The objective of this work was to characterize and study the factors affecting the production and the application of sakacin G produced by Lactobacillus sakei CWBI-B1365. After a first screening, four bacteriocin producing lactic acid bacteria were selected. The structural genes for some of the bacteriocins were identified in the genome of these strains by amplification using specific PCR primers. Lb. sakei CWBI-B1365 has then been specifically studied. Sakacin G was identified as the only bacteriocin produced. Sequencing of a part of the sakacin G gene clusters involved in the bacteriocin production allowed to identify an original genetic organization. The pH, the temperature, the carbon source used and its concentration, as well as the meat extract quantity, strongly influence sakacin G production. Sakacin G was produced and showed antilisterial activity in beef. To obtain antilisterial activity in poultry meat, it was necessary to combine Lb. sakei CWBI-B1365 and Lb. curvatus CWBI-B28, a sakacin P producer.

Nisin is a naturally occurring food antimicrobial that inhibits many Grampositive pathogens, including Listeria monocytogenes, a bacterial pathogen responsible for ~500 deaths in the U.S. annually. Factors known to counteract the nisin activity in a food matrix include: antimicrobial interaction with food components, insolubility, protease inactivation, and target cell-driven envelope modifications. Encapsulating nisin in liposomes can help protect nisin functionality by regulating its introduction to the external environment. The objectives of this study were to determine the encapsulation efficiency (%EE) of nisin within liposomes as a function of encapsulation method and the capacity of liposomal nisin to inhibit L. monocytogenes in fluid milk. Phosphatidylcholine (PC) and phosphotidyl-DL-glycerol (PG) were used to prepare three lipid molar formulations: PC, PC/PG 7:3, and PC/PG 6:4 (mol.%). Liposomes were formulated to entrap the self-quenching fluorophore calcein and nisin. Unencapsulated analyte was removed via size-exclusion chromatography, and percent EE was determined. To determine antilisterial activity of liposomes, fluid milk samples containing L. monocytogenes (4 log10 CFU/mL) in combination with liposomal or unencapsulated nisin at 50 IU/mL were mixed and aerobically stored at 5 degrees C and 20 degrees C. Surviving L. monocytogenes were enumerated via plating on a non-selective microbiological medium after 0, 1, 3, 6, 12, 24, 48, and 72 hours of incubation. Encapsulation of nisin via extrusion resulted in a mean EE% of 84.20%, 77.33% and 80.78% for PC, PC/PG 7:3, and PC/PG 6:4 liposomes, respectively. Freeze-thaw cycling formed liposomes without detectable fluorophore entrapment. L. monocytogenes populations grew to 5 log10 CFU/mL after 72 hours at 5 degrees C and 8 log10 CFU/mL at 20 degrees C after 48 hours. Unencapsulated nisin exerted statistically greater inhibition of Listeria in skim milk compared to liposomal nisin, regardless of incubation temperature. No statistically significant differences in Listeria populations exposed to free or encapsulated nisin in whole milk were observed at either incubation temperature. Results indicate storage temperature and presence of milk fat exert greater influence then nisin delivery (free vs. encapsulated) over Listeria inhibition. Further research is needed to confirm these findings and develop more effective means of liposome entrapment of nisin for the inhibition of foodborne bacterial pathogens.

