Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1976.

Marburg, Universiẗat, Diss., 2004.

Le premier objectif de ce travail a été d’étudier l’implication des lipopeptides de Bacillus spp. dans la colonisation de la rhizosphère de tomates. Alors que seules les souches produisant de la surfactine sont capables de coloniser un milieu synthétique, toutes les souches testées, colonisent la rhizosphère de tomates avec une plus ou moins bonne efficacité quelque soit leur propriété de production de lipopeptide(s). L’efficacité de la colonisation des racines de tomates est principalement espèce-dépendante. Ce n’est que quand une souche est déjà une bonne colonisatrice que la surfactine semble améliorer cette propriété. Le deuxième objectif a été de tester l’effet des lipopeptides surfactine et mycosubtiline contre le phytopathogène obligatoire de la laitue : Bremia lactucae. À l’échelle du laboratoire, la mycosubtiline à 100 mg/L réduit le pourcentage de plantes infestées de 70 %. La surfactine ne montre aucun effet contre le champignon. Un mélange de mycosubtiline et de surfactine à 50 mg/L chacun diminue le pourcentage de plantes infestées de 65 %. Il semble diminuer le nombre de spores par plante infestée alors que cette propriété n’est pas remarquée avec les autres traitements. L’utilisation de la mycosubtiline dans une serre de culture limite la maladie aux symptômes les moins sévères et protège les plantes saines d’une contamination croisée. L’action des lipopeptides dans la colonisation des racines par Bacillus spp. n’avait jamais été validée in situ. De même, c’est la première fois que l’activité des lipopeptides est testée contre un phytopathogène obligatoire. / The first aim of this work was to study the role played by the lipopeptides of Bacillus spp. in the colonization of the tomato rhizosphere. While only the strains producing surfactin are able to colonize a synthetic agar medium, all the strains are able to colonize the rhizosphere of tomatoes with a more or less good efficiency, whatever the lipopeptide(s) they have the capability to produce. The efficiency of the colonization of the tomato rhizosphere is species-dependant. However, surfactine seems to improve the efficiency of only the good colonizing strains. The second aim of this thesis was to test the effect of surfactin and mycosubtilin against a biotrophic parasite of lettuce: Bremia lactucae. Used at 100 mg/L, mycosubtilin reduces the percentage of infested plants of 70 %. Surfactin does not have effect against the fungy. A mixture of mycosubtilin and surfactin at both 50 mg/L decreases the percentage of infested plants of 65 %. This mixture seems to reduce the number of spore per infested plant while this property was not found for the other treatments. The use of mycosubtilin in a greenhouse confines the disease to the lowest classes of severity and protects the healthy plants from a cross contamination.The efficiency of lipopeptides of Bacillus spp. in root colonization by these bacteria was never tested in situ before. Furthermore, this is the first time that the activity of lipopeptides is validated against an obligate phytopathogen.

Orientador: Nilva Kazue Sakomura / Resumo: Doenças entéricas se apresentam como um dos maiores problemas da produção avícola. Historicamente, antibióticos promotores de crescimento (APC’s) têm sido usados para controlar essa situação. No entanto, as preocupações a respeito de resistência bacteriana a antibióticos têm levado a proibições regionais para reduzir o uso de APC’s. Alternativamente, microrganismos administrados diretamente na dieta como por exemplo os probióticos, podem impactar positivamente sobre a microbiota intestinal, prevenindo problemas entéricos que, inevitavelmente culminam em perdas no desempenho das aves. Outro ponto importante a ser considerado quando se fala em desafio sanitário é o consumo de ração dos animais. A utilização de modelos matemáticos que consideram o fator desafio para modelagem do consumo ainda é escasso. Nesse sentido, essa dissertação foi conduzida com dois principais propósitos: 1) investigar os efeitos de Bacillus amyloliquefaciens CECT 5940 como probiótico (DFM) sozinho ou em associação com bacitracina metileno disalicilato (BMD) em frangos de corte sobre desafio de patógeno entérico. 2) modelar o consumo relativo de animais sobre condição de desafio sanitário e integrar esse modelo dentro de um modelo mecanicista de simulação, o Broiler Growth Model (BGM). Assim, permitindo a modelagem do desempenho de animais sob condição de desafio sanitário. Para isso, um total de 1.530 frangos de corte machos Cobb500 com um dia de idade foram casualizados em cinco tratamentos, com nove r... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Enteric diseases appear one of the biggest problems of poultry production. Historically, antibiotics growth promoter (AGPs) have been used to control this framework. However, concerns about antibiotic resistance bacteria have led regional bans for reducing the use of AGPs. Alternatively, microorganisms directly supplemented in diets such as probiotics, may exhibit a positive impact on intestinal microbiota avoiding enteric problems that inevitably causes loss on broilers performance. Another important point to be considered when talking about sanitary challenge is the feed intake. The use of mathematical models that consider the challenge factor for modelling in this feed intake is still scarce. In this sense, this project was conducted with two main purposes: 1) to investigate the effects of Bacillus amyloliquefaciens CECT 5940 as a probiotic (DFM) alone or in combination with bacitracin methylene disalicylate (BMD) in broilers on enteric pathogen challenge. 2) Model the relative feed intake of animals under sanitary challenge condition and to integrate this model into the mechanistic simulation model, the Broiler Growth Model (BGM). Thus, allowing the modelling of performance of animals under sanitary challenge condition. For such, a total of 1,530-day-old male Cobb500 chicks, were randomly assigned to five treatments, with nine replicate pens with 34 birds each. Treatments included positive control (PC, basal diet without additives or challenge); negative control (NC, basa... (Complete abstract click electronic access below) / Mestre

