Conselho Nacional de Desenvolvimento Científico e Tecnológico / The food industry has changed in recent decades the focus of the production, since the eating habits of consumers have been directed to practical, fast and tasty foods. So, breaded frozen chicken were created, one of the biggest hits of the fast food industry. This class of pre-made and frozen products allows for long-term storage and can select species of microorganisms capable of growing in low temperatures, especially filamentous psychrophilic fungi. Despite annual losses are estimated at 1 to 1.5 % by fungal spoilage of frozen chicken nuggets, rare are the available scientific data. The objective of this study was to conduct a general mycological investigation of a processing industry of frozen chicken nuggets, analyzing the raw materials, the products in different processing steps and the ambient air of each unit of operation. It was also analyzed the effect of heating treatments applied in the manufacture of the nugget on inactivation of Penicillium commune (NGT 16/12), Penicillium polonicum (NGT NGT 23/12 and 33/12), Penicillium glabrum (NGT 29/12 and NGT 35/12), Penicillium solitum (NGT 30/12) and Penicillium crustosum (NGT 51/12), main species related to deterioration of these products. The flour exhibited counts between 101 and 104 CFU/mL, predominating species P. polonicum, Aspergillus flavus, Aspergillus candidus, Penicillium citrinum and Eurotium amstelodami. The following processed samples showed a steady reduction in scores for 101 CFU/g, with a predominance of P. polonicum. In the other hand, regarding the samples of final product analyzed, 10% were contaminated by P. glabrum, with was also the most predominant species of spoilers in the air environment. The results show that the P. polonicum (NGT 23/12), P. commune (NGT 16/12), P. solitum (NGT 30/12) and P crustosum (NGT 51/12) were able to survive to the heat treatments applied (fried by immersion in oil at 195-200 °C for 6 seconds), and baking in oven at 120-130 °C until the internal temperature reached 70 °C when inoculated in the frozen chicken nuggets. Additionally, it was observed that P. polonicum (NGT 23/12), was the most heat resistant species, recovering counts of 104 CFU/g after frying for 6 minutes and 30 seconds of cooking, having 2,02 log CFU/g reduced at 72 °C and 3,29 log CFU/g reduced at 78 °C in the internal of the product during the baking. According to the results it was observed that both the flour used to manufacture breaded as the air industry environment pose a hazard, then strategies must be taken to reduce the presence of fungal spores in this points, possible sources of these fungal contamination. / A indústria de alimentos tem modificado nas últimas décadas o foco de produção, uma vez que os hábitos alimentares dos consumidores foram alterados, em busca de alimentos práticos, rápidos e saborosos. Logo, foram criados os empanados congelados de frango, um dos maiores sucessos da indústria de fast food. Esta classe de produtos pré-prontos e congelados permite a armazenagem por longos períodos o que seleciona espécies de micro-organismos capazes de se desenvolver em condições de baixas temperaturas, com destaque para os fungos filamentosos psicrófilos. Apesar das perdas anuais serem estimadas em 1 a 1,5 % por deterioração fúngica de empanados congelados de frango, raros são os dados científicos disponíveis. O objetivo deste estudo foi realizar uma investigação micológica geral de uma indústria processadora de empanados congelados de frango, analisando as matérias-primas, os produtos originados e o ar ambiente de processamento. Assim como, o efeito dos tratamentos térmicos aplicados na fabricação dos empanados sob Penicillium commune (NGT 16/12), Penicillium polonicum (NGT 23/12 e NGT 33/12), Penicillium glabrum (NGT 29/12 e NGT 35/12), Penicillium solitum (NGT 30/12) e Penicillium crustosum (NGT 51/12), principais espécies relacionadas a deterioração destes produtos. As amostras de farinha apresentaram contagens entre 101 e 104 UFC/mL, predominando as espécies de P. polonicum, Aspergillus flavus, Aspergillus candidus, Eurotium amstelodami e Penicillium citrinum. As amostras seguintes ao processamento apresentaram uma constante redução nas contagens para 101 UFC/g, com predominância de P. polonicum. Já nas amostras analisadas do produto final, 10% apresentaram contaminação por P. glabrum, espécie também predominante no ar ambiente da fábrica. Os resultados demonstraram que o P. commune (NGT 16/12), P. polonicum (NGT 23/12), P. solitum (NGT 30/12) e P. crustosum (NGT 51/12) são capazes de sobreviver aos tratamentos térmicos aplicados (fritura por imersão com óleo a 195-200 °C por 6 segundos) e cozimento em forno à 120-130 °C até alcançar a temperatura interna de 70 °C, quando inoculados nos empanados congelados de frango. Adicionalmente, foi verificado que o P. polonicum (NGT 23/12), espécie mais resistente aos tratamentos, manteve-se estável com média de contagem fúngica de 104 UFC/g desde a fritura até 6 minutos e 30 segundos de assamento, tendo 2,02 log UFC/g de redução com 72 °C e 3,29 log UFC/g de redução com 78 °C no interior do produto durante o assamento. De acordo com os resultados apresentados foi observado que tanto as farinhas utilizadas na fabricação dos empanados quanto o ar ambiente da indústria representam um perigo de contaminação, assim como foi verificado a sobrevivência de algumas das principais espécies deteriorantes de empanados aos tratamentos térmicos aplicados na indústria, logo estratégias devem ser adotadas para a redução de esporos fúngicos em possíveis fontes de contaminação e a necessidade de novas adequações dos tratamentos para eliminação de fungos filamentosos.

