Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Lactic acid bacteria (LAB) play a vital role in reducing wine acidity and also contributing to its aroma and flavour. However, they can also be responsible for many wine spoilage problems that compromise the quality and value of wine. While Oenococcus oeni contributes positive characteristics to the sensory properties of wine, certain species of the genera, Lactobacillus and Pediococcus can affect the wholesomeness of wine by producing undesirable compounds, such as biogenic amines and ethyl carbamate. Chemical preservatives like sulphur dioxide (SO2) are used to prevent the growth of spoilage micro-organisms during the winemaking process. SO2 also acts as a reducing agent and maintains the benefits of antioxidant properties of the polyphenols of wine. However, there is a worldwide demand to reduce SO2 levels due to the increasing health related risks and other factors. All these considerations have increased the interest in research to look for new preservation strategies, and LAB-produced bacteriocins seem to be a potential alternative that has been explored in the last decade. Various types of bacteriocins have been identified and characterized. However, there are few reports on bacteriocins produced by LAB of oenological origin or on bacteriocins present in the finished wine. The present study screened 155 LAB isolates from the IWBT culture collection for bacteriocin production. The isolates originated from South African red wines undergoing spontenous malolactic fermentation (MLF). Eight strains (5%) were identified to be producers, as evidenced by strong inhibition zones formed against sensitive organisms on agar plates. The producers demonstrated a broad spectrum of antimicrobial activity by inhibiting Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes and Pediococcus pentosaceus strains. Some of these bacterial genera are important in winemaking since they are potential wine spoilage bacteria. Hence these strains and/or the bacteriocins they produce could possibly find application in the food fermentation industry. The physiological results, biochemical tests and sugar fermentation profiles all gave the same results for the seven isolates, which were indicative of enterococci. The identification through 16S rRNA gene sequencing revealed that the seven tested isolates were all Enterococccus faecium. RAPD-PCR fingerprinting gave the same profile for the seven strains confirming that they were all identical on genetic level. Determining the molecular weight using SDS-PAGE showed the peptides to be below 4.6 kDa in size. PCR amplification of the enterocin P gene, sequencing and BLAST search results confirmed that all eight strains contained the enterocin P gene from Ent. faecium. The enterocin tested in this study was heat stable at 100°C (30 min), but lost 50% of its activity at 121°C (15 min). Factors such as bacteriocin production and heat resistance are among many that enable enterococci to be dominant in fermented products such as dairy foods or meat. Therefore, enterococci producing bacteriocins have potential applications in various foods and fermented products. The pH tests showed enterocin to be active over a broad pH range (2-10). Enterocin activity over a wide pH range make them potentially more suitable as natural preservatives of foods and fermented products where products are acidified or pH decreases due to natural LAB present. They also have potential applications in oenological process where pH levels are as low as 3 and 4. Proteolytic enzyme treatments with lysozyme, lipase, lyticase and catalase could not inhibit enterocin activity. This indicated that their antimicrobial activity was independent of lipid or carbohydrate moieties or hydrogen peroxide. α-Chymotrypsin and proteinase K inactivated enterocin, which indicated that the compound was proteinaceous in nature. Bacteriocin production tested in two of the isolates, #16.3 and 128.1, coincided with the exponential growth phase which occurred after 6 hours of incubation at 30°C, which was an indication of primary metabolite kinetics. The highest production of 400 AU/ml was observed after eight hours and was maintained for several hours (46 hours) in the stationery phase. The bactericidal effect of the cell free supernatants from #16.3 and 128.1 against the sensitive