O objetivo deste estudo foi determinar os efeitos de doses dietéticas crescentes de um produto comercial com atividade amilolítica (AmaizeTM, Alltech Inc., Nicholasville, KY, EUA) na ingestão e digestibilidade aparente total de nutrientes, índice de seleção, fermentação ruminal, produção e composição do leite, perfil metabólico, balanço de energia e nitrogênio em vacas no terço médio de lactação. Foram utilizadas vinte e quatro vacas multíparas da raça Holandesa (162,29 ± 107,96 dias em lactação e 31,60 ± 6,51 kg/d de produção de leite, no início do experimento), sendo que 8 delas possuíam cânulas ruminais, distribuídas em seis quadrados Latinos 4 × 4 contemporâneos e balanceados de acordo com a produção de leite, dias em lactação e o peso corporal dos animais. Os períodos experimentais tiveram duração de 14 dias de adaptação aos tratamentos e 7 dias de amostragem. Os tratamentos foram: dieta basal sem adição de enzima amilolítica ou controle (CON), e dieta basal com adição de 150, 300 ou 450 FAU/kg de MS da dieta (A150, A300 ou A450, respectivamente). Uma FAU (unidade de amilase fúngica) é a quantidade de enzima capaz de dextrinizar amido solúvel na taxa de 1 g/h a 30°C e pH de 4,8. A ingestão média esperada do produto comercial pelos animais foi de 7,37; 14,45; e 21,97 g/d nos tratamentos A150, A300 e A450, respectivamente. Os tratamentos não influenciaram a ingestão de MS e de nutrientes, como também o índice de seleção. Os tratamentos não influenciaram a digestibilidade do amido; porém, a inclusão de enzimas amilolíticas aumentou linearmente a digestibilidade de proteína bruta e tendeu a aumentar linearmente a digestibilidade da MS. Os tratamentos com atividade amilolítica não afetaram o pH e a concentração de amônia no fluído ruminal. A adição de enzimas amilolíticas aumentou linearmente a produção de iso-valerato no rúmen. Além disso, os tratamentos não influenciaram a produção e composição do leite, assim como a eficiência alimentar (kg leite / ingestão de MS) dos animais. A enzima amilolítica aumentou linearmente o peso corporal. Apesar de não alterar a composição do leite, a suplementação com enzima amilolítica diminuiu linearmente a excreção de nitrogênio no leite. As doses crescentes de enzima amilolítica tenderam a diminuir linearmente a eficiência de síntese de proteína microbiana. Não foram observadas diferenças nas concentrações séricas de glicose, ureia, e enzimas que indicam lesões hepáticas. A suplementação com doses crescentes enzima amilolítica não afetou a eficiência alimentar, produção de propionato e de proteína microbiana no rúmen, e perfil metabólico de vacas em lactação. No entanto, os tratamentos aumentaram linearmente a digestibilidade de proteína bruta e o peso corporal das vacas / The objective of the current study was to determine the effects of increasing dietary doses of a commercial product with amylolytic activity (AmaizeTM, Alltech Inc., Nicholasville, KY, USA) on nutrient intake and total apparent digestibility, sorting index, ruminal fermentation, milk yield and composition, serum metabolic profile, energy and nitrogen utilization of midlactating dairy cows. Twenty-four multiparous Holstein cows (162.29 ± 107.96 days in milk and 31.60 ± 6.51 kg/d milk yield, before starting the experiment), in which 8 were ruminally cannulated, were used in a replicated 4 × 4 Latin experiment design. The squares were contemporaneous and balanced for milk production, days in milk and live weight of cows. The experimental periods consisted of 14 days to treatments adaptation and 7 days for sampling. Treatments were composed of: basal diet with no enzyme or control (CON), and basal diet with addition of 150, 300 or 450 FAU/kg diet DM (A150, A300 or A450, respectively). One FAU (fungal amylase unit) is able to dextrinize soluble starch at rate of 1g/h on 30°C and pH 4.8. The expected average intake of the commercial product for treatments A150, A300 and A450 were 7.37, 14.45 and 21.97 g/d, respectively. Treatments did not influence the DM and nutrient intake, as well as the sorting index. Treatments did not alter starch digestibility; however, they linearly increased the crude protein digestibility and tended to linearly increase the DM digestibility. Treatments with amylolytic activity did not affect the pH and ammonia concentrations of ruminal fluid. The addition of amylolytic enzyme linearly increased the iso-valerate production in the rumen. In addition, treatments