In this study, a new bacteriocin isolated from a Leuconostoc mesenteroides subsp. cremoris strain was purified to homogeneity by pH mediated cell adsorption-desorption, solid phase extraction with Amberlite XAD-16, cation-exchange chromatography and hydrophobic interaction chromatography. The purification resulted in an electrophoretically pure protein that was smaller than 6 kDa as judged by SDS-PAGE. The bacteriocin was found to be very hydrophobic and cationic and could be adsorbed by synthetic calcium silicate due to its cationic and especially hydrophobic nature. It was observed that this bacteriocin was sensitive to &amp / #945 / -amylase in addition to proteinase K, trypsine, pepsine and chymotrypsine and had a bactericidal mode of action with a concomitant cell lysis. The results indicated that bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was more stable to combined effect of pH and heat than nisin, lacticin 481, lacticin 3147 and lactococcin G and was bactericidal between pH 2.0-12. It was found that the bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was stable to organic solvents and could be extracted with chloroform containing solvents efficiently for purification. The bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was found to have a strong inhibitory activity against Listeria innoqua, Listeria monocytogenes, Bacillus cereus, Enterococcus faecalis, Lactobacillus delbrueckii, Lactobacillus cremoris, other Leuconostoc meenteroides strains and gram-negative bacterium Pseudomonas fluorescens. Some of the insensitive bacteria were observed to be sensitive when high concentration of the bacteriocin was used.

No description available.

No description available.

Several obstacles to widespread use of bacteriocins in food have been identified, including lack of specific, rapid quantitation methods, and little data on their efficacy in food systems. The first objective of this study was to develop a specific, rapid quantitation method for bacteriocins that did not rely on bioassays and their associated limitations. Phage display was chosen to reduce reliance on continued use of animals and produce antibodies to the bacteriocins leucocin A, piscicolin 126, and brochocin-A. Although the antibody libraries generated by phage display were not successful for antibody production, a strong immune response to leucocin A and piscicolin 126 was observed in mice. The second objective of the study was to determine the efficacy of brochocin-C against Clostridium botulinum in a model system and in a vacuum-packaged, chopped and formed pork product stored at refrigeration temperature. Group I Cl. botulinum was not controlled by brochocin-C, and was found to inactivate brochocin-C and several class IIa bacteriocins by proteolysis. Cell counts revealed that Group II Cl. botulinum was controlled by brochocin-C in a model meat system, but was not controlled in the chopped and formed pork product. Powdered smoke and NaCl in the pork product had a synergistic interaction against Group II Cl. botulinum, as shown by minimum inhibitory concentration testing. The choice of media for isolation of Cl. botulinum from the chopped and formed pork product was important, as the presence of background microflora isolated from the meat was found to impact growth of Group II Cl. botulinum on plating media. In the presence of the background microflora, which were identified by 16S rDNA sequencing as carnobacteria and staphylococci, inclusion of phosphate in the plating medium was found to allow growth of Cl. botulinum. Other nutrients such as magnesium, sulphur, or increased protein sources added to the medium had no effect on growth of Cl. botulinum. Two of the background microflora strains, Carnobacterium maltaromaticum MH3 and Staphylococcus pasteuri EIV-21, inhibited Cl. botulinum, while one strain, C. maltaromaticum MH2, stimulated growth of Cl. botulinum. / Food Science and Technology

Durch die wachsende Anzahl von multiresistenten Bakterien, die auch durch den Mißbrauch von Antibiotika als Masthilfsmittel in der Tierzucht entstanden sind, erlangen alternative Methoden zur Bekämpfung bakterieller Infektionen ihre Bedeutung zurück. Diese Arbeit befaßt sich mit zwei Substanzen, um Infektionen mit Yersinia (Y.) enterocolitica einzudämmen. Es wurde in dieser Arbeit ein Bacteriocin aus Y. enterocolitica isoliert und charakterisiert. Das Reinigungschema folgte den Strategien der Phagenaufreinigung, angeschlossen wurde zur Überprüfung der Reinheit ein Gelfiltrationsschritt. Die Eigenschaften des gereinigten Enterocoliticins wurden in vitro und in vivo getestet. Im Zellkulturversuch zeigte sich das Enterocoliticin in der Abtötung von an eukaryonte Zellen adhärierten Bakterien als sehr wirksam, in