Orientadores: Helena Maria Andre Bolini, Rosires Deliza / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T19:13:21Z (GMT). No. of bitstreams: 1 Marcellini_AlineMotadeBarros_M.pdf: 1250175 bytes, checksum: dbebba72665ad354430068a9e091ff20 (MD5) Previous issue date: 2006 / Resumo: O abacaxi é uma fruta apreciada em todo mundo, devido às suas distintas características sensoriais, podendo ser consumido ¿in natura¿ ou processado de diversas maneiras, sendo o suco a forma de processamento mais popular. O Brasil é o quarto maior produtor mundial de abacaxi, e nos cinco últimos anos a exportação do suco da fruta aumentou cerca de sete vezes. Associado à produção deste está o tratamento térmico, que afeta a qualidade sensorial, produzindo alterações desagradáveis ao produto. Nos últimos anos, os consumidores têm procurado por alimentos que mantenham as características sensoriais e nutricionais do produto, aliado à garantia da segurança microbiológica. A Alta Pressão Hidrostática (APH) é uma tecnologia inovadora, capaz de inativar microorganismos patogênicos e deteriorantes, enquanto minimiza a perda da qualidade sensorial e nutricional do alimento. Este trabalho teve como objetivo processar suco de abacaxi (Ananás comosus (L.) Merril, variedade Smooth Cayenne) através da tecnologia de APH aplicada à polpa do fruto e avaliar as características microbiológicas, sensoriais e a aceitabilidade do produto obtido. Os dados foram analisados através da Análise de Variância (ANOVA), testes de médias de Tukey, Análise de Componentes Principais (ACP), Mapa Interno da Preferência, ¿Cluster Analysis¿, bem como Mapa Externo da Preferência utilizando o programa estatístico SAS e XLSAT. O processamento a APH da polpa de abacaxi por 300MPa e 5 min à 25oC, demonstrou ser eficaz para a produção de polpa com vida útil de 14 dias, estando de acordo com os parâmetros determinados pela legislação brasileira. Os resultados obtidos na determinação de concentração de polpa e doçura ¿ideais¿ para a formulação do suco de abacaxi foram 56,5% e 7,0%, respectivamente. A Análise Descritiva Quantitativa demostrou a similaridade sensorial entre as amostras de suco de abacaxi obtida através da polpa ¿in natura¿ e da polpa tratada por APH (300MPa/5 min/25oC) e que de forma geral, possuem diferença significativa (p<0,05) das amostras comerciais estudadas. Os atributos sabor de suco de abacaxi natural, cor característica de suco de abacaxi natural, aroma característico de suco de abacaxi natural, consistência e presença de fibras visual e percebida na boca apresentaram maior importância para a caracterização dos produtos obtidos através da polpa ¿in natura¿ e da polpa tratada por APH (300MPa /5 min/ 25oC). O teste de aceitação comprovou que as amostras de suco de abacaxi obtidas através da polpa ¿in natura¿ e da polpa tratada por APH (300MPa/5 min/25oC), apresentaram aceitação significativamente (p<0,05) superior às amostras comerciais avaliadas, e sem diferença (p<0,05) entre si, sendo preferidas por segmentos específicos de consumidores. Os atributos que apresentaram maior importância na caracterização dos produtos obtidos através da polpa ¿in natura¿ e da polpa tratada por APH (300MPa/5 min/25oC) dirigiram a preferência de alguns segmentos de consumidores / Abstract: Pineapple is a well appreciated fruit worldwide due to its distinct sensory characteristics, to be consumed ¿in natura¿ or processed in many different ways, juice being the most popular industrialized product. Brazil is the fourth world pineapple producer, presenting a seven fold increase in pineapple juice exportation in the last five years. Thermal treatment associated to juice production affects its sensory quality, resulting in unacceptable changes in the industrialized product. Nowadays consumers are searching for Technologies which preserve nutritional and sensory characteristics of food, as well as guarantee its microbiological safety. High Hydrostatic Pressure (HHP) is an innovative technology capable of inactivating pathogenic and deteriorative microorganisms while minimizing loss of sensory and nutritional product quality. The objectives of this study were to process pineapple juice (Ananas comosus (L.) Merril, Smooth Cayenne variety) through HHP technology applied to the fruit puree and to investigate the microbiological and sensory characteristics of the obtained juice, as well as its acceptability. Data were analyzed through Analysis of Variance (ANOVA); Tukey¿s mean tests; Principal Components Analysis (PCA); Internal Preference Mapping; Cluster Analysis, as well as External Preference Mapping, using statistical programs as SAS and XLSTAT. HHP processing of pineapple puree at 300 MPa for 5 minutes at 25ºC resulted in the production of pineapple puree with a shelf-life of 14 days, in accordance to microbiological parameters established by Brazilian Legislation. Results found in the determination of ideal pineapple puree concentration and ideal sweetness for pineapple juice formulation were 56.5% and 7.0%, respectively. Quantitative Descriptive Analysis (QDA) revealed a sensory similarity between the pineapple juice samples obtained from the utilization of ¿in natura¿ puree and of HHP treated puree (300MPa/5min/25ºC), both showing a statistically significant difference (p<0,05) compared to the commercial pineapple juice samples evaluated in this study. The following sensory attributes: natural pineapple juice flavor; characteristic natural pineapple juice color; characteristic natural pineapple juice aroma; consistency and visual and mouth-perceived fiber presence were considered the most important concerning the sensory characterization of both juices obtained from the utilization of ¿in natura¿ puree and of HHP treated puree (300MPa/5min/25ºC). The acceptance test established that the pineapple samples obtained from the utilization of ¿in natura¿ puree and of HHP treated puree (300MPa/5min/25ºC) showed higher statistically significant acceptance (p<0,05) in relation to the evaluated commercial samples, bearing no statistical significant difference (p<0,05) between them; each one being preferred by specific consumer segments. The sensory attributes which mostly contributed to the characterization of pineapple juices obtained from the utilization of ¿in natura¿ puree and of HHP treated puree (300MPa/5min/25ºC) drove the preference in some consumer segments / Mestrado / Consumo e Qualidade de Alimentos / Mestre em Alimentos e Nutrição

