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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

S. thermophilus est une bactérie lactique largement utilisé dans l'industrie laitière et, comme toute bactérie, doit s'adapter à des environnements variés tels que le lait, le yaourt et même le tractus digestif, après que le produit ait été ingéré. Les systèmes à deux composants (TCS) constituent un des mécanismes essentiels qu'utilisent les bactéries pour percevoir et s'adapter à des changements environnementaux. D'un point de vue structural, les TCS sont constitués de deux composants: un " senseur " ou protéine histidine kinase (HK) qui s'auto-phosphoryle en réponse à un stimulus puis transfère son groupement phosphate au " response regulator " (RR), le deuxième composant. Celui-ci se comporte alors le plus souvent comme un régulateur transcriptionnel permettant une réponse physiologique adaptée. Afin de mieux comprendre ces phénomènes de régulation impliqués dans la réponse aux changements environnementaux, nous avons étudié la contribution de chacun des 8 TCS de Streptococcus thermophilus LMD-9 à son adaptation dans le lait. Ainsi, des études transcriptionnelles effectuées sur des cultures en lait montrent que tous les RR sont exprimés, à des niveaux et profils d'expression différents. Nous avons noté en coculture avec Lactobacillus bulgaricus, le partenaire de Streptococcus thermophilus dans le yaourt, une induction de l'expression de 4 RR qui atteint, pour rr02 et rr09, un facteur 6. Nous avons construit par ailleurs des mutants négatifs pour 7 des 8 RR de S. thermophilus et montré l'essentialité de RR05, un orthologue de YycF chez B. subtilis ou de or de WalR chez S. aureus. Pour les 7 autres mutants RR, l'absence d'un seul gène rr n'impacte pas suffisamment la croissance du streptocoque en lait. Enfin, la détermination du régulon du TCS06 par des études post-génomiques a permis de montrer que ce système est impliqué dans la résistance à la bacitracine en modulant entre autres la voie de biosynthèse du polysaccharide à rhamnose (RGP).

Argonautes (AGOs) are present in all domains of life. Like their eukaryotic counterparts, archaeal and eubacterial AGOs adopt a similar global architecture and bind small nucleic acids. In many eukaryotes, AGOs, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. We find that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15–18 nt DNA guides derived from the chromosomal region where replication terminates, and TtAgo complexed to short DNA guides enhances target finding and prefers to bind targets with full complementarity. Additionally, TtAgo associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of both gyrase and TtAgo activity slows growth and produces long, segmented filaments in which the individual bacteria are linked by DNA. Furthermore, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. Finally, at physiologic temperature in vitro, we find TtAgo possesses highest affinity for fully complementary targets. We propose that terminus-derived guides binding in such a fashion localize TtAgo, and that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.