eukaryonte Zellen eingewanderte Bakterien wurden hingegen nicht abgetötet. Aufgrund dieser vielversprechenden Ergebnisse wurde der Therapieansatz im Mausmodell angewendet. Das Mausmodell ist für Y. enterocolitica ein bereits erprobtes Modell. Die Tiere wurden oral infiziert, um den natürlichen Infektionsweg nachzustellen, das Enterocoliticin wurde ebenfalls oral verabreicht. Die Infektion wurde durch die Enterocoliticingabe nur unwesentlich beeinflußt, auch gelang der Nachweis des Enterocoliticins weder im Gastrointestinaltrakt noch in den Faeces. Die Therapie der infizierten Mäuse gelang auf diese Weise nicht. Weiterhin wurde ein Yersinia-Phage aus Schweinegülle isoliert, gereinigt und charakterisiert. Es handelt sich um einen T4-ähnlichen, virulenten Phagen mit einer Genomgröße von ca. 50 kbp und einem weiten Wirtsspektrum in Yersinia, das sogar speziesübergreifend ist. Da der Phage bei 37°C die Wirtszelle lysiert und durch seine hohe Wirksamkeit in vitro erschien der Phage von seinen Eigenschaften her zur Phagentherapie als geeignet. Es wurden analog zum Enterocoliticin-Tierexperiment Mäuse mit Y. enterocolitica oral infiziert, diesen Tieren wurde der Phage auf unterschiedlichen Wegen und zu unterschiedlichen Zeitpunkten appliziert. Die Tiere zeigten bei der parenteralen Gabe keinerlei Unverträglichkeitserscheinungen, bei der oralen Gabe wurde der Magensaft zuvor abgepuffert. Der Therapieerfolg im Vergleich zur Kontrollgruppe war wenig vielversprechend, es zeigten sich sehr ähnliche Infektionsverläufe in Kontroll-und Therapiegruppe. Diese beiden untersuchten Alternativwege zur Behandlung von Yersiniosen erwiesen sich bei den angewendeten Methoden bislang als nicht erfolgreich, dennoch muß auf diesem Gebiet weitergeforscht werden, wie erfolgreiche Therapieansätze aus anderen Tiermodellen zeigen. Es wirken nur wenige Phagen im Organismus als Therapeutikum, dennoch müssen diese Untersuchungen unternommen werden, um im Organismus wirksame, antibakterielle Substanzen zu finden und um einen Alternativweg zur Antibiotikumtherapie zu entwickeln. / The increasing number of multi-resistant bacteria, which resulted also from the abuse of antibiotics as mast additives in animal breeding, alternative methods regain importance for the combat at bacterial infections back. In this work two substances were investigated to restrict infections with Yersinia (Y.) enterocolitica. In this study a bacteriocin from Y. enterocolitica, designated enterocoliticin, was isolated and characterized. The purification strategy followed protocols of phage isolation. The purified preparations were examined by final gel filtration step. The properties of the purifed enterocoliticin were tested in vitro and in vivo. In a cell culture assay enterocoliticin was able to kill bacteria adherent to eukaryontic cells very effectively, however, bacteria invaded into eukaryotic cells were not affected. Due to these results enterocoliticin was applied in a mouse-infection-model in a therapeutic attempt. The mouse infection model is a well established system for infections with Y. enterocolitica. The animals were orally infected with Yersinia, and the enterocoliticin was orally applied, too. The infection was only insignificantly influenced by enterocoliticin. In addition of enterocoliticin was not detected succeeded in the gastro-intestinal-tract or in the faeces. The therapy of the infected mice did not succeed in this way. Furthermore, a Yersinia phage from pig manure was isolated and characterized. It is a T4 phage like virulent phage, containing a genom of approx. 50 kbp and posessing a wide host-range in Yersinia. Because of lytic properties of the phage at 37°C and his high effectiveness in vitro the phage appeared to be for phage therapy experiments. Similarly to the enterocoliticin experiment mice were infected with Y. enterocolitica orally, these animals were treated with the phage on different application routes and different time points. The animals did not show any incompatibilities upon parenteral gift. Before oral administration of the phage the gastric juice was buffered. Therapy outcome in comparison to the control group was little promising, it revealed a very similar infection process in control group and therapy group. These two investigated alternative ways for the control of Yersinia infections did not prove successful with the applied methods, however, further research must be carried out as successful therapeutical experiments from other animal models showed. It has to be considered that not all phages are appropriate as therapeutical agent however, more studies must be conducted to find more appropriate substances, which may work as effective antibacterial substances to develop alternative ways to antibiotic therapy.

