ABSTRACT CHARACTERIZATION AND IDENTIFICATION OF BACTERIOCINS FROM TWO LACTOCOCCUS LACTIS SUBSP. LACTIS STRAINS Ak&ccedil / elik, Oya M.S., Department of Biotechnology In this study, bacteriocins from two L. lactis subsp. lactis isolates of Turkey origin designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after &amp / #61537 / -amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of partially purified bacteriocins were determined by SDS-polyacrylamide gel electrophoresis. PCR amplification was carried out with different primers for the detection of structural genes of lactococcal bacteriocins. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Association of the bacteriocin production with plasmid DNA was examined by using acriflavine as a plasmid curing agent. Plasmid profiles of the wild type and its non-bacteriocin producing mutants were determined by using the alkali lysis method followed by agarose gel electrophoresis. The genetic nature of industrially important characteristics of Lactococcus lactis strains were investigated through gene transfer studies via conjugation. According to the results of plasmid curing and conjugal transfer trials, it was concluded that in Lactococcus lactis subsp. lactis OC1 strain a 39,7 kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity. In Lactococcus lactis subsp. lactis OC2 strain, on the other hand, a 16 kb plasmid appeared to be responsible for lacticin 481 production and lactose fermentation.

Dissertation (PhD)--University of Stellenbosch, 2009. / Bibliography. Also available via the Internet.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Listeria monocytogenes é um microrganismo patogênico psicrotrófico e consequentemente de grande importância para alimentos conservados em refrigeração. Vários estudos têm demonstrado que é possível utilizar antimicrobianos naturais, como bacteriocinas e compostos fenólicos, para retardar a multiplicação desse patógeno em produtos cárneos refrigerados. Entretanto, sabe-se que a atividade antimicrobiana desses compostos pode ser influenciada pelos componentes da matriz alimentar. Esse trabalho de mestrado pretendeu avaliar a influência dos ingredientes de lingüiça frescal de suíno na ação inibitória de óleo essencial de orégano (OEO) e nisina sobre Listeria monocytogenes. Foram realizados testes com diferentes modelos alimentares preparados com carne suína moída adicionada de cada um dos ingredientes utilizados na fabricação de lingüiça frescal (sais de cura, açúcar, antioxidante, emulsificante, condimentos e NaCl) separadamente, comparando-se os resultados com os obtidos no modelo contendo todos os ingredientes simultaneamente. Pretendeu-se também avaliar a interferência do tempo de contato da nisina e óleo essencial de orégano com os diferentes modelos alimentares na ação antimicrobiana sobre L. monocytogenes. Os resultados indicaram que os antimicrobianos foram capazes de inibir a multiplicação de L. monocytogenes em todos os modelos analisados armazenados à 5ºC por até 72h. Comparando-se todos os modelos compostos por carne adicionada de apenas um dos ingredientes, a maior atividade antilisterial foi observada no modelo composto de carne com antioxidante. Entretanto, essa atividade inibitória foi menor do que a obtida no modelo contendo todos os ingredientes em conjunto. Quanto à influência do tempo prévio de contato entre os antimicrobianos e os modelos estudados na ação antilisterial, observou-se que na formulação com todos os ingredientes a ação foi mais intensa quando o contato foi de 2h. Entretanto, neste modelo o efeito mais duradouro foi obtido com 12h de contato prévio. Nos demais modelos, o maior efeito foi obtido no tempo zero. Os resultados permitem concluir que cada um dos ingredientes da formulação da lingüiça contribui com uma parcela na ação antilisterial do OEO e nisina nesse produto, e que essa ação é influenciada pelo tempo de contato prévio entre os modelos e os agentes antimicrobianos. / Listeria monocytogenes is psychrotrophic pathogen and, consequently, very important for cold-preserved foods. Many studies have demonstrated that natural antimicrobials such as bacteriocins and phenolic compounds can be used to inhibit the growth of this pathogen in refrigerated meat products. However, it is known that the antimicrobial activity of these antimicrobials may be influenced by components of the food matrix. The aim of the present study was to evaluate the influence of the ingredients of \"lingüiça\" in the inhibitory activity of O.E.O. and nisin against L. monocytogenes. The study was carried out with different food models prepared with minced raw pork meat added separately of each ingredient used for manufacturing \"lingüiça\" (curing salts, sugar, antioxidants, emulsifiers, condiments and NaCl), and results were compared to those obtained in a model containing all ingredients simultaneously. The study also aimed to evaluate the interference of the contact time between the food models and the antimicrobials on the effect against L. monocytogenes. Results indicated that the antimicrobials added to the models were able to inhibit the growth of the pathogen at 5ºC up to 72h. Comparing the models, the highest antilisterial activity was observed in the model containing pork meat and antioxidants. However, this inhibitory activity was lower than that observed in the model with all ingredients. What concerns the influence of contact time in antilisterial activity, we observed that in the model containing all ingredients the antilisterial activity was more intense after 2h of contact, but the more lasting effect was observed after 12h of contact. In the other models, the most intense effect was noted in time zero. The conclusions of the study are that each ingredient used in manufacture of \"lingüiça\" has its share in the antilisterial activity of nisin and OEO in this product and that this activity is influenced by the contact time between the food matrix and the antimicrobials.

