Rhamnolipids are surface-active molecules produced by Pseudomonas aeruginosa as congener mixtures. They are considered “green” alternatives to synthetic surfactants used in many applications. Optimizing yield and controlling congener distribution are necessary steps for successful commercialization. Studies have noted that vegetable oils, composed of a mixture of fatty acids, increase rhamnolipid yield. The physiological explanation for this is not yet understood. Furthermore the exact effects of various fatty acid components in the oils on rhamnolipid production have not been reported. The first part of the dissertation was to investigate rhamnolipid biosynthesis when fatty acid substrates are present. A combination of stable isotope tracing and gene expression assays were used to identify rhamnolipid lipid precursors and potential lipid metabolic pathways used in rhamnolipid synthesis. Result suggests that an octanoyl-CoA intermediate of β-oxidation is diverted from β-oxidation to de novo fatty acid synthesis via a “bypass route”, and is incorporated into either a 2-carbon or a 4 carbon β-ketoacyl- ACP, which can then be recognized by the RhlA enzyme for the biosynthesis of rhamnolipid lipid moiety. The second part of the dissertation focuses on studying how fatty acid substrates of different chain length (C₁₂ to C₂₂) and saturation (C(18:1) and C(18:2)) affect rhamnolipid yield, carbon conversion rate, and congener distribution. Results showed that stearic acid significantly increased rhamnolipid yield. A positive linear correlation between the mass percent of stearic acid used and the carbon conversion rate was observed. For all treatments, the RhaC₁₀C₁₀ was the most abundant and RhaC₁₀C(12:1) was the least abundant of the major congeners produced. However, the relative amounts of RhaC₁₀C₈ and RhaC₁₀C₁₂ congeners were dependent on several factors. In general, fatty acid substrates with relatively short chain length (C₁₂ and C₁₄), the unsaturated fatty acid C(18:2), and longer cultivation times resulted in a higher RhaC₁₀C₈/ RhaC₁₀C₁₂ ratio. The studies presented here demonstrate that the medium composition, in particular the organic substrate component, can affect rhamnolipid biosynthesis, yield, and congener distribution. Furthermore, this work presents evidence that C₁₈ fatty acids as co-substrates increase rhamnolipid yield by draining rhamnolipid intermediates directly from the β-oxidation pathway.

Rhamnolipid (RL) biosurfactants have been considered "green" alternatives to synthetic surfactants. Here, systematic studies of monorhamnolipids (mRLs) and their synthetic analogues are performed to characterize their interfacial and solution behaviors as surfactants. Chemical structure-surface activity relationships of rhamnolipids were probed using surface tension measurements on RLs and a series of their synthetic analogues designed by "truncation modification." Based on our study on RLs and the rationally-designed RL analogues, the key structural factor responsible for the excellent surface activity performance of rhamnolipids is the presence of the rhamnose moiety in the headgroup. As a result, rhamnopyranosides (RhEs), the simplest surfactants with a rhamnose moiety in the headgroup, show surface activity comparable to the bioproduced mRLs. The purified mixture of mRLs harvested from Pseudomonas aeruginosa ATCC 9027 was mixed with a nonionic surfactant Tween-20 (TW) and studied by surface tension measurements at pH 8. The experimental values of CMC show deviation from the theoretical values predicted by ideal solution theory, which is hypothesized to be due to a shape change from rod-shaped to spherical as the mole fraction of TW is increased. The hypothesis about the shape change is supported by dynamic light scattering results, regular solution theory, and packing parameter theory. Polarization modulated-infrared reflection-absorption spectroscopy (PM-IRRAS) has been used to characterize the orientation of the synthetic rhamnolipid Rha-C18-C18 at the air-water interface. Although rhamnolipids exhibit pH-dependent micellization, their orientation at the air-water interface is not affected by pH. The average tilt angle of their alkyl chains is determined to be ~45° at a surface pressure π = 40 mN/m which decreases to 36° when Pb²⁺ is present in the subphase. Assisted by molecular modeling, the packing of mRLs at the air-water interface is believed to be dominated by the packing of their large hydrophilic headgroups. Finally, the adsorption isotherm of mRLs on hydrophobic polyethylene surfaces was generated by ATR-FTIR from solutions of different pH, which were then fit to a Frumkin adsorption model to yield the thermodynamic adsorption parameters, the adsorption equilibrium constant and the interaction parameter. mRLs strongly adsorb to d-PE, and the adsorption is pH dependent.

