Despite the fact that Glycosyl Hydrolase Family 32 present 4 467 enzyme entries, only 14 of them have been characterized structurally. From the ten protein crystal structures deposited for Bifidobacterium adolescentis ATCC 15703 at PDB just one enzyme is related to the processing of non-digestible sugars and there is no structure of a β-fructofuranosidase. In this research we studied the biochemical properties and the structural features of a recombinant β-fructofuranosidase (BaFFse) from the healthy gut bacteria B. adolescentis ATCC 15703 (gen BAD_1325) heterologously expressed in Escherichia coli Rosetta. The enzyme was purified by nickel ion affinity chromatography and molecular exclusion chromatography; the purification process was judged by denaturing SDS-PAGE gel. Sucrose was used as a substrate for the enzyme activity assays and the amount of reducing sugars, detected by Dinitrosalycilic acid, was taken as indicator of the optimum conditions of hydrolysis for the enzyme. BaFFase crystal, grown in PEG 8K 25% (w/v) and buffer MES 0.1M pH 6.5, was diffracted at 2.44 Å and processed using the CCP4 program package. The enzyme presented a classical four-stranded five-bladed β-propeller and a C-terminal β-sandwich characteristic from the GH 32 family; however, connected to the β-propeller through a loop of 38 residues, BaFFase also presented an N-terminal β-sandwich domain, which sequence (residues 3-100 from BaFFase) did not match with any protein sequence when aligned against PDB database. Assays with Gel filtration calibration, DLS and SAXS showed that the enzyme was a stable homodimer in solution. Based on the superposition of structures using the a β-fructofuranosidase from B. longum KN29.1 we could deduced the three key aminoacids involved in the transferring of fructosyl moieties by BaFFase. A nucleophile attack is performed by the carboxylate of Asp 131, forming the fructose BaFFase intermediate; Glu 375 donates a proton, acting as an acid base catalyst and Asp 269 stabilizes the transitions state in the fructosyl transferring activity. This is the first GH32 oligomeric enzyme belonging to the bacteria kingdom. We have described a novel additional β-sandwich domain for a GH32 enzyme that increases the region of contact to form a dimer. This is the first β-fructofuranosidase crystal structure from the microorganism B. adolescentis ATCC 15703. / O presente trabalho disserta sobre os estudos das propriedades bioquímicas e as características estruturais de uma β-frutofuranosidase recombinante (BaFFse) da bactéria Bifidobacterium adolescentis ATCC 15703 (gen BAD_1325) presente em intestinos saudáveis. A proteína foi expressa heterologamente em Escherichia coli Rosetta. A enzima foi purificada por cromatografia de afinidade (íons de níquel) e cromatografia de exclusão por massa molecular; o processo de purificação foi avaliado por gel desnaturante tipo SDS-PAGE. A sacarose foi usada como substrato para os ensaios de atividade enzimática e a quantidade de açúcares redutores, detectados por ácido dinitrosalicílico, foi tomada como indicador das condições ótimas de hidrólise para a enzima. Cristais de BaFFase, crescidos em solução contendo PEG 8 k em tampão Hepes pH 6,5, foram difratados a uma resolução de 2,44 Å e processados utilizando o pacote de programas CCP4. A enzima apresentou um clássico enovelamento tipo composto por cinco pás de quatro-fitas β cada e um domínio C-terminal sanduíche-β característico da família GH32; no entanto, ligado ao β-propeller, através de um loop de 38 resíduos, a BaFFase apresentou um inédito domínio N-terminal sanduíche-β (resíduos 3-100 de BaFFase) ainda sem precedentes, quando alinhado contra a base de dados PDB. Ensaios de gel filtração, DLS e SAXS mostraram que a enzima se apresenta como um homodímero estável em solução. Com base na superposição estrutural, utilizando uma β-frutofuranosidase de B. longum KN29.1, foi possível inferir os três aminoácidos essenciais envolvidos na transferência de unidades de frutosil pela BaFFase. Um ataque nucleofílico é realizado pelo grupo carboxílico do Asp131, formando um intermediário frutose-BaFFase; o Glu375 doa um próton, atuando como um catalisador ácido-base e o Asp269 estabiliza o estado transições na atividade de transferência frutose. Um novo domínio sanduíche-β adicional para uma enzima GH32 é descrito. Esse domínio é responsável pelo aumento da região de contato e essencial para a formação do homodímero. Esta é a primeira estrutura cristalina da β-frutofuranosidase do microrganismo B. adolescentis ATCC 15703, além de ser a primeira enzima GH32 descrita neste estado oligomérico pertencente ao reino das bactérias.