As bactérias probióticas, além de diversos possíveis efeitos benéficos à saúde do consumidor, podem apresentar um potencial para bioenriquecer alimentos através da produção de compostos de interesse nutricional e para a saúde durante a etapa de fermentação. Nesse sentido, a produção de vitaminas naturais por bactérias láticas, incluindo as probióticas, emerge como alternativa promissora ao uso de vitaminas sintéticas para a fortificação de alimentos. O uso de substratos vegetais para estimular a produção de vitaminas do grupo B por probióticos compreende uma solução sustentável para o reaproveitamento desse material oriundo, principalmente, do processamento de frutas. Os subprodutos de frutas são compostos por diversas substâncias bioativas e a incorporação desses ingredientes para o desenvolvimento de alimentos com alto valor agregado representa uma solução sustentável para o seu reaproveitamento. Sendo assim, o presente trabalho tem como objetivo avaliar a influência da adição de subproduto de fruta no bioenriquecimento de um produto de soja fermentado tipo frozen yogurt probiótico com riboflavina (vitamina B2). Para este fim, a capacidade de diferentes cepas de Streptococcus thermophilus, Lactobacillus spp. e Bifidobacterium spp. em produzir riboflavina foi avaliada, utilizando-se um meio de cultura desprovido desta vitamina (Riboflavin Assay médium, RAM). Paralelamente, foi avaliada a capacidade dessas cepas em fermentar os subprodutos de uva (Vitis spp.) e de caju (Anacardium occidentale L.). De acordo com os resultados de produção de riboflavina em RAM e de fermentação dos subprodutos de fruta, foram selecionadas as cepas para o desenvolvimento do produto fermentado de soja tipo frozen yogurt (FY). Dentre as cepas testadas, os Streptococcus thermophilus (ST-M6, TA-40 e TH-04) e o probiótico Lactobacillus fermentum PCC apresentaram potencial para produzir riboflavina em meio RAM. As cepas PCC e ST-M6 mostraram-se capazes de fermentar os subprodutos de caju e de uva (atingindo 8,25 e 8,40 log UFC/mL, respectivamente, para PCC e ST-M6 com subproduto de uva e 4,82 e 8,38 log UFC/mL, respectivamente, para PCC e ST-M6 com subproduto de caju em 24h). Por outro lado, as cepas TH-4 e TA-40 fermentaram apenas o subproduto de uva (atingindo 6,87 e 7,24 log UFC/mL, respectivamente, em 24h). Sendo assim, foram selecionadas as cepas de St. Thermophilus ST-M6, TH-4 e TA-40 e a de Lactobacillus fermentum PCC para a aplicação nos produtos visando o seu bioenriquecimento. O FY suplementado com 1% de subproduto de uva foi preparado, utilizando-se as cepas em co-cultura totalizando 3 combinações, ou seja, entre Lb. Fermentum PCC e as 3 cepas de St. Thermophilus. As formulações de FY foram capazes de atingir uma concentração de riboflavina de 8,40 e de 5,18 &#181;g/mL (combinando TA-40 e PCC) em 60 dias, respectivamente, sem e com o subproduto de uva. Já a associação de STM6 e PCC, sem o subproduto, resultou na menor concentração obtida (2,30 &#181;g/mL), atingindo 5,62 &#181;g/mL na presença do subproduto. Dessa maneira aplicação do subproduto de uva para estimular a produção de riboflavina só foi efetiva para a formulação que combinou St. Thermophilus ST-M6 e Lb. Fermentum PCC. / Probiotic bacteria, in addition to several possible beneficial effects on the consumers health, may present a potential to bioenrich food through the production of compounds of nutritional and health interest during fermentation. In this sense, the production of natural vitamins by lactic acid bacteria, including probiotics, emerges as a promising alternative to the use of synthetic vitamins for food fortification. The use of vegetable substrates to stimulate the production of B-group vitamins by probiotics comprises a sustainable solution for the reuse of this material coming mainly from fruit processing. Fruit by-products are composed of various bioactive substances and the incorporation of these ingredients into the development of high value-added foods represents a sustainable solution for their reuse. Therefore, the present study aims to evaluate the influence of the addition of fruit by-product on the bio-enrichment of a probiotic frozen yogurt-like fermented soy product with riboflavin (vitamin B2). For this purpose, the ability of different strains of Streptococcus thermophiles, Lactobacillus spp. and Bifidobacterium spp. in producing riboflavin was evaluated using a culture medium devoid of this vitamin (Riboflavin Assay Medium, RAM). In addition, we evaluated the ability of these strains to ferment the grape (Vitis spp.) and cashew (Anacardium occidentale L.) by-products. According to the results of riboflavin production in RAM and fermentation of fruit by-products, strains were selected for the development of a fermented frozen yogurt-like (FY) soybean product. Among the strains tested, Streptococcus thermophiles (ST-M6, TA-40, and TH-04) and the probiotic Lactobacillus fermentum PCC had the potential to produce riboflavin in RAM medium. The strains PCC and ST-M6 were able to ferment the cashew and the grape byproducts (reaching 8.25 and 8.40 log CFU/mL, respectively, for PCC and ST-M6 with the grape by-product, and 4.82 and 8. 38 log CFU/mL, respectively, for PCC and STM6 with cashew by-product in 24h). On the other hand, TH-4 and TA-40 fermented only the grape by-product (reaching 6.87 and 7.24 log CFU/mL, respectively, in 24h). Therefore, the strains St. thermophiles ST-M6, TH-4, and TA-40 and the strain Lactobacillus fermentum PCC were selected for application in the products for bioenrichment. The FY supplemented with 1% of grape by-product was prepared using the co-culture strains comprising three combinations between Lb. fermentum PCC and the three strains of St. thermophiles. The FY formulations were capable of producing 8.40 and 5.18 &#181;g/mL (combination of TA-40 and PCC) in 60 days, respectively, without and with the grape by-product. However, the association of ST-M6 and CCP, without the by-product, resulted in the lowest concentration obtained (2.30 &#181;g/mL), achieving 5.62 &#181;g/mL in the presence of the by-product. Thus, the application of the grape byproduct in order to stimulate the production of riboflavin was only effective for the formulation that combined St. thermophiles ST-M6 and Lb. fermentum PCC.