This dissertation examines the bacterial diversity of hyperarid and arid regions of the Atacama Desert, Chile, as a first step towards understanding the global biogeochemical significance of arid-land microbial communities. The specific objectives were to characterize bacterial diversity and infer the possible metabolic potential of these bacterial communities, and to evaluate the influence of moisture exposure on community structure. In addition, the strengths and limitations of available tools for probing microbial diversity and activity in terrestrial ecosystems were characterized for their application to extreme oligotrophic communities. Preliminary PCR-DGGE analysis of a west-east elevational transect from the Pacific Ocean near Antofagasta to the western slopes of the central Andes indicated that bacterial communities along this transect belonged to two distinct community types: 1) hyperarid (700 - 2000 m) and 2) arid (2500 - 4500 m) communities that included both vegetated and unvegetated regions. Subsequent diversity analysis of these two regions revealed novel but distinct communities in both regions. A greater diversity was observed in the unvegetated arid regions than in the unvegetated hyperarid areas. The unvegetated arid sites were characterized by a bacterial community harboring a combination of radiotolerant and halotolerant heterotrophs as wells as diverse phylotypes closely related to chemolithoautotrophs. These rare phylotypes may be uniquely adapted to arid ecosystems. Molecular tools evaluated for community diversity analysis included PCR-DGGE, Sanger-clone and 454-pyrosequencing analysis of 16S rRNA gene libraries, and the use of reverse transcriptase quantitative PCR (RT-qPCR) for quantifying the impact of environmental variables on the metabolic activity of a specific organism. These techniques were evaluated using the ecosystems of the Atacama Desert as well as model ecosystems designed to address specific questions. Molecular tools are invaluable to the study of microbial ecology because they facilitate the study of fastidious organisms that are difficult or impossible to culture, but the analysis presented in this dissertation demonstrates that each of these methods has limitations and biases which must be acknowledged to avoid inaccurate conclusions from skewed results. The most complete picture of the taxonomic and functional profile of a microbial community is obtained by employing a combination of molecular techniques.

Os ramnolipídeos (RLs) são biossurfactantes glicolipídeos que podem ser produzidos por diferentes espécies bacterianas. P. aeruginosa produz RLs ricos em 3-hidroxidecanoato, já o RL produzido por B. thailandensis apresenta elevada proporção de 3-hidroxitetradecanoato. A enzima RhlA sintetiza o 3-(3-hidroxialcanoiloxi)alcanoato (HAA), porção lipídica de RLs, e apresenta diferenças estruturais nas espécies de P. aeruginosa e B. thailandensis. Esse trabalho concentrou-se na clonagem e expressão heteróloga dos genes rhlA da linhagem P. aeruginosa LFM634 e Burkholderia thailandensis E264. Os HAAs e RLs produzidos pelas linhagens recombinantes foram caracterizados. Além disso, genes quiméricos foram sintetizados com a finalidade de modificar a especificidade das enzimas RhlAs. Os resultados obtidos neste trabalho demonstraram que a expressão de genes rhlA de diferentes espécies modificou a composição dos HAAs e/ou RLs produzidos em P. aeruginosa, B. thailandensis e E. coli. Os dados indicam que a enzima RhlA desempenha um papel importante na composição dos HAAs produzidos, porém o metabolismo bacteriano também é responsável pela composição desses tensoativos. Além disso, os resultados sugerem que a enzima RhlB, responsável pela ligação de ramnose ao HAA, também apresenta diferenças de especificidade. Quando as enzimas quiméricas foram avaliadas, detectou-se um comportamento semelhante à RhlA de B. thailandensis indicando que a estrutura de RhlA de P. aeruginosa é muito específica para funcionar adequadamente junto com a enzima RhlB. Por fim, as propriedades tensoativas demonstraram diferenças