Orientadores: Marcia Regina Braga, Marcia Maria Camargo de Morais / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T04:21:10Z (GMT). No. of bitstreams: 1 Galvao_DaianeFelbergAntunes_D.pdf: 15313845 bytes, checksum: aa218f685858df886eb7062bfe4337dc (MD5) Previous issue date: 2014 / Resumo: Fruto-oligossacarídeos (FOS) são frutanos de baixo peso molecular produzidos por microorganismos. O interesse em FOS vem aumentando uma vez que eles são considerados ingredientes funcionais benéficos à saúde humana. Com o objetivo de analisar como a produção de FOS e a composição da parede celular de fungos filamentosos é afetada pela fonte de carbono, os fungos Fusarium solani (URM 3338) e Neocosmospora vasinfecta (URM 3329) foram cultivados em meios contendo cinco fontes de carbono diferentes (sacarose, inulina, glucose, frutose ou glucose mais frutose, todos a 1%) e coletas foram realizadas aos 5, 10 e 15 dias de crescimento. A partir do meio de cultivo filtrado foram analisados o pH, teores de açúcar total, açúcares redutores e proteínas, a presença de FOS e atividades enzimáticas invertásica e inulinásica. A partir do micélio, a biomassa foi quantificada e a parede celular foi isolada e sua composição em açúcares neutros, ácidos urônicos e quitina analisada. Foi avaliada também a expressão relativa de genes de síntese de parede celular b-1,3-glucano sintase e quitina sintases. Os dois fungos utilizaram todas as fontes de carbono crescendo nas diferentes condições. Atividade de hidrólise foi detectada no meio contendo sacarose ou inulina para o fungo F. solani, gerando glucose, frutose e fruto-oligossacarideos como produtos havendo utilização dos monossacarídeos. O micélio deste fungo apresentou alterações visíveis no crescimento em meio sólido apenas no meio com frutose, mas foi observada igual quantidade de quitina da parede celular deste fungo quando crescido por cinco dias em sacarose e inulina, mas em menor quantidade com relação aos demais meios. As análises de expressão relativa de genes mostraram indução do gene da b-1,3-glucano sintase e repressão do gene quitina sintase 5 em sacarose e inulina com relação a condição frutose. Estes dados sugerem que a alteração na composição da parede celular do F. solani pode ter relação com a secreção de enzimas nos meios sacarose e inulina. Para N. vasinfecta, quando crescido em sacarose foi observada atividade de transfrutosilação, com a liberação de glucose e síntese de 1-cestose (FOS) no meio. Transfrutosilação também foi observada no meio que teve inulina como fonte de carbono. O micélio deste fungo apresentou alterações visíveis em meio sólido nas condições frutose e inulina, sendo mais hialino do que nas demais condições. A quantidade de quitina na parede celular deste fungo crescido por cinco dias foi maior nas condições frutose e inulina com relação às demais. As análises de expressão relativa de genes mostraram indução dos genes de quitina sintase 4 e 5 nestas duas condições em relação à sacarose. A partir dos resultados, pode-se concluir que as fontes de carbono oferecidas foram utilizadas pelos fungos, que as mesmas afetaram a composição de açúcares da parede celular e a expressão de genes de síntese de componentes da parede e que estes fungos são promissores para a produção de FOS, pois possuem enzimas que hidrolisam a inulina, além de enzimas que sintetizam oligossacarídeos a partir de sacarose por transfrutosilação / Abstract: Fructooligosaccharides (FOS) are low molecular weight fructans produced by microbes and plants. Interest in FOS has been increasing since they are considered as functional food ingredients with benefical effects in human nutrition. With the aim of examining how the production of FOS and the composition of the cell wall of filamentous fungi are affected by the carbon source, Fusarium solani (URM 3338) and Neocosmospora vasinfecta (URM 3329) were cultured in media containing five different carbon sources (sucrose, inulin, glucose, fructose or glucose plus fructose) and samples were taken at 5, 10 and 15 days of growth. From the filtered culture medium, pH, total carbohydrates, reducing sugars and proteins, the presence of FOS and inulinase and invertase activities were analyzed. Mycelium biomass was measured and the cell wall was isolated and its composition in neutral sugars, uronic acids and chitin analyzed. The expression of b-1,3-glucan synthase and chitin synthase genes was also evaluated. Both fungi utilized all the carbon sources for growing. In sucrose- and inulin-containing media, hydrolytic activity was detected in F. solani generating glucose, fructose and FOS as products. When grown on solid culture media, visible changes were observed in mycelium of this fungus only in fructose, but the amount of chitin in the cell wall was higher in the sucrose and inulin-containing media when compared to other carbon sources. The