Rhamnolipid biosurfactants are investigated to determine their role in biological processes, and for discovery of novel, more biocompatible applications in areas related to medicine, agriculture, and environment. Fundamental understanding of the physical and chemical properties of rhamnolipids is needed. Thus, systematic studies on solution and interfacial properties on rhamnolipids from P.aeruginosa ATCC 9027 were undertaken. A purification protocol was developed and a thorough qualitative and quantitative speciation analysis was performed with high performance liquid chromatography-mass spectrometry (HPLC-MS), nuclear magnetic resonance (NMR) spectroscopy and HPLC with evaporative light scattering detection (ELSD). Acid-base properties of the mixture of 30 monorhamnolipid congeners, including both saturated and unsaturated species, were characterized at concentrations below and above the CMC at fixed ionic strength using potentiometry and attenuated total reflectance-Fourier transform infrared (ATRFTIR) spectroscopy. A pKₐ of 4.3 was found for concentrations below 50 μM and a pKₐ of 5.6 above 100 μM. The pKₐ is dictated by aggregation in solution. Molecular areas of monorhamnolid monolayers at the air-water interface are strongly influenced by protonation state, and increase from 31 to 109 Ų/molecule as pH increases from 4 to 8.5 and as ionic strength decreases. Adsorption isotherms of monorhamnolipids on γ-Al₂O₃ were investigated as a function of pH using ATR-FTIR spectroscopy and Frumkin model fits, from which K(ads) values of 1.20 (± 0.10) x 10⁵ M⁻¹ at pH 4.0, 2.14 (± 0.51) x 10⁴ M⁻¹ at pH 6.3 and 1.31 (± 0.09) x 10³ M⁻¹ at pH 8.6 were obtained. Interaction parameters were positive at all pH values. Cooperative adsorption is driven by hydrophobic interactions (physisorption) at any pH including hydrogen bonding and electrostatic interactions. Chemisorption was also observed at high pH values. Formation constants for monorhamnolipid-Pb²⁺ and monorhamnolipid-Cd²⁺ complexes were determined using differential pulse polarography in the low μM concentration range. A modified Lingane equation was developed to account for monorhamnolipid adsorption on the Hg surface. β values for adsorbed metal complexes are ~10^3.2 and ~10^0.8 for Pb²⁺ and Cd²⁺, respectively, compared to previously published β values of 10^8.58 and 10^6.89, respectively. Evidence for 1:1 and 2:1 monorhamnolipid-metal complexes was provided by electrospray ionization-mass spectrometry.

The purpose of this research has been to explore the native monorhamnolipid (mRL) mixture produced by Pseudomonas aeruginosa ATCC 9027, as well as newly- synthesized monorhamnolipid diastereomers (R,R)-Rha-C10-C10, (R,S)-Rha-C10-C10, (S,S)-Rha-C10-C10 and (S,R)-Rha-C10-C10, to understand their aggregation and phase behavior in aqueous solution. This work is in response to the consideration that biosurfactants offer a "greener" alternative to conventional surfactants. Relationships between chemical structure of the diastereomers and surface activity were studied using surface tension measurements. It was found that the critical aggregation concentration (CAC) values are lower for deprotonated (R,S)-Rha-C10-C10 and higher for protonated (R,S)-Rha-C10-C10 compared to the other diastereomers or the native mRL mixture. Furthermore, the 1:1:1:1 diastereomeric mixture has the overall lowest cross-sectional area for deprotonated rhamnolipids. Dynamic light scattering (DLS) was used to study the hydrodynamic radii (Rh) of the mRL aggregates and the four diastereomers in aqueous solution. In all five surfactants studied, each observed at pH 8.0 and 4.0, three aggregate populations were observed. For all deprotonated rhamnolipids the micelle, at a hydrodynamic radius of ~2 nm, was found to be in the highest abundance where the two lower abundant other population take on a lamellar aggregate structure. Data to support this were collected using fluorescence probing techniques. For the surfactants in the protonated state, pH 4.0, it was found that all form primarily lamellar structures, also confirmed using fluorescence probing. The average aggregation numbers (Nagg) of the micelles for the deprotonated native mRL mixture and the four diastereomers were studied using steady-state and time-resolved fluorescence quenching measurements. Somewhat unexpectedly, the Nagg values were observed to be a strong function of the rhamnolipid concentration for all systems. At low concentrations, pre-micellar aggregates with aggregation numbers too small for micelles were observed. A critical concentration is identified at which a critical aggregation number is defined; this is proposed to be the smallest fully-formed micelle in solution with values of ~25-30 molecules/micelle for the