quando há modificações na composição dos 3-hidroxiácidos incorporados aos RLs. / Rhamnolipids (RLs) are glycolipid biosurfactants that can be produced by different bacterial species. P. aeruginosa produces RLs rich in 3-hydroxydecanoate, whereas the RL produced by B. thailandensis presents a high proportion of 3-hydroxytetradecanoate. RhlA enzyme synthesizes 3-(3-hydroxyalkanoyloxy)alkanoate (HAA), lipid portion of RLs, and presents structural differences in P. aeruginosa and B. thailandensis. HAAs and RLs produced by the recombinant strains were characterized. In addition, chimeric genes were synthesized for the purpose of modifying the specificity of the RhlAs enzymes. The results obtained in this study demonstrated that the expression of rhlA genes from different species modified the composition of HAAs and/or RLs produced by P. aeruginosa, B. thailandensis and Escherichia coli. The data also strongly suggest that the composition of the RLs produced depends on the 3-hidroxiacids supplies by the cellular metabolism. Futhermore, the results suggest that the RhlB enzyme that binds to the molecule of HAA a d-TDP-L-rhamnose, also exhibits differences in specificity. When the chimeric enzymes were evaluated, a RhlA-like behavior of B. thailandensis was detected indicating that the RhlA structure of P. aeruginosa is very specific to function properly together with the RhlB enzyme. Finally, the results also indicate differences in the tensoactive properties of the RLs with different compositions.

No description available.

No description available.

As bactérias patogênicas são os principais agentes que contaminam alimentos e podem prejudicar a saúde humana. Para tentar combater e controlar a contaminação de alimentos investigam-se novos compostos que apresentam atividade antimicrobiana. O ramnolipídeo (RL) é um biossurfatante (BS) produzido por Pseudomonas spp. que apresenta elevada biodegradabilidade e, baixa toxicidade além de potencial antimicrobiano. O objetivo desse trabalho foi estudar a atividade antimicrobiana do RL frente às bactérias patogênicas Gram positivas, Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) e Gram negativas, Escherichia coli (EHEC) (ATCC 43895) e Salmonella enterica (ATCC 13076) além de contribuir na elucidação do mecanismo de ação destes compostos. Os testes de susceptibilidade ao RL foram realizados a partir da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) utilizando a técnica de micro-diluição. O efeito do pH sobre a atividade antimicrobiana foi avaliado na faixa de pH 5 a 9. Para avaliação do mecanismo de ação foram realizados ensaios de permeabilidade celular, espectroscopia de infravermelho e hidrofobicidade celular. O RL apresentou atividade antimicrobiana para as bactérias B. cereus em CIM 19,5 &#956g/mL e CBM 39,1 &#956g/mL, e para L. monocytogenes CIM 156,2 &#956g/mL e CBM 312,5 &#956g/mL. Para B. cereus apresentou efeito bactericida a partir de 30 minutos na CBM, e para L. monocytogenes em 8 horas de incubação com o RL na CBM. As bactérias Gram negativas E. coli e S. enterica mostraram-se resistentes ao RL. O pH influenciou a ação antimicrobiana do RL sendo mais efetivo em pH mais ácidos. O tratamento com RL promoveu redução da hidrofobicidade da superfície celular das bactérias sensíveis. Os espectros infravermelhos evidenciaram alterações na composição química da membrana/parede celular principalmente para bactérias Gram positivas. A permeabilidade da membrana celular aumentou de acordo com o aumento da concentração de RL. A atividade antimicrobiana do RL foi evidenciada para as bactérias Gram positivas sendo mais sensíveis B. cereus e L. monocytogenes. Os resultados obtidos neste trabalho sugerem que o RL promove alterações na permeabilidade e composição química da membrana celular bacteriana sendo um agente potencial para controle de bactérias Gram positivas de importância alimentar. / Pathogenic bacteria are main agents that contaminate food and are harmful to human health. The search for new compounds to combat and control food pathogens is of increasing interest. Rhamnolipid (RL) is a biosurfactant (BS) typically produced by Pseudomonas spp., showing high biodegradability, low toxicity and antimicrobial activity. This study aimed to evaluate the antimicrobial activity of RL against the food pathogenic Gram positive bacteria Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) and Gram negative, Escherichia coli (EHEC) (ATCC 43895) and Salmonella enterica (ATCC 13076) and also contribute to the elucidation of RL mechanism of action. Susceptibility tests were performed by determination of the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using the broth microdilution method. The effect of pH on antimicrobial action was also investigated ranging from 5 to 9. Mechanism of action was studied using membrane permeability, infrared spectroscopy and cell hydrophobicity assays. The MIC value for B. cereus was 19.5 &#956g/mL and MBC was 39.1 &#956g/mL. L. monocytogenes was inhibited at concentration 156.2 &#956g/mL showing MBC of 312.5 &#956g/mL. B. cereus presented bactericidal effect after 30 minutes and for L. monocytogenes after 8 hours. The Gram-negative E. coli and S. enterica were resistant to RL. The pH influence antimicrobial activity of the RL showing decreasing MIC values at acidic conditions. Cell hydrophobicity was reduced by RL for the sensitive bacteria. Infrared spectroscopy showed that RL induced changes in chemical composition of cell membrane/ wall especially for the Gram positive bacteria. Cell permeability also increases as RL concentration increases. Antimicrobial activity of RL was evidenced for Gram positive bacteria and the most sensitive were B. cereus and L. monocytogenes. The results of this study suggest that rhamnolipid biosurfactant promotes changes in the permeability and membrane chemical composition showing potential to control foodborne Gram positive bacteria.

Made available in DSpace on 2016-06-02T19:55:33Z (GMT). No. of bitstreams: 1 4672.pdf: 1519614 bytes, checksum: a835ffbdf3a94ccbfaa789a3611d3aa6 (MD5) Previous issue date: 2012-09-28 / Universidade Federal de Sao Carlos / Biosurfactants are a surface-active chemical compounds synthesized by microorganisms. These compounds have many advantages when compared to their chemically synthesized counterparts as specific action, low toxicity, higher biodegradability, effectiveness at extreme temperatures, pH and strength ionic. They appear as promising candidates to replace chemical surfactants produced from petrochemicals. The use of renewable and low cost substrates such as agro based industrial wastes is one of the attractive strategies for economical large scale biosurfactants production. In this work, it was evaluated glycerol, a co-product of biodiesel production, as carbon source for biosurfactant production. Two microorganisms, Pseudomonas aeruginosa MSCIC02 and Bacillus subtilis LAMI009, both isolated from environmental sources, were used thorough this work. In the first part of the work experiments were carried out in shake flasks using P. aeruginosa. The results showed that the increase in nitrogen source (sodium nitrate) and the decrease in the carbon source (glycerin) favored rhamnolipids production. In the range studied, the maximum biosurfactant concentration obtained was 2.3 g⋅L-1 (C/N ratio 12). The effect of nitrogen concentration on the biosynthesis of rhamnolipids and pH behavior as a function of the nitrate concentration in the cultures indicated that this strain probably carried a denitrification route favoring the production of rhamnolipids. Experimental runs carried out in bioreactor indicated that the integrated process of production and separation/concentration by fractionation in bubble column equipment caused many operation problems, such as the drag cell, and reducing the concentration of rhamnolipids to 0.4 g⋅L-1in the reaction medium. The kinetics of product formation was evaluated by two models. The Luedeking-Piret model was not able to represent the process. The model proposed by MERCIER et. al. (1992) could adequately describe the rhamnolipids production from P. aeruginosa