Thesis (MTech (Food Technology))--Cape Peninsula University of Technology, 2016. / In South Africa malnutrition exists due to inadequate dietary intake of micronutrients which is one of the major causes of vitamin deficiencies leading to disease. The treatment of malnutrition over the past years has been a considerable burden on the South African economy. Therefore, food fortification is one of the current strategies used to minimize malnutrition by increasing the nutritional value of staple foods. Commercial dairy products and pharmaceutical nutritional products (food supplements) in South Africa have been developed and produced for affluent consumers. Hence the need to develop an affordable fortified dairy product for the majority of South Africans prompted this study aimed at using a “naturally” fortified kefir beverage with vitamin B12 and folate to increase B-vitamins levels. Since Propionibacterium freudenreichii and Streptococcus thermophilus are known to be good producers of vitamin B12 and folate, respectively, and propionibacteria has the ability to grow symbiotically in the presence of lactic acid bacteria, the inclusion of these organisms with the kefir grains was an achievable objective. In order to conduct the analysis of vitamin B12 and folate in the samples, sample extraction and HPLC assay techniques were developed. The extraction of vitamin B12 and folate were achieved by using KCN extraction buffer and the trienzymatic method, respectively. The samples were also subjected to purification and concentration using solid phase extraction for optimum results. All standards and samples were flushed with nitrogen gas and stored for a maximum of 2 weeks at –20°C to prevent B-vitamin deterioration. The HPLC assembly for the vitamin B12 analysis included a Luna C18 column and a diode array detector (DAD) for the detection and quantification. For the folate analysis it included a Zorbax SB-C18 and Luna C18 columns in tandem and the fluorescence detector (FLD) was used for the detection and quantification of THF, 5-CH3-THF and 5-CHO-THF, while the DAD was used for PGA and pteroyltri-γ-L-glutamic acid concentration in the samples.

<p>An introduction to bacterial polysaccharides and the methods for structural determination are described in the first two parts of the thesis.</p><p>In a structural elucidation of bacterial polysaccharides NMR experiments are important as is component analysis. A short description of immunochemical methods such as enzyme immunoassays is included. Two NMR techniques used for interaction studies, trNOE and STD NMR, are also discussed. </p><p>The third part of the thesis discusses and summarizes the results from the included papers. The structures of the exopolysaccharides produced by two lactic acid bacteria are determined by one- and two dimensional NMR experiments. One is a heteropolysaccharide produced by <i>Streptococcus thermophilus</i> and the other a homopolysaccharide produced by <i>Propionibacterium freudenreichii</i>. The structure of an acidic polysaccharide from a marine bacterium with two serine residues in the repeating unit is also investigated. The structural and immunological relationship between two O-antigenic polysaccharides from <i>Escherichia coli</i> strain 180/C3 and O5 is discussed and investigated. Finally, interaction studies of an octasaccharide derived from the <i>Salmonella enteritidis</i> O-antigen and a bacteriophage are described which were performed with NMR experiments.</p>

An introduction to bacterial polysaccharides and the methods for structural determination are described in the first two parts of the thesis. In a structural elucidation of bacterial polysaccharides NMR experiments are important as is component analysis. A short description of immunochemical methods such as enzyme immunoassays is included. Two NMR techniques used for interaction studies, trNOE and STD NMR, are also discussed. The third part of the thesis discusses and summarizes the results from the included papers. The structures of the exopolysaccharides produced by two lactic acid bacteria are determined by one- and two dimensional NMR experiments. One is a heteropolysaccharide produced by Streptococcus thermophilus and the other a homopolysaccharide produced by Propionibacterium freudenreichii. The structure of an acidic polysaccharide from a marine bacterium with two serine residues in the repeating unit is also investigated. The structural and immunological relationship between two O-antigenic polysaccharides from Escherichia coli strain 180/C3 and O5 is discussed and investigated. Finally, interaction studies of an octasaccharide derived from the Salmonella enteritidis O-antigen and a bacteriophage are described which were performed with NMR experiments.

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.

We have carried out structural studies on bovine pancreatic phospholipase A2 (BPLA2) and two proteins involved in molybdenum cofactor (Moco) biosynthesis pathway. In addition, molecular-dynamics simulations and other analyses have been performed to corroborate the findings obtained from the crystal structures. Crystal structures of the three active-site mutants (H48N, D49N and D49K) of BPLA2 were determined to understand the mechanism by which the mutant H48N is able to catalyze the reaction of phospholipid hydrolysis and to see the effect of the loss of Ca 2+ ion in the active site of D49N and D49K mutants. We found that Asp49 could possibly play the role of a general base instead of His48 in the case of the H48N mutant. In the case of D49N and D49K mutants, the active site of the enzyme is perturbed, whereas the overall tertiary structure of these mutants is intact. In addition, a total of 24 invariant water molecules were identified in all of the crystal structures of BPLA2 available in its archive, PDB. Out of these, four water molecules are essential for the catalytic activity, whereas, the remaining water molecules play a role in the stability of the enzyme. In addition, structural studies on two proteins MoaC and MogA involved in Moco biosynthesis pathway have been carried out. For the first time, crystal structure of MoaC bound with GTP molecule has been reported. The gene id TTHA0341, which is mentioned as MoaB in the CMR database, was annotated as MogA based the comparative analysis of sequences and structures (with the present work and the structures available in the literature). The role of N-and C-termini of MoaB and MogA proteins were proposed that these residues might stabilize the substrate and/or product molecule in the active site. In addition, the residues involved in the oligomerization are compared with MD simulations. The molecular docking studies show that MoaB proteins show more preference to GTP than ATP. The comparison of the two active (MPT and AMP-binding) sites revealed that MPT-binding site is preferred over AMP-binding site for nucleotide binding.