culture of Lactobacillus pentosus DSM 20314 was clearly demonstrated by complete inhibition of growth for most of the experimental period, while the control increased exponentially throughout the experiment. In conclusion, this study has confirmed the isolation and identification of Ent. faecium strains from wine, a genus that is rarely found in the wine environment. Although one can speculate on the origin of this bacterium in the wine e.g. human handling and contaminated water, these bacterial isolates produced enterocin P which have antimicrobial action against wine-related LAB genera and therefore have a potential role in wine spoilage control. / AFRIKAANSE OPSOMMING: Melksuurbakterieë (MSB) speel ‘n belangrike rol in die redusering van die suurgehalte van wyn en dra ook by tot die aroma en smaak daarvan. Hulle kan egter ook verantwoordelik wees vir vele wynbederfprobleme wat die gehalte en waarde van wyn negatief beïnvloed. Hoewel Oenococcus oeni positiewe karaktertrekke aan die sensoriese eienskappe van wyn verleen, kan sekere spesies van die genus, Lactobacillus en Pediococcus, die heilsaamheid van wyn beïnvloed deur ongewenste verbindings, soos biogeniese amienes en etielkarbamaat, te produseer. Chemiese preserveermiddels, soos swaweldioksied (SO₂), word gebruik om die groei van bederfmikro-organismes tydens die wynbereidingsproses te voorkom. SO₂ fungeer ook as ‘n reduseermiddel en onderhou die voordele van die antioksidant eienskappe van die poli-fenole van wyn. Daar is egter ‘n wêreldwye vraag na die redusering van SO₂-vlakke as gevolg van die toename in gesondheidsverwante risiko’s en ander faktore. Al hierdie oorwegings het belangstelling in die navorsing van nuwe preserveringstrategieë laat toeneem en MSB-geproduseerde bakteriosiene lyk na ‘n potensiële alternatief wat in die laaste dekade ondersoek word. Verskeie tipes bakteriosiene is geïdentifiseer en getipeer. Daar is egter nog weinig gerapporteer oor bakteriosiene wat deur MSB van wynkundige oorsprong geproduseer is of oor bakteriosiene wat in afgeronde wyn teenwoordig is. Die huidige studie het 155 MSB isolate van die Instituut vir Wynbiotegnologie se kultuurversameling vir bakteriosien-produsering gegradeer. Agt stamme (5%) is as produseerders geïdentifiseer, soos gestaaf is deur sterk inhibisiesones wat teen sensitiewe organismes op agarplate gevorm het. Die produseerders het ‘n breë spektrum van antimikrobiese aktiwiteit by inhiberende Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes en Pediococcus pentosaceus stamme gedemonstreer. Sommige van hierdie bakteriese genera is belangrik in wynbereiding, omdat dit potensiële wynbederfbakterieë is. Hierdie isolate en/of die bakteriosiene wat dit produseer, kan dus moontlik toepassing in die voedselfermentasiebedryf vind. Die fisiologiese resultate, biochemiese toetse en suikerfermentasieprofiele het almal dieselfde resultate vir die sewe isolate, wat indikatief van enterococci was, gelewer. Die identifisering deur 16S rRNA-basispaaropeenvolging het onthul dat die sewe getoetste isolate almal Enterococccus faecium was. RAPD-PKR-vingerafdrukke het dieselfde profiel vir die sewe rasse gelewer, wat bevestig dat die rasse almal identies op genetiese vlak was. Deur die molekulêre gewig vas te stel deur middel van SDSPAGE, het dit getoon dat die peptiede kleiner as 4.6 kDa in grootte is. PKR-amplifikasie van die enterosien-P geen, die bepaling van basispaaropeenvolging en BLAST-soekresultate het bevestig dat al agt rasse die enterosien-Pgeen van Ent. faecium bevat. Die enterosien wat in hierdie studie getoets is, was hitte-stabiel teen 100°C (30 min), maar het 50% van sy aktiwiteit teen 121°C (15 min) verloor. Faktore soos bakteriosienproduksie en hittebestandheid, is van die vele faktore wat enterococci in staat stel om dominant in gefermenteerde produkte, soos suiwelprodukte of vleis te wees. Enterococci wat bakteriosiene produseer het dus potensiële toepassings in verskeie kossoorte en gefermenteerde produkte. Die pH-toetse het getoon dat enterosien-P oor ‘n breë pH spektrum (2-10) aktief was. Enterosienaktiwiteit oor ‘n wye pH spektrum maak dit potensieel meer geskik as natuurlike preserveermiddels vir kossoorte en gefermenteerde produkte waar produkte versuur word of die pH afneem as gevolg van natuurlike MSB wat teenwoordig is. Dit het ook potensiële toepassings in enologiese