L'utilisation de souches de levures sélectionnées pour réaliser la FA est une pratique très répandue en œnologie. Après le développement de l'utilisation de levains de souche pure de Saccharomyces, l'innovation est aujourd'hui dans la mise en œuvre de levains mixtes de Saccharomyces et de non-Saccharomyces qui permettent de diversifier les produits finaux obtenus. La problématique réside dans l'existence d'interactions entre les souches rendant difficile la maîtrise de la fermentation. T. delbrueckii présente dans la flore indigène du mout de raisin est une des levures non-Saccharomyces les plus appropriées pour entrer dans la composition de ces levains mixtes. En effet, elle présente une bonne capacité fermentaire et peut permettre d'augmenter la complexité aromatique du vin ou encore de réduire son acidité volatile. L'objectif de ce travail était d'étudier les interactions pendant la FA entre des souches sélectionnées pour l'oenologie : une T. delbrueckii et une S. cerevisiae. Pour cela des expériences ont été réalisées dans des milieux synthétiques simulant le moût de raisins blancs. Le comportement des souches pures a tout d'abord été caractérisé. Il a été montré que la souche S. cerevisiae avait de meilleures performances fermentaires d'un point de vue cinétique que la souche T. delbrueckii. Toutefois, T. delbrueckii a montré des capacités acceptables pour épuiser les sucres et surtout a permis d'obtenir des profils aromatiques différents. Le comportement vis-à-vis de l'oxygénation des moûts de ces deux levures est assez semblable, T. delbrueckii étant cependant beaucoup plus sensible à ce paramètre que S. cerevisiae. L'interaction entre ces deux levures a ensuite était étudiée dans un bioréacteur à membrane sous anaérobie stricte dans différentes conditions : composition en azote assimilable du milieu et stratégie d'inoculation. Il a été clairement mis en évidence que T. delbrueckii était affectée par la présence de S. cerevisiae. Le type d'interaction soupçonné est celui d'amensalisme lié à l'excrétion par S. cerevisiae d'un constituant toxique pour T. delbrueckii. Dans ces conditions, la stratégie d'inoculation recommandée est l'ensemencement séquentiel des levures : T. delbrueckii en début de fermentation, puis l'ajout de S. cerevisiae 48 h après. Ceci permet à T. delbrueckii de se développer et d'exprimer son potentiel aromatique avant que S. cerevisiae ne soit introduit pour assurer une fin rapide de la fermentation. Toutefois, nous avons montré que même dans ces conditions, l'implantation de T. delbrueckii n'était pas garantie car, le moût n'étant pas stérile, une présence, même faible, de S. cerevisiae dans la flore naturelle peut inhiber sa croissance. Par ailleurs, il a été mis en évidence que dans les mouts à faible teneur en azote initial, ce constituant pouvait être épuisé au moment de l'inoculation de S. cerevisiae. Dans ces conditions, S. cerevisiae ne peut se développer et l'achèvement de la fermentation est alors problématique. / The use of the selected yeast strains to realize the alcoholic fermentation is very prevalent practice in vinification. After the development of utilization of the preparation of pure Saccharomyces cerevisiae strain, the innovation is now to apply the mixte starter cultures of Saccharomyces and non-Saccharomyces that allow to diversifying the obtained final products. The problem resides in the existence of interactions between the strains giving the difficulty to controle the fermentation. Torulaspora delbrueckii present in the indigenous flora of the grape must is one of the most appropriate non-Saccharomyces yeasts to enter in the composition of these multistater cultures. In fact, this strain has been presented a good fermentative capacity and could allow to increasing not only the aromatic complexity of wine, but also to reducing its volatile acidity. The objective of our work is to study the interactions during the alcoholic fermentation between the selected strains for enology: one T. delbrueckii and one S. cerevisiae. For this reason, the experiments were realized in the synthetic mediums simulated to the white grape must. The behaviours of the pure strains were firstly characterized. It was shown that the S. cerevisiae strain had the best fermentative performances, a critical point in comparaison with the T. delbrueckii strain. Nevetheless, T. delbrueckii showed the acceptable capacities to exhaust the sugars and especially to allow us to obtain the different aromatic profiles to that of S. cerevisiae. The behaviour via the oxygenation to the musts of these two yeasts is enough close, T. delbrueckii being however much more sensible to that