O presente trabalho teve como objetivo verificar a viabilidade da cepa probiótica Bifidobacterium animalis subsp. lactis Bb-12 incorporado em margarina, suplementada com inulina, concentrado protéico de soro (WPC) e concentrado de caseína (CMP), bem como avaliar as características do produto e a resistência do probiótico às condições simuladas do trato gastrintestinal humano. Foram produzidos 7 diferentes tipos de margarinas de mesa (60% de lipídios: 60 % de óleo de palma + 40% de óleo de canola), empregando-se um modelo de mistura, onde inulina, WPC e CMP foram as variáveis estudadas. Uma formulação controle foi produzida (M8), sem adição desses ingredientes. A utilização da mistura do óleo de palma com óleo de canola favoreceu nutricionalmente as formulações, fornecendo produtos contendo ácidos graxos essenciais em sua composição e ausência de ácidos graxos trans. As formulações M1 a M7, exceto a formulação M2 após o 21º dia de armazenamento, apresentaram populações satisfatórias de Bb-12 para um alimento probiótico, com populações acima de 6 log UFC/g durante 35 dias de armazenamento. Margarinas suplementadas com inulina apresentaram populações satisfatórias durante todo o armazenamento, atingindo populações de 8,01 log UFC/g ao 35º dia (M1). Além disso, M3 e M6, revelaram populações de Bb-12 de 6,87 log UFC/g e 7,27 log UFC/g (dia 35), respectivamente. Por outro lado, M8 não foi caracterizada como margarina probiótica, uma vez que apresentou populações abaixo de 6 log UFC/g, já ao 1º dia de armazenamento. Embora WPC seja utilizado em pesquisas para aumentar a viabilidade de probióticos em alimentos, a suplementação de margarina com WPC sem inulina ou CMP não resultou em populações satisfatórias de Bb-12, apresentando decréscimo de 7,82 (dia 1) para 4,64 log UFC/g (M2, dia 35) (p<0,05). Durante todo o ensaio de resistência in vitro, Bb-12 apresentou sobrevivência significativamente superior (p<0,05) em M1 e revelou populações acima de 6 log UFC/g após 6h de ensaio mesmo ao 28º dia. As populações observadas para M2 diminuíram drasticamente durante o ensaio in vitro (5 log UFC/g após 2h no dia 7). Para as outras formulações, as populações de Bb-12 diminuíram 2 log UFC/g após 2h de ensaio in vitro. Entretanto, M1, M2 e M5 (dias 14 e 28) revelaram aumento significativo nas populações de Bb-12 (p<0,05) entre a fase gástrica (2h) e a segunda fase entérica (6h). As margarinas suplementadas com inulina, principalmente M1, revelaram decréscimo significativo no pH durante todo o armazenamento (p<0,05). Entretanto, isto não afetou a qualidade sensorial dos produtos, uma vez que não foram detectadas diferenças significativas entre as formulações após 7 e 14 dias de armazenamento (p>0,05). A suplementação de margarina com inulina e CMP garantiu populações apropriadas de Bb-12 durante o armazenamento estudado pelo menos até o 28º dia. Além disso, contribuiu para sua sobrevivência durante o ensaio de resistência in vitro. Os resultados revelaram que a margarina apresenta-se como uma matriz alimentar adequada para administração de Bb-12, principalmente quando a inulina foi adicionada. / This study aimed to determine the viability of probiotic Bifidobacterium animalis subsp. lactis Bb-12 incorporated in margarine, with inulin, whey protein concentrate (WPC) and caseinomacropeptide (CMP) supplementation. In addition, the in vitro resistance of Bb-12 incorporated in margarine and related properties were evaluated. Seven margarine-making trials (60% of fat: 60% of palm oil +40% canola oil) were produced, using a mixture model, where inulin, WPC and CMP were the variables studied. Also, a control formulation without these ingredients was manufactured. The use of blending palm oil with canola oil improved the margarine formulations nutritionally, providing products containing essential fatty acids in its composition and absence of trans fatty acids. The formulations M1 to M7, except M2 after 21 days of storage, revealed satisfactory Bb-12 populations for a probiotic food, with counts above 6 log CFU/g during 35 days of storage at 5±1ºC. Margarines supplemented with inulin presented suitable Bb-12 populations throughout the whole storage period, reaching up to 8 log CFU/g by the end of storage (M1). Also, M3 and M6, revealed Bb-12 populations of 6.87 log CFU/g and of 7.27 log CFU/g (day 35), respectively. In contrast, M8 was not characterized as probiotic