Made available in DSpace on 2014-06-11T19:28:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-06-03Bitstream added on 2014-06-13T18:35:00Z : No. of bitstreams: 1 bonini_m_me_botfca.pdf: 503952 bytes, checksum: 1553a755d247df6ec4852dd6104b7a70 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Empresa Privada / O presente trabalho teve por objetivo avaliar a produção e a sensibilidade de 48 isolados de Xanthomonas axonopodis pv. citri e de 14 isolados de Xanthomonas spp. à bacteriocinas. Estudos foram realizados para verificar o efeito da temperatura, tempo de incubação e do tipo do meio de cultura sobre a produção de bacteriocina por isolados de X. axonopodis pv. citri. Todos isolados de X. axonopodis pv. citri não foram sensíveis às bacteriocinas produzidas por eles, não sendo essas afetadas pelo meio BDA, nutriente ágar + NaCl e YPDA, nos diferentes tempos e temperaturas de incubação. Porém, isolados de X. axonopodis pv. passiflorae foram sensíveis às bacteriocinas produzidas por 25 isolados de avaliados e o isolado de X. campestri pv. campestris e o de X. axonopodis pv. manihotis apresentaram sensibilidade variável. Dos 25 isolados de X. axonopodis pv. citri apenas cinco não foram inibidos pelas bacteriocinas produzidas por dois isolados de X. axonopodis pv. passiflorae. As bacteriocinas produzidas pelos isolados de X. axonopodis pv. citri (FDC-806) e de X. axonopodis pv. passiflorae (Mar 2850-A) foram termolábeis e resistentes à lisozima e sensíveis a DNAse. A bacteriocina produzida pelo isolado de X. axonopodis pv. passiflorae foi resistente à ação de proteinase K, tripsina e RNAse enquanto que a produzida pelo isolado de X. axonopodis pv. citri foi sensível a essas enzimas. / The objective of this work was to evaluate the production and sensitivity of 48 Xanthomonas axonopodis pv. citri strains and 14 Xanthomonas spp. strains to bacteriocins. A number of studies were carried out to verify the effect of temperature, incubation time, and type of culture medium on bacteriocine yield by X. axonopodis pv. citri strains. None of the X. axonopodis pv. citri strains were sensitive to the bacteriocins produced by themselves, and were not affected by the PDA, nutrient agar + NaCl, and YPDA media, at the different incubation times and temperatures studied. However, X. axonopodis pv. passiflorae and strains were sensitive to the bacteriocins produced by the 25 Xac strains evaluated, while a X. campestri pv. campestris and X. axonopodis pv. manihotis strains showed variable sensitivity. Of the 25 X. axonopodis pv. citri strains, only five were not inhibited by the bacteriocins produced by the two X. axonopodis pv. passiflorae strains. The bacteriocins produced by X. axonopodis pv. citri (FDC-806) and X. axonopodis pv. passiflorae (Mar 2850-A) were thermolabile and resistant to lysozyme and sensitive to DNAse. The bacteriocin produced by the X. axonopodis pv. passiflorae was resistant to the action of proteinase K, trypsin and RNAse, while the bacteriocin produced by the X. axonopodis pv. citri isolate was sensitive to those enzymes.