Lipossomas são estruturas auto-organizadas adequadas para encapsular e proteger substâncias antimicrobianas de interações com componentes dos alimentos. O presente estudo teve por objetivo desenvolver lipossomas contendo nisina e bacteriocina produzida por Bacillus sp. P34 (BLS P34) para o controle de Listeria monocytogenes em produtos lácteos. Os lipossomas desenvolvidos nesse trabalho foram preparados utilizando fosfatidilcolina de soja parcialmente purificada. Inicialmente, a encapsulação de nisina foi avaliada através de duas metodologias: fase reversa e hidratação do filme. Com isso, observou-se que a metodologia de hidratação do filme foi a mais adequada para a continuação dos estudos devido à manutenção integral da atividade antimicrobiana da bacteriocina após a encapsulação. BLS P34 foi encapsulada nas mesmas condições. Os lipossomas contendo nisina ou BLS P34 foram estáveis por 24 dias, apresentando as seguintes características: tamanho entre 130 e 160 nm e polidisperdidade entre 0,22 e 0,35, determinados por espalhamento de luz; morfologia esférica, determinada por microscopia eletrônica; eficiência de encapsulação de 94,12% para a nisina e 100% para a BLS P34, determinados por ultra-filtração; potencial zeta de -55 mV para a nisina e -24 mV para a BLS P34. A nisina livre manteve 100% de sua atividade antimicrobiana inicial após 24 dias, enquanto que a nisina encapsulada apresentou 25% de atividade antimicrobiana residual. Não houve diferença na atividade antimicrobiana de BLS P34 livre e encapsulada durante o armazenamento. O modo de ação de BLS P34 encapsulada contra Listeria monocytogenes demonstrou que não ocorre fusão entre o lipossoma e a bactéria, sendo necessária a liberação do peptídeo para a ação antimicrobiana. A ação antimicrobiana das bacteriocinas livres e encapsuladas sobre leite e queijo Minas frescal contaminados artificialmente com L. monocytogenes foi investigada. A nisina livre foi mais eficiente na inibição do patógeno em leite desnatado armazenado a 30ºC. Entretanto, em temperatura de refrigeração, nisina livre e encapsulada exerceram efeito bactericida. BLS P34 livre e encapsulada mantiveram as contagens de células viáveis de L. monocytogenes sempre abaixo do controle em leite desnatado e integral armazenados a 30ºC e 7ºC. Em queijo Minas frescal, todos os tratamentos reduziram a população do patógeno em comparação ao controle, por 21 dias. Porém, a encapsulação de nisina e BLS P34 promoveram melhor efeito inibitório do que as bacteriocinas livres após 10 dias de armazenamento do queijo. A partir desses resultados evidencia-se o potencial da tecnologia de encapsulação de peptídeos antimicrobianos no controle de L. monocytogenes em produtos lácteos. / Liposomes are promising self-organized structures able to encapsulate and protect antimicrobial substances of the interactions with food components. This study aimed to develop liposomes containing nisin and the bacteriocin produced by Bacillus sp. P34 (BLS P34) for control of Listeria monocytogenes in dairy products. The liposomes developed in this work were prepared using partially purified soybean phosphatidylcholine. Initially, the encapsulation of nisin was evaluated by two methods: reverse phase and film hydration. It was observed that the film hydration method was the most suitable for further studies due to the overall maintenance of the antimicrobial activity after encapsulation. BLS P34 was encapsulated in the same conditions. The liposomes containing nisin or BLS P34 were stable for 24 days, showing the following characteristics: size between 130 and 160 nm and polydispersity between 0.22 and 0.35 determined by light scattering; spherical morphology, determined by electron microscopy; encapsulation efficiency of 94.12% for nisin and 100% for BLS P34, as determined by ultra filtration; zeta potential of -55 mV for nisin and -24 mV for BLS P34. Free nisin retained 100% of its original antimicrobial activity after 24 days, while the encapsulated nisin showed 25% residual antimicrobial activity. There was no difference in antimicrobial activity of free and encapsulated BLS P34 during storage. The mode of action of encapsulated BLS P34 against Listeria monocytogenes showed that no fusion occurs between liposomes and bacteria, being necessary to release the peptide for antimicrobial activity. The antimicrobial action of free and encapsulated bacteriocins on milk and Minas cheese artificially contaminated with L. monocytogenes was investigated. Free nisin was more effective in inhibiting of the pathogen in skim milk stored at 30°C. However, under refrigeration, free and encapsulated nisin exerted bactericidal effect. Viable cell counts of L. monocytogenes were always below the control in whole and skimmed milk stored at 30 and 7°C in presence of free and encapsulated BLS P34. In Minas cheese, all treatments reduced the pathogen population in comparison to control, for 21 days. However, the encapsulation of nisin and BLS P34 promoted better inhibitory effect than the free bacteriocins after 10 days storage of cheese. These results show the potential of liposomes containing nisin or BLS P34 on the control of L. monocytogenes in dairy products.