expression b-1,3-glucan synthase gene was induced and chitin synthase 5 gene repressed on sucrose and inulin media. N. vasinfecta showed transfructosilation activity when was grown in sucrose, with release of glucose and synthesis of 1-kestose (FOS) in the culture medium. Transfructosilation was also observed in the inulin-containing medium. The mycelium showed visible changes when the fungus was cultured in solid medium with fructose or inulin as carbon sources. The amount of chitin in the cell wall of this fungus when grown for five days in inulin or fructose was higher in comparison to other carbon sources. The analysis of gene expression showed induction of chitin synthase 4 and 5 genes in these two conditions in relation to sucrose. From the results it can be concluded that the carbon sources affected growth, enzymic activity, composition of the cell wall and gene expression in F. solani and N. vasinfecta, and that these fungi are promising organisms for FOS production since they secrete enzymes that hydrolyze inulin or synthesize oligosaccharides from sucrose by transfructosylation / Doutorado / Biologia Celular / Doutora em Biologia Celular e Estrutural

Abstract Flocculation plays a critical role in the activated sludge process, where flocs are removed by settling and where unsatisfactory flocculation is resulting in poor effluent quality. Control and operation of the process is also challenging as it is sensitive to external and internal disturbances. Furthermore, stricter environmental demands are also being placed on wastewater treatment and discharge quality thus solutions are needed to improve the current systems. A novel optical monitoring method employing a tube flow and a CCD camera was developed to characterize the flocs and filaments of the sludge, and the method was tested on samples from full-scale activated sludge plants. An online device operating on the same principle was also developed and this was tested over a period of eight months at municipal wastewater treatment plant. Optical monitoring was employed in the laboratory to study the breakage of activated sludge flocs. Based on the image analysis data, in the industrial plant the major breakage process was large-scale fragmentation. In the two municipal plants, it was surface erosion. The flocs had more filaments and were more irregular in shape in the industrial plant, which could be the reason for the large-scale fragmentation. The effect of floc morphology on the effluent clarity of the activated sludge process was studied in the industrial and municipal activated sludge plants by optical monitoring over periods of three months and eight months, respectively. The changes in floc morphology took place slowly in both plants. Four major factors that correlated with the purification results were the size and shape of the flocs and the quantities of small particles and filaments. The image analysis results suggested that the settling problem that occurred during the test periods in the industrial plant was caused by dispersed growth, whereas that in the municipal plant was caused by filamentous bulking. In conclusion, it is possible to use the developed method online in order to analyse the state of flocculation. Thus the method could be useful when developing online monitoring applications for quantifying floc characteristics and for diagnosing the causes of settling problems in the wastewater treatment plants. / Tiivistelmä Aktiivilieteprosessissa flokkulaatiolla on merkittävä rooli, sillä muodostuneet flokit poistetaan prosessista laskeutuksen avulla. Siten huono flokkulaatio johtaa puhdistetun jäteveden kiintoainemäärän lisääntymiseen. Prosessin säätö ja operointi on kuitenkin hankalaa, sillä aktiivilieteprosessi on herkkä ulkoisille ja sisäisille häiriöille. Jätevedenpuhdistukseen liittyvät ympäristövaatimukset ja päästöehdot vesistöihin ovat myös tiukentuneet, joten uusia menetelmiä tarvitaan parantamaan nykyisiä prosesseja. Tässä työssä kehitettiin uusi, optinen kuvantamismenetelmä karakterisoimaan flokkeja ja rihmoja. Menetelmä hyödyntää putkivirtausta ja CCD-kameraa ja sitä testattiin aktiivilietelaitosten näytteillä. Lisäksi kehitettiin samaa periaatetta noudattava online-laitteisto, jota testattiin kahdeksan kuukauden ajan. Optista kuvantamista testattiin laboratoriossa flokkien hajoamistutkimuksessa. Kuva-analyysitulosten perusteella kahden kunnallisen aktiivilietelaitoksen flokit hajosivat pintaeroosioon perustuvan mallin mukaan ja teollisen aktiivilietelaitoksen flokit hajosivat fragmentaatiomallin mukaan. Teollisen aktiivilietelaitoksen flokeissa oli enemmän rihmoja ja ne olivat epäsäännöllisemmän muotoisia, mikä voi olla syynä flokkien fragmentaatioon. Flokkien morfologian vaikutus jäteveden puhdistustuloksiin tutkittiin teollisessa (kolmen kuukauden ajan) ja kunnallisessa (kahdeksan kuukauden ajan) aktiivilietelaitoksessa optisella kuvantamismenetelmällä. Molemmissa laitoksessa muutokset flokkien morfologiassa tapahtuivat hitaasti. Neljä tärkeintä tekijää, jotka korreloivat puhdistustulosten kanssa, olivat flokkien koko ja muoto sekä pienten partikkelien ja rihmojen määrä. Kuva-analyysitulosten perusteella laskeutumisongelma teollisessa jätevesilaitoksessa johtui flokinmuodostajabakteerien liian pienestä määrästä ja kunnallisessa jätevesilaitoksessa rihmamaisten bakteerien liikakasvusta. Yhteenvetona voidaan todeta, että kehitettyä menetelmää on mahdollista käyttää online-mittarina sekä sen avulla voidaan arvioida flokkulaation tilannetta. Siten menetelmää on mahdollista hyödyntää flokkien ominaisuuksien karakterisoinnissa ja arvioidessa jätevedenkäsittelylaitoksen laskeutumisongelmien aiheuttajaa.