strain. Emulsifying capacity of the cell-free culture medium was assessed by the emulsification index (EI24). The biosurfactant produced was able to emulsify vegetable oils as well as mineral oils. EI24 greater than 55% was reached. In the second part of the work experimental data from Bacillus subtilis LAMI009 cultivated in shake flasks showed that the growth of this strain was dependent on iv the medium supplementation with yeast extract. A change in culture medium was implemented in order to reduce the length of the lag phase. The use of inorganic nitrogen sources showed that both ammonium nitrate and ammonium sulphate reached similar values of surfactin concentration and volumetric productivity. It was obtained 35 mg⋅L-1 and 6.1 mg⋅L-1⋅h-1, respectively. Surface tension of the cell-free culture medium was similar for both nitrogen sources. The minimal value obtained was 29.7 mN⋅m-1. Sodium nitrate was found to be an adequate nitrogen source for cell growth. However, in these assays low productivity and low surface tension reduction were obtained when compared to the other nitrogen sources evaluated. The supplementation of the culture medium with yeast extract improves the surfactin concentration (60.0 mg⋅L-1) and volumetric productivity (5.2 mg⋅L-1⋅h-1). In this assay the surface tension reached 28.1 mN⋅m-1. The inoculum size had a great influence on cell growth and production of surfactin. When 2% (v/v) of inoculum was used the surfactin concentration and volumetric productivity obtained were 148.2 mg⋅L-1 e 14.22 mg⋅L-1⋅h-1, respectively. The search for genes responsible for production of lipopeptides surfactin and iturine indicated the presence of the genes lpa14 and ituD in B. subtilis LAMI009 genome. Analysis of the chromatography profile of methanol extracts of the lipopeptides from culture medium with ammonium nitrate and sodium nitrate as nitrogen source showed characteristic peaks of the surfactin and iturine. Thereby, it is believed that this strain is a co-producer of both surfactin and iturine. Emulsifying capacity of the cell-free culture medium showed higher stability with the media that employed ammonium nitrate and sodium nitrate as nitrogen source. It was obtained EI24 of 65% with n-hexadecane and 45% with kerosene. The acid precipitation of biosurfactant from the cell-free culture medium showed that this prepurification step promoted an increase in the emulsifying capacity of the mixture of lipopeptides synthesized by B. subtilis LAMI009. The aqueous solution of crude biosurfactant was able to emulsify naphthenic oils, vegetable oils, and an aromatic hydrocarbon. Values of EI24 greater than 65% were obtained. Emulsions formed with naphthenic oils were more stable according to droplet-size distribution. The smaller the size of droplets, the more stable was the emulsion. / Biossurfactantes são compostos químicos tensoativos sintetizados por microrganismos. Estes compostos possuem muitas vantagens quando comparados com seus equivalentes sintetizados quimicamente como ação específica, baixa toxicidade, alta biodegradabilidade, efetividade em condições extremas de temperatura, pH e força iônica. Apresentam-se como substitutos promissores aos surfactantes químicos derivados da indústria do petróleo. A utilização de substratos renováveis e de baixo custo, como os resíduos agroindustriais, consiste em um dos fatores mais importantes para a viabilização econômica da produção destes compostos em escala industrial. Neste trabalho avaliou-se o uso da glicerina, um coproduto da produção de biodiesel, como fonte de carbono para produção de biossurfactante. Dois microrganismos, Pseudomonas aeruginosa MSIC02 e Bacillus subtilis LAMI009, ambos isolados a partir de amostras ambientais, foram empregados neste trabalho. Na primeira parte do trabalho experimentos realizados em frascos agitados com a P. aeruginosa mostraram que o aumento da produtividade de ramnolipídeos foi favorecido pelo aumento da concentração da fonte de nitrogênio (nitrato de sódio) e pela redução da concentração da fonte de carbono (glicerina). Na faixa estudada