Foi estudada uma bacteriocina produzida por uma linhagem de B. cereus 8A, isolado de solo da região Sul do Brasil. Na primeira etapa de estudo determinaram-se as condições básicas de produção de bacteriocina com amplo espectro de ação denominada de Cereína 8A. Observou-se que durante a fase estacionária ocorre o máximo da sua produção, iniciando sua síntese no final da fase exponencial. As condições de maior produção foram a 30º C, agitação e contínua e numa faixa de pH de 7,0-8,5. A bacteriocina bruta inibiu várias bactérias indicadoras, como Listeria monocytogenes, Clostridium perfringens e Bacillus cereus. O teste de termoestabilidade mostrou a perda de atividade quando submetida a uma temperatura a partir de 87º C. Verificou-se a resistência da bacteriocina bruta frente à tripsina e papaína, mas não frente à proteinase K e pronase E. B. cereus e L. monocytogenes foram utilizadas como bactérias indicadoras para a determinação do modo de ação, após a determinação da dose bactericida de 200 UA mL-1 e 400 UA mL-1 respectivamente. A Cereína 8A demonstrou uma ação inibidora em culturas de Escherichia coli e Salmonella Enteritidis, quando tratadas com EDTA. A atividade esporicida foi observada contra esporos de B. cereus após tratamento com 400 UA ml -1. A análise da biomassa de L. monocytogenes e B. cereus após tratamento com a Cereína 8A, através da espectrofotometria de infravermelho determinou alteração no perfil, correspondente à fração dos ácidos graxos da membrana celular bacteriana. A substância peptídica foi separada por meio da precipitação com sulfato de amônio, extração com 1-butanol e aplicação em coluna de cromatografia por troca iônica tipo Q-Sepharose. A Cereína 8A purificada mostrou maior sensibilidade a proteases e ao calor e um peso molecular de aproximadamente 26 kDa. O espectro ultravioleta foi típico de um polipeptídeo e o espectro de infravermelho indica presença de grupamentos NH, acil e ligações peptídicas na sua estrutura. Uma hipótese do mecanismo de ação seria a desestruturação da membrana celular pela abertura de poros.

Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-05T06:30:19Z (GMT). No. of bitstreams: 1 Figueiredo_CamilaAlexandrinaVianade_D.pdf: 9514972 bytes, checksum: 602b147a415d93d261e4b82f68a9f117 (MD5) Previous issue date: 2005 / Resumo: Esse trabalho investiga o efeito protetor do tratamento com diferentes doses (50, 100 e 250 mg/Kg) do extrato padronizado de Chá Verde (GTE) em camundongos infectados com Listeria monocytogenes (LM). A eficácia terapêutica foi avaliada para uma dose letal de LM por sete dias consecutivos com diferentes doses de GTE. A proteção significativa do extrato foi verificada durante a fase aguda da infecção e um mecanismo de ação foi proposto a partir da análise dos efeitos do GTE sobre o crescimento e a diferenciação de progenitores hematopoéticos para granulócitos e macrófagos (CFU-GM) da medula óssea e do baço, sobre a atividade estimuladora de colônias (CSA) do soro, sobre a atividade lítica de células NK, sobre a proliferação de linfócitos e sobre a produção de citocinas de padrão Thl (IFN-y) / Th2 (IL-10) em animais normais e infectados com LM. Os resultados demonstraram que a resistência dos animais infectados com uma dose letal de LM tratados com GTE está relacionada com a modulação da mielossupressão e diminuição da hematopoese extramedular produzidas pela infecção. Também foi verificado um aumento da atividade lítica de células NK, da capacidade proliferativa de linfócitos e da polarização da resposta imunológica para Thl. O GTE não apresentou toxicidade sobre o estroma medular e estimulou a geração de progenitores para granulócitos-macrófagos em sistema de cultura líquida de longa duração (LTBMC). O mecanismo de ação da atividade imunomoduladora do GTE ainda não está completamente definido. No entanto, os mecanismos de ação apresentados neste trabalho são de fundamental importância para a atividade antibacteriana in vivo do GTE / Abstract: In the present study we investigated the protective effects of the oral administration of Green tea (GTE) (50, 100 and 250mg/Kg) in mice infected with Listeria monocytogenes (LM). We demonstrated that GTE protects mice from a lethal dose of LM when administered prophylactically for seven consecutive days. To determine the mechanism of action of GTE, we examined the effect of GTE on the number of granulocyte and macrophage colony-forming units (CFU-GM) from bone marrow and spleen, on the colony-stimulating activity (CSA) of serum, on natural killer (NK) cell activity, on lymphocyte proliferation and on Thl/Th2-based cytokine production in normal and LM infected mice. Myelosuppression and extramedular hematopoiesis/splenomegaly were the major features of LM infection, in association with NK cell activation and alteration of lymphocyte proliferation. The resistance of mice infected with a lethal dose of LM but treated prophylactically with 100 and 250mg/Kg of GTE could be related to the modulation of myelosuppression and extramedular hematopoesis induced by the infection, and to an increase in NK cell activity, lymphocyte proliferation and polarization of the Thl-based immune response. The GTE extract was not toxic to either the stromal cell layer or to GM progenitors generated from long-term bone marrow culture (LTBMC). In this way, the mechanism of action through GTE exerts its pharmacological effect is not completely elucidated, although the parameters mentioned here are essential for its antibacterial effect in vivo / Doutorado / Doutor em Farmacologia

Orientador: Arnaldo Yoshiteru Kuaye / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T02:48:18Z (GMT). No. of bitstreams: 1 Rocha_JoaoAndersonKeiti_M.PDF: 6430386 bytes, checksum: 2a5815761af437dc46567523865e2440 (MD5) Previous issue date: 2004 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Tecnologia de Alimentos / Mestre em Tecnologia de Alimentos

Determina la presencia de bacterias patógenas y en particular de Listeria monocytogenes en las ensaladas de pollerías del distrito de Los Olivos, Lima, Perú. Utiliza muestras de ensalada obtenidas de 83 pollerías. El análisis microbiológico se realiza en base a la Norma Técnica Peruana Nº 071 (MINSA/DIGESA, 2003). Utiliza el método de ICMSF- 2000 para el recuento de bacterias aerobias mesófilas. Emplea los métodos de FDA-BAM para la numeración de Coliformes totales y Escherichia coli, así como el recuento de Staphylococcus aureus, detección de Listeria monocytogenes y Salmonella sp. Encuentra que el 96,39% (80) de las muestras de ensaladas eran no aptas para el consumo humano, el 84,34% (70) presenta más de 105 UFC/g de Aerobios Mesófilos, el 95,18% (79) más de 102 NMP/g de Coliformes totales, el 24,10% (20) más de 10 NMP/g de E. coli, y el 21,69% (18) más de 10 UFC/g de S. aureus. Respecto a las bacterias patógenas, el 7,23% (06) presentó Listeria spp., el 3,61% (03) Listeria monocytogenes y el 14,46% (12) Salmonella sp. Los resultados obtenidos demuestran que un elevado porcentaje de ensaladas frescas que se expenden en las pollerías del distrito Los Olivos es no apto para el consumo humano, y un porcentaje significativo está contaminado con microorganismos patógenos, constituyendo un serio riesgo para la salud de los comensales. / Tesis