Nisina, um antimicrobiano produzido por cepas de Lactococcus lactis subsp. Lactis, pode ser utilizada como preservante em alimentos e, possivelmente, como agente antimicrobiano. Para aumentar sua produção por Lactococcus lactis ATCC 11454 em meio MRS (pH = 6,5) realizamos ensaios em agitador rotativo (100 rpm/ 30 °C/ 36h) a partir de 3 grupos de ensaios, designados por desenho fatorial de dois níveis (2 (4-1) no grupo 01) e 2(3) nos grupos 2 e 3. As concentrações de sacarose (2,2 a 15 g/L), asparagina (7,5 a 75 g/L), fosfato de potássio (6 a 18 g/L) e Tween 80 (1,0 a 6,6 g/L) foram adicionadas ao meio MRS (pH = 6,5). A produção de nisina foi acompanhada a partir do método de difusão em ágar a partir de utilização de Lactobacillus sake ATCC 9924 como microorganismo sensível. Os melhores níveis de nisina atingidos foram de 1,6 x 104 AU/mL (grupo 2) a 1,8 x 104 AU/mL (grupo 1) e obtidos para os níveis máximos de sacarose e asparagina. Dependendo das proporções entre as concentrações dos outros nutrientes, quando a concentração final de sacarose variou entre 0,33 g/L a 3,64 g/L, o pH oscilou entre 5,10 to 6,01, o acido lático produzido variou de 1,19 g/L a 4,44 g/L e a concentração celular variou de 0,99 a 4,184 mg/L. produção de nisina mostrou-se independente das velocidades de crescimento, as quais variaram de 0,0070 h-1 a 0,0401 h-1. A analise de regressão mostrou se adequar a um modelo polinomial quadrático: Log AU/mL = 1,98905 - 0,213558 x2 + 1,80028 x3 - 1,40776 x4 + 0,23436 x1 x2 + 0,35936 x1 x3 +1,47217 x3 x4 (equação principal). Onde os índices representam as concentrações de sacarose; asparagina; fosfato e tween 80 respectivamente. A análise de variância foi realizada para os parâmetros fixados para demonstrar a adequação do modelo proposto. (x são os índices dos nutrientes adicionados). / Nisin, an antimicrobial substance tha is produced by Lactococcus lactis, can be utilized as a preservative and therapeutic agent. To improve its production by Lactococcus lactis ATCC 11454 in MRS broth (pH =6.5), in rotatory shaker (100 rpm/ 30 °C/ 36h), three groups of assays, set up by a fractional factorial design of two-levels (2 (4-1) in group 01), and 2(3) in groups 2 and 3 were carried out. The minimum and maximum concentrations of sucrose (5 to 12.5 g/L), asparagine (7.5 to 75 g/L), potassium phosphate (6 to 18 g/L) and Tween 80 (1.0 to 6.6 g/L) were added to the MRS broth (pH = 6.5). The nisin production was accompanied through the agar diffusion assay using Lactobacillus sake ATCC 9924 as a sensitive microorganism. The best nisin activities ranged from 1.67 x 104 AU/mL (group 2) to 1.84 x 104 AU/mL (group 1) and were obtained for maximum levels of sucrose and asparagine, dependending of the proportions of the other nutrients concentrations, when the final sucrose content varied from 0.33 g/L to 3.64 g/L, the pH value oscillated beTween 5.10 to 6.01, the produced lactic acid was ranged from 1.19 g/L to 4.44 g/L and the dry-cell content was about 0.99 to 4,184 mg (DCW)/L. Nisin production was shown to be independent of growth rates, which ranged from 0.0070 h-1 to 0.0401 h-1. The regression analysis attained a quadratic polynomial model: Log AU/mL = 1.98905 - 0.213558 x2 + 1.80028 x3 - 1.40776 x4 + 0.23436 x1 x2 + 0.35936 x1 x3 +1.47217 x3 x4 (main equation). The analysis of variance performed to the fitted parameters of the independent variables was performed for checking model fitting results adequacy. (x are the index for the nutrients added).