margarine, since it showed Bb-12 populations below 6 log CFU/g on day 1. Even though whey protein is largely employed in probiotic foods, margarine supplementation with WPC without inulin or CMP did not lead to Bb-12 satisfactory populations, decreasing from 7.82 (day 1) to 4.64 log CFU/g (M2, day 35) (p<0.05). During the whole in vitro assays, Bb-12 survived significantly better (p<0.05) in M1 and revealed populations above 6 log CFU/g after 6h even after 28 days. M2 populations decreased drastically during the in vitro assays for all storage period tested (reduction of 5 log CFU/g after 2h of in vitro assays on day 7 and populations of 2.8 log CFU/g after 6h). For the other formulations, Bb-12 populations decreased 2 log CFU/g after 2h of the in vitro assays. However, for M1, M2 and M5 (on day 14 and 28) the populations of Bb-12 increased significantly (p<0.05) between the gastric phase (2h) and the enteric phase (6h). Formulations containing inulin, mainly M1, showed a significant decrease in pH values during the whole storage period (p<0.05). However, this ingredient did not affect the sensory quality of products, since no significant differences between formulations after 7 and 14 days of storage were observed (p>0.05). The supplementation of margarine with inulin and CMP guaranteed appropriate Bb-12 populations during storage for at least 28 days, and also contributed for its survival throughout the in vitro assays. Therefore, margarine might be considered an appropriate food matrix for Bb-12 survival, mainly when inulin is also added.

The aim of this study was to develop probiotic microcapsules of Bifidobacterium animalis and Lactobacillus acidophilus evaluating the survival of the microencapsulated probiotics under simulated gastrointestinal conditions and availability during storage at different temperatures, with further application to the production of Italian salami evaluating their effects on the physicochemical, microbiological and sensory characteristics of salami. Survival assays were conducted to evaluate the resistance of the microencapsulated probiotic to simulated gastrointestinal conditions and availability during 120 days of storage at 4°C and 25 ° C, besides to morphological analysis of the microcapsules. The microcapsules showed spherical shape with relatively smooth and continuous surface without cracks, protecting the probiotics from simulated gastrointestinal conditions when compared to free probiotics, remained with greater viability after 120 days of storage at 4°C for both microorganisms. After probiotic microcapsules were added to the production of Italian salami, generating three treatments: T1 control and T2 and T3 with the addition of microcapsules of B. animalis and L. acidophilus, respectively. Analyzes of water activity, pH, moisture, weight loss, coliform at 45ºC, Staphylococcus coagulase positive and Salmonella spp. detection were realized and monitoring the microencapsulated probiotic cultures, all analyzes were conducted during 120 days; beyond sensory analysis of salami after 30 days and monthly up to 120 days. There were no significant difference (p>0,05) between the treatments in relation to physicochemical, microbiological and sensory analyses. The treatments T2 e T3 maintained counts of B. animalis and L. acidophilus above 6 log CFU.g-1 to 90 days. Therefore, the production of probiotic Italian salami is possible. However, it is suggested that probiotics salamis should be designed such that the microorganisms can maintain their availability until the end of the shelf life of the product, either by changing the structure of the microcapsule or increasing the microorganism initial number or reducing the shelf life of the product. / O objetivo geral deste trabalho foi desenvolver microcápsulas probióticas de Bifidobacterium animalis e Lactobacillus acidophilus avaliando a sobrevivência dos probióticos microencapsulados sob condições gastrointestinais simuladas e a viabilidade durante armazenamento sob diferentes temperaturas, com posterior aplicação à produção de salames tipo Italiano avaliando seus efeitos sobre as características físico-químicas, microbiológicas e sensoriais do salame. Ensaios de sobrevivência foram conduzidos para avaliar a resistência dos probióticos microencapsulados às condições gastrointestinais simuladas e a viabilidade durante 120 dias de armazenamento a 4ºC e 25ºC, além da