native mRL mixture and all four diastereomers. Thus, the aggregation properties of the native mRL mixture and the four diastereomers are generally similar at this critical concentration. However, the increase in aggregation number above this critical point varies for all the surfactants, where the (R,R)-Rha-C10-C10 diastereomer has the greatest increase in monomers with concentration and the (S,S) and (R,S) diastereomers have the lowest. The increased observed aggregation number is consistent with the computational work showing increased growth with concentration for the deprotonated rhamnolipid micelles. Furthermore, these computations have confirmed the ability of protonated rhamnolipids to form lamellar structures in solution. Fluorescence probing work was also used to study rhamnolipid phase behavior as a function of varying solution conditions, such as ionic strength, solution pH, surfactant concentration, and temperature. Steady-state fluorescence methods are utilized to probe the surfactant microenvironment using the polarity-sensitive dyes prodan and laurdan. By dissolving the dye within the rhamnolipid aggregates and observing their solvatochromic behavior, all surfactants studied are known to form more micellar aggregates at high pH and low surfactant concentration, whereas elongation of the micelle is observed at low pH and moderate to high surfactant concentration.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-19Bitstream added on 2014-06-13T20:24:23Z : No. of bitstreams: 1 lovaglio_rb_dr_rcla.pdf: 1731223 bytes, checksum: 43dfc85c18edb74e4fc520e7b8f963ac (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os biossurfactantes são metabólitos secundários que apresentam atividade de superfície, podendo ser aplicados em diferentes processos industriais, como produção de fármacos, compostos químicos, cosméticos, biorremediação de ambientes poluídos por petróleo e derivados e recuperação terciária do petróleo. A maior vantagem destes compostos quando comparado aos surfactantes sintéticos reside na sua diversidade estrutural, baixa toxicidade e alta biodegradabilidade. Apesar das inúmeras vantagens apresentadas pelos biotensoativos, eles não são comercialmente utilizados devido ao alto custo de produção. O objetivo deste trabalho foi estudar a produção de ramnolipídios por mutantes de P. aeruginosa a partir de resíduos industriais e óleos vegetais, bem como caracterizar as propriedades de solução desses biossurfactante, além de verificar a influência da fonte de carbono na produção de homólogos de ramnolipídios por P. aeruginosa. Na primeira etapa as fermentações foram conduzidas em tubos de ensaio, após a seleção dos micro-organismos com potencial para alta produção, os mesmos foram testados em frascos Erlenmeyer. O mutante com potencial para elevada produção foi avaliado, junto com a linhagem selvagem, em fermentadores. A linhagem selecionada e a utilização do meio de cultura livre de cálcio contendo óleo de girassol como fonte de carbono acarretou em um aumento de 70% na produção de ramnolipídios. A adição de etanol às soluções de ramnolipídios comprovou o potencial desse tensoativo para serem aplicados na indústria de cosméticos, já que as propriedades de tensão superficial, agregação e emulsificação foram mantidas nas três concentrações de álcool adicionadas. Além disso, verificou-se que a mudança da fonte de carbono acarreta alteração nos tipos ácidos graxos e proporções de homólogos de ramnolipídios produzidos por P. aeruginosa LBI 2A1 / The biosurfactants are secondary metabolites that exhibit surface activity and can be applied in different industrial processes such as production of pharmaceuticals, chemicals, cosmetics, bioremediation of environments polluted by oil and derivatives and tertiary recovery of oil. The greatest advantage of these compounds compared to synthetic surfactants lies in their structural diversity, low toxicity and high biodegradability. Despite the numerous advantages provided by biosurfactants, they are not commercially used due to the high cost of production. The present study aimed to evaluate the production of rhamnolipids by mutants of P. aeruginosa using industrial waste and vegetable oils as carbon source, as well as to characterize the properties of these biosurfactant solutions and investigate the influence of carbon source in the production of homologous rhamnolipds by P. aeruginosa. In the first stage fermentations were conducted in test tubes, after selection of microorganisms with potential for high production they were tested in Erlenmeyer flasks. The mutant with the potential for increased production was assessed, along with the wild strain in bioreactors. The selected strain and the calcium free medium used with sunflower oil as carbon source resulted in an 70% increase in the production of rhamnolipids. The addition of ethanol to the rhamnolipids solutions proved the potential of this biosurfactant to be applied in the cosmetic industry, since the properties of surface tension, emulsification and aggregation were held in all three concentrations of ethanol added. Moreover, it was found that changing the carbon source caused alteration in the types of fatty acids and rhamnolipids counterparts proportions produced by P. aeruginosa LBI 2A1