did not influence the milk yield and composition, as well as the milk production efficiency (kg of milk / DM intake) of animals. Treatments linearly increased the live weight and maintenance energy utilization of cows. Despite the enzyme supplementation did not alter the milk composition, it linearly decreased the milk nitrogen excretion. Treatments tended to linearly decreased the efficiency of microbial protein synthesis. No differences were observed on serum metabolic profile of animals, including concentrations of glucose, urea and enzymes which indicate hepatic damage. The supplementation with increasing doses of amylolytic enzymes did not affect the milk production efficiency, ruminal propionate production and microbial protein synthesis, and serum metabolic profile of mid-lactating dairy cows. However, treatments linearly increased the crude protein digestibility and live weight of cows

Orientador: Hélia Harumi Sato / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-20T21:23:26Z (GMT). No. of bitstreams: 1 Castro_RuannJanserSoaresde_M.pdf: 2807315 bytes, checksum: e6965e5648d4556cb18db9b7869a58c4 (MD5) Previous issue date: 2012 / Resumo: As proteases constituem um dos mais importantes grupos de enzimas produzidos comercialmente, apresentando diversas aplicações nas indústrias de alimentos e farmacêutica. A utilização de proteases na hidrólise enzimática de proteínas para obtenção de peptídeos com propriedades antioxidantes tem recebido grande notoriedade nas pesquisas científicas. Nesse contexto, o presente trabalho visou estudar a produção e caracterização bioquímica de protease de Aspergillus oryzae LBA 01 obtida por processo fermentativo em estado sólido e avaliar a aplicação desta protease e de preparações comerciais na hidrólise de proteínas para obtenção de hidrolisados com atividade antioxidante. A maior produção de protease por A. oryzae LBA 01 foi observada em meio de cultivo composto de farelo de trigo, peptona (2,0% p/p) e extrato de levedura (2,0% p/p) sob as seguintes condições: 50,0% de umidade inicial, inóculo de 107 esporos.g-1 e incubação a 23°C por 72h. A caracterização bioquímica, realizada por planejamento experimental, mostrou que a protease apresentou maior atividade na faixa de pH 5,0-5,5 e 55-60°C, e estabilidade no intervalo de pH 4,5-6,0 após 1h de tratamento na faixa de temperatura de 35-45°C. Proteína isolada de soja, soro de leite e clara de ovo apresentaram aumento expressivo nas suas propriedades antioxidantes quando hidrolisadas com diferentes proteases microbianas. A aplicação de protease comercial Flavourzyme® 500L, obtida de A. oryzae, para a hidrólise de proteína isolada de soja, resultou na obtenção de hidrolisados com maior atividade antioxidante quando comparados aos hidrolisados preparados com as proteases de A. oryzae LBA 01 e a protease comercial Alcalase® 2.4L de Bacillus licheniformis. As condições de hidrólise, definidas a partir de delineamento composto central rotacional (DCCR), foram: concentração de substrato de 90,0 mg.mL-1 e adição de 70,0 U de protease por mL de mistura reacional (U.mL-1), resultando em 775,17 e 11,83 Trolox EQ µmol.g-1, para os ensaios de ORAC e DPPH, respectivamente. Os hidrolisados de soro de leite com maior capacidade antioxidante foram obtidos com a protease de A. oryzae LBA 01. A adição de 70,0 U.mL-1 de protease a solução de soro de leite 80,0 mg.mL-1, resultou em 424,32 e 16,39 Trolox EQ µmol.g-1, para os ensaios de ORAC e DPPH, respectivamente. Na preparação de hidrolisados de proteínas de clara de ovo, a utilização de 30,0 mg.mL-1 de substrato e 20,0 U.mL-1 da protease comercial Flavourzyme® 500L de A. oryzae, resultou em 1.193,12 e 19,05 Trolox EQ µmol.g-1 para os ensaios de ORAC e DPPH, respectivamente. Os maiores valores de atividade antioxidante, para os três substratos, foram detectados entre 30 e 180 minutos de incubação, onde o grau de hidrólise variou de 40,0 a 66,0%. Os resultados obtidos mostraram que a preparação de protease de A. oryzae LBA 01 obtida por fermentação em estado sólido e posterior concentração por precipitação com sulfato de amônio, diálise e liofilização, apresentou atividade enzimática semelhante às preparações comerciais avaliadas, tendo, portanto, potencial para aplicação na hidrólise proteica. A hidrólise enzimática, nas condições de estudo avaliadas, aumentou de 2 a 23 vezes a capacidade antioxidante de proteína isolada de soja, soro de leite e clara de