a concentração máxima de biossurfactante obtida foi de 2,3 g⋅L-1 (razão C/N de 12). O efeito da concentração de nitrogênio sobre a biossíntese de ramnolipídeos e o comportamento do pH em função da concentração de nitrato durante os cultivos indicou que esta cepa possivelmente realizou uma rota denitrificante favorecendo a produção de ramnolipídeos. Os cultivos realizados em biorreator indicaram que o processo de produção integrado a extração/concentração por fracionamento em coluna de bolhas acarretou diversos problemas operacionais, como o arraste de células, e a redução da concentração de ramnolipídeos no meio reacional para 0,4 g⋅L-1. Foram avaliados dois modelos cinéticos de formação de produto para os ensaios realizados. O modelo de Luedeking-Piret não apresentou boa representatividade do processo. O modelo proposto por MERCIER et al. (1992) mostrou-se mais adequado para representar a produção de ramnolipídeos pela cepa estudada. A avaliação da capacidade emulsificante do meio de cultivo livre de células mostrou que o biossurfactante produzido pela P. aeruginosa teve um desempenho eficiente, sendo capaz de emulsificar óleos de origem vegetal e mineral e atingir índice de emulsificação (IE24) maior que 55 %. Na segunda parte do trabalho, cultivos realizados em frascos agitados para avaliação da produção de biossurfactantes lipopeptídeos por B. subtilis LAMI009 indicaram que o crescimento desta cepa foi dependente da suplementação do meio com extrato de levedura. Uma adaptação ao meio de fermentação foi necessária para eliminar a extensa fase lag durante o processo fermentativo. A utilização de fontes de nitrogênio inorgânicas mostrou que tanto o nitrato de amônio quanto o sulfato de amônio apresentaram valores de concentração de surfactina e produtividade volumétrica da ordem de 35 mg⋅L-1 e 6,1 mg⋅L-1⋅h-1, respectivamente. A tensão superficial do meio de cultivo livre de células também foi semelhante para ambas fontes de nitrogênio, cujo valor mínimo foi 29,7 mN⋅m-1. O nitrato de sódio foi fonte de nitrogênio adequada para o crescimento celular, entretanto apresentou baixa produtividade quando comparado com as demais fontes de nitrogênio avaliadas. Com a suplementação do meio de cultivo com extrato de levedura ii obteve-se maior concentração de surfactina (60,0 mg⋅L-1) e produtividade volumétrica (5,2 mg⋅L-1⋅h-1) e menor tensão superficial (28,1 mN⋅m-1) relativamente ao meio de cultivo contendo fonte de nitrogênio inorgânica. O tamanho do inóculo exerceu grande influência sobre a concentração de surfactina e a produtividade volumétrica. Quando se utilizou 2% (v/v) de inóculo a concentração de surfactina e a produtividade volumétrica alcançaram valores de 148,2 mg⋅L-1 e 14,22 mg⋅L-1⋅h-1, respectivamente. A pesquisa de genes responsáveis pela produção dos lipopeptídeos surfactina e iturina indicou a presença dos genes lpa14 e ituD no genoma da linhagem B. subtilis LAMI009. A avaliação do perfil cromatográfico dos extratos metanólicos de lipopeptídeos obtidos a partir dos cultivos com as fontes de nitrogênio nitrato de amônio e nitrato de sódio apresentou picos característicos de outro lipopeptídeo além da surfactina, a iturina,. Portanto, acredita-se que esta linhagem é uma co-produtora de surfactina e iturina. A capacidade emulsificante do meio de cultivo livre de células apresentou maior estabilidade com os cultivos com nitrato de amônio e nitrato de sódio, obtendo-se IE24 de 65 % com n-hexadecano e 45 % com querosene. A separação do biossurfactante por precipitação ácida a partir do meio de cultivo livre de células mostrou que esta etapa de pré-purificação promoveu um aumento da capacidade emulsificante da mistura de lipopeptídeos sintetizada por B. subtilis LAMI009. A solução aquosa do biossurfactante bruto foi capaz de emulsificar óleos naftênicos, óleos vegetais e um hidrocarboneto aromático, apresentando IE24 maiores que 65 % com os óleos avaliados. As emulsões formadas com óleos naftênicos, utilizados como base para lubrificantes, foram mais estáveis. Quanto menor o tamanho das gotas mais estável foi a emulsão formada.