Ce travail porte sur le développement de systèmes d’encapsulation à base de polysaccharides et de molécules antimicrobiennes de nature protéique comme le lysozyme du blanc d’œuf et la nisine produite par la bactérie Lactococcus lactis. La coacervation complexe de l’alginate ou la pectine (deux polysaccharides anioniques) avec le lysozyme ou la nisine, chargés positivement sur une large gamme de pH, serait une solution pour protéger ces molécules et assurer une libération contrôlée lors de la conservation d’un aliment. Les polysaccharides utilisés ont été choisis en fonction de leur sensibilité aux enzymes produites par les bactéries cibles. L’ensemble des travaux menés à différentes échelles a mis en évidence (i) le rôle de certains facteurs physico-chimiques (ratio, pH, force ionique,…) sur les interactions mises en jeu entre le lysozyme ou la nisine et l’alginate ou la pectine, (ii) l’influence de ces facteurs sur les propriétés des complexes formés et (iii) le rôle des polysaccharides sélectionnés dans la stabilisation de la structure du lysozyme ou de la nisine lors du séchage par atomisation. Enfin, la structure et l’activité antimicrobienne des films obtenus par voie solvant (casting) et des microcapsules obtenues par atomisation sont étudiées en relation avec les propriétés des complexes formés. Ce travail a permis une meilleure compréhension des mécanismes impliqués dans la formation des complexes peptides/polysaccharides ou protéines/polysaccharides, leur résistance au séchage par atomisation ainsi que leur capacité à protéger et à libérer des molécules actives / This work deals with the development of encapsulation systems based on polysaccharides and antimicrobial molecules of protein nature such as hen egg-white lysozyme and nisin produced by Lactococcus lactis. Complex coacervation of alginate or pectin (two anionic polysaccharides) with lysozyme or nisin, positively charged over a wide range of pH, would be a solution to protect these molecules and ensure their controlled release during food preservation. The used polysaccharides were chosen according to their sensitivity to the enzymes produced by the targeted bacteria. The whole of this work performed at various levels showed (i) the impact of some physicochemical factors (ratio, pH, ionic strength,…) on the interactions between lysozyme or nisin and alginate or pectin, (ii) the influence of these factors on the properties of the formed complexes, and (iii) the role of the selected polysaccharides in stabilizing the structure of lysozyme or nisin during spray-drying. Finally, the structure and the antimicrobial activity of films produced by casting and microcapsules obtained by spray-drying were studied in relation to the properties of the formed complexes. This work makes it possible to contribute to a better understanding of the various mechanisms implied in the formation of peptide/polysaccharide or protein/polysaccharide complexes, their resistance to spray-drying and their ability to protect and release active molecules

Os produtos cárneos prontos para consumo estão entre os principais alimentos associados a surtos de listeriose, uma doença causada por Listeria monocytogenes. Este estudo objetivou avaliar a ação antimicrobiana do lactato de sódio e de duas formulações comerciais à base de nisina (produtos A e B) em mortadela, e do lactato de sódio e fumaça líquida em salsicha, experimentalmente contaminadas com um pool de seis cepas de L. monocytogenes, durante o armazenamento à vácuo à 8 ºC. Os aditivos com melhor atividade anti-listeria também foram avaliados quanto ao controle de bactérias láticas (BAL) e bolores e leveduras em produto cárneo, durante o armazenamento à 8 ºC. O produto A é uma mistura de nisina encapsulada, nisina livre e extrato de alecrim, enquanto o produto B é uma mistura de nisina livre e extrato de alecrim. L. monocytogenes foi