Magnaporthe grisea est responsable de la pyriculariose du riz, principale maladie de cette céréale. L’entrée du champignon dans la plante hôte se fait via l’appressorium. La différenciation de cette structure résulte d’une réorientation génétique et métabolique, et nécessite une régulation génétique fine. Une étude transcriptomique comparant les stades mycélium et appressorium a permis de montrer qu’environ 1300 ORFs sont surexprimées au stade appressorial. Ce transcriptome a permis l’identification de 32 gènes codant pour des facteurs de transcription pour lesquels dix mutants de délétion ont été générés et caractérisés. L’étude de leur pouvoir infectieux a révélé que le mutant délété du gène TF7 présente une pathogénie réduite de 70% sur plant d’orge. De plus, ce mutant est incapable de former des appressoria sur membrane artificielle sauf en présence d’un inducteur chimique (1,16- hexadecanediol). Par ailleurs, lorsque les appressoria sont formés, ils éclatent au bout de 14 heures. Cette altération peut être compensée par l’addition de sorbitol comme osmoprotecteur. Ce mutant est hypersensible à la Nikkomycine Z, un inhibiteur de la chitine synthase suggérant une altération du métabolisme pariétal. Un transcriptome différentiel réalisé à partir d’appressoria sauvages et mutés différenciés sur membrane de Téflon a révélé que des gènes impliqués dans le métabolisme de la chitine sont sous-exprimés dans le mutant ΔTf7. Le facteur de transcription Tf22 dont la délétion conduit à une réduction de 70% de la pathogénie sur riz a également fait l’objet d’une attention particulière. En effet, la recherche d’homologie a montré la présence de deux protéines homologues à Tf22 chez les deux champignons phytopathogènes S. nodorum et C. nicotianae et au-delà de la conservation d’un cluster potentiel de gènes du métabolisme secondaire, identifié chez C.nicotianae. La caractérisation du mutant a montré que l’expression de ce cluster potentiel est régulée négativement par le gène TF22 / Magnaporthe grisea is responsible for rice blast, the major disease of rice. The entry of the fungus in the host plant is via a specialized cell called appressorium. The differentiation of this structure results from a genetic and metabolic shift, and requires fine control mechanisms. A transcriptomic study comparing vegetative mycelium and appressorium mature stage, characteristic of the pre-penetration step was realized. In order to identify new regulatory networks specific of the appressorial differentiation, we focused on 32 genes encodi. Ten deletion mutants of transcription factor genes were generated and characterized. The study of their infectivity revealed that the TF7 gene deleted mutant has a reduced pathogenicity of 70% on barley plant resulting from an inability to penetrate the plant surface. Moreover, unlike the parental strain, this mutant is unable to form appressoria on artificial membrane except in the presence of a chemical inducer (1.16-hexadecanediol). Moreover, when appressoria are formed, they burst after 14 hours. This alteration can be compensated by a sorbitol solution acting as an osmoprotectant. This mutant is hypersensitive to nikkomycin Z, a chitin synthase inhibitor suggesting an alteration of parietal metabolism. A differential transcriptome was conducted comparing wild and mutated appressoria differentiated on Teflon membrane revealed that genes involved in chitin metabolism are dependent on the transcription factor Tf7. A second transcription factor Tf22 whose, deletion leads to a reduction of 70% of pathogenesis on rice, has also been studied. Indeed, the homology search showed the presence of two proteins homologous to Tf22 for S. nodorum and C. nicotianae. Beyond the conservation of the transcription factor, we observed the conservation of a potential cluster of genes of secondary metabolism identified in C.nicotianae. The characterization of the mutant revealed that expression of this potential cluster is negatively regulated by the gene TF22

Les métaux lourds et les radionucléides sont présents dans différents écosystèmes du monde à cause de contaminations naturelles ou des activités anthropiques. L’utilisation de micro-organismes pour restaurer ces écosystèmes pollués, processus connu sous le nom de bioremédiation, suscite beaucoup d’intérêt, spécialement aux pH proches de la neutralité. Les minéraux de fer qui encroûtent les bactéries neutrophiles du fer, notamment les Oxydes de Fer Biogéniques (BIOS en anglais), ont une structure très faiblement cristalline, qui en plus de leur grande surface et réactivité font d’eux d’excellents supports pour le piégeage de polluants inorganiques. Dans cette thèse nous avons étudié les différents mécanismes de piégeage de l’uranium uranium par la bactérie neutrophile Sphaerotilus natans, choisie comme modèle bactérien de micro-organismes du fer capables de filamenter en formant des gaines. S. natans peut croître sous forme de cellules individuelles ou formant des filaments. Ces derniers ont été utilisés pour étudier la biosorption d’U(VI) et sa sorption sur les BIOS. De plus, la sorption d’U(VI) sur les analogues abiotiques de ces minéraux de fer a été testée. Afin d’utiliser les filaments de S. natans pour piéger l’U(VI), il était nécessaire d’identifier les facteurs induisant la filamentation de S. natans. L’influence de l’oxygène a été établie en utilisant des techniques de biologie moléculaire et nos résultats ont démontré que tandis qu’en condition d’oxygène saturé elle croît sous forme de cellules individuelles, une diminution modérée d’oxygène à ~ 3 mg O2.L-1 la fait croître sous la forme désirée, des filaments de S. natans.Les BIOS attachés aux filaments de S. natans ainsi que ses analogues abiotiques ont été analysés pas XAS au seuil K du Fe. Les deux matériaux identifiés sont des phosphates de fer(III) amorphes avec une faible proportion de fer(II), qui présentent une réactivité élevée pour le piégeage de polluants inorganiques. L’EXAFS au seuil LIII de l’U a montré la même structure pour les couches O, tandis que celles P, Fe et C étaient différentes en fonction des sorbants. Une étude intégrée qui combine des techniques expérimentales avec des calculs de spéciation a permis de décrire les isothermes d’adsorption de l’U(VI) en utilisant un modèle de complexation de surface. Ces résultats suggèrent que les groupes phosphoryles et carboxyles sont les groupes fonctionnels principaux pour la biosorption d’U(VI) par des filaments de S. natans. Les résultats de cette thèse vont aider à comprendre les processus contrôlant l’immobilisation de l’U(VI), soit par la biosorption sur S. natans, la sorption sur les BIOS ou la sorption sur les phosphates de fer, et en conséquence le devenir de l’U en conditions neutres / Heavy metals and radionuclides are present in some ecosystems worldwide due to natural contaminations or anthropogenic activities. The use of microorganisms to restore those polluted ecosystems, a process known as bioremediation, is of increasing interest, especially under near-neutral pH conditions. Iron minerals encrusting neutrophilic iron-related bacteria, especially Bacteriogenic Iron Oxides (BIOS), have a poorly crystalline structure, which in addition to their large surface area and reactivity make them excellent