As bactérias patogênicas são os principais agentes que contaminam alimentos e podem prejudicar a saúde humana. Para tentar combater e controlar a contaminação de alimentos investigam-se novos compostos que apresentam atividade antimicrobiana. O ramnolipídeo (RL) é um biossurfatante (BS) produzido por Pseudomonas spp. que apresenta elevada biodegradabilidade e, baixa toxicidade além de potencial antimicrobiano. O objetivo desse trabalho foi estudar a atividade antimicrobiana do RL frente às bactérias patogênicas Gram positivas, Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) e Gram negativas, Escherichia coli (EHEC) (ATCC 43895) e Salmonella enterica (ATCC 13076) além de contribuir na elucidação do mecanismo de ação destes compostos. Os testes de susceptibilidade ao RL foram realizados a partir da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) utilizando a técnica de micro-diluição. O efeito do pH sobre a atividade antimicrobiana foi avaliado na faixa de pH 5 a 9. Para avaliação do mecanismo de ação foram realizados ensaios de permeabilidade celular, espectroscopia de infravermelho e hidrofobicidade celular. O RL apresentou atividade antimicrobiana para as bactérias B. cereus em CIM 19,5 &#956g/mL e CBM 39,1 &#956g/mL, e para L. monocytogenes CIM 156,2 &#956g/mL e CBM 312,5 &#956g/mL. Para B. cereus apresentou efeito bactericida a partir de 30 minutos na CBM, e para L. monocytogenes em 8 horas de incubação com o RL na CBM. As bactérias Gram negativas E. coli e S. enterica mostraram-se resistentes ao RL. O pH influenciou a ação antimicrobiana do RL sendo mais efetivo em pH mais ácidos. O tratamento com RL promoveu redução da hidrofobicidade da superfície celular das bactérias sensíveis. Os espectros infravermelhos evidenciaram alterações na composição química da membrana/parede celular principalmente para bactérias Gram positivas. A permeabilidade da membrana celular aumentou de acordo com o aumento da concentração de RL. A atividade antimicrobiana do RL foi evidenciada para as bactérias Gram positivas sendo mais sensíveis B. cereus e L. monocytogenes. Os resultados obtidos neste trabalho sugerem que o RL promove alterações na permeabilidade e composição química da membrana celular bacteriana sendo um agente potencial para controle de bactérias Gram positivas de importância alimentar. / Pathogenic bacteria are main agents that contaminate food and are harmful to human health. The search for new compounds to combat and control food pathogens is of increasing interest. Rhamnolipid (RL) is a biosurfactant (BS) typically produced by Pseudomonas spp., showing high biodegradability, low toxicity and antimicrobial activity. This study aimed to evaluate the antimicrobial activity of RL against the food pathogenic Gram positive bacteria Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) and Gram negative, Escherichia coli (EHEC) (ATCC 43895) and Salmonella enterica (ATCC 13076) and also contribute to the elucidation of RL mechanism of action. Susceptibility tests were performed by determination of the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using the broth microdilution method. The effect of pH on antimicrobial action was also investigated ranging from 5 to 9. Mechanism of action was studied using membrane permeability, infrared spectroscopy and cell hydrophobicity assays. The MIC value for B. cereus was 19.5 &#956g/mL and MBC was 39.1 &#956g/mL. L. monocytogenes was inhibited at concentration 156.2 &#956g/mL showing MBC of 312.5 &#956g/mL. B. cereus presented bactericidal effect after 30 minutes and for L. monocytogenes after 8 hours. The Gram-negative E. coli and S. enterica were resistant to RL. The pH influence antimicrobial activity of the RL showing decreasing MIC values at acidic conditions. Cell hydrophobicity was reduced by RL for the sensitive bacteria. Infrared spectroscopy showed that RL induced changes in chemical composition of cell membrane/ wall especially for the Gram positive bacteria. Cell permeability also increases as RL concentration increases. Antimicrobial activity of RL was evidenced for Gram positive bacteria and the most sensitive were B. cereus and L. monocytogenes. The results of this study suggest that rhamnolipid biosurfactant promotes changes in the permeability and membrane chemical composition showing potential to control foodborne Gram positive bacteria.

This work deals with the current topic of carrier systems. Since the biggest problem is the passage of hydrophobic drugs through the bloodstream, or through universal body barriers (eg blood-brain), it is necessary to chemically modify these carriers in order to be able to administer hydrophobic substances effectively. Based on a literature search, several systems are designed and subsequently studied, in which there is a presumption of possible use for carrier systems and at the same time they have biosurfactants incorporated in them due to their ability to solubilize hydrophobic molecules. The theoretical part of this work will describe the individual biosurfactants, the process of their production, their physicochemical properties, and the possibility of their use in carrier systems. At the same time, the individual carrier systems, the procedure of their preparation, the possibilities of their use are described, and their advantages and disadvantages are also compared. In the practical part, the screening of both individual substances and their mutual interactions, as well as methods used to study the emerging structures is then performed. The study of molecular interactions is primarily performed using the technique of dynamic light scattering. The next part describes the optimization of hydrogel formation with incorporated biosurfactants in their structure and then the formed gels are subjected to rheological and solubilization tests. The study of the internal structure of these gels is performed using a scanning electron microscope.