capaz de se multiplicar em todas as formulações de mortadela ao longo do período de armazenamento. Entretanto, o produto A exerceu efeito antimicrobiano contra este patógeno. Após 10 dias de armazenamento, as contagens de L. monocytogenes nas mortadelas formuladas com o produto A foram 3 log UFC/g inferiores às contagens observadas nas demais formulações avaliadas (produto B, lactato de sódio e controle), além disso, o produto A foi capaz de reduzir a velocidade de multiplicação deste microrganismo. Em relação às salsichas tratadas com fumaça líquida, as contagens de L. monocytogenes, no oitavo dia de armazenamento foram 3 log UFC/g inferiores às contagens de L. monocytogenes nas salsichas formuladas com lactato de sódio e controle. Ao final do período de armazenamento (28 dias) observou-se diferença de 2 log UFC/g nas contagens de L. monocytogenes entre as salsichas formuladas com lactato de sódio + fumaça líquida e as demais formulações. A fumaça líquida foi capaz de inibir a multiplicação de bolores e leveduras por 21 dias de armazenamento, observando-se diferença de 2 log UFC/g entre as contagens observadas nas salsichas imersas em fumaça líquida e não imersas. As contagens de BAL permaneceram baixas ao longo do período de armazenamento (< 2 log UFC/g). Também não foi observada alteração nos valores de pH e atividade de água dos produtos cárneos, o que permite concluir que as diferenças observadas nas contagens de L. monocytogenes e bolores e leveduras são devidas à ação dos antimicrobianos adicionados aos produtos cárneos. A partir destes resultados é possível concluir que o produto A e a fumaça líquida são uma barreira adicional para o controle de L. monocytogenes em mortadela e salsichas, respectivamente, durante armazenamento a 8ºC. / Ready-to-eat meat products are among the foods most frequently associated to outbreaks of listeriosis, a disease caused by Listeria monocytogenes. The objective of the present study was to evaluate the antimicrobial activity of sodium lactate and two nisin-based commercial products (A and B) in bologna, and sodium lactate and liquid smoke in frankfurters, experimentally contaminated with a pool of six strains of L. monocytogenes, during storage at 8 ºC under vacuum. The antimicrobial with better activity against L. monocytogenes was also evaluated against yeasts and molds and lactic acid bacteria. Product A is a mixture of encapsulated nisin, free nisin and rosemary extract, and product B is a mixture of free nisin and rosemary extract. L. monocytogenes was able to grow in all bologna formulations during storage. However, product A was capable of reducing the growth rate of the microorganism. Besides, L. monocytogenes populations in bologna formulated with product A were 3 log CFU/g lower than the population observed for control samples, after 10 days of storage at 8 ºC. Regarding frankfurter samples, the use of liquid smoke alone or in combination with sodium lactate resulted in a 3 log CFU/g difference between L. monocytogenes counts found in control and treated samples, after 8 days of storage at 8 ºC. However, after 28 days only the samples treated with liquid smoke combined with sodium lactate presented lower counts than controls (2 log CFU/g). Liquid smoke was also able to inhibit the growth of yeasts and molds in frankfurters, for 21 days of storage at 8 ºC. Low counts of lactic acid bacteria (< 2 log CFU/g) were detected in frankfurters during the storage. There was no variation on pH and water activity values in the meat products during storage period; therefore, the differences found in counts of L. monocytogenes in the meat products can be attributed to the presence of the antimicrobials added in the formulations. Hence, product A and liquid smoke appear to be an additional barrier to control L. monocytogenes in bologna and frankfurters, respectively, during storage at 8 ºC.