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-04Bitstream added on 2014-06-13T19:43:43Z : No. of bitstreams: 1 delfino_tpc_dr_jabo.pdf: 382159 bytes, checksum: 8e7833c07c682a4bbf8eee1668ce058f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A avicultura comercial tem como objetivo obter alta produtividade a baixo custo e oferecer ao consumidor produto de qualidade. Uma bactéria patogênica que tem preocupado o setor avícola nos últimos tempos é a Salmonella. Neste sentido, o presente trabalho objetivou caracterizar seis isolados de Lactobacillus acidophilus em combinação com antimicrobianos na inibição de Salmonella Enteritidis e Salmonella Gallinarum. O experimento foi desenvolvido nas dependências da FCAV/UNESP – Campus de Jaboticabal. Para tanto foram conduzidos quatro estudos, sendo que dois foram dedicados em selecionar uma bactéria probiótica produtora de bacteriocina e avaliar a interação de substâncias antimicrobianas como EDTA, àcido acético e Nisina “in vitro” sobre a capacidade inibitória de Salmonella Enteritidis e Salmonella Gallinarum em diferentes tempos e o terceiro e quarto experimento estudam o potencial de aplicação do probiótico e do antimicrobiano em aves. O isolado de Lactobacillus acidophilus C1, demonstrou ser produtor de bacteriocina e inibir a multiplicação de Salmonella Enteritidis e Salmonella Gallinarum. Dentre os antimicrobianos testados o que apresentou efeito na eliminação de Salmonella Enteritidis foi a combinação de Ácido Acético + nisina e Ácido Acético + Lactobacillus acidophilus C1. O uso do probiótico no estudo em aves reduziu a excreção de Salmonella sp, mas não sua eliminação no trato intestinal das aves, já que é possível constatar sua presença no tratamento controle. A combinação de nisina e ácido acético não levou a redução da multiplicação de Salmonella sp. nas aves. O estudo permitiu verificar que existe um bom potencial de aplicação do probiótico citado, assim como o uso de alguns antimicrobianos na segurança alimentar. / The commercial poultry aims at obtaining high productivity at low cost and offering quality products to the consumer. A pathogenic bacterium which has worried the poultry sector in recent times is Salmonella. Therefore, the present study aimed at characterizing six isolates of Lactobacillus acidophilus in combination with antimicrobials in the inhibition of Salmonella enteritidis and Salmonella gallinarum. The experiment was developed in the dependencies of FCAV/UNESP – Campus of Jaboticabal. In order to do this, four studies were conducted, being two of them dedicated to select a bacteriocin-procucer probiotic bacterium and evaluate the interaction of antimicrobial substances such as EDTA, acetic acid and “in vitro”Nisin on the inhibitory capacity of Salmonella enteritidis and Salmonella gallinarum in different periods and the third and fourth experiment study the potential of application of the probiotic and antimicrobial in poultry. The isolate of Lactobacillus acidophilus C1, showed to be bacteriocin producer and inhibit the multiplication of Salmonella enteritidis and Salmonella gallinarum. Among the antimicrobial tested, the one which presented effect in the elimination of Salmonella enteritidis, was the combination of Acetic Acid + nisin and Acetic Acid + Lactobacillus acidophilus C1. The use of the probiotic in the study of birds reduced the counting of Salmonella sp, but not its elimination in the intestinal tract of the birds, as we can see its presence in the control treatment. The combination of nisin and acetic acid did not show the reduction of the multiplication of Salmonella sp. in birds. The study has shown that there is a good potential of application of the mentioned probiotic, as well as the use of some antimicrobial in food safety.

