficas isoladas inicialmente puderam ser classificadas como heterocarióticas. A partir destas, o produto de fusão híbrido foi obtido na forma de setores que exibiam complementação entre as marcas genéticas das parentais. Através da análise do híbrido, pode ser evidenciada ligação entre os genes nicl, 0lv2, bwnl, amy, pro. Houve distribuição ao acaso dos grupos de ligação, semelhante ao esperado para um diplóide intra - específico sugerindo alto grau de homologia cromossômica entre as duas espécies. Os mesmos critérios de caracterização utilizados com sucesso para separar linhagens haplóides de diplóides. nos cruzamentos intra-específicos foram adotados e também nesse caso mostraram resultados satisfatórios / Abstract: The present work with Aspergillus was done aiming to study the following: 1- Occurrence of the parasexual cycle;2- Occurrence of interspecific hybridization with A. niger.3- Amyloglucosidase production of the parental and derived strains, including auxotrophic mutants, diploids and the interspecific hybrid. During the first stage of the work, it was observed that the NRRL 3112 strain of A. awamori is unstable because it spontaneously segregates pro conidia (deficient for proline synthesis) and produces sectors. The last characteristic is also observed in pro derivatives and it was supposed that it is independent from pro conidia segregation. These characteristics are not expected from a clone and together with other evidences (Benlate segregation, differential susceptibility to acetone, variation in number of nuclei per conidia and conidial diameter), it was suggested that there is a partial or total duplication of the genome. Auxotrophic and morphological mutants of A. awamori were induced by ultraviolet light and selected by using total isolation and filtration enrichment methods. An increase of 12 times in the mutant frequency was observed when the last method was employed. Ethidium bromide resistant mutants were also isolated only after ultraviolet induction. Diploid strains were readily obtained and could easily be' separated from haploid strains by conidia diameter, Benlate segregation and nuclear DNA content. Segregation analysis indicated linkage between etb1 gre1 bwn1, mor1, arg1 and leu1, mor2. Because pab1 marker segregated independently from all others it was suggested at least 4 linkage groups for A. awamori. Only heterokaryotic colonies were detected when A. awamori and A. niger protoplasts were fused and plated in selective medium. The low frequency (0,6%) and the heterokaryotic nature of the colonies could probably be attributed to: 1) low protoplasts number and 2) toxic effect to the fusogenic agent to A. niger protoplasts. Hybrid colonies were isolated after several transfers in selective medium. The hybrid nature of these colonies was established by the same criteria used in the intraspecies crossing. Segregation analysis indicated a high level of chromosomal homology between the 2 species and it was possible to suggest linkage between nic1 olv2 genes of niger and bwn, amy, pro of awamori. As it was evident from the use of limit dextrin and a specific inhibitor. glucoamylase is the main enzyme activity detected by the usual assay procedure. It has also been detected that high. frequency of pro conidia in A. awamori is correlated with the low level of enzyme production / Mestrado / Mestre em Genetica

Orientador : Fumio Yokoya / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-19T02:11:52Z (GMT). No. of bitstreams: 1 Acosta_LilianaAgudelo_M.pdf: 2209949 bytes, checksum: 34d6759159e1044a10a95ec9339c90be (MD5) Previous issue date: 1994 / Resumo:Foi acompanhado o comportamento de vinte linhagens de Aspergillus niger na produção de ácido cítrico, quando cultivadas em diferentes formas e meios de fermentação. Observou-se que as exigências nutricionais de Aspergillus niger para a produção de ácido cítrico dependem da linhagem e do processo do cultivo. Dentre as linhagens estudadas e nas condições experimentais - fermentação em superfície e submersa em meio natural, escolheram-se as cepas FTPT 906 para a fermentação em superfície, FTPT 488 para fermentação submersa