ovo, mostrando-se um processo eficaz para obtenção de peptídeos com atividade antioxidante / Abstract: Proteases are one of the most important groups of enzymes produced commercially, with several applications in the food and pharmaceutical industries. The use of proteases in the enzymatic hydrolysis of proteins to obtain peptides with antioxidant properties has gained great notoriety in scientific research. In this context, the main objectives of the present study were to optimize the production of the protease from Aspergillus oryzae LBA 01 by solid state fermentation, and to determine its biochemical characteristics. The application of this protease and of commercial preparations to protein hydrolysis, and the study of the antioxidant properties of the hydrolysates obtained, was evaluated. The optimum fermentation medium was composed of wheat bran, 2.0% (w/w) peptone and 2.0% (w/w) yeast extract, and the conditions for maximum protease production were an initial moisture content of 50.0%, an inoculum level of 107 spores.g-1 and incubation at 23°C for 72h. The biochemical characterization, evaluated using an experimental design, showed that the enzyme was most active in the pH range 5.0-5.5 and 55-60°C. The enzyme was stable from pH 4.5 to 6.0 after 1h incubation at 35-45°C. Soy protein isolate, bovine whey protein and egg white protein exhibited increases in antioxidant activity when hydrolyzed with the different microbial proteases. For the hydrolysis of soy protein isolate, application of the commercial protease Flavourzyme® 500L from A. oryzae resulted in hydrolysates with greater antioxidant activity as compared to hydrolysates prepared with the protease from A. oryzae LBA 01 and the commercial protease Alcalase® 2.4L from Bacillus licheniformis. The hydrolysis conditions, as defined by a central composite rotational design (CCRD), were: 90.0 mg.mL-1 substrate concentration plus 70.0 U of protease per mL of reaction mixture (U.mL-1), which resulted in 775.17 and 11.83 Trolox EQ µmol.g-1 as determined by the ORAC and DPPH assays, respectively. For the whey protein hydrolysates, the greatest antioxidant activity was obtained with the protease from A. oryzae LBA 01. According to the CCRD, the use of 80.0 mg.mL-1 of bovine whey protein and 70.0 U.mL-1 of protease resulted in 424.32 and 16.39 Trolox EQ µmol.g-1, respectively, as determined by the ORAC and DPPH assays. For the egg white protein, hydrolysis with 20.0 U.mL-1 of Flavourzyme® 500L from A. oryzae with 30.0 mg.mL-1 substrate concentration, resulted in 19.05 and 1,193.12 Trolox EQ µmol.g-1, respectively, as determined by the ORAC and DPPH assays. The maximum antioxidant activities were obtained in the range from 30 to 180 min of hydrolysis, with a degree of hydrolysis of about 40.0-66.0%. The results showed that the protease preparation from A. oryzae LBA 01 obtained by solid state fermentation produced enzymatic activity similar to that of the commercial preparations, and was an attractive enzyme to apply in protein hydrolysis. Under the conditions evaluated in this study, enzymatic hydrolysis resulted in a 2.0- to 23.0-fold increases in antioxidant activity for the soy protein isolate, bovine whey protein and egg white protein, being an effective process to obtain peptides with antioxidant activity / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

Xylanase was purified from the crude culture of Aspergillus oryzae DSM1863 by sephadex G200 and DEAE – cellulose ion exchange chromatography. The molecular mass of the purified xylanase determined by SDS–PAGE was 21 kDa with a specific activity of 6768 U/mg towards 1% (w/v) of birch wood xylan. The optimum temperature was observed at 60°C. The enzyme was thermostable in the temperature range of 37-50°C with a high residual activity of 62-74% (650.6- 775.9 U/mg protein). / Enzyme xylanase được tinh sạch từ dịch lên men của chủng Aspergillus oryzae DSM1863 sau khi qua cột sắc ký lọc gel sephadex G200 và sắc ký trao đổi ion DEAE – cellulose. Khối lượng phân tử của enzyme xylanase tinh sạch được xác định bằng điên di đồ SDS- PAGE. Xylanase tinh sạch có kích thước là 21 kDa với hoạt tính đặc hiệu đạt 6768 U/mg sau khi được xác định với nồng độ cơ chất là 1% birch wood xylan. Nhiệt độ tối ưu để enzyme hoạt động mạnh nhất là 60°C. Enzyme xylanase khá bền nhiệt. Hoạt tính của enzyme vẫn còn duy trì 62-74% (hoạt tính đặc hiệu đạt 650.6-775.9 U/mg protein) sau khi 8 giờ ủ ở 37-50°C.