scavengers for inorganic pollutants. In this PhD work we studied the different mechanisms of uranium scavenging by the neutrophilic bacterium Sphaerotilus natans, chosen as a model bacterium for iron-related sheath-forming filamentous microorganisms. S. natans can grow as single cells and filaments. The latter were used to investigate U(VI) biosorption and U(VI) sorption onto BIOS. In addition, uranium sorption onto the abiotic analogues of such iron minerals was assessed. In order to use S. natans filaments for U(VI) scavenging, it was necessary to identify factors inducing S. natans filamentation. The influence of oxygen was ascertained by using molecular biology techniques and our results revealed that while saturated oxygen conditions resulted in single cell growth, a moderate oxygen depletion to ~ 3 mg O2.L-1 led to the desired filamentous growth of S. natans. BIOS attached to S. natans filaments as well as the abiotic analogues were analysed by XAS at Fe K-edge. Both materials were identified as amorphous iron(III) phosphates with a small component of Fe(II), with a high reactivity towards scavenging of inorganic pollutants. In addition, EXAFS at the U LIII-edge revealed a common structure for the O shells, while those for P, Fe and C were different for each sorbent. An integrated approach combining experimental techniques and speciation calculations made it possible to describe U(VI) adsorption isotherms by using a surface complexation model. These results suggested the role of phosphoryl and carboxyl groups as the main functional groups involved in the U(VI) biosorption by S. natans. The results of this PhD work will help to better understand the processes governing U(VI) immobilization, either by S. natans biosorption, sorption onto BIOS or sorption onto iron phosphates, an thus the fate of uranium in near-neutral pH environments

Made available in DSpace on 2017-06-01T18:20:46Z (GMT). No. of bitstreams: 1 daniele_gilvanise_souza.pdf: 2151048 bytes, checksum: cf75e4ab2690e7269aca6ee3a4e5f76b (MD5) Previous issue date: 2015-12-01 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / One of the biggest challenges in biotechnological production is to produce high value-added products at a low cost. In this context, the filamentous fungus Cunninghamella elegans presents in its cell wall large amounts of chitin and chitosan, but is also able to produce biosurfactants. Chitin and chitosan has a vast field of biotechnological applications, and the bioremediation has been used in the removal and recovery of different waste, pollutant biotransformation and textile effluent discoloration. These biopolymers have linear structures with monomeric units &#946;-1,4-N-acetyl-D-glucosamine and &#946;-1,4-D-glucosamine, respectively. Furthermore, the surfactants are compounds synthesized by micro-organisms having properties such as reducing surface and interfacial tension, emulsification, solubilization and dispersion phases, being widely applied in the petrochemical industry. Studies with C. elegans UCP/WFCC 0542 were performed in order to evaluate their biotechnological potential for the production of chitin, chitosan and biosurfactant with the use of agroindustrial residues (corn steep liquor and soybean oil waste), using a central composite design rotational 2². The biopolymers chitin and chitosan were obtained by alkali-acid treatment with 1M sodium hydroxide, and subsequent use of 2% acetic acid. The surface-active properties of the biosurfactant were evaluated by measuring the surface tension of the metabolic liquid cell-free. Biomass production by C. elegans was 8.12 g/L with yields of 0.095 mg/g chitin and 0.036 mg/g of chitosan with a deacetylation degree of 87.44% in the proposed condition. The biosurfactant obtained in condition 8 of planning with 2.15% of corn steep liquor and 5.22% soybean oil waste has demonstrated the best surface tension with 28.20 mN/m-1 and showed stability against to different environmental conditions, having anionic character and its preliminary biochemical composition suggests that the isolated biosurfactant consists of proteins and lipids. Also this proved effective in the removal of petroleum derivatives hydrophobic compounds, removing 55.15% of motor oil, 71.42% of crude petroleum, 77.46% of kerosene and 96.41% of diesel oil in sand beach. Biosurfactant toxicity tests with Brassica oleracea seeds proved their non-toxic nature. The results show the biotechnological potential of C. elegans from alternative and low cost agroindustrial substrates, allowing its use in bioremediation process in environmental recovery. / Um dos grandes desafios na produção biotecnológica é a produção de insumos de alto valor agregado a um baixo custo. Neste contexto, o fungo filamentoso Cunninghamella elegans apresenta em sua parede celular grandes quantidades de quitina e quitosana, como também é capaz de produzir biossurfactantes. A quitina e quitosana apresentam um vasto campo de aplicações biotecnológicas, e na biorremediação vem sendo utilizado na remoção e recuperação de diferentes resíduos, biotransformação de poluentes e descoloração de efluente têxtil. Estes biopolímeros possuem estruturas lineares, com unidades monoméricas &#946;-1,4-N-acetil-D-glicosamina e &#946;-1,4-D-glicosamina, respectivamente. Por outro lado, os biossurfactantes são compostos sintetizados por micro-organismos, apresentando propriedades como a redução da tensão superficial e interfacial, emulsificação, solubilização e dispersão de fases, sendo muito aplicado na indústria petroquímica. Estudos com Cunninghamella elegans UCP/WFCC 0542 foram realizados com objetivo de avaliar o seu potencial biotecnológico para a produção de quitina, quitosana e biossurfactante com a utilização de resíduos agroindustriais (milhocina e óleo de soja pós-fritura), através de um delineamento central composto rotacional de 2². Os biopolímeros quitina e quitosana foram obtidos através de tratamento álcali-ácido, com hidróxido de sódio 1M, e posterior emprego de ácido acético a 2%. As propriedades tensoativas do biossurfactante foram avaliadas pela determinação da tensão superficial do líquido metabólico livre de células. A produção de biomassa por C. elegans foi de 8,12 g/L de com rendimentos de 0,095 mg/g de quitina e 0,036 mg/g de quitosana, com um grau de desacetilação de 87,44%, na condição proposta. O biossurfactante obtido na condição 8 do planejamento com 2,15% de milhocina e 5,22% de óleo de soja pós-fritura demonstrou a melhor tensão superficial com 28,20 mN/m-1 e apresentou estabilidade frente a diferentes condições ambientais, possuindo caráter aniônico e sua composição bioquímica preliminar sugere que o biossurfactante isolado seja constituído por proteínas e lipídeos. Também este se mostrou eficiente na remoção de compostos hidrofóbicos derivados do petróleo, com remoção de 55,15% de óleo de motor, 71,42% de petróleo bruto, 77,46% de querosene e 96,41% de óleo diesel em areia de praia. Testes de toxicidade do biossurfactante com sementes de Brassica oleracea provaram seu caráter atóxico. Os resultados obtidos demonstram o potencial biotecnológico de C. elegans a partir substratos agroindustriais alternativos e de baixo custo, possibilitando o seu emprego em processo de biorremediação na recuperação ambiental.