No description available.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2009. / Surfactants are compounds that reduce interfacial surface tension, resulting in detergency, emulsifying, foaming and dispersing properties. Surfactants produced via biochemical processes (biosurfactants) form a niche market with their low toxicity, biodegradability and high specificity attributes. Biosurfactants have recently received considerable attention owing to their potential as biomedical molecules. In this study a knowledge base was established for the development of a process which produces biosurfactants for use as antimicrobial agents. Specifically, rhamnolipid biosurfactants were produced from Pseudomonas aeruginosa and tested for antimicrobial activity against target organisms. Accurate and reproducible analyses for the quantification of rhamnolipids and antimicrobial activity were developed. The amount of rhamnolipid was determined indirectly by measuring the rhamnose concentration. A novel HPLC method as well as an orcinol colorimetric method were developed for rhamnose measurement. In order to obtain accuracy with the orcinol method it was found that samples must be extracted at least three times prior to the analysis. An examination of literature on rhamnolipid production showed that many studies used colorimetric methods without extraction. Antibacterial activity was quantified by zone clearing around wells of supernatant in soft agar containing the target organism Mycobacterium aurum. This target organism is especially important in a South African context, since it is used to indicate possible susceptibility of tuberculosis to antibiotics. This method was developed for antibacterial testing, after a standard disk diffusion method proved to be ineffective. Antifungal activity of rhamnolipids was evaluated against the fungus Botrytis cinerea, by growing a lawn of fungus on a plate and adding rhamnolipid. The factors influencing rhamnolipid production were studied by growing different Pseudomonas aeruginosa strains from the ATCC culture collection, namely ATCC 9027 and ATCC 27853 as well as a locally isolated strain under different media conditions. The initial focus was on production of biosurfactants in media containing glucose as substrate. Alkanes were subsequently investigated as an alternative substrate, since they are readily available in South Africa as byproducts from the petrochemical industry. The rhamnolipids produced from the culture collection strains were evaluated for their antibacterial activity against Mycobacterium aurum. A number of key factors were identified which were important for the development of a rhamnolipid production process. Of critical importance were the media conditions. Good production was achieved on glucose media containing a phosphate limitation, pH buffering around neutral pH and a high carbon concentration (2 % carbon). When Pseudomonas aeruginosa ATCC 9027 was cultured on this medium (a minimal salts phosphate limited medium with a Tris buffer), it produced 1.31 g/l rhamnose, equivalent to 4.0 g/l rhamnolipid. This rhamnolipid concentration is 2.7-fold higher that of 1.47 g/l reported in the literature with the same strain (cultured on a different phosphate limited medium The particular strain also proved to be a factor which influenced the yield of rhamnolipids. A rhamnose concentration of 0.43 g/l was obtained with Pseudomonas aeruginosa ATCC 27853 grown on MSM+Tris medium, compared to 1.31 g/l produced by Pseudomonas aeruginosa ATCC 9027 on the same medium. The most promising strain and medium, Pseudomonas aeruginosa ATCC 9027 and MSM+Tris medium, were evaluated under controlled conditions in an instrumented bioreactor. Nearly double the rate of growth and production were obtained in the bioreactor, indicating that production time can be shortened considerably under controlled conditions. However, when compared to shake flask studies, only a 4 % increase in growth and a 5 % increase in rhamnolipid production were achieved in the bioreactor, indicating that the yield was limited by the media components or process conditions. With media containing hexadecane as sole carbon source, negligible rhamnolipid production was achieved. Slow growth was observed and