Listeria monocytogenes (L. monocytogenes) are Gram-positive, facultatively anaerobic and intracellular bacilli, occupying a wide range of ecological niches and are responsible for a number of serious infections in man. Primarily transmitted to humans through contaminated food stocks, L. monocytogenes invade mammalian cells in a phagosome, escaping and growing in the cell cytoplasm. Currently, there is a great deal of information about pathogenesis of L. monocytogenes, however, much less is known about the physiology of the bacteria. In particular, very little is known about the physiology during intracellular growth and even less about host cell physiology and changes in response to infection. The focus of this research was to address these issues using a multidisciplinary approach, utilising multiple biological techniques. The catabolic metabolism of L.monocytogenes was elucidated using mutagenesis and protein purification studies. The results are not completely conclusive; however, it was shown that unlike in Escherichia coli, L.moncytogenes may not be dependent on fermentation enzymes Ldh and Pflb during anaerobic growth. Instead anaerobic respiration is hypothesised, utilising a putative fumarate reductase with fumarate as a terminal electron acceptor. The putative fumarate reductase gene was purified and confirmed to have enzymatic activity.External and internal metabolism of HeLa cells, and the effect of L.monocytogenes infection was elucidated by mass spectrometry. The external metabolomic studies proved inconclusive. The internal metabolomic studies show that a number of key amino acids are being sequestered by L.monocytogenes during the course of an infection. Also, the studies show that a large number of carbon compounds are being sequestered by L.monocytogenes, pointing to a complex carbon metabolism for L.monocytogenes during intracellular growth. A targeted analysis of the nitrogen metabolism of L.monocytogenes has shown that L.monocytogenes may utilise a number of nitrogen compounds with glutamine and glutamate being particularly important. The ability to synthesise glutamine de novo is shown to be essential for normal intracellular growth.

The first aim of this study was to determine the effect of irrigation intervals on the survival of L. monocytogenes on spray irrigated broccoli under field trial conditions, and subsequent survival of the pathogen on broccoli during postharvest processing procedures. The nonpathogenic L. innocua was used as surrogate organism to L. monocytogenes. Broccoli in the field was treated with irrigation water inoculated with L. innocua, during intervals over a period of five weeks and the growth and survival of the organism was monitored weekly. L. innocua numbers remained similar over intervals that received consecutive inoculations and L. innocua numbers decreased by at least 2.3 log cfu/g after inoculation ceased, which showed an inoculation effect and that time had an influence on organism survival. Cessation of irrigation before harvest was found to effectively reduce pathogen contamination levels on the crop, whilst repeated irrigation with contaminated water contributes to maintenance of L. innocua as well as elevated total microbial counts on the broccoli. A lack of correlation between the L. innocua counts and the recorded environmental temperatures in the field, including temperature and relative humidity, suggested that survival is not solely dependent on and influenced by, nor can it be predicted by these parameters. It was found that the presence of high levels of contamination (with, in this case L. innocua) in irrigation water used for vegetable crops, can be associated with an increased microbial population on the crop surface. Secondly, the effect of processing on organism survival post-harvest was assessed. Washing with water caused a 1 log reduction of L. innocua, whilst washing with 200 ppm chlorinated water facilitated a further 1 log reduction. Cooking reduced L. innocua numbers on broccoli by an average of 1.1 log units and aerobic plate counts by between 1 and 2 log units. A combined treatment of washing with chlorine, storage in MAP (5% CO2, 5% O2) for two days at 4°C and final microwave heating resulted in the lowest pathogen numbers, causing a 5.13 log cfu/g log reduction. Therefore, even though chlorine isprocessing, it does not suffice alone to eliminate pathogens (with L. innocua being representative of L. monocytogenes) from vegetables, just as MAP storage is only effective as part of a hurdle procedure. Cooking is essential in destroying L. innocua present on broccoli and to ensure vegetables that are safe for consumption in terms of pathogenic exposure. With this knowledge on the behaviour of L. monocytogenes on broccoli, the risk associated with the application of contaminated irrigation water to fresh produce can be better understood and the hazard managed. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Food Science / unrestricted