Submitted by Biblioteca Central (biblioteca@unicap.br) on 2018-02-19T17:29:12Z No. of bitstreams: 1 maria_cleudenora_cassia_mororo.pdf: 880158 bytes, checksum: ea675b48ea15f324c1021f82c3bd3508 (MD5) / Made available in DSpace on 2018-02-19T17:29:12Z (GMT). No. of bitstreams: 1 maria_cleudenora_cassia_mororo.pdf: 880158 bytes, checksum: ea675b48ea15f324c1021f82c3bd3508 (MD5) Previous issue date: 2017-08-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES# / #2075167498588264571# / #600 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq# / #-2555911436985713659# / #600 / Cellulases (E.C. 3.2.1.4) are enzymes responsible for the degradation of cellulose, are molecules capable of accelerating chemical reactions and breaking the chemical bonds between glucose units. Cellulases correspond to the complex consisting of three enzymes endoglucanases, exoglucanases and beta-glucosidases, with diverse applications, being the microbial biotechnological processes responsible for a great part of the world economy, yet the costs of production are still very high. In this context, 25 strains of filamentous fungi (Ascomycetes) isolated from mangrove sediments of Rio Formoso, PE, Brazil, were investigated to investigate the production potential of the enzymes of the cellulolytic complex. The initial studies were carried out by selecting the fungi with the highest enzymatic activity, through the detection of cellulolytic activity in solid synthetic medium, with carboxymethylcellulose (CMC) as the substrate. The results indicated the presence of the cellulase enzyme through the formation of halo in 3 strains of the genus Trichoderma, 3 strains of the genus Aspergillus and 1 strain of the genus Penicillium. The most representative enzymatic indices were those of Penicillium sp. UCP 0279 with Index of 2,2, followed by Aspergillus flavus UCP 1413 with enzymatic index of 1,7. Submerged fermentations were carried out to evaluate the endoglucanase activity, exoglucanase and β-glycosidase, using agroindustrial residues, tangerine peel, pineapple peel, pineapple crown, wheat bran and corn bran as substrate. The results indicated a CMCase activity of 20.2 IU / mL for Penicillium sp. UCP 0279, with wheat bran as substrate in 72 h of fermentation and an activity of 18.3 IU / mL in 24 h with the pineapple crown. For the Aspergillus flavus UCP 1413, the yield was 14.9 IU / mL and 14.5 IU / mL with the residues of corn bran and pineapple peel respectively, and both results were obtained with 24 h of fermentation. The FPase activity for Penicillium sp. UCP 0279, using pineapple peel as substrate had 45.5 IU / mL and the tangerine peel 42.8 IU / mL, both in fermentation at 48 h. For A. flavus UCP 1413 the pineapple crown presented 25.0 IU / mL enzymatic activity in 24 h and the pineapple peel 14.4 U / mL at the same time. In the activity of the enzyme β-glycosidase, Penicillium sp. UCP 0279 showed a production of 18.2 IU / mL in 24 h, with the pineapple crown residue and the pineapple peel had 9.1 IU / mL in 48 h. The A. flavus UCP 1413 presented with 96 h of fermentation an activity of 16.9 U / mL and 14.5 U / mL, with wheat bran and corn bran, respectively. / As celulases (E.C. 3.2.1.4) são enzimas responsáveis pela degradação da celulose, são moléculas capazes de acelerar reações químicas e realizar a quebra das ligações químicas existentes entre as unidades de glicose. As celulases correspondem ao complexo constituído por três enzimas endoglucanases, exoglucanases e beta-glicosidases, com diversas aplicações, sendo os processos biotecnológicos microbianos responsáveis por uma grande parcela da economia mundial, contudo os custos de produção ainda são muito elevados. Neste contexto, foi realizada a bioprospecção de 25 linhagens de fungos filamentosos (Ascomycetes) isolados de sedimentos de mangue do município Rio Formoso, PE, Brasil, investigando o potencial de produção das enzimas do complexo celulolítico. Os estudos iniciais foram realizados selecionando os fungos com maior atividade enzimática, através da detecção da atividade celulolítica em meio sintético sólido, tendo como substrato a carboximetilcelulose (CMC). Os resultados indicaram a presença da enzima celulase através da formação do halo em 3 linhagens do gênero Trichoderma, 3 linhagens do gênero Aspergillus e 1 linhagem do gênero Penicillium. Os índices enzimáticos mais representativos foram os de Penicillium sp. UCP 0279 com Índice de 2,2, seguido de Aspergillus flavus UCP 1413 com índice enzimático de 1,7. Em seguida, foram realizadas fermentações submersas para avaliação da atividade endoglucanase, exoglucanase e β-glicosidase, utilizando resíduos agroindustriais, casca de tangerina, casca de abacaxi, coroa de abacaxi, farelo de trigo e farelo de milho como substrato. Os resultados indicaram uma atividade para CMCase de 20,2 UI/mL para Penicillium sp. UCP 0279, com farelo de trigo como substrato em 72 h de fermentação e com a coroa de abacaxi observou-se uma