the stationary phase had not been reached even after 2 weeks of growth. It was shown that in glucose media rhamnolipid production only commenced in the stationary phase. Since the stationary phase was not reached during growth on hexadecane, rhamnolipids, which are known to increase the availability of alkanes through emulsification and solubilisation, could not be produced. A strategy was devised to accelerate growth on alkane media. A dual substrate medium containing both glucose and hexadecane was investigated. It was hypothesised that growth would be promoted by glucose leading to rhamnolipid production, which would then increase the uptake of hexadecane. Rhamnolipid was produced in the dual substrate experiments, but the hexadecane uptake was still poor. This was suggested to be due to the exposure of the cells to glucose in the inoculum or test flask, which hampered the ability of the cells to utilise hexadecane. It was reasoned that the ability to utilise hexadecane was determined by the cell hydrophobicity, which was influenced by the exposure to hydrophilic or hydrophobic substrates. Rhamnolipids from Pseudomonas aeruginosa ATCC 9027 and ATCC 27853 were shown to have antibacterial activity against Mycobacterium aurum. The largest zone of clearing of 45 mm was obtained with 4 g/l rhamnolipid from Pseudomonas aeruginosa ATCC 9027. The activity was shown to be directly related to the rhamnolipid concentration, highlighting the importance of maximising the biosurfactant yield when developing a process for the production of rhamnolipids as antimicrobial agents. Antifungal activity tests against Botrytis cinerea were inconclusive. Future studies should expand the antimicrobial application of rhamnolipids by testing their activity against a larger range of target organisms. In order to maximise the rhamnolipid yield in future studies, a fed batch process is proposed which would increase the cell density thereby increasing rhamnolipid production and prolonging the stationary phase, which was found to be the phase associated with rhamnolipid production. Different feeding strategies should be investigated, depending on the kinetics of substrate consumption. It is desirable to feed the smallest volume of substrate that is necessary with a high concentration in order to keep the dilution rate low and maximise the product concentration. A factorial design is recommended for this purpose. Further studies with alkanes as carbon source should be conducted using strains that have been maintained and cultured on media containing alkanes as sole carbon source. Alternative biosurfactant producing strains should also be investigated, which have higher natural cell hydrophobicities.

Surfactantes de origem química e biológica são substâncias anfipáticas que se localizam em interfaces e diminuem a tensão intererficial. Este trabalho objetivou isolar microrganismos do gênero Pseudomonas, a partir de amostras ambientais; selecionar, dentre os isolados, aqueles que são capazes de produzir biossurfactante e caracterizar os biossurfactantes encontrados de acordo com suas propriedades. Foram isoladas 1713 linhagens, destas, 944 foram capazes de crescer em meio PIA. A análise do sobrenadante para presença de ácidos graxos normalmente encontrados em ramnolipídios demonstrou que em 31 linhagens, das 95 que apresentaram atividades tensoativas, foi detectada a presença de 3-hidroxidecanoato (3HD) e outras 56 não apresentaram picos característicos de ácidos graxos característicos de ramnolipídios. Foram selecionadas 10 linhagens para caracterização dos biossurfactantes produzidos por elas. Foi confirmada a produção de ramnolipídios por 3 linhagens e foi possível identificar um novo composto com tensoatividade, o 2-acetilfurano. / Biological and chemical surfactants are anphipatic compounds whitch reduce interfaces tension. The present work aimed to isolate microorganisms of samples from diverse environments, chosing among the isolated ones, those capaple of producing biosurfactants and characterizing it according to some of their properties. From 1713 isolated strains, 944 were capable of growing in PIA medium. The supernatant analysis for the presence of fatty acids usually found in rhamnolipds showed that 31 of the 87 strains that presented tensoactive activities, had 3-hydroxyalkanoates (3HD) detected in their supernatant. Other 56 strains did not present characteristic rhamnolipid fatty acids peaks. Based in this resoults 10 isolates were selected for a better characterization of their biosurfactants. The producton os RHLs were confirmed for 3 diferent strains. It was possible to identify a new compound called 2-acethylfuran that was not mentioned by other authors as a possible biosurfactant or as produced by Pseudomonas.