The aim of this study was to evaluate the action of edible coating the corn starch-based embedded with the bacteriocin nisin in the conservation of fresh cut fruit salad. The salads were composed of mango, papaya and pineapple. After processing the minimum salads were submitted to the following treatments: uncoated fruit salad (control); Corn starch coating without Nisin; and corn starch coating with Nisin. All samples were packed in polyethylene terephthalate (PET) packages for 12 days at 5 ± 1 ° C and 80% RH. At 0, 3, 6, 9,12 physicochemical analysis of soluble solids content were held, pH, titratable acidity, ascorbic acid content, phenolic compounds, carotenoids, color, loss of weight and Polyphenol oxidase activity of enzymes. The microbiological analysis included aerobic mesophilic count, molds and yeasts, and survival analysis of Listeria monocytogenes inoculated in minimally processed fruit salads plus the added edible coating or not nisin. The fruit salads treated with edible coating nisin and showed significantly less mass loss, and increased vitamin C content when compared with control treatment samples. Coated fruits showed also significantly lower levels of soluble solids and lower activity of polyphenol oxidase. There was no significant variation in relation to the analysis of pH, acidity, carotenoids and color among treatments. It was observed increase in mesophilic aerobic count in all samples but, on samples coated with starch and nisin, it was found that the microbial population was statistically lower than the other treatments. Samples of fruit salad coated with corn starch, had significantly lower counts of yeasts and molds until the 6th day of storage. The presence of nisin resulted in significant reduction in the number of viable cells of the bacterium L. monocytogenes inoculated in output of fruit, unchecked behavior in samples of other treatments during the 12 days of storage at 5 ° C. The results of this study demonstrate that the use of corn starch coating and nisin combined with good processing practices and minimum control of storage conditions (temperature and humidity) was effective in preserving minimally processed fruit salads. Once a positive influence on the physical and chemical parameters evaluated, and obtained the best results in relation to maintaining the microbiological quality of salads for up to 12 days. / O objetivo do presente trabalho foi avaliar a ação do revestimento comestível a base de amido de milho incorporado com a bacteriocina nisina, na conservação de salada de frutas minimamente processadas. As saladas foram compostas por manga, mamão e abacaxi. Após processamento o mínimo as saladas foram submetidas aos seguintes tratamentos: salada de frutas sem revestimento (controle); revestimento de amido de milho sem Nisina; e revestimento de amido de milho com nisina. Todos os produtos foram acondicionados em embalagens de tereftalato de polietileno (PET) por 12 dias a 5±1°C e 80% UR. Nos tempos 0, 3, 6, 9,12 foram realizadas análises físico-químicas de teor de sólidos solúveis, pH, acidez titulável, teor de ácido ascórbico, compostos fenólicos, carotenoides, cor, perda de massa fresca e atividade da enzima polifenol oxidase. As análises microbiológicas incluíram contagem de aeróbios mesófilos, bolores e leveduras, e a análise da sobrevivência de Listeria monocytogenes, previamente inoculadas nas saladas de frutas minimamente processadas acrescida do revestimento comestível adicionado ou não de nisina. As saladas de frutas tratadas com revestimento comestível e nisina apresentaram perda de massa significativamente menor, e maior teor de vitamina C quando comparadas com as amostras do tratamento controle. Frutos revestidos apresentaram também, teores significativamente menores de sólidos solúveis e menor atividade da enzima polifenol oxidase. Não foi observada variação significativa em relação às análises de de pH, acidez, carotenoides e cor entre os tratamentos estudados. Foi verificado aumento na contagem de microrganismos aeróbios mesófilos em todas as amostras porém, em amostras revestidas com amido e nisina, verificou-se que população microbiana foi estatisticamente menor que nos demais tratamentos. Amostras de salada de fruta revestidas com amido de milho, apresentaram contagens significativamente menores de bolores e leveduras até o 6º dia de armazenamento. A presença da nisina resultou em redução significativa no número de células viáveis da bactéria L. monocytogenes inoculada em salda de frutas, comportamento não verificado nas amostras dos demais tratamentos durante os 12 dias de estocagem a 5±1ºC. Os resultados obtidos neste estudo demonstram que a utilização de revestimento de amido de milho e nisina, associado a boas práticas de processamento mínimo e controle das condições de armazenamento (temperatura e umidade), foi eficiente na conservação de saladas de frutas minimamente processadas. Uma vez que interferiu positivamente nos parâmetros físico-químicos avaliados, e obteve os melhores resultados em relação a manutenção da qualidade microbiológica das saladas por até 12 dias.