prosessering waar pH-vlakke so laag as 3 en 4 is. Proteolitiese ensiembehandelings met lisosiem, lipase, litikase en katalase kon nie enterosienaktiwiteit inhibeer nie. Daar is getoon dat hul antimikrobiese aktiwiteit onafhanklik was van lipiede, koolhidraatdele óf waterstofperoksied. α-Chymotripsien en proteïenase-K het enterosien onaktief gemaak, wat getoon het dat die samestelling proteïenagtig van nature is. Bakteriosienproduksie wat in twee van die stamme #16.3 en 128.1 getoets is, het ooreengestem met die eksponensiële groeifase wat na 6 ure van inkubasie teen 30°C plaasgevind het, en wat ‘n aanduiding is van primêre metabolitiese kinetika. Die hoogste produksie van 400 AU/ml is na agt ure waargeneem en is vir etlike ure (46 uur) in die stasionêre fase gehandhaaf. Die bakterie-dodende effek van die selvrye supernatant van #16.3 en 128.1 teenoor die sensitiewe kultuur van Lactobacillus pentosus DSM 20314 is duidelik gedemonstreer deur totale inhibisie van groei vir die grootste deel van die eksperimentele periode, terwyl die kontrole eksponensieel deur die hele eksperiment toegeneem het. Hierdie studie het dus die isolering en identifisering van Ent. faecium-stamme, ‘n genus wat baie selde gevind word in ‘n wynomgewing, vanuit wyn bevestig. Alhoewel daar gespekuleer kan word oor die oorsprong van hierdie bakterie in wyn bv. menslike hantering en besmette water, het hierdie rasse wel enterosien geproduseer en daarom die potensiaal om ‘n rol te speel in beheer teen verskeie bederf-MSB-genera. / TIA, NRF and THRIP

Após a constatação da escassez de estudos realizados com vegetais crus na busca por novas estirpes de bactérias láticas (BAL) produtoras de bacteriocinas e diante do potencial tecnológico da aplicação destas cepas tanto como agentes de conservação em alimento, bem como cultura probiótica em alimentos funcionais, este estudo objetivou isolar e identificar cepas de bactérias láticas potencialmente bacteriocinogênicas de amostras de rúcula obtidas no comércio local de São Paulo, SP - Brasil, identificar e caracterizar as bacteriocinas produzidas pelos isolados e avaliar o potencial probiótico dos isolados testando sua sobrevivência no modelo dinâmico do trato gastrointestinal TNO gastro-Intestinal Model - TIM-1 disponível no TNO (The Netherlands Organization for Applied Scientific Research) divisão Quality of Life (Zeist, Holanda). A produção de bacteriocinas neste modelo também foi avaliada, comparando-se com L. sakei 2a, também produtora de bacteriocinas e ainda avaliou-se a interferência na viabilidade de E. faecium LMA1. A cepa Lactococcus lactis subsp. lactis MK02R de rúcula produziu uma bacteriocina sensível à enzimas proteolíticas, termoestável e não influenciada pelo pH, sendo capaz de inibir Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii e Listeria Monocytogenes de diferentes grupos sorológicos. Os ensaios genéticos utilizando primers Nisf e Nisr confirmaram que a bacteriocina MK02R é uma nisina, apresentando uma alteração dos aminoácidos no peptídeo líder em relação às nisinas A, Z, Q, F e U, porém com a estrutura do peptídeo maduro idêntica ao da nisina F. Estes resultados foram confirmados por espectrometria de massas de amostras purificadas por HPLC. L. lactis MK02R resistiu à passagem no modelo dinâmico TIM-1, apresentando uma alta capacidade de sobreviver nas condições simuladas do trato gastrointestinal humano. Entretanto, não foi capaz de causar a redução no número de E. faecium LMA1. Em contrapartida, L. sakei 2a, mesmo apresentando uma sobrevivência menor, foi capaz de causar uma redução de 70% na população de E. faecium LMA1 no ambiente simulado do TGI. Não foi detectada atividade residual da ação antimicrobiana das bacteriocinas produzidas por L. lactis MK02R ou L. sakei 2a após a passagem pelo modelo dinâmico TIM-1. Estes resultados evidenciam a possível aplicação de L. lactis MK02R como um agente de controle biológico na conservação de alimentos e também como uma cultura potencialmente probiótica. / Given the scarcity of studies performed with raw vegetables addressing the search for new bacteriocinogenic strains of lactic acid bacteria (LAB) and considering the technological application of these strains as food preservatives and probiotic cultures in functional foods, this study was aimed at isolation and identification of bacteriocinogenic LAB strains from