Na natureza, os microorganismos podem apresentar forma de vida planctônica ou podem estar aderidos a superfícies formando comunidades conhecidas como biofilmes. A formação de biofilmes na indústria alimentícia é uma constante preocupação visto que os microorganismos aderidos podem causar contaminações persistentes, levando a deterioração do alimento e a transmissão de doenças. Uma alternativa para evitar a adesão bacteriana e a formação de biofilmes é o pré-condicionamento de superfícies com biosurfatantes, que são compostos tensoativos de origem microbiana capazes de alterar as propriedades físico-químicas e conseqüentemente modificar as interações entre a bactéria e a superfície. Os biosurfatantes, surfactina obtida de Bacillus subtilis e ramnolipídeo de Pseudomonas aeruginosa, foram testados quanto a capacidade de evitar a adesão e remover biofilmes de bactérias patogênicas de interesse alimentar. Foram avaliadas culturas individuais e mistas de Staphylococcus aureus, Listeria monocytogenes, e Salmonella Enteritidis utilizando-se como modelo superfícies de poliestireno. O pré-condicionamento da superfície com surfactina na concentração de 0,25% reduziu a adesão de Salmonella Enteritidis e Listeria monocytogenes em 42%, enquanto que o tratamento com ramnolipídeo a 1% reduziu a adesão de Listeria monocytogenes e Staphylococcus aureus ao poliestireno em 57,8% e 67,8% respectivamente. O condicionamento com os biosurfatantes não se mostrou eficiente na redução da adesão das culturas mistas das bactérias se comparado aos resultados obtidos para as culturas individuais. O poliestireno condicionado com os biosurfatantes apresentou redução na hidrofobicidade devido ao caráter aniônico destas moléculas. A repulsão eletrostática e a redução das interações hidrofóbicas promovidas pelo condicionamento do poliestireno com ramnolipídeo foram fatores determinantes na atividade antiadesiva observada para L. monocytogenes e S. aureus, entretanto os resultados obtidos para a superfície tratada com surfactina sugerem que outros parâmetros influenciaram nos resultados observados. Após 2 h de contato a surfactina na concentração de 0,1% promoveu a remoção de 63,7% do biofilme de S. aureus, 95,9% do biofilme de L. monocytogenes, 35,5% do biofilme de S. Enteritidis e 58,5% do biofilme da cultura mista das três bactérias. Já o ramnolipídeo na concentração de 0,25% removeu 58,5% do biofilme de S. aureus, 26,5% do biofilme de L. monocytogenes, 23,0 % do biofilme de S. Enteritidis e 24% do biofilme da cultura mista após 2 h contato. De modo geral, o aumento do tempo de contato e da concentração dos biosurfatantes reduziu a remoção dos biofilmes. A surfactina e o ramnolipídeo demonstraram potencial para uso como agentes anti-adesivos assim como para a remoção de biofilmes de bactérias patogênicas de importância alimentar. / In nature, microrganisms can live as planktonic cells or can be found living in communities attached in surfaces forming biofilms. Biofilm represents a great concern for food industry, since it can be a source of persistent contamination that can lead to food spoilage and the transmission of diseases. To avoid the adhesion of bacteria and the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants that are microbial compounds that can modify the physico-chemical properties of the surfaces changing bacterial interactions and consequently adhesion. The biosurfactants, surfactin obtained from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated as agents to avoid the adhesion and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Staphylococcus aureus, Listeria monocytogenes e Salmonella Enteritidis were studied using polystyrene as the model surface. The pre-conditioning with surfactin 0,25% reduces in 42,0% the adhesion of L. monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1,0% reduced in 57,8% the adhesion of L. monocytogenes and in 67,8% the adhesion of S. aureus to polystyrene. The conditioning of surface with biosurfactants was less effective to avoid adhesion of mixed cultures of the bacteria when compared with the results obtained for individual cultures. The polystyrene surface conditioned with the biosurfactants showed a reduction in the hydrophobicity due to the anionic character of the molecules. The electrostatic repulsion and the reduction on hydrophobic interactions promoted by the conditioning of surface with rhamnolipids were determinant factors to explain the anti-adhesive activity observed for L. monocytogenes and S. aureus, however, the data obtained with surfactin suggest that other parameters have influenced the results observed. After 2 h contact with surfactin at 0,1% concentration, the pre-formed biofilms of S. aureus were reduced by 63,7%, L. monocytogenes biofilms were reduce by 95,9% , S. Enteritidis biofilms by 35,5% and the mixed culture biofilm by 58,5%. The rhamnolipids at 0,25% concentration removed 58,5% of biofilm of S. aureus, 26,5% of the biofilm of L. monocytogenes, 23,0% the biofilm of S. Enteritidis and 24,0% the biofilm of the mixed culture after 2 h of contact. In general, the increase in concentration of biosurfactants and in the time of contact decreases the biofilm remove percentage. These results demonstrate that surfactin and rhamnolipids present potential to be used as agents to control the attachment and to disrupt biofilms of food-borne pathogens.