Xanthomonas oryzae pv. oryzae (Xoo) est une bactérie phytopathogène responsable de la bactériose vasculaire du riz, maladie pouvant engendrer de fortes pertes de rendement à travers le monde. La course à l'armement entre la bactérie et sa plante hôte correspond d'une part à la mise en place de la virulence par le microorganisme et d'autre part en la résistance du végétal face à l'agression. Comprendre les mécanismes par lesquels Xoo accompli son cycle infectieux est d'une importance cruciale pour le développement futur de nouvelle méthode de luttes. Plusieurs approches complémentaires ont été mises en uvre afin de caractériser des éléments associés au pouvoir pathogène de Xoo.Dans un premier temps nous avons effectué une analyse protéomique comparative. Cette approche a permis l'identification chez une souche Africaine de Xoo d'un jeu de protéines induites par HrpX et susceptibles de jouer un rôle dans la virulence. Dans un second temps, l'implication de deux peptides dans la virulence Xoo a été étudiée. Le premier de ces peptides, supposé être le facteur d'avirulenceAvrXa21, a fait l'objet d'une caractérisation fonctionnelle et phylogénique. Le second peptide est synthétisé par un cluster NRPS, similaire à l'un de ceux présent chez Xanthomonas albilineans. Afin d'élucider l'importance de la molécule synthétisée par cette voie pour Xoo, une étude préliminaire impliquant la mutation d'un élément régulateur des NRPS a été effectuée. En dernier lieu, des informations nouvelles ont été apportées sur la topologie de la protéine membranaire HrcR qui est une composante essentielle du système de sécrétion de type III chez la plupart des bactéries appartenant au genre Xanthomonas. / Xanthomonas oryzae pv. oryzae (Xoo) is the agent of bacterial leaf blight BLB in rice, a disease which causes considerable yield losses throughout the world. In the arms race underlying the interactions between the microorganism and the host, the presence of virulence factors in the former parallels that of resistance factors in the latter. Understanding the mechanisms of Xoo's infectious cycle is of paramount importance for the elaboration of new fighting strategies to combat BLB. To achieve this, several complementary approaches to characterize components of Xoo's pathogenicity have been employed.First, we performed comparative proteomics that allowed us to identify novel HrpX-induced candidate pathogenicity factors of an African Xoo strain. Second, the involvement of two peptides in Xoo's pathogenicity has been investigated. One was speculated to be the avirulence factor AvrXa21 and has been characterized both functionally and phylogenetically. The other one was found to be synthesized by a Non-Ribosomal Peptide Synthetase (NRPS), reminescent to NRPS genes found in Xanthomonas albilineans. In order to determine the role of NRPS-mediated synthesis in Xoo virulence, we studied a strain carrying a mutated regulatory gene of the NRPS pathway. Finally, we provide new information on the topology of the HrcR membrane protein which is a conserved component of the type III secretion system of most Xanthomonas.

Host colonization is mediated through the secretion of effector proteins in order to neutralize host immune responses. However, the mechanism of the effector delivery during biotrophic invasion is not well defined in M. oryzae. In this thesis, I define the role of the exocyst complex, an evolutionarily conserved octameric protein complex involved in vesicle docking to the plasma membrane (composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84), during infection-related development in M. oryzae. Like other filamentous fungi, M. oryzae, exocyst components localize to the vegetative hyphal tip distinct from the Spitzenkörper. However, at the initial stage of infection-related development all the exocyst components localise as a ring at the cortex of the appressorium and re-assembles around the appressorium pore in an actin-dependent manner in mature appressoria. I report that the septin network is required for the transition of exocyst ring from periphery to the appressorium pore. Deletion of Exo70 and Sec5 showed significant reduction in protein secretion and plant infection. I show that Sec6 is required for the exocyst assembly around the appressorium pore and effector secretion from the appressorium. I report that, during biotrophic invasion, effectors are secreted through a distinct pathway. Apoplastic effectors, Bas4 and Slp1 are secreted via a Golgi-dependent pathway while secretion of cytoplasmic effectors, Pwl2 and Bas1 meditates through a Golgi-independent pathway in which exocyst components Exo70 and Sec5 are involved.