samples of rocket salad obtained in the local market of São Paulo, SP - Brazil, subsequent characterization of the bacteriocins produced by these LABs and evaluation of their probiotic potential by testing their survival in the dynamic gastrointestinal model TNO gastro- Intestinal-Model - TIM-1, available at the TNO (Netherlands Organization for Applied Scientific Research) Quality of Life division (Zeist, Netherlands). The studies in the TIM-1 model were also done with another bacteriocinogenic strain L. sakei 2a for comparison, evaluating their interference on the viability of E. faecium LMA1. The bacteriocin produced by strain Lactococcus lactis subsp. lactis MK02R isolated from rocket salad was sensitive to proteolytic enzymes, heat-stable and not influenced by the pH. The bacteriocin inhibited the growth of Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii the primers Nisf and Nisr indicated that the bacteriocin produced by the strain MK02R is a nisin, with a change in the amino acid sequence of the leader peptide when compared to nisin A, Z, Q, U and F, but with the structure of the mature peptide homologous to that of nisin F. These results were confirmed by mass spectrometry of purified samples obtained by HPLC. L. lactis MK02R withstood the test in the dynamic model TIM-1, presenting capability to survive in the simulated conditions of the human gastrointestinal tract. However, the strain was not able to cause a reduction in the number of E. faecium LMA1. On the other hand, L. sakei 2a, even presenting lower survival, was able to cause 70% reduction in the population of E. faecium LMA1 in the gut simulated environment. No residual antimicrobial activity of bacteriocin produced by L. lactis MK02R or L. sakei 2a was detected after the transit through the dynamic model TIM-1. These results demonstrate the possible application of L. lactis MK02R both as a biocontrol agent in food preservation and as a potentially probiotic culture.

Após a constatação da escassez de estudos realizados com vegetais crus na busca por novas estirpes de bactérias láticas (BAL) produtoras de bacteriocinas e diante do potencial tecnológico da aplicação destas cepas tanto como agentes de conservação em alimento, bem como cultura probiótica em alimentos funcionais, este estudo objetivou isolar e identificar cepas de bactérias láticas potencialmente bacteriocinogênicas de amostras de rúcula obtidas no comércio local de São Paulo, SP - Brasil, identificar e caracterizar as bacteriocinas produzidas pelos isolados e avaliar o potencial probiótico dos isolados testando sua sobrevivência no modelo dinâmico do trato gastrointestinal TNO gastro-Intestinal Model - TIM-1 disponível no TNO (The Netherlands Organization for Applied Scientific Research) divisão Quality of Life (Zeist, Holanda). A produção de bacteriocinas neste modelo também foi avaliada, comparando-se com L. sakei 2a, também produtora de bacteriocinas e ainda avaliou-se a interferência na viabilidade de E. faecium LMA1. A cepa Lactococcus lactis subsp. lactis MK02R de rúcula produziu uma bacteriocina sensível à enzimas proteolíticas, termoestável e não influenciada pelo pH, sendo capaz de inibir Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii e Listeria Monocytogenes de diferentes grupos sorológicos. Os ensaios genéticos utilizando primers Nisf e Nisr confirmaram que a bacteriocina MK02R é uma nisina, apresentando uma alteração dos aminoácidos no peptídeo líder em relação às nisinas A, Z, Q, F e U, porém com a estrutura do peptídeo maduro idêntica ao da nisina F. Estes resultados foram confirmados por espectrometria de massas de amostras purificadas por HPLC. L. lactis MK02R resistiu à passagem no modelo dinâmico TIM-1, apresentando uma alta capacidade de sobreviver nas condições simuladas do trato gastrointestinal humano. Entretanto, não foi capaz de causar a redução no número de E. faecium LMA1. Em contrapartida, L. sakei 2a, mesmo apresentando uma sobrevivência menor, foi capaz de causar uma redução de 70% na população de E. faecium LMA1 no ambiente simulado do TGI. Não foi detectada atividade residual da ação antimicrobiana das bacteriocinas produzidas por L. lactis MK02R ou L. sakei 2a após a passagem pelo modelo dinâmico TIM-1. Estes resultados evidenciam a possível aplicação de L. lactis MK02R como um agente de controle biológico na conservação