análise morfológica das microcápsulas. As microcápsulas apresentaram forma esférica, com superfície contínua relativamente lisa e sem fissuras, protegendo os probióticos das condições gastrointestinais simuladas quando comparado a probióticos livres, permanecendo com maior viabilidade após 120 dias de armazenamento à 4ºC, para ambos os micro-organismos. Após, as microcápsulas probióticas foram adicionadas à produção de salame tipo Italiano, gerando três tratamentos, sendo T1 o controle e T2 e T3 com adição de microcápsulas de B. animalis e L. acidophilus, respectivamente. Foram realizadas análises de atividade de água, pH, umidade, perda de peso, contagem de coliformes à 45ºC e Staphylococcus coagulase positiva e detecção de Salmonella spp. e o acompanhamento das culturas probióticas microencapsuladas, todas as análises ocorreram durante os 120 dias; além da análise sensorial do salame após 30 dias e mensalmente até 120 dias. Através dos resultados constatou-se que não houve diferença significativa (p>0,05) entre os tratamentos com relação às análises físico-químicas, microbiológicas e sensoriais. Os tratamentos T2 e T3 mantiveram contagens de B. animalis e L. acidophilus acima de 6 log UFC.g-1 até 90 dias. Portanto, é possível a produção de salames tipo Italiano com propriedades probióticas. No entanto, sugere-se que salames probióticos devem ser projetados de tal forma que os micro-organismos possam manter sua viabilidade até o final do prazo de validade do produto, seja alterando a estrutura da microcápsula ou aumentando a carga microbiana inicial ou reduzindo a vida de prateleira do produto.

Les cellules de notre système immunitaire différencient les antigènes du Soi, envers lesquels elles ne doivent pas engendrer de réponse immunitaire effectrice, et les pathogènes qu’elles doivent éliminer. Outre les antigènes du Soi, le système immunitaire doit également tolérer des antigènes de l’environnement non pathogènes comme les aliments et les bactéries de la flore commensale qui colonisent l’intestin. Au sein de cette flore se trouvent des bactéries « probiotiques » dont certaines souches ont un effet préventif et/ou curatif dans le cadre d’allergies, de maladies inflammatoires ou même de cancers. Ces effets seraient au moins en partie dus à une action des probiotiques ou de leurs métabolites sur des cellules du système immunitaire. Les cellules dendritiques (DC) ont une grande plasticité qui leur permet, selon les signaux qu’elles perçoivent, de générer soit une réponse immunitaire effectrice pour éliminer les pathogènes soit une tolérance en induisant des lymphocytes T régulateurs. On les retrouve notamment au niveau de la muqueuse intestinale où elles sont susceptibles d’interagir avec les probiotiques. Nous avons analysé l’impact d’un surnageant de fermentation d’un milieu laitier simplifié par la bactérie probiotique Bifidobacterium breve C50 (BbC50sn) sur les DC humaines in vitro : ce surnageant entraîne la maturation de ces cellules (DC-BbC50sn) ainsi qu’une forte production d’IL-10 et une augmentation de leur survie via le TLR-2. L’analyse des gènes transcrits dans les DC-BbC50sn par la technique des puces à ADN met en évidence l’expression de gènes codant des molécules tolérogènes comme ILT-3, ILT-4 et PDL-1. De plus, nous montrons que les DC-BbC50sn induisent des lymphocytes T (LT) régulateurs fonctionnels in vitro. Ces LT régulateurs secrètent de l’IL-10 et du TGF-ß et nécessitent une activation spécifique d’alloantigène par des DC pour exercer leur activité suppressive. Nous avons également montré l’induction de LT régulateurs ayant des caractéristiques différentes par des DC traitées avec d’autres ligands de TLR-2 et TLR-4. Nous démontrons donc que BbC50sn peut avoir des capacités régulatrices au travers de son action sur les cellules dendritiques humaines en induisant des lymphocytes T régulateurs in vitro. Ces résultats représentent une base rationnelle de son utilisation en clinique. / The immune system protects our organism by removing pathogen bacteria and viruses while tolerating non-pathogenic antigens from self, environment and commensal bacteria. Some commensal bacteria called “probiotics” have been shown to exert beneficial effects on the host health. Recent studies demonstrated that these probiotic bacteria could act on immune cells either directly or via their metabolites. Dendritic cells (DC) are able to induce either