Intoxicações alimentares são uma das causas mais significativas de mortalidade em países desenvolvidos e em desenvolvimento, sendo as contaminações bacterianas as responsáveis pela maioria dos casos. Dentre elas destacam-se as causadas por espécies de Staphylococcus, que são nocivas tanto pela infecção do organismo hospedeiro, quanto pela intoxicação por enterotoxinas termoestáveis presentes em alimentos contaminados e mal acondicionados. Além disso, a capacidade de S. aureus formarem biofilmes confere maior proteção contra agentes de controle. Neste contexto, torna-se importante desenvolver novos métodos visando inibir estes agentes patogênicos. Uma alternativa ao emprego de conservantes sintéticos é a utilização de biossurfatantes, como os ramnolipídeos (RL) que, além de apresentarem alta estabilidade a temperatura, pH e concentração salina, apresentam baixa toxicidade e são biodegradáveis. O objetivo deste trabalho foi avaliar o efeito do pH na atividade antimicrobiana dos RL sobre células planctônicas e sésseis de Staphylococcus aureus (ATCC 8095) e comparar seu efeito ao do dodecil sulfato de sódio (SDS). Os resultados mostraram que o pH exerce grande influência na ação dos surfatantes, sendo mais efetiva em valores de pH menores. Em pH 5, os RL apresentaram ação bactericida (CBM=19,5 mg L-1) superior ao SDS (CBM=39,1 mg L-1), sendo capazes de inibir e remover 80% dos biofilmes em concentrações de 156,2 mg L-1 e reduzir em cerca de 40 % a hidrofobicidade da superfície celular de S. aureus. Em valores de pH maiores a ação dos RL não superou a do SDS, porém estes ainda mostraram resultados promissores, principalmente na remoção de biofilmes evidenciado pela microscopia confocal. A espectroscopia FTIR revelou que quando em pH 6, 7 e 8 S. aureus, possivelmente, induz alterações em sua membrana celular a fim de diminuir a sensibilidade aos surfatantes. As imagens de MEV mostraram que os RL promovem deformações nas células, podendo levá-las a ruptura. O aumento da força iônica, promovido pela adição de NaCl no meio, favoreceu a ação antimicrobiana dos RL, o que torna a aplicação dos RL em alimentos bastante promissora. / Food poisoning can be considered one of the most significant causes of mortality in developed and developing countries with bacterial contamination being responsible for the majority of cases. Among them are those caused by Staphylococcus species that can beharmful, both from the infection of the host organism as the intoxication by thermostable enterotoxins present in contaminated and poorly conditioned food. In addition, the ability to form biofilms gives S. aureus greater protection against control agents. Under this context, it becomes important to develop new methods to inhibit those pathogens. An alternative to the use of synthetic preservatives are biosurfactants such as rhamnolipids (RL), which shows high stability to temperature, pH and salt concentration along with the fact that they have low toxicity and are biodegradable. The aim of this study was to evaluate the effect of pH on the antimicrobial activity of RL on planktonic and sessile Staphylococcus aureus cells (ATCC 8095) and compare its effect to that of the sodium dodecyl sulfate (SDS). The results showed that pH exerts a great influence on the action of surfactants, with greater effectiveness at lower pH. At pH 5, RL presented higher bactericidal action (CBM = 19.5 mg L-1) than SDS (CBM = 39.1 mg L-1), also being able to inhibit and remove 80% of biofilms at concentrations of 156. 2 mg L- 1 and reduce the hydrophobicity of S. aureus cell surface by about 40%. At higher pH values, the action of RL did not exceed that of SDS, neverthelessthey showed promising results, mainly on the removal of biofilms evidenced by confocal microscopy. FTIR spectroscopy revealed that at pH 6, 7 and 8,S. aureus possibly induces changes in its cell membrane in order to decrease the sensitivity to surfactants. The MEV images showed that the RL cause deformations in the cells, which can lead to rupture. The increase in ionic strength, promoted by the addition of NaCl in the medium, favored the antimicrobial action of RL, which makes the application of RL in foods very promising.