de alimentos e também como uma cultura potencialmente probiótica. / Given the scarcity of studies performed with raw vegetables addressing the search for new bacteriocinogenic strains of lactic acid bacteria (LAB) and considering the technological application of these strains as food preservatives and probiotic cultures in functional foods, this study was aimed at isolation and identification of bacteriocinogenic LAB strains from samples of rocket salad obtained in the local market of São Paulo, SP - Brazil, subsequent characterization of the bacteriocins produced by these LABs and evaluation of their probiotic potential by testing their survival in the dynamic gastrointestinal model TNO gastro- Intestinal-Model - TIM-1, available at the TNO (Netherlands Organization for Applied Scientific Research) Quality of Life division (Zeist, Netherlands). The studies in the TIM-1 model were also done with another bacteriocinogenic strain L. sakei 2a for comparison, evaluating their interference on the viability of E. faecium LMA1. The bacteriocin produced by strain Lactococcus lactis subsp. lactis MK02R isolated from rocket salad was sensitive to proteolytic enzymes, heat-stable and not influenced by the pH. The bacteriocin inhibited the growth of Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii the primers Nisf and Nisr indicated that the bacteriocin produced by the strain MK02R is a nisin, with a change in the amino acid sequence of the leader peptide when compared to nisin A, Z, Q, U and F, but with the structure of the mature peptide homologous to that of nisin F. These results were confirmed by mass spectrometry of purified samples obtained by HPLC. L. lactis MK02R withstood the test in the dynamic model TIM-1, presenting capability to survive in the simulated conditions of the human gastrointestinal tract. However, the strain was not able to cause a reduction in the number of E. faecium LMA1. On the other hand, L. sakei 2a, even presenting lower survival, was able to cause 70% reduction in the population of E. faecium LMA1 in the gut simulated environment. No residual antimicrobial activity of bacteriocin produced by L. lactis MK02R or L. sakei 2a was detected after the transit through the dynamic model TIM-1. These results demonstrate the possible application of L. lactis MK02R both as a biocontrol agent in food preservation and as a potentially probiotic culture.

Growing consumer awareness of the health benefits associated with fruits and vegetables and demand for easy to prepare products has prompted the development of a wide variety of minimally processed fruits and vegetables. Minimally processed fruits and vegetables are often peeled, cut, or diced which compromise the produces’ natural protective barriers, exposing a nutrient rich medium and providing an ideal environment for the growth of microorganisms, including foodborne pathogens. The germination conditions of sprout vegetables consisting of relatively high temperatures and humidity, low light and abundance of nutrients are also conducive to the proliferation of foodborne pathogens. Recent outbreaks and recalls indicate additional measures are needed to improve food safety and maintain the integrity of the food industry. The objective of this research was to evaluate the efficacy of Lactic Acid Bacteria (LAB) against E. coli O157:H7, L. monocytogenes, and Salmonella spp. on apple slices and alfalfa sprouts and it’s influence on product quality. Apple slices inoculated with E. coli O157:H7, L. monocytogenes, and Salmonella spp. (each at 104 CFU/g) were treated with Lb. plantarum alone and in combination with Pediococcus acidophilus and P. pentosaceus (LPP) (107 CFU/g) while alfalfa seeds were inoculated with L. monocytogenes and Salmonella spp. (each at 101 CFU/g and 103 CFU/g) and treated with LPP (107 CFU/g). The growth of the microorganisms on the apple slices was assessed during five and seven days of storage at 4◦C and 20◦C, respectively. Growth on alfalfa seeds was reported during five days of sprouting at 20◦C. Populations of LAB were maintained between 7.0 log CFU/g and 8.0 log CFU/g throughout storage and sprouting on the sliced apples and alfalfa seeds, respectively. Although LAB had no significant effect on pathogen populations on apple slices during storage at 4°C (p > 0.05), populations were significantly different at 