an effective or a tolerogenic immune response depending on the environment signals. They can be found in the intestinal mucosa where they could interact with probiotic bacteria. We demonstrated that a bacteria-free fermentation product of Bifidobacterium breve C50 (BbC50sn) induced human DC maturation with high IL-10 production in vitro and prolonged their survival. The BbC50sn action on dendritic cells was mediated via the TLR-2 pathway. The DNA microarray analysis showed that BbC50sn-DC produced high levels of mRNA corresponding to genes encoding tolerogenic molecules such as ILT-3, ILT-4 and PDL-1. We also highlighted that these BbC50sn-DC could induce functional regulatory T cells in vitro. These regulatory T cells needed an alloantigen specific activation to exert their suppressive activity and didn’t act through a T cell-T cell contact. These regulatory T cells secreted IL-10 and TGF-ß; however, these cytokines didn’t appear to mediate the suppressive activity. We also showed that other dendritic cells treated with TLR-2 and TLR-4 ligands could induce regulatory T cells different from those induced by BbC50sn-DC. BbC50sn is thus able to exert a regulatory effect through this action on human dendritic cells by inducing regulatory T cells in vitro. As far as we know, it is the first demonstration of regulatory T cell induction by a probiotic derivative product. These results represent a rational basis for BbC50sn use in clinics.

Probiotics are living organisms which exert a beneficial health effect when consumed in sufficient numbers. Consumer interest in probiotics has increased dramatically in recent years prompting an increase in production and development of functional foods. One major problem is the decreased viability of probiotic bacteria during functional food production and storage and subsequent digestion due to environmental stresses. The most common probiotic strains belong to the genus Lactobacillus or Bifidobacterium. Due to the anaerobic nature of these bacteria, they lack the required defense mechanisms for oxidative stress inherent in aerobic microorganisms. This study examined the oxidative stress responses of six strains of Bifidobacterium, which are commonly used as probiotics in functional foods.The first phase of the study investigated the innate and inducible hydrogen peroxide (H2O2) stress response of Bifidobacterium longum strains NCC2705 and D2957, Bifidobacterium longum ssp. infantis ATCC 15697, and Bifidobacterium animalis ssp. lactis strains BL-04, DSM10140 and RH-1. Strains were screened for survival at increasing concentrations of H2O2 and lethal and sublethal concentrations were determined for each. In the second phase, B. animalis ssp. lactis strains BL-04 and DSM10140 and B. longum strains NCC2705 and D2957 were treated with a sublethal H2O2 concentration and RNA samples were collected for transcriptome analysis after 5 min and either 20 or 60 min. Statistical analysis was performed to identify genes that increased or decreased in expression during H2O2 treatment compared to control cells.Results showed that survival was species and strain dependent and that strains which naturally survived higher H2O2 concentrations had a larger number of differentially expressed genes early on during H2O2 exposure. Some of the protective genetic systems that were activated during H2O2 stress are mechanisms which perform basic cellular functions under normal conditions such as deoxuynucleotide synthesis. Under stress conditions, these systems can be used to detoxify oxidative free radicals. Also a number of genes involved in sugar transport and energy production for the cell showed increased expression, which reveals the increased energy needs of the cells during oxidative stress.During testing, it was found that two B. animalis ssp. lactis strains, BL-04 and DSM10140, had differing levels of survival and gene expression during H2O2 exposure despite having almost identical genome sequences. It was determined that one possible cause of the differences was a genetic deletion in a gene that allows the cell to incorporate extracellular fatty acids into the cell membrane instead of synthesizing them.Results from this project have increased the understanding of oxidative stress responses in bifidobacteria and highlighted possible methods to increase bacterial survival during food manufacture, storage, and human digestion.