atividade de 18,3 UI/mL em 24 h. Para o Aspergillus flavus UCP 1413, a produção foi de 14,9 UI/mL e 14,5 UI/mL com os resíduos de farelo de milho e de casca de abacaxi respectivamente, e ambos os resultados foram obtidos com 24 h de fermentação. A atividade FPase para Penicillium sp. UCP 0279, usando casca de abacaxi como substrato apresentou 45,5 UI/mL e a casca de tangerina 42,8 UI/mL, ambos em fermentação a 48 h. Para A. flavus UCP 1413 a coroa de abacaxi apresentou 25,0 UI/mL de atividade enzimática em 24 h e a casca de abacaxi 14,4 U/mL no mesmo tempo. Na atividade da enzima β-glicosidase o Penicillium sp. UCP 0279 apresentou uma produção de 18,2 UI/mL em 24 h, com o resíduo da coroa de abacaxi e com a casca de abacaxi apresentou 9,1 UI/mL em 48 h. O A. flavus UCP 1413 apresentou com 96 h de fermentação uma atividade de 16,9 U/mL e 14,5 U/mL, com farelo de trigo e farelo de milho respectivamente.

O gene TrSNF1, homólogo aos membros da subfamília das proteínas serina-treonina quinases ativadas por AMP (AMPK) e relacionadas a SNF1, foi isolado do fungo filamentoso trichoderma reesei. A seqüência de aminoácidos putativa possui um domínio de quinase com 42% de identidade e 59% de similaridade com outras proteínas quinases da mesma subfamília. Em S. cerevisiae a SNFl é essencial para a expressão de genes reprimidos por glicose, em resposta a privação de glicose do meio de cultura. A expressão de TrSNFl em levedura mutante para SNF1, restaura a função de SNF1. A expressão de um antisense de TrSNFl em T. reesei causa um atraso na expressão do gene regulado por glicose, CBHI. Além disso, em experimento utilizando matrizes de DNA foi possível observar uma alteração da tendência global da expressão gênica entre a cepa selvagem e a cepa antisense. A observação da homologia estrutural com proteínas quinase da mesma subfamília, a similaridade funcional com SNFl de S. cerevisiae, e a alteração no padrão da expressão gênica in vivo, sugerem que TrSNFl pode estar envolvido na regulação do metabolismo de carboidratos em T. reesei. / A gene homologue to the members of the AMP-activated/SNFl protein kinase subfamily, TrSNF1, was isolated from the filamentous fungus Trichoderma reesei. The predicted protein of 692 amino acids has a kinase domain, that share 42 % identity and 59 % similarity to that of serine/threonine protein kinase of this family. In Saccharomyces cerevisiae, the SNFl protein kinase is required for expression of glucose repressed genes in response to withdrawal of glucose from the medium. Expression of the Trichoderma reesei SNF1-related sequence in yeast SNFl mutant restores SNFl function. The TrSNFl antisense expression in T. reesei causes a control alteration in the glucose-regulated gene CBHI. The observed structural identity with the AMP-activated/SNFl protein kinase subfamily, and the functional similarity to the yeast SNFl suggest that the TrSNFl may be involved in the regulation of sugar metabolism in Trichoderma reesei.

Não consta resumo na publicação. / The gene encoding actin in the cellulolytic filamentus fungus Trichoderma reesei has been isolated and sequenced. The nucleotide sequence reveals that the gene is composed of 6 exons separated by 5 introns within the coding region. The positions of the introns were predicted by comparison of sequence homology to the genes coding for actin with known amino acid sequence and by identification of splice-site signal sequences. The actin protein of Trichoderma reesei shows extensive homology to the actins of other fungi E. nidulans, 95% , T. lanuginosus, 92% and S. pombae. The T. reesei actin promoter has a CT-rich region, CAAT and GC. There is no obvious TATA sequence in the T. reesei actin promoter. The absence of TATA-like sequence were also observed in anothers genes of T. reesei. An important aspect in molecular biology of filamentous fungi is the analysis, under a specific metabolic events, of the mechanism(s) regulating the expression of constitutive and induced genes. The filamentous fungus Trichoderma reesei is considered to be one of the most efficient producer of cellulase, and it serves as a model system for enzymatic cellulose hydrolysis. Expression of the cellulase genes are stringently regulated by the carbon source. Growth on cellulose results in induction of the cellulase transcripts, whereas glucose strongly represses their expression. The availability of a constitutive expressed genes of T. reesei provides not only important information regarding the molecular biology of the fungi, but also is essential for a better understanding of the mechanism(s) controlling the expression of the cellulase transcripts. Under inductive process of the of the major cellulase transcript (cbh1) and its repression by glucose, actin mRNA is constitutively expressed. The present results should be useful for further structural and functional analysis of the elements involved in inductive and constitutive expression of cellulase and actin transcripts.