20°C (p < 0.05). Populations of L. monocytogenes in the presence of Lb. plantarum and LPP were 1.84 log CFU/g and 2.84 log CFU/g less than the controls after five days of storage at 20°C (p < 0.05). Populations of E. coli O157:H7 in the presence of Lb. plantarum and LPP were 1.83 log CFU/g and 1.86 log CFU/g less than the control after one and three days of storage, respectively. Finally, populations of Salmonella spp. were 0.86 log CFU/g less than populations in the absence of LPP after three days of storage. LPP had a significant effect on the growth of L. monocytogenes and Salmonella spp. on alfalfa seeds (p < 0.05). After five days of sprouting, populations of L. monocytogenes at an initial concentration of 101 CFU/g and 103 CFU/g on seeds treated with LPP were approximately 4.5 log CFU/g and 1.0 log CFU/g less than the untreated seeds, respectively. Populations of Salmonella spp. at an initial concentration of 101 CFU/g and 103 CFU/g were 1.0 log CFU/g less than the control. Overall, on apple slices the combination of Lb. plantarum with P. acidophilum and P. pentosaceus demonstrated greater efficacy than Lb. plantarum alone and reduction of L. monocytogenes by Lb. plantarum and LPP was greater than Salmonella spp. and E. coli O157:H7 on apple slices and alfalfa seeds, alike. LAB had a minimal effect on the quality of the apple slices and alfalfa seeds. LAB could be an effective strategy in reducing pathogen populations at abusive temperatures and germination conditions without influencing the quality of minimally processed fruit and vegetables.

A produção de etanol nas destilarias brasileiras é baseada na atividade fermentativa da levedura Saccharomyces cerevisiae que utiliza o caldo da cana-de-açúcar e/ou o melaço como substrato. Bactérias contaminantes da fermentação alcoólica competem com as leveduras pelos açúcares, afetando o rendimento do sistema produtivo e, consequentemente, causando perdas econômicas significativas às usinas. Biofilmes formados nos tanques de fermentação alcoólica agem como reservatórios de bactérias, contribuindo para contaminações persistentes e de difícil controle. Os biofilmes proporcionam aos seus habitantes certo grau de proteção contra diversas ameaças do meio, incluindo a ação dos antibióticos. Desta forma, o conhecimento da comunidade bacteriana dos biofilmes é fundamental para as medidas que visam o controle das contaminações na produção do bioetanol. No primeiro estudo, a composição e dinâmica da comunidade bacteriana foram determinadas pela análise de sequências do gene 16S rRNA de biofilmes com diferentes períodos de crescimento, correspondentes aos estágios iniciais de estabelecimento destes biofilmes dentro dos tanques de fermentação alcóolica Os resultados mostraram que estas comunidades foram compostas predominantemente pelas bactérias ácido-lácticas (LAB), com destaque para o gênero Lactobacillus. A visualização da estrutura dos biofilmes por microscopia eletrônica de varredura evidenciou que estes são formados por bactérias e leveduras (biofilmes mistos). No segundo estudo, a suscetibilidade aos antimicrobianos (monensina, virginiamicina e beta-ácido derivado do lúpulo) e a capacidade de formação de biofilmes em culturas puras foram avaliadas para isolados de Lactobacillus spp. provenientes de biofilmes (células sésseis) e de vinho bruto (células planctônicas) coletados dos tanques de fermentação. A partir dos resultados foi possível observar que as diferenças na suscetibilidade aos antimicrobianos e na habilidade de formar biofilmes foram estirpe-dependentes e que, em alguns casos, o perfil apresentado para algumas espécies mostrou-se relacionado à fonte de isolamento. Este foi o primeiro estudo sobre biofilmes contaminantes da fermentação alcoólica, em escala industrial, para a produção de etanol a partir da cana-de-açúcar / Bioethanol production in Brazilian distilleries is based on fermentative activity of the yeast Saccharomyces cerevisiae which uses sugarcane juice and/or molasses as a substrate. Bacterial contaminants of alcoholic fermentation compete with yeasts for sugars, affecting ethanol yield and consequently causing relevant economic losses to the fuel ethanol industry. Biofilms formed into fermentors act as bacterial reservoirs, contributing to persistent contaminations that are difficult to control. Biofilms provide a certain degree