Os ramnolipídeos (RL) são biossurfatantes que apresentam características de grande interesse para a indústria de alimentos, tais como alta biodegradabilidade, baixa toxicidade e propriedades emulsionantes. Este trabalho teve como objetivo avaliar a atividade antimicrobiana do ramnolipídeo comercial (RL-Com - Jeneil Co.) e do ramnolipídeo produzido e purificado no Laboratório de Biotecnologia Microbiana (RL-LBM) sobre culturas de Listeria monocytogenes, através da determinação da concentração inibitória mínima (CIM) utilizando o método de microdiluição em caldo. Os valores de CIM dos RLs variaram entre 78,1 μg mL-1 e 2500 μg mL-1 sendo que a CIM predominante foi de 2500 μg mL-1. Dentre as 32 L. monocytogenes testadas 68,7% foram sensíveis ao RL-LBM e 90,6% sensíveis ao RL-Com. O efeito do ramnolipídeo sobre L. monocytogenes mostrou-se predominantemente bacteriostático. O RL aumentou a permeabilidade celular de L. monocytogenes, contudo este efeito não foi relacionado à sua atividade antimicrobiana. A interação entre os antimicrobianos RL-Com e nisina foi avaliada sobre dois isolados de L. monocytogenes com sensibilidades diferentes ao RL, o L17 menos sensível, com CIM de 2500 μg mL-1, e o L12 mais sensível, com CIM de 156,2 μg mL-1. O índice CIF obtido para os isolados foi de 0,078 e 0,18 , para L17 e L12 respectivamente, caracterizando o efeito sinérgico da combinação entre RL e nisina. As curvas de sobrevivência dos isolados L12 e L17 mostraram que a interação entre a nisina e o RL foi bactericida em concentrações menores que as obtidas individualmente para cada antimicrobiano. Para a L12 a combinação de 78,1 μg mL-1 de RL e 160 UI mL-1 de nisina reduziu completamente a população em 30 min de tratamento, e para a L17 a combinação de 156,2 μg mL-1 de RL e 320 UI mL-1 de nisina foi bactericida após 2 h. Os resultados obtidos neste trabalho demonstraram que o ramnolipídeo possui potencial como agente de controle de L. monocytogenes assim como efeito sinérgico quando combinado à nisina. / The rhamnolipids (RL) are biodegradable biosurfactants which have low toxicity and surface activity properties that can be useful for food processing industries. The objective of this study was to evaluate the susceptibility of Listeria monocytogenes with two rhamnolipids products: the rhamnolipid from P. aeruginosa LBI that was produced and purified in the Microbial Biotechnology Laboratory (RLMBL) and a commercial product (RL-Com - Jeneil Co.). Susceptibility tests were performed by the minimal inhibitory concentration (MIC) using the micro-broth dilution technique. The MIC values varied from 78.1 μg mL-1 to 2500 μg mL-1 and the 2500 μg mL-1 concentration was the predominant value . Among the 32 tested cultures, 68.7% were susceptible to RL-MBL and 90.6% to RL-Com. Results showed that the rhamnolipid activity was primarily bacteriostatic. The RL increases the membrane permeability of L. monocytogenes, however this effect was not directly related to its antimicrobial activity. The combined effect of nisin and RL-Com was evaluated against two wild-type isolates of L. monocytogenes, L12 more sensitive (MIC 156.2 μg mL-1) and L17 less sensitive (2500 μg mL-1). The FIC index for the isolates were 0.18 and 0.078 for L12 and L17 respectively, indicating a particular synergistic effect. The survival curve of isolates L12 and L17 showed that the combination between nisin and RL was bactericidal at lower concentration than for the individual antimicrobials. For L12 isolate 78.1 μg mL-1 of RL and 160 UI mL-1 of nisin eliminated the population after 30 min of incubation. The combination of 156.2 μg mL-1 of RL and 320 UI mL-1 of nisin reduced completely L17 population after 2 h of incubation. This work demonstrated the potential antimicrobial activity of rhamnolipids against L. monocytogenes, as well as the synergistic effect of this biosurfactant with nisin.