parameter than . cerevisiae. The interactions between these two yeasts were then studied in a membrane bioreacteur under strict anaerobie in different conditions: composition in assimilable nitrogen of the medium and strategy of inoculation. It has been clearly demonstrated that T. delbrueckii has been affected by the presence of S. cerevisiae. The suspected type of this interaction is the amensalism one bound to a toxic compound excreted by S. cerevisiae. In these conditions, the recommended inoculation strategy is the sequential culture of these yeasts: T. delbrueckii at the beginning of the fermentation, then the addition of S. cerevisiae after 48 h. This allows T. delbrueckii to develop and express its potentiel of aromatic production before the S. cerevisiae is introduced to assure a rapid finish of the fermentation. However, we showed that even in these conditions, T. delbrueckii growth has been not guaranteed because of, since the must is not sterilized, a presence even small of S. cerevisiae in the natural flore can inhibite the croissance of the former. It has been also demonstrated that in the must with low intitial nitrogen content, this compound could be exhausted at the moment of the S. cerevisiae inoculation. In these conditions, S. cerevisiae can not develop and the achievement of the fermentation is yet problematic.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-05T12:37:57Z No. of bitstreams: 1 2011 - Simone de Souza Fernandes.pdf: 745580 bytes, checksum: 7836d760ce3ea36d3e5602714715cd42 (MD5) / Made available in DSpace on 2016-10-05T12:37:57Z (GMT). No. of bitstreams: 1 2011 - Simone de Souza Fernandes.pdf: 745580 bytes, checksum: 7836d760ce3ea36d3e5602714715cd42 (MD5) Previous issue date: 2011-05-27 / Yogurt is a fermented milk resulting from a mutual interaction between the lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. For yoghurt quality assurance, the cell numbers of each microorganism, should not be less than 1x107per gram, therefore a relative ratio lactobacilli and streptococci should be 1:1, although a 1:2 ratio is also accepted. During storage of yoghurt exposed for sale, post-acidification may occur, changing the ratio of the two microorganisms. The objectives of this research were to evaluate yoghurts of four different manufacturers named A, B, C and D, in relation to the number and balance between streptococii and lactobacilli during the storage and its relation with acidity and pH. In this way, yoghurts with up to 20 days of manufacturing (band I) and more than 20 days (band II) were evaluated. Better recovery of L. delbruckii subsp. bulgaricus and S. thermophilus was observed with LB and M17 media respectively, which were used to determine the relative ratio of the two microorganisms. An imbalance in the lactobacilli numbers which were lower than that of streptococci was verified and considered inadequate, in two out of four commercial brands. In yoghurts from the manufacturer A, there was a significant reduction in the number of lactobacilli from band I to band II leading to an increase in the relative ratio of streptococii to lactobacilli. There was no significant difference in the counts of streptococci or bacilli from one band to another with the other three brands of yoghurts. The acidity of yoghurts from manufacturer D revealed significantly higher (P< 0.05) than the others, although it did not result in an increased pH reduction. All samples attended the legislation in relation to total lactic acid bacteria counts, acidity, pH and mould and yeast count / Iogurte ? um leite fermentado resultante de uma intera??o microbiana mutualista entre as bact?rias l?cticas Streptococcus thermophilus e Lactobacillus delbrueckii subsp. bulgaricus. Para que a qualidade do iogurte seja garantida o n?mero de c?lulas destes dois microrganismos individualmente, n?o deve ser inferior a 1x107 por grama, portanto uma propor??o relativa de lactobacilos e estreptococos de 1:1 sendo a propor??o 1:2 tamb?m aceita. Durante o armazenamento dos iogurtes expostos para venda podem ocorrer p?s-acidifica??o e modifica??o na propor??o dos dois microrganismos. Os objetivos desta pesquisa foram avaliar iogurtes de quatro diferentes fabricantes as quais foram denominados A, B, C, D quanto ao n?mero e equil?brio entre as duas bact?rias l?cticas durante o armazenamento, e ao mesmo tempo determinar as caracter?sticas f?sico-quimicas (pH e acidez) e contagem de bolores e leveduras. Para tanto, adotou-se duas faixas de avalia??o, faixa I (at? 20 dias de fabrica??o) e faixa II (ap?s 20 dias). Foi observada melhor recupera??o L. delbruckii subsp. bulgaricus e S. thermophilus, respectivamente