1. The major phospholipids extracted from Bacillus amylolique - faciens were cardiolipin, phosphatidylycerol and phosphatidylethanolamine. 2. The distribution of these phospholipids between the two halves of the cytoplasmic membrane bilayer was studied using phospholipase C ( B. cereus ), phospholipase A2 ( Crotalus ) and the non - penetrating chemical probe trinitrobenzenesulphonic acid ( TNBS ). After treatment of intact protoplasts of B. amylolique - faciens with either phospholipase, approximately 70 % of total membrane phospholipid was hydrolysed ; specifically approximately 90 %, 90 % and 30 % of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin respectively. Under these conditions, protoplasts remained intact and sealed. However, when protoplasts that were permeabilized by cold shock treatment were incubated with either of the phospholipases, up to 80 % of cardiolipin was hydrolysed and phosphatidylglycerol and phosphatidylethanolamine were hydrolysed virtually to completion. In intact cells, 92 % of the phosphatidylethanolamine could be labelled with TNBS under conditions in which the reagent did not penetrate the membrane to any significant extent. 3. These results suggest that 70 % of total phospholipid of this bacillus exists in the outer half of the bilayer. The distribution of phosphatidylethanolamine in this bilayer is highly asymmetric with it being located predominantly in the outer half. The results with phospholipases suggest that the distributions of cardiolipin and phosphatidylglycerol are also asymmetric but independent confirmation or this is required. 4. The fatty acid composition of cells grown at different temperatures was investigated. When cells were grown at 30 ° C, branched - chain saturated fatty acids made up over 80 % of the total fatty acids. Saturated straight - chain fatty acids made up the bulk of the remainder. Less than 1 % of the total fatty acids were unsaturated. Decrease in growth temperature was accompanied by an increase in the ratio of branched to straight - chain fatty acids and a marked increase in the level of unsaturation of branched - chain fatty acids. 5. When cells of this organism, grown at 30 ° C, were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower temperatures or addition of Tween - 80 to cells caused the critical temperature zone for cold shocking to be significantly lowered. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock. 6. The role of lipids in the process of extracellular enzyme secretion was studied using cerulenin, an antibiotic known to inhibit fatty acid synthesis in microorganisms. Cerulenin inhibited the secretion of alpha - amylase and protease in washed cell suspensions by 80 % and 75 % respectively over 3 hours. The effect was a general one since secretion of all protein species into the medium was drastically reduced by the antibiotic. At the concentration of cerulenin used ( 100 . µ g / ml ), [ 14C ] - acetate incorporation into cellular lipid was inhibited by approximately 50 % but total cellular protein and RNA synthesis were virtually unaffected. The inhibitory effect of cerulenin on alpha - amylase and protease secretion could be partially reversed if cell suspensions were supplemented with either fatty acids prepared from the lipids extracted from B. amyloliquefaciens, or various individual pure fatty acids. These results suggest that fatty acid synthesis may be required for protein secretion by this organism. 7. Attempts were made to detect precursors to extracellular enzymes either associated with the cells or in the culture medium, employing immunological techniques. These experiments, however, were not successful. / Thesis (Ph.D.)--Department of Biochemistry, 1979.