of protection for their inhabitants against some environmental threats, including antibiotics. Thus, understanding bacterial community within biofilms is essential for actions to control contaminations in bioethanol production. In the first study, composition and dynamic of bacterial community were determined by 16S rRNA gene sequences analysis of biofilms with different growth periods, corresponding to initial stages of biofilm establishment in fermentation tanks. Results showed that these communities were dominated by lactic acid bacteria (LAB), mainly of the genus Lactobacillus. Visualization of biofilm structure by scanning electron microscopy revealed a mixed-species biofilm composed by bacteria and yeasts. In the second study, susceptibility to antimicrobials (monensina, virginiamicina and beta-acids from hops) and capacity to form biofilm in pure culture were evaluated for Lactobacillus spp. isolated from biofilms (sessile cells) and wine (planktonic cells) collected from fermentors. The results showed that differences in the susceptibility to antimicrobials and the ability to form biofilms were strain-specific and, in certain cases, the response of some species was related to the isolation source. This was the first investigation of contaminant biofilms from sugarcane-based alcoholic fermentation on an industrial scale

A produção de etanol nas destilarias brasileiras é baseada na atividade fermentativa da levedura Saccharomyces cerevisiae que utiliza o caldo da cana-de-açúcar e/ou o melaço como substrato. Bactérias contaminantes da fermentação alcoólica competem com as leveduras pelos açúcares, afetando o rendimento do sistema produtivo e, consequentemente, causando perdas econômicas significativas às usinas. Biofilmes formados nos tanques de fermentação alcoólica agem como reservatórios de bactérias, contribuindo para contaminações persistentes e de difícil controle. Os biofilmes proporcionam aos seus habitantes certo grau de proteção contra diversas ameaças do meio, incluindo a ação dos antibióticos. Desta forma, o conhecimento da comunidade bacteriana dos biofilmes é fundamental para as medidas que visam o controle das contaminações na produção do bioetanol. No primeiro estudo, a composição e dinâmica da comunidade bacteriana foram determinadas pela análise de sequências do gene 16S rRNA de biofilmes com diferentes períodos de crescimento, correspondentes aos estágios iniciais de estabelecimento destes biofilmes dentro dos tanques de fermentação alcóolica Os resultados mostraram que estas comunidades foram compostas predominantemente pelas bactérias ácido-lácticas (LAB), com destaque para o gênero Lactobacillus. A visualização da estrutura dos biofilmes por microscopia eletrônica de varredura evidenciou que estes são formados por bactérias e leveduras (biofilmes mistos). No segundo estudo, a suscetibilidade aos antimicrobianos (monensina, virginiamicina e beta-ácido derivado do lúpulo) e a capacidade de formação de biofilmes em culturas puras foram avaliadas para isolados de Lactobacillus spp. provenientes de biofilmes (células sésseis) e de vinho bruto (células planctônicas) coletados dos tanques de fermentação. A partir dos resultados foi possível observar que as diferenças na suscetibilidade aos antimicrobianos e na habilidade de formar biofilmes foram estirpe-dependentes e que, em alguns casos, o perfil apresentado para algumas espécies mostrou-se relacionado à fonte de isolamento. Este foi o primeiro estudo sobre biofilmes contaminantes da fermentação alcoólica, em escala industrial, para a produção de etanol a partir da cana-de-açúcar / Bioethanol production in Brazilian distilleries is based on fermentative activity of the yeast Saccharomyces cerevisiae which uses sugarcane juice and/or molasses as a substrate. Bacterial contaminants of alcoholic fermentation compete with yeasts for sugars, affecting ethanol yield and consequently causing relevant economic losses to the fuel ethanol industry. Biofilms formed into fermentors act as bacterial reservoirs, contributing to persistent contaminations that are difficult to control. Biofilms provide a certain degree of protection for their inhabitants against some environmental threats, including antibiotics. Thus, understanding bacterial community within biofilms is essential for actions to control contaminations in bioethanol production. In