Os ramnolipídeos (RL) são biossurfatantes que apresentam características de grande interesse para a indústria de alimentos, tais como alta biodegradabilidade, baixa toxicidade e propriedades emulsionantes. Este trabalho teve como objetivo avaliar a atividade antimicrobiana do ramnolipídeo comercial (RL-Com - Jeneil Co.) e do ramnolipídeo produzido e purificado no Laboratório de Biotecnologia Microbiana (RL-LBM) sobre culturas de Listeria monocytogenes, através da determinação da concentração inibitória mínima (CIM) utilizando o método de microdiluição em caldo. Os valores de CIM dos RLs variaram entre 78,1 μg mL-1 e 2500 μg mL-1 sendo que a CIM predominante foi de 2500 μg mL-1. Dentre as 32 L. monocytogenes testadas 68,7% foram sensíveis ao RL-LBM e 90,6% sensíveis ao RL-Com. O efeito do ramnolipídeo sobre L. monocytogenes mostrou-se predominantemente bacteriostático. O RL aumentou a permeabilidade celular de L. monocytogenes, contudo este efeito não foi relacionado à sua atividade antimicrobiana. A interação entre os antimicrobianos RL-Com e nisina foi avaliada sobre dois isolados de L. monocytogenes com sensibilidades diferentes ao RL, o L17 menos sensível, com CIM de 2500 μg mL-1, e o L12 mais sensível, com CIM de 156,2 μg mL-1. O índice CIF obtido para os isolados foi de 0,078 e 0,18 , para L17 e L12 respectivamente, caracterizando o efeito sinérgico da combinação entre RL e nisina. As curvas de sobrevivência dos isolados L12 e L17 mostraram que a interação entre a nisina e o RL foi bactericida em concentrações menores que as obtidas individualmente para cada antimicrobiano. Para a L12 a combinação de 78,1 μg mL-1 de RL e 160 UI mL-1 de nisina reduziu completamente a população em 30 min de tratamento, e para a L17 a combinação de 156,2 μg mL-1 de RL e 320 UI mL-1 de nisina foi bactericida após 2 h. Os resultados obtidos neste trabalho demonstraram que o ramnolipídeo possui potencial como agente de controle de L. monocytogenes assim como efeito sinérgico quando combinado à nisina. / The rhamnolipids (RL) are biodegradable biosurfactants which have low toxicity and surface activity properties that can be useful for food processing industries. The objective of this study was to evaluate the susceptibility of Listeria monocytogenes with two rhamnolipids products: the rhamnolipid from P. aeruginosa LBI that was produced and purified in the Microbial Biotechnology Laboratory (RLMBL) and a commercial product (RL-Com - Jeneil Co.). Susceptibility tests were performed by the minimal inhibitory concentration (MIC) using the micro-broth dilution technique. The MIC values varied from 78.1 μg mL-1 to 2500 μg mL-1 and the 2500 μg mL-1 concentration was the predominant value . Among the 32 tested cultures, 68.7% were susceptible to RL-MBL and 90.6% to RL-Com. Results showed that the rhamnolipid activity was primarily bacteriostatic. The RL increases the membrane permeability of L. monocytogenes, however this effect was not directly related to its antimicrobial activity. The combined effect of nisin and RL-Com was evaluated against two wild-type isolates of L. monocytogenes, L12 more sensitive (MIC 156.2 μg mL-1) and L17 less sensitive (2500 μg mL-1). The FIC index for the isolates were 0.18 and 0.078 for L12 and L17 respectively, indicating a particular synergistic effect. The survival curve of isolates L12 and L17 showed that the combination between nisin and RL was bactericidal at lower concentration than for the individual antimicrobials. For L12 isolate 78.1 μg mL-1 of RL and 160 UI mL-1 of nisin eliminated the population after 30 min of incubation. The combination of 156.2 μg mL-1 of RL and 320 UI mL-1 of nisin reduced completely L17 population after 2 h of incubation. This work demonstrated the potential antimicrobial activity of rhamnolipids against L. monocytogenes, as well as the synergistic effect of this biosurfactant with nisin.