Rice blast disease is caused by the filamentous ascomycete fungus Magnaporthe oryzae and is the most destructive disease of cultivated rice. The pathogen elaborates a specialized infection structure called the appressorium. The morphological and physiological transitions that lead to appressorium formation of M. oryzae are stimulated through perception of environmental signals and are tightly regulated by cell cycle checkpoints. External stimuli are internalized by a variety of intracellular MAP kinase signaling pathways, and the major pathway regulating appressorium morphogenesis and plant infection is the Pmk1 MAP kinase signaling pathway. The central kinase, Pmk1, is required for appressorium morphogenesis and the homeobox and C2/H2 Zn-finger domain transcription factor, called Mst12, is required for appressorium formation and tissue invasion. The Mst12 null mutant is able to form melanised appressoria, but it is non-pathogenic. To understand the mechanism of appressorium morphogenesis and penetration peg formation, genome-wide comparative transcriptional profiling analysis was performed for the Δpmk1 and Δmst12 mutant using RNA-seq and HiSeq 2000 sequencing. This thesis reports the identification of gene sets regulated by the Pmk1 signalling pathway and defines the sub-set of these genes regulated by Mst12. I show that a hierarchy of transcription factors is likely to operate downstream of Pmk1 to regulate the main processes required for appressorium morphogenesis and plant infection. I also report the role of Mst12 in cytoskeletal re-organisation and show that it is necessary for septin-dependent F-actin polymerisation at the base on the appressorium prior to plant infection. This is consistent with the major transcriptional changes observed by RNA-seq. The thesis also reports experiments that strongly suggest that appressorium mediated plant penetration is regulated by an S-phase checkpoint which operates independently of the conventional DNA damage and repair response, and the Cds1 and Chk1 checkpoint kinases. Transcriptional profiling results are consistent with the S-phase checkpoint operating downstream of the Pmk1 MAP kinase signalling pathway. An integrated model for the operation of the Pmk1/Mst12 signalling pathways and the hierarchical control of appressorium morphogenesis in the rice blast fungus is presented.

Os frutooligossacarídeos (FOS) são oligômeros de frutose, cujas unidades frutosil estão ligadas na posição β-(2→1) na molécula de sacarose. Esses açúcares, de baixa caloria, são classificados com prebióticos, não são cariogênicos, podem ser usados por diabéticos, são de 0,4 a 0,6 vezes menos doce que a sacarose, sendo amplamente utilizados pelas indústrias farmacêutica e de alimentos como açúcares funcionais. Apesar dos FOS serem produzidos naturalmente por enzimas presentes em diversos vegetais, são disponibilizados comercialmente por meio da produção sintética, utilizando enzimas de origem microbiana como as frutosiltransferases (FTases, E.C.2.4.1.9) e sacarose como principal substrato. Diante deste contexto, o presente trabalho teve como objetivo a produção de FTases extracelular e micelial de Aspergillus oryzae IPT-301 por fermentação submersa aeróbia utilizando meio de cultura sintético, assim como a caracterização e estudos cinéticos das enzimas produzidas. Também foram investigados os efeitos da temperatura e pH do meio reacional nas atividades enzimáticas mediante técnica de planejamento experimental. Para a produção de FTases foi necessário o cultivo do micro-organismo em meio de cultura estéril e a fermentação foi conduzida em agitador orbital do tipo shaker. Com o caldo de fermentação filtrado e o micélio úmido foi possível determinar as atividades de transfrutosilação (quantidade de enzima necessária para produzir 1 µmol de FOS por minuto nas condições experimentais) e hidrolítica (quantidade de enzima necessária para liberar 1 µmol de frutose por minuto nas condições experimentais) extracelular e micelial, respectivamente, para as diferentes condições experimentais avaliadas. A concentração máxima de biomassa celular obtida foi de 9,35 ± 1,26 g.L-1 em 48 h de fermentação, sendo que em 76 h, houve a produção de 7,51 ± 1,57 g.L-1, período em que ocorreu a acidificação do caldo fermentado (pH 4,82). As condições nas quais a enzima extracelular obteve maior atividade de transfrutosilação foi aquela produzida em 64 h de fermentação, incubada na faixa de pH 4,5-6,0, temperatura reacional de 50 °C, concentração de sacarose a partir de 296,0 g.L-1, apresentando estabilidade entre 30 e 35 ºC e em pH 6,0. Por outro