e FTPT 1762 como cepa aleatória, porque demonstraram maior capacidade de produção de ácido cítrico. Verificou-se a necessidade de tratamento do melaço de cana-de-açúcar com óxido de cálcio (CaO) e ferrocianeto de potássio (FCN), visando a remoção e precipitação de íons que contaminam os substratos evitando, assim, sua ação inibidora na fermentação cítrica. Observou-se que a quantidade de FCN requerida em excesso é determinado principalmente pela linhagem do fungo e qualidade do substrato. Aspergillus niger FTPT 906 demonstrou, nas condições de fermentação em superfície em meio natural, maior capacidade de produção de ácido cítrico. A adição de íons ferro e cobre em conjunto ao meio de cultura não favorece a produção de ácido; o mesmo aconteceu quando adicionou-se íon cobre no processo. 0 crescimento celular não foi influenciado pela adição de íons metálicos no meio de cultura. / Abstract: Twenty citric acid producing strains of Aspergillus niger were tested regarding their capacity to produce citric acid in various media composition under surface and submerged growth conditions. Nutritional requeriment for their growth and acid production varied from strain to strain. Strain FTPT 906 was selected for further studies by surface culture method because of its hability to produce highest amount of acid. Strain FTPT 488 produced highest amount of acid in submerged culture and strain FTPT 1762 produced reazonable amounts of acid in both cultivation methods. The treatment with calcium oxide and potassium ferrocianate (FCN) was requered for the precipitation of metallic ions from molasses media to reduce inhibition of citric acid production. The excess of FCN required for highest citric acid production varied according to the fungus strain and qualites of substrate. Addition of iron and copper ions to the media did not enhance acid production. The growth of fungus was not affected by those metallic ions. / Mestrado / Mestre em Ciência de Alimentos

In den vergangenen Jahrzehnten kam es durch den Einsatz massiv immunsupprimierender Therapien zu einer steigenden Inzidenz invasiver Mykosen. Die durch eine Infektion mit A. fumigatus ausgelöste invasive Aspergillose stellt eine lebensbedrohliche Erkrankung für Patienten mit hämatologischen Malignomen oder nach hämatopoetischer Stammzelltransplantation dar. Für die Behandlung solcher Erkrankungen ist eine frühzeitige Diagnosestellung unabdingbar. Da invasive diagnostische Maßnahmen bei den betroffenen Patienten jedoch häufig kontraindiziert sind, werden neue Biomarker und nicht invasive Diagnoseverfahren intensiv beforscht. Ein kürzlich beschriebener Ansatz zur Primär- und Verlaufsdiagnostik bei Patienten mit invasiven pulmonalen Aspergillosen und Mukormykosen ist die Verwendung des auf aktivierten T-Zellen exprimierten Aktivierungsmarkers CD154 zur durchflusszytometrischen Quantifizierung A. fumigatus-spezifischer T-Helfer-Zellen. Die Detektion dieser Zellen mit dem in der Literatur beschriebenen Protokoll erfordert die Isolation von PBMCs und duldet vor der Verarbeitung der Proben in einem spezialisierten Labor nur kurze präanalytische Lagerungszeiten. Dies stellt einen limitierenden Faktor für die klinische Verwendbarkeit des beschriebenen Assays dar. Die vorliegende Dissertationsschrift beschäftige sich damit, den beschriebenen Assay zur Detektion A. fumigatus-spezifischer T-Zellen, hinsichtlich seiner Präanalytik und klinischen Anwendbarkeit eingehender zu evaluieren und zu optimieren. Zunächst konnte gezeigt werden, dass mittels Verdünnung und Agitation der zur PBMC Isolation verwendeten Blutproben eine Verlängerung des präanalytischen Zeitfensters zwischen Blutentnahme und Aufbereitung auf bis zu 4 h möglich ist, ohne dass dabei die Sensitivität des CD154-basierten T-Zell-Assays beeinträchtigt wird. Weiterhin konnte die Verwendung eines Vollblut-basierten Protokolls, das auf die zeitaufwendige Isolation von PBMCs verzichtet, etabliert werden. Hinsichtlich seiner Detektionsleistung zeigte sich das Vollblutprotokoll dem PBMC Protokoll grundsätzlich überlegen. Für verschiedene Stimulationsperioden konnte ein gegenüber dem PBMC Standardprotokoll