A nisina é uma bacteriocina produzida por Lactococcus factis subsp. lactis e seu uso é permitido como conservador de alimentos em diversos países. Em 1998, o Ministério da Agricultura e Abastecimento do Brasil, em atitude pioneira, autorizou a aplicação de 200 ppm de Nisaplin® em solução de ácido fosfórico grau alimentício na superfície de produtos cárneos embutidos, tais como salsichas, como alternativa tecnológica para aumentar a segurança microbiológica e a vida-de-prateleira desses produtos. Este trabalho avaliou a eficiência deste tratamento na inibição da deterioração microbiana e controle da multiplicação de Listeria monocytogenes em salsichas tratadas com nisina conforme preconizado, e armazenadas sob refrigeração (8°C) e abuso de temperatura (12°C). Salsichas foram coletadas em uma planta processadora da cidade de São Paulo e submetidas a tratamento por imersão em solução de 200 ppm de Nisaplin® em ácido fosfórico grau alimentício a 0,1 % durante 1 minuto. Para o estudo da eficiência do tratamento sobre Listeria monocytogenes, as salsichas foram contaminadas experimentalmente após o tratamento através de imersão em suspensões contendo L. monocytogenes Scott A, resultando em dois níveis de contaminação superficial denominados baixo (103-104UFC/cm2) e alto (106-107 UFOcm2). Controles não tratados e não inoculados foram realizados concomitantemente. As amostras foram embaladas a vácuo e armazenadas a 8°C e 12°C durante 42 dias. A enumeração das populações de microrganismos aeróbios mesófilos, bactérias láticas, microrganismos psicrotróficos e L. monocytogenes foi realizada semanalmente até o final do período de estocagem. Os resultados indicam que não há diferença estatisticamente significativa entre as populações de bactérias aeróbias mesófilas, psicrotróficas e bactérias láticas em produtos tratados e não tratados com solução de Nisaplin® e armazenados a 8°C ou a 12°C. A redução das populações de L. monocytogenes em salsichas tratadas com Nisaplin® quando a contaminação superficial foi de 103 a 104UFC/cm2 e o produto foi armazenado a 8°e foi pequena (<1 log UFC/cm2), porém estatisticamente significativa. Esses resultados indicam serem necessárias medidas adicionais para garantir a segurança do produto. / Nisin is a bacteriocin produced by Lactococcus lactis subsp lactis and its use is permitted as a food preservative in severaI countries. In 1998, the Brazilian Ministry of Agriculture authorized the application of 200 ppm nisin (Nisaplin®) on the surface of meat products by immersion or spraying, as a technological alternative to increase safety and shelf-life of cooked frankfurters. This work intended to evaluate the effectiveness of this treatment on inhibition of microbial spoilage and control of growth of Listeria monocytogenes in frankfurters during storage under refrigeration (8°C) and temperature abuse (12°C). Frankfurters from a local meat industry were submerged for 1 min in nisin solution (200 ppm) in 0.1% phosphoric acid and experimentally contaminated with low (103-104 CFU/cm2) and high (106-107CFU/cm2) levels of L. monocytogenes. Non-treated and non-inoculated controls were also prepared. Counts of lactic acid bacteria, aerobes (mesophilic and psychrotrophic) and L. monocytogenes were carried out weekly up to 42 days, in four genuine repetitions. Results available indicate that the differences of counts of mesophilic and psychrotrophic aerobes, lactic acid bacteria in nisin-treated and non-treated controls are not significant. Reduction of L. monocytogenes populations in nisin-treated frankfurters experimentally contaminated with low (103-104CFU/cm2) leveis of L. monocytogenes stored under refrigeration (8°C) was low (<1 log UFC/cm<SUP2), but statistically significant. Results indicate that additional measures are necessary to ensure the safety of these products.

No description available.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2009. / Multidrug resistant strains of Staphylococcus aureus is presenting an increasing threat, especially immune compromised individuals. Many of these strains have developed resistance to newly approved drugs such as quinupristin-dalfopristin, linezolid and daptomycin. The search for alternative treatment, including bacteriocins (ribosomally synthesized antimicrobial peptides) of lactic acid bacteria is increasing . Lactococcus lactis subsp. lactis F10, isolated from freshwater catfish, produced a new nisin variant active against clinical strains of S. aureus. The operon encoding nisin F is located on a plasmid and the structural gene has been sequenced. The lantibiotic is closely related to nisin Z, except at position 30 where valine replaced isoleucine. The antimicrobial activity of nisin F against S. aureus was tested in the respiratory tract of Wistar rats. Non-immunosuppressed and immunosuppressed rats were intranasally infected with S. aureus K and then treated with either nisin F or sterile physiological saline. Nisin F protected immunosuppressed rats against S. aureus, as symptoms of an infection were only detected in the trachea and lungs of immunosuppressed rats treated with saline. The safety of intranasally administered nisin F was also evaluated and proved to have no adverse side effects. The potential of nisin F as an antimicrobial agent to treat subcutaneous skin infections was evaluated by infecting C57BL/6 mice with a bioluminescent strain of S. aureus (Xen 36). Immunosuppressed mice were treated with either nisin F or sterile physiological saline 24 h and 48 h after infection with subcutaneously injected S. aureus Xen 36. Histology and bioluminescence flux measurements revealed that nisin F was ineffective in the treatment of deep dermal staphylococcal infections. Non-infected and infected mice treated with nisin F had an influx of polymorphonuclear cells in the deep stroma of the skin tissue. This suggested that nisin F, when injected subcutaneously, may have modulated the immune system. Nisin F proved an effective antimicrobial agent against S. aureus-related infections in the respiratory tract, but not against subcutaneous infections. The outcome of nisin F treatment thus depends on the route of administration and site of infection.