lado, a FTase micelial mostrou sua máxima atividade quando produzida em 72 h de fermentação, incubada na faixa de pH 4,5-6,0, temperatura reacional entre 45-55 °C, concentração de substrato igual a 470,6 g.L-1, indicando estabilidade para faixas de pH entre 6,0-8,0 e temperatura entre 30-40 ºC. A FTase extracelular apresentou cinética michaeliana em relação à concentração de substrato, exibindo valores de Vmax igual a 16,23 U.mL-1 e Km de 50,41 g.L-1 , enquanto a FTase micelial ajustou-se satisfatoriamente ao Modelo de Hill, cujos valores dos parâmetros Vmax, K0,5 e n foram iguais a 342,23 U.g-1 e 234,73 g.L-1 e 1,41, respectivamente. O estudo da influência do tempo e da temperatura reacional na síntese de FOS mostrou que a FTase extracelular produziu maior concentração de FOS a 50 °C, enquanto a FTase micelial a 40°C. A otimização do processo para a obtenção de FOS comprovou que a zona ótima da FTase extracelular (em que elevada atividade de transfrutosilação e baixa atividade hidrolítica são esperadas) ocorreu nas faixas de temperatura entre 45-50 °C e de pH entre 5,5-6,75. Para a FTase micelial, apenas a atividade de transfrutosilação ajustou-se satisfatoriamente ao modelo quadrático com interação, cuja zona ótima ocorreu em temperaturas superiores a 46 °C e valores de pH abaixo de 6,5. Os resultados obtidos atestaram que o fungo se destacou como fonte produtora de FTases e, estas, por sua vez, mostraram-se promissoras para a obtenção de FOS em escala laboratorial. / Fructooligosaccharides (FOS) are fructose oligomers in which the fructosyl units are bound in β-(2→1) position of sucrose molecule. These sugars, that present properties such as low caloric value and non-cariogenicity, are classified as prebiotics and can be used in diabetic products; they are 0.4 to 0.6 times less sweet than sucrose, being widely applied in the pharmaceutical and food industries as functional sugars. Despite the natural production of FOS by enzymes present in various vegetables, they are commercially available by synthetic production, using microbial enzymes such as fructosyltransferases (FTases, E.C.2.4.1.9) and sucrose as main substrate. In face of this context, the objective of the present study was the production of extracellular and mycelium FTases by Aspergillus oryzae IPT-301 from aerobic submerged fermentation using synthetic growth media, as well as the characterization of the enzymes produced and the conduction of kinetic studies. It was also investigated the temperature and pH effects of the growth media in the enzymatic activities through experimental planning. For FTases production was necessary the use of sterile growth media, and the fermentation was carried out in rotary shaker. With the filtered broth and the humid mycelium it was possible to determine the transfructosylation and hydrolytic activities of the extracellular and mycelium enzymes in different experimental conditions; the transfructosylation activity was defined as the amount of enzyme necessary to produce 1 µmol of FOS per minute under the experimental conditions, while the hydrolytic activity was defined as the amount of enzyme necessary to release 1 µmol of fructose per minute under the experimental conditions. The maximum biomass concentration (9.35 ± 1.26 g.L-1) was obtained in 48 h of fermentation; in 76 h there was a biomass production of 7.51 ± 1.57 g.L-1 and the acidification of the fermented broth (pH 4.82). The conditions in which was observed the maximum transfructosylation activity of the extracellular enzyme were 64 h of fermentation, pH range of 4.5 to 6.0, reactional temperature of 50 °C and sucrose concentration from 296.0 g.L-1; the thermal stability was between 30 and 35 °C and pH 6.0. On the other hand, mycelium FTase had its optimum in 72 h of fermentation, pH range of 4.5 to 6.0, reactional temperature between 45-55 °C and substrate concentration of 470.6 g.L-1; the thermal stability was between 30 and 40 °C and pH range 6.0 to 8.0. Extracellular FTase presented michaelian kinetic according to substrate concentration, showing Vmax and Km values of 16.23 U.mL-1 and 50.41 g.L-1, while micelial FTase fitted to Hill Model, whose Vmax, K0,5 e n values was 342.23 U.g-1 e 234.73 g.L-1 and 1.41, respectively. The study of reaction’s time and temperature showed that extracellular FTase produced a maximum concentration of FOS at 50 °C, while the micelial one at 40 °C. The process optimization for FOS production verified that the optimum zone for extracellular FTase, in which are expected high transfructosylation and low hydrolytic activities, occurred in temperature range of 45 - 50 °C and pH range of 5.5 - 6.75. For mycelium FTase, only the transfructosylation activity presented a satisfactory adjustment to the quadratic model with interaction, in which the optimum zone occurred in temperatures above 46 °C and pH values below 6.5. The results obtained showed that the fungus stood out as source of FTase, and these enzymes presented themselves as promising sources for FOS production in laboratorial scale. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