com os meios LB e M17 que foram usados para determina??o da propor??o relativa dos dois microrganismos. Foi verificado um desequil?brio no n?mero de lactobacilos que foi inferior ao de cocos e, considerado inadequado, em duas das quatro marcas comerciais. Nos iogurtes da marca A houve redu??o significativa no n?mero de lactobacilos da faixa I para faixa II levando a um aumento na propor??o de cocos relativa ao de bacilos. Nos iogurtes dos tr?s outros fabricantes n?o houve diferen?a significativa nas contagens de cocos ou de lactobacilos de uma faixa para outra. Tamb?m n?o foi observada diferen?a significativa de acidez e pH relacionados ao tempo de prazo comercial nas faixas I e II nas quatro marcas de iogurte analisadas. A acidez dos iogurtes do fabricante D foi significativamente mais elevada (P<0,05) que a dos demais, embora n?o tenha resultado em maior redu??o de pH nestes produtos. Todas as amostras analisadas estavam em conformidade com a legisla??o vigente no que se refere ao m?nimo exigido de bact?rias l?ticas totais que ? de 1x107 UFC/mL. Tamb?m estavam em conformidade com rela??o ? acidez, pH e contagem de bolores e leveduras. Palavras-chave: leite

Yogurt is a characteristic fermented dairy product of Turkey and Bulgaria and its popularity has been increasing all over the world. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) are used together as starter culture in production of yogurt. The objective of this study was to isolate and characterize yogurt cultures from traditionally produced yogurts (i.e. produced without using commercial starter cultures) and to search the genotypic diversity within traditional S. thermophilus isolates. Yogurt cultures were isolated from traditionally produced yogurts collected from different regions of Turkey and identified biochemically. Acidification ability of the isolates was examined and the cultures giving best acidifying rates were further subjected to a selection in terms of their acetaldehyde production ability. Then, phage resistance and proteolytic activity of chosen isolates were tested. Finally, twenty-five L. bulgaricus and twenty-two S. thermophilus isolates were selected as cultures having best technological properties. Furthermore, subtyping studies were carried out to indicate strain diversity among isolates. S. thermophilus was selected as target organism for subtyping in this study. Clustered regularly interspaced short palindromic repeats (CRISPR) loci are highly polymorphic genetic regions, which are composed of partially palindromic direct repeats interspaced by sequences called spacers. In order to characterize S. thermophilus isolates genotypically, CRISPR1 locus of the isolates were analyzed. Additionally, nineteen isolates selected after CRISPR1 analysis were characterized using multilocus sequence typing (MLST). This provided to compare CRISPR1 analysis with MLST as a typing method. According to CRISPR1 analysis S. thermophilus isolates were grouped into 6 main clusters with a total of 15 sub-clusters. MLST results demonstrated an evolutionary relationship among these strains compatible with that derived from the CRISPR1 analysis.

This diploma thesis deals with the study of the utilization of Raman spectroscopy and Raman tweezers for analysis and isolation of polyhydroxyalkanoates (PHA) producing bacteria. Using gas chromatography with FID detection, we determined the polyhydroxybutyrate (P(3HB)) content of the PHA biomass of bacterial strains Burkholderia cepacia, Halomonas halophila, Cupriavidus necator H16 and its mutant strain Cupriavidus necator H16/PHB-4 and Lactobacillus delbrueckii, which is not a producer of polyhydroxyalkanoates but this bactrea was selected as representative of Gram-positive bacteria. Subsequently, thanks to Raman microspectroscopy, Raman tweezers and FT-IR spectrometer in combination with Raman FT-module, we were able to confirm or disprove the presence of P(3HB) in bacteria. Furthermore, the thesis describes Cupriavidus necator H16, which is a model organism for the production of P(3HB), and his mutant strain Cupriavidus necator H16/PHB-4. The bacterial strain Cupriavidus necator H16 was cultivated in a production mineral medium of various nitrogen contents, while cultivation was also carried out in liquid Nutrient Broth. By this cultivation we were able to reach various P(3HB) content in bacterial biomass, the spectra were subsequently compared with the spectrum of the bacterial strain Cupriavidus necator H16/PHB-4. Raman spectroscopy is well used to characterize the composition of individual bacterial cells, is a fast, versatile, and virtually non-invasive tool for studying cells.