the first study, composition and dynamic of bacterial community were determined by 16S rRNA gene sequences analysis of biofilms with different growth periods, corresponding to initial stages of biofilm establishment in fermentation tanks. Results showed that these communities were dominated by lactic acid bacteria (LAB), mainly of the genus Lactobacillus. Visualization of biofilm structure by scanning electron microscopy revealed a mixed-species biofilm composed by bacteria and yeasts. In the second study, susceptibility to antimicrobials (monensina, virginiamicina and beta-acids from hops) and capacity to form biofilm in pure culture were evaluated for Lactobacillus spp. isolated from biofilms (sessile cells) and wine (planktonic cells) collected from fermentors. The results showed that differences in the susceptibility to antimicrobials and the ability to form biofilms were strain-specific and, in certain cases, the response of some species was related to the isolation source. This was the first investigation of contaminant biofilms from sugarcane-based alcoholic fermentation on an industrial scale

Parmi les bactéries lactiques, Lactococcus lactis est la plus utilisée en fabrication fromagère. Actuellement, les ferments lactiques sont majoritairement choisis pour leurs propriétés acidifiantes, protéolytiques et aromatiques. Un autre paramètre majeur est le potentiel redox (Eh). En effet, un Eh réducteur est souvent associé à une bonne qualité aromatique. L’activité réductrice de L. lactis pourrait donc être un nouveau paramètre à prendre en compte dans la maitrise du Eh dans la fabrication des produits laitiers fermentés. Néanmoins, les mécanismes impliqués dans l’activité réductrice de L. lactis demeurent encore inconnus. L’objectif de ce présent travail de thèse a été de les découvrir. Tout d’abord, nous avons développé des milieux de culture gélosé de discrimination redox utilisant des sels de tétrazolium pour cribler une banque de mutants aléatoires de L. lactis. Ceci a permis de démontrer la participation partielle de la chaine de transport d’électrons (Ménaquinones) dans l’activité réductrice de L. lactis. Ensuite, l’approche biochimique nous a permis de déterminer les composés biochimiques principaux contribuant à la diminution du Eh vers des valeurs très réductrices. La présence de groupements thiols exofaciaux est responsable du Eh réducteur atteint par L. lactis. Enfin, l’analyse protéomique utilisant un marquage spécifique des protéines thiols de surface a mis en évidence la présence d’une dizaine de protéines exposant des groupements thiols exofaciaux potentiellement impliquées dans l’activité réductrice de L. lactis. Les thiols sont connus pour être de très puissants antioxydants ce qui confère à L. lactis un intérêt supplémentaire à prendre en considération dans l’élaboration des produits laitiers fermentés. / Among the Lactic Acid Bacteria, Lactococcus lactis is the most used in cheese making. Nowadays, starters are used mainly for their acidifying, proteolytic and flavor properties. Another important parameter is the redox potential (Eh). Indeed, reducing Eh is often related to good flavor properties. The reducing activity of L.lactis should be therefore a new parameter to take into account in the monitoring of Eh during dairy fermented products making. Nevertheless, the mechanisms involved in the reducing activity of L.lactis are still unknown. The aim of this work was to understand them. First of all, we have developed tetrazolium salts agar plate media in order to screen a random bank of mutants of L. lactis on their redox capacities. These media allowed us to demonstrate the partial implication of the electron transport chain (Menaquinone) in the reducing activities of L. lactis. Secondly, we have determined the biochemical compounds involved in the decrease of Eh to very reducing values thanks to a biochemical approach. Exofacial thiol groups are mainly responsible for the reducing Eh reached by L.lactis. Lastly, a proteomical analysis using a specific staining of thiols surface proteins revealed the presence of about ten proteins displaying thiols exofacials groups. These proteins might be involved in the reducing activity of L.lactis. Thiols are known to be very strong antioxidants which confer to L. lactis an additional interest to consider in dairy products making.