Serrated tussock (Nassella trichotoma) and Giant Parramatta Grass (Sporobolus fertilis) are among the most noxious weeds in Australia. Both cause problems in pasture and there are limited control measures relying heavily on the herbicide flupropanate. With the recent confirmation of flupropanate resistance in serrated tussock and the report of suspected flupropanate resistance in Giant Parramatta Grass (GPG), this option appeared to be under threat.The aims of this thesis were to determine the extent of flupropanate resistance in serrated tussock and GPG in Australia and to understand the genetics of flupropanate resistance in serrated tussock. This thesis also documents the GPG resistance to 2,2-DPA and investigates a fungal pathogen, Nigrospora oryzae, as a potential biocontrol agent for GPG. A local paddock survey determined the spread and extent of flupropanate resistance in serrated tussock within 5 km of the original resistant site. The pot-dose method of assessing resistance identified plants resistant to flupropanate up to 3.5 km from the original site found in Victoria. Seeds from these plants showed 0-100% resistance, with sensitive plants often having a low („T5%) level of resistant seed. These results indicate the movement of flupropanate resistance through seeds or pollen and shows that its spread occurred within one year of detection. A national mail survey confirmed the massive impacts of serrated tussock across Australia, with annual serrated tussock costs ranging from $15,000 to $16,000 per year per respondent. This survey also identified the widespread infestation of this weed in a variety of land use patterns, from pasture to native grasslands, and the decrease in the value of farmland as a result. Heritability studies using controlled breeding experiments indicated a strong involvement of a maternal component in the inheritance of flupropanate resistance in serrated tussock, with a minor proportion of resistance heritable through pollen. GPG plants and seedlings were tested for flupropanate and 2,2-DPA resistance.Seedlings tested for flupropanate resistance were highly resistant (tolerating 33-39 times more than sensitive biotypes). With 2,2-DPA, resistant GPG plants did not die even at 14 times the field rate and resistant seedlings also showed 5-6 times more resistance than the sensitive biotype. The study has confirmed that flupropanate and 2,2-DPA resistance now exists in GPG.The potential of Nigrospora oryzae, a pathogenic fungus, as a biocontrol agent for GPG was determined. Mature plants and seedlings of GPG were inoculated with conidia of N. oryzae using three treatments (run-off, crown, and spray). Inoculated plants were smaller, with greater proportions of dead leaves (70% with the run-off a nd crown treatments and 53% with the spray treatment) than the control plants. GPG seedlings inoculated with N. oryzae were stunted and showed greater proportions of necrotic leaves in all the treatments than the control. There is potential to develop N. oryzae as a mycoherbicide to control GPG and further testing is warranted.

No description available.

Egg is one of the major food allergens that affects 1.6~3.2% of the infants and young children population. The objective of this study is to reduce the egg white IgE binding ability by lactobacilli or Aspergillus oryzae fermentation. Modifications of egg white proteins during fermentation were analyzed by Ninhydrin method, Ellman method, SDS-PAGE, ELISA, and MALDI-TOF-MS. Tryptone supplementation and acidification are necessary to grow lactobacilli in egg white. Egg whites were fermented by L sanfranciscensis, L. sakei, and L. delbrueckii subsp. delbrueckii individually for 96 h; and Aspergillus oryzae for 120 h. The IgE binding ability of egg white was significantly reduced (~50%) by L. delbrueckii subsp. delbrueckii after 48 h of incubation and almost eliminated by Aspergillus oryzae after 24 h of inoculation. In addition to slight modification of ovomucoid (the dominant egg allergen), no substantial protein degradation was observed during fermentation. / Food Science and Technology