Zvýšená konzumace fermentovaných mléčných výrobků v posledních letech vede k zavádění nových výrobků na trh. Funkční a probiotické produkty jsou v dnešní době také hojně rozšířeny. Cílem diplomové práce bylo posoudit mikrobiální kvalitu komerčních jogurtů a nefarmakologických probiotických výrobků. Bylo zkoumáno 24 vzorků jogurtů a 8 vzorků probiotických mléčných výrobků. Pozornornost byla zaměřena zvláště na kvantifikaci mikroorganismů v jednotlivých výrobcích a na sledování viability buněk. Většina mléčných produktů obsahovala počty odpovídající minimální požadované hodnotě 1x107cfu/ml. Nicméně byly objeveny produkty, které neobsahovaly živé bakterie nebo obsahovaly jen jejich menší množství. To platí zejména pro běžné jogurty. Probiotické mléčné výrobky v zásadě obsahovaly větší množství bakterií než jogurty, protože je u nich navíc požadováno 1x106cfu/ml minimálního množství specifických bakterií jiných než jogurtových startovacích kultur. Isoláty z fermentovaných mléčných výrobků byly identifikovány jako Streptococcus thermophilus and Lactobacillus spp. Isoláty byly poté rozlišeny na jednotlivé kmeny pomocí RAPD použitím tří různých primerů. Byla zjištěna velká různorodost mezi kmeny rodu Lactobacillus spp. používaných různými podniky.

Made available in DSpace on 2015-03-26T13:51:43Z (GMT). No. of bitstreams: 1 texto completo.pdf: 248917 bytes, checksum: 84bc05c660ca867229222e12c0fa08ec (MD5) Previous issue date: 2003-03-31 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Phylogenetic analysis of Lactobacillus UFV H2b20 based on the 16S rDNA sequence positions this isolate among the Lactobacillus delbrueckii subspecies. It confirms the classification by DNA/DNA hybridization. Comparisons of the substituted bases along the 16S rDNA sequence show that most are probably caused by 5-methylcytosine deamination that results in GC/AT transition. Presence of polymorphic copies of the 16S rRNA gene in Lactobacillus delbrueckii UFV H2b20 were confirmed by PCR-DGGE. Five distinct bands of rDNA amplified sequences confirm at least five different copies of the gene. The intensity of one of the bands allows the inference of two identical copies. The DGGE profile of this isolate was different from those of other L. delbrueckii strains, providing a mean to distinguish it in mixed cultures. Analysis of the nucleotide sequences along the rDNA region revealed the presence of putative insertion sequences. One of them, 915 bp long, was characterized and denominated ISLdH2b20. It presents a single ORF that encodes a putative transposase with some homology to the one of ISL5 of L. delbrueckii subsp. bulgaricus. All the characteristic elements of an IS were located as well as the putative regulatory sequences of the transposase. / O estudo de filogenia baseado em seqüências de rDNA de Lactobacillus UFV H2b20 mostrou que esse microrganismo foi agrupado entre as subespécies de Lactobacillus delbrueckii. Este resultado confirma a nova classificação do isolado, baseada na hibridização DNA-DNA. A análise das substituições de bases mostra que a maioria é causada pela desaminação da 5-metilcitosina, o que causa uma transição GC/AT. A técnica PCR-DGGE de L. delbrueckii UFV H2b20 demonstrou resultados satisfatórios na análise de polimorfismos do operon rrn em microrganismos isolados. Foram encontradas cinco bandas diferentes para o L. delbrueckii UFV H2b20, confirmando a presença de cinco cópias distintas do rDNA 16S nesse microrganismo e possivelmente seis no total. O isolado L. delbrueckii UFV H2b20 apresentou um perfil de bandas diferente das outras linhagens de Lactobacillus, o que poderá ser utilizado para diferenciá-lo dos demais, uma vez que outros métodos baseados em PCR, como RAPD, mostram-se pouco discriminatórios. A análise das seqüências da região do rDNA 16S revelou uma seqüência de inserção putativa, de 915 pb, que foi caracterizada e denominada ISLdH2b20. Ela apresenta uma única ORF e codifica uma transposase putativa que apresenta homologia com a transposase de ISL5. Todos os elementos característicos de uma IS foram identificados bem como as seqüências regulatórias putativas da transposase.