Dispersion is a natural part of a biofilm life cycle in many bacterial species. Dispersion occurs when bacteria revert from a stationary, sessile state to a free-swimming, planktonic state and are freed from a biofilm. Bacterial biofilms consist of proteins, polysaccharides, and extracellular DNA that together make up the extracellular polymeric substances. Surrounded by this mucus-like substance, sessile cells can be extremely difficult to eradicate as compared to the planktonic form of Listeria monocytogenes. Biofilms are robust due to increased surface adherence, inhibition of diffusion of harmful compounds, and increased genetic diversity that exists within a biofilm. As a result, traditional biofilm removal methods are often inadequate; and a novel method for the eradication of Listeria monocytogenes biofilms is needed. Here it is shown that two known biofilm dispersal agents, nitric oxide and cis-2-Decenoic acid, do not induce dispersion in Listeria monocytogenes strain LM23. Nitric oxide and cis-2-Decenoic acid do not influence planktonic cell numbers or biofilm biomass. Ten carbohydrates were screened for their influence on biofilm biomass for use in investigation into natural biofilm dispersion in Listeria monocytogenes strain LM23. Carbohydrate source can significantly increase or decrease biofilm biomass as compared to glucose. Natural biofilm dispersion in Listeria monocytogenes remains inconclusive, yet warrants further investigation. Changes in planktonic cells numbers, sessile cell numbers, and biofilm biomass were tracked under static growth conditions, and suggested a possible dispersion event. However, treatment of biofilms with spent media and observation using scanning electron microscopy did not clarify the results obtained. This research deems the nitric oxide donors, molsidomine (N- (ethoxycarbonyl)-3-(4-morpholinyl)-sydnone imine) and MAHMA NONOate (6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine), as well as cis-2-Decenoic acid as ineffective in inducing biofilm dispersion. It also brings about new research questions into natural biofilm dispersion in Listeria monocytogenes.

The objective of this study was to evaluate the efficacy of buffered vinegar in a marinade solution on inhibiting Listeria monocytogenes growth on cooked broiler breast meat. Broiler breasts were vacuum-tumbled for 30 min in a marinade consisting of dry (0%, 0.4% DV, 0.6%DV, and 0.8%) or liquid vinegar (1.5%), sodium chloride, sodium tripolyphospate, and water. The chicken breasts were then cooked to an internal temperature of 75°C. The breast meat was inoculated with L. monocytogenes, placed into modified atmosphere packaging, and stored at 2°C plus/minus 2 for 0-60 days. L. monocytogenes growth was stable on treatments for up to 30 days. However, from 35 to 60 days, the buffered vinegar treatments had fewer L. monocytogenes counts (P<0.05) than the control treatment. In addition, the 0.8 % DV and 1.5 % LV treatments had fewer than 2.0 log counts of L. monocytogenes after 60 days of storage.

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. The infectious process depends upon survival in high bile salt conditions encountered throughout the gastrointestinal tract, including the gallbladder. However, it is not clear how bile salt resistance mechanisms are induced, especially under physiologically relevant conditions. This study sought to determine how L. monocytogenes responds to bile salts under anaerobic conditions. The study found resistance to be strain specific and not dependent upon virulence. Changes in the expressed proteome were analyzed using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. A general response among virulent and avirulent strains found significant alterations in intensity of cell wall associated proteins, DNA repair proteins, protein folding chaperones and oxidative response proteins. Strain viability was correlated with an initial osmotic stress response followed by strain specific proteins associated with biofilm formation in EGDe and a transmembrane efflux pump in F2365.