reproduzierbarer Konversionsfaktor ermittelt werden, welcher einen Vergleich von Ergebnissen bei alternierender Durchführung von PBMC- und Vollblut-Assay bei den selben Patienten möglich macht. Das Protokoll erlaubt die Kombination von Stimulations- und Transportzeit unter Verwendung eines auf 37 °C temperierten Transportgefäßes. Eine alleinige Stimulation bei Raumtemperatur zeigte sich hingegen nicht erfolgreich. Die Anwendung des Assays am Point-of-Care wird durch die Verwendung vorbereiteter, marktüblicher Blutentnahmeröhrchen möglich, welche bis zum Zeitpunkt der Verwendung eingefroren gelagert werden können. Eine Analyse der Material- und Arbeitskosten ergab eine Reduktion der Gesamtkosten für die Verwendung des Vollblut-basierten Protokolls von bis zu 22 % pro Probe. Prinzipiell kann davon ausgegangen werden, dass der entwickelte Assay mit jedem Peptid-Antigen durchführbar ist, das in konstanter Qualität bezogen oder generiert werden kann und einen immunogenen Effekt aufweist, ohne dabei mit anderen verwendeten Reagenzien zu interagieren. Weiterhin wurde in der vorliegenden Arbeit geprüft, wie sich T-Zell-inhibitorische Substanzen auf die Testergebnisse auswirken. Hierbei fand sich eine nicht unwesentliche Anzahl falsch-negativer Testergebnisse. Die Ergebnisse der vorliegenden Dissertationsschrift zeigen, dass eine suffiziente und sensitive Bestimmung A. fumigatus-spezifischer T-Zellen im Rahmen eines Vollblut-basierten Protokolls möglich ist. Eine Ausweitung des Assays für andere Infektionserreger, sowie die Entwicklung Brefeldin A-freier Protokolle wären wünschenswert. Ein mögliches Einsatzgebiet stellen klinisch infektiologische Studien oder umwelt- und arbeitsmedizinische Fragestellungen dar. / Over the past decades, the use of immunosuppressive therapies has led to an increasing incidence of invasive mycoses. Specifically, invasive aspergillosis, most commonly caused by Aspergillus fumigatus, is a life-threatening disease in hematopoietic stem cell transplant recipients and patients with hematologic malignancies. An early confirmation of the diagnosis is pivotal in order to improve therapeutic outcomes. As invasive diagnostic procedures are often contraindicated in high-risk patients, non-invasive diagnostic modalities and novel biomarkers for invasive mold infections are intensively studied. A recently published method to diagnose and monitor invasive pulmonary aspergillosis utilizes flow cytometric quantification of mold-reactive T-helper cells by CD154 upregulation, an activation marker expressed on activated T cells. The published standard methodology to quantify these cells is based on isolated peripheral blood mononuclear cells (PBMCs). Among several limitations hampering the applicability of this method in the clinical routine, our group has previously shown that PBMC-based mold-reactive T-cell quantification is highly susceptible to pre-analytic delays. Therefore, my thesis focused on the optimization of pre-analytic sample handling prior to PBMC isolation in order to improve the detection efficacy of A. fumigatus-specific T-helper cells. Using agitation and dilution of blood samples prior to PBMCs isolation, the pre-analytic storage period could be extended to 4 hours without observing an adverse impact on assay sensitivity. To overcome the many logistical limitations of PBMC-based assays, my thesis further focused on the evaluation and optimization of a whole blood-based method to quantify A. fumigatus-specific T-helper cells. Therefore, blood collection tubes containing costimulatory antibodies and an A. fumigatus mycelial lysates were prepared and inoculated with heparinized whole blood from healthy adults. Frequencies of CD154+ A. fumigatus-specific T-helper cells were then compared with PBMC-based detection rates. Using optimized stimulation schemes for both matrices, significantly higher specific T-cell detection rates were achieved by the whole blood-based method. Excellent correlation and reproducible conversion factors between whole blood- and PBMC-based results were observed. Using frozen ready-to-use test tubes, detection rates of specific T cells were comparable