Thesis (Ph. D.)--University of Adelaide, Dept. of Biochemistry, 1984. / Includes bibliographical references (leaves 118-130).

Thesis (Ph.D. 1979) from the Department of Biochemistry, University of Adelaide.

Em anos recentes, surgiram numerosos casos de intoxicação alimentar envolvendo patógenos emergentes. Estes casos levaram a um aumento da preocupação com a preservação dos alimentos minimamente processados e com a segurança alimentar. Este fato está induzindo a pesquisa por inibidores para estes patógenos e fatores para prolongar a vida de prateleira de produtos alimentícios. Entre as novas alternativas na preservação está a utilização de peptídeos antimicrobianos produzidos por bactérias. No presente trabalho uma bactéria identificada como Bacillus amyloliquefaciens LBM 5006 isolada de solos de mata Atlântica de Santa Catarina foi selecionada dentre outros microrganismos e sua capacidade de produzir antimicrobianos foi avaliada. O extrato bruto da cultura do isolado LBM 5006 foi caracterizado, sendo ativo contra importantes bactérias patogênicas e deteriorantes como Listeria monocytogenes, Bacillus cereus, Erwinia carotovora, Escherichia coli, dentre outras. Houve maior produção do antimicrobiano quando a bactéria foi propagada em caldo infusão de cérebro e coração (BHI) a 37o C durante 48 h. Após concentração, a atividade antimicrobiana resistiu ao tratamento com enzimas proteolíticas. A atividade antimicrobiana foi verificada em pHs ácidos, sendo inibida em pH 9 e 10. O extrato foi purificado por meio de cromatografia de gel filtração e extração com butanol. O teste qualitativo de ninidrina, juntamente com a espectroscopia de infravermelho e ultravioleta, feitos com a substância purificada revelou que o antimicrobiano possui natureza protéica. O antimicrobiano apresentou um efeito bacteriostático contra 106 UFC/mL de Listeria monocytogenes na concentração de 25 AU/ml. / In recent years, an increase in food poisoning cases involving emerging pathogens have been described. These cases have increased the preoccupation with the preservation of minimally processed food products and food safety. This fact is leading the research for inhibitory substances and for factors to extend the shelflife of food products. Among the new preservation alternatives is the utilization of antimicrobial peptides produced by bacteria. In present work a bacterium identified as Bacillus amyloliquefaciens LBM 5006 isolated from Atlantic Forest soils of Santa Catarina was selected among other microorganisms and its capacity to produce antimicrobials was evaluated. The crude culture supernatant of the strain LBM 5006 was characterized, being active against important pathogenic and spoilage bacteria like Listeria monocytogenes, B. cereus, E. carotovora, E. coli, among other. Maximum production of antimicrobial activity was observed when the bacteria was propagated in Brain Heart Infusion (BHI) at 37o C for 48 h. After concentration, the antimicrobial activity was resistant to proteolytic enzymes. Antimicrobial activity was verified in acid pHs, but it was lost at pH 9 and 10. The supernatant was purfied by gel filtration chromatography and butanol extraction. The qualitative ninhydrin test, together with infrared and ultraviolet spectroscopy of purified substance revealed that the antimicrobial has a peptide moiety. A dose of 25 UA/mL caused a bacteriostatic effect against 106 UFC/mL Listeria monocytogenes.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. / Made available in DSpace on 2012-10-18T12:26:38Z (GMT). No. of bitstreams: 0 / Extraiu-se uma substância antimicrobiana de uma cultura 18-24 h de Bacillus amyloliquefaciens em Caldo Triptona de Soja suplementado com Extrato de Levedura a 0,6% (TSB-YE - OXOID) por precipitação com 20% de saturação de sulfato de amônio, ressuspensão em tampão fosfato pH 7,2, diálise em membrana 3,5kD (Spectra/Por) e esterilização por filtração (membrana 0,22mm - Millipore). Esta substância foi caracterizada segundo sua sensibilidade a temperatura (50, 75 e 100°C) e pH (2,0 a 9,0). Para determinação de sua natureza química empregaram-se as enzimas (Sigma) protease de S. griseus (P6911), protease de A. saitoi (P2143), a-quimotripsina (C4129) e pepsina (P6887) a 50mg/mL e a a-amilase Termamyl 120 L (Novo Nordisk) a 1% v/v. Para verificação da manutenção da atividade após tais ensaios, o extrato foi aplicado em poços preparados em placas de Ágar Triptona de Soja suplementado com Extrato de Levedura a 0,6% (TSA-YE - OXOID) pré-semeadas com 0,1 mL de uma cultura 105 UFC/mL de Listeria monocytogenes. Determinou-se sua concentração mínima inibitória (CMI) sobre uma cultura 104 UFC/mL de L. monocytogenes e seu peso molecular por eletroforese em gel de poliacrilamida (SDS-PAGE). Como resultado dos testes de caracterização, a substância apresentou-se estável a 50°C, perdendo sua atividade gradualmente a 75°C e rapidamente a 100°C. Também foi estável em valores de pH de 2,0 a 9,0. Perdeu parcialmente sua atividade quando tratada com as enzimas protease de S.griseus, protease de A. saitoi e a-amilase, indicando uma estrutura glicoproteica. A CMI foi 25% v/v e a substância agiu de forma bacteriostática. A eletroforese indicou um peso molecular inferior a 6.500 Daltons.