Oxygen toxicity is considered significant in the poor survival of probiotic bacteria such as Lactobacillus acidophilus and Bifidobacterium spp. in yoghurts. This study investigated methods to protect these bacteria from oxygen exposure. To confirm the accuracy of the reported survival estimates of L. acidophilus or Bifidobacterium spp. in yoghurts, the reliability of several enumeration media was evaluated with different commercial yoghurts. None of the media however, was found reliable thereby casting doubts on the reported cell numbers of probiotic bacteria in yoghurts. After much research,it was found that although oxygen can be detrimental to L. acidophilus and Bifidobacterium spp.in culture broths, it may not be significant for their poor survival in yoghurts. Nevertheless, techniques such as oxidative stress stress adaption, alternative packaging materials and microencapsulation as investigated in this study, can serve as general protective techniques to help yoghurt manufacturers in maintaining the recommended numbers of probiotic bacteria in their products. This would eventually assist in the efficient delivery of probiotic health benefits to yoghurt consumers. / Doctor of Philosphy (PhD)

Reproduction de : Thèse de doctorat : Sciences de la Vie et de la Santé : Lille 1 : 2004. Reproduction de : Thèse de doctorat : Sciences de la Vie et de la Santé : Universitatea "Al. I. Cuza", Iasi : 2004. / Thèse en cotutelle. N° d'ordre (Lille 1) : 3479. Titre provenant de la page de titre du document numérisé. Bibliogr. p. 196-223.

Conjugated linoleic acid (CLA) is predominantly found in foods of ruminant origin such as milk and processed cheese, and has gained much interest recently due to its beneficial health and biological effects on animals and humans. / The bioconversion of linoleic acid (LA) and linolenic acid (LNA) by a selected Bifidobacterium from healthy infant feces was studied. Bifidobacterium breve JKL03 had the ability to convert linolenic acid (0.2 mg/ml) to CLNA in fermentation of skim milk medium for 24 h up to a yield of 72.0% (up to 74.7% under aerobic conditions) and linoleic acid (0.2 mg/ml) into CLA by fermentation in skim milk medium for 24 h up to a yield of 23.9% (up to 28.0% under aerobic conditions). / B. breve JKL03 was also co-fermented with Lactobacillus acidophilus (NCFMRTM strain), a commonly added starter culture, to observe the resulting effects on growth during fermentation for yogurt production. Fermentation of LNA in skim milk with B. breve JKL03 and L. acidophilus (NCFM) maintained high CLNA production level. On the other hand, CLA production in the same media with both strains did not exhibit as high level as with the single B. breve. / These results are important for the advancement of knowledge on the production of CLA and CLNA in dairy products and for knowledge on the basic metabolic mechanisms for such conversion.

Access to abstract permanently restricted to Ball State community only. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science

Thesis (Ph.D.)--University of Western Sydney, 2005. / A thesis submitted to the University of Western Sydney, College of Health and Science, Centre for Plant and Food Science, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.