to those observed with freshly prepared tubes. Taken together, these results suggest that our whole blood protocol provides a robust, highly sensitive, and cost-effective method for mold-reactive T-cell quantification. Our protocol allows for point-of-care sample stimulation and may contribute to better assay standardization for multi-center studies of mold reactive T-cell assays. Lastly, my thesis focused on the impact of T-cell-inhibitory immunosuppressive pharmacotherapy on A. fumigatus specific T-cell quantification. My results revealed significantly reduced mean detection rates of specific T-cells and considerable inter-individual variation in assay reliability upon exposure of PBMC samples to cyclosporine A, mycophenolic acid, and prednisolone. These findings suggest that false-negative results need to be considered when performing this test in immunosuppressed patient cohorts and inspired further studies of our group to attenuate the detrimental impact of immunosuppressive agents on assay performance by enhanced costimulation.

Aspergillus flavus is a pathogenic fungus causing maize ear rot disease and producing aflatoxins that are carcinogenic to humans and animals. Characterizing maize host resistance mechanisms and prioritizing candidate resistance genes are important to the development of resistant maize germplasm. This study investigated transcriptomics approaches and statistics methods on the identification of maize host resistance genes. Full-length cDNA libraries were also constructed and evaluated for gene cloning and functional analysis. This study established important sources for functional studies on differentially expressed genes and for identification of allelic gene forms to characterize gene polymorphisms and facilitate the development of DNA markers.

Aflatoxins are a secondary metabolite produced by the fungus Aspergillus flavus. A. flavus has been known to infect several crops including tree nuts, peanuts, rice, cotton and maize. Aflatoxins have been found to cause tumors with aflatoxin B1 being the most carcinogenic biologically produced substance known to man. Therefore, the FDA has restricted the amount of aflatoxin in maize for human consumption to 20 ppb (ng/g). An estimated $225 million are lost each year in the United States due to aflatoxin contamination in maize crops alone. Agriculture is a vital part of Mississippi’s economy, and maize is one of its largest crops. The purpose of this research is to track the correlations between aflatoxin accumulation and Aspergillus flavus fungal biomass for the first several weeks after inoculation, as well as the spreading of the fungus and the aflatoxin throughout the inoculated ear of maize. This will allow for better understanding of the pathogen-host interactions and how the fungus progresses over time. GA209 x T173 is the aflatoxin accumulation susceptible maize hybrid, GA209 x Mp313E is the susceptible and resistant hybrid, and Mp717 x Mp313E is the resistant maize hybrid to aflatoxin accumulation. These maize hybrids were each inoculated with toxin producing Aspergillus flavus NRRL 3357 and water as a control 21 days after silk maturation. Collections of the inoculated maize cobs were made 3, 7, 14, 21, 28, 35, and 60 days after inoculation. Maize samples were collected and analyzed for aflatoxin and DNA concentration. The extracted aflatoxin was analyzed using an LC/MS. The fungal biomass was determined by performing quantitative real time polymerase chain reaction (PCR). GA209xT173 and Mp717xMp313E showed no aflatoxin production two days after inoculation. The resistant maize hybrid lead in aflatoxin accumulation the last two years but had the least amount of fungal biomass for second and third years of the experiment The production of aflatoxin seems to begin decelerating after 21 days after inoculation. Resistance characteristics are more to prevent fungal infection. Fungal biomass was significantly higher in the susceptible hybrid GA209xT173 compared to the other hybrids. However, fungal spread was significantly higher in Mp313ExT173 and Mp717xMp313E.

No description available.

No description available.