Em anos recentes, surgiram numerosos casos de intoxicação alimentar envolvendo patógenos emergentes. Estes casos levaram a um aumento da preocupação com a preservação dos alimentos minimamente processados e com a segurança alimentar. Este fato está induzindo a pesquisa por inibidores para estes patógenos e fatores para prolongar a vida de prateleira de produtos alimentícios. Entre as novas alternativas na preservação está a utilização de peptídeos antimicrobianos produzidos por bactérias. No presente trabalho uma bactéria identificada como Bacillus amyloliquefaciens LBM 5006 isolada de solos de mata Atlântica de Santa Catarina foi selecionada dentre outros microrganismos e sua capacidade de produzir antimicrobianos foi avaliada. O extrato bruto da cultura do isolado LBM 5006 foi caracterizado, sendo ativo contra importantes bactérias patogênicas e deteriorantes como Listeria monocytogenes, Bacillus cereus, Erwinia carotovora, Escherichia coli, dentre outras. Houve maior produção do antimicrobiano quando a bactéria foi propagada em caldo infusão de cérebro e coração (BHI) a 37o C durante 48 h. Após concentração, a atividade antimicrobiana resistiu ao tratamento com enzimas proteolíticas. A atividade antimicrobiana foi verificada em pHs ácidos, sendo inibida em pH 9 e 10. O extrato foi purificado por meio de cromatografia de gel filtração e extração com butanol. O teste qualitativo de ninidrina, juntamente com a espectroscopia de infravermelho e ultravioleta, feitos com a substância purificada revelou que o antimicrobiano possui natureza protéica. O antimicrobiano apresentou um efeito bacteriostático contra 106 UFC/mL de Listeria monocytogenes na concentração de 25 AU/ml. / In recent years, an increase in food poisoning cases involving emerging pathogens have been described. These cases have increased the preoccupation with the preservation of minimally processed food products and food safety. This fact is leading the research for inhibitory substances and for factors to extend the shelflife of food products. Among the new preservation alternatives is the utilization of antimicrobial peptides produced by bacteria. In present work a bacterium identified as Bacillus amyloliquefaciens LBM 5006 isolated from Atlantic Forest soils of Santa Catarina was selected among other microorganisms and its capacity to produce antimicrobials was evaluated. The crude culture supernatant of the strain LBM 5006 was characterized, being active against important pathogenic and spoilage bacteria like Listeria monocytogenes, B. cereus, E. carotovora, E. coli, among other. Maximum production of antimicrobial activity was observed when the bacteria was propagated in Brain Heart Infusion (BHI) at 37o C for 48 h. After concentration, the antimicrobial activity was resistant to proteolytic enzymes. Antimicrobial activity was verified in acid pHs, but it was lost at pH 9 and 10. The supernatant was purfied by gel filtration chromatography and butanol extraction. The qualitative ninhydrin test, together with infrared and ultraviolet spectroscopy of purified substance revealed that the antimicrobial has a peptide moiety. A dose of 25 UA/mL caused a bacteriostatic effect against 106 UFC/mL Listeria monocytogenes.