Os benefícios à saúde oferecidos pelos leites fermentados probióticos podem ser ampliados pela adição de polpa de frutas vermelhas uma vez que estas apresentam reconhecidas propriedades funcionais. Ácido elágico e elagitaninos presentes nas frutas apresentam função fisiológica e metabólica na alimentação. Porém, a estabilidade destes compostos bioativos em produtos probióticos durante armazenamento refrigerado ainda não foi estudada. Sendo assim, este projeto tem por finalidade investigar o comportamento do ácido elágico em bebidas lácteas simbióticas suplementadas com L-triptofano e polpa de frutas vermelhas durante armazenamento refrigerado. O estudo foi realizado através de um delineamento experimental do tipo simplex centróide para modelagem de misturas. Foram preparadas 16 bebidas lácteas divididas em duas diferentes matrizes de ensaios segundo: (i) enriquecimento ou não da bebida láctea com triptofano e (ii) composição em frutas vermelhas do preparado de frutas. As amostras de bebida láctea foram avaliadas quanto as suas características físico-químicas (pH, reologia e atributos de cor), microbiológicas, microestrutura e quanto ao teor de ácido elágico. As bebidas lácteas apresentaram reduzidas pós-acidificação sem variações significativas para as bebidas contendo triptofano. As mesmas bebidas apresentaram melhora significativa na viabilidade da bactéria probiótica Lactobacillus acidophilus ao longo do período estudado. Os parâmetros de cor se mostraram estáveis nas bebidas lácteas durante o armazenamento refrigerado. O comportamento reológico das bebidas lácteas foi semelhante e adição de polpa de fruta proporcionou um efeito benéfico na consistência e viscosidade dos produtos. Foi possível estimar o conteúdo ótimo de ácido elágico nas bebidas lácteas simbióticas pela metodologia de superfície de resposta. As bebidas lácteas adicionadas de polpa de morango apresentaram maiores teores de ácido elágico, principalmente nas bebidas contendo triptofano. As fotos micrografias obtidas através da microscopia eletrônica de varredura permitiram avaliar a interação dos componentes da matriz das bebidas lácteas simbióticas de forma satisfatória. / The health benefits provided by probiotic fermented milk can be enhanced by the addition of native fruit pulp as they have recognized functional properties. Ellagic acid and ellagitannins present in fruis show physiological and metabolic function. Identification of these bioactive compounds in the formulation of symbiotic fermented beverages supplemented with L-tryptophan and red fruit pulp is innovative. Therefore, this project aimed to investigate the behaviour of ellagic acid in symbiotic lactic beverages supplemented with L-tryptophan and red pulp fruits during cold storage. Through an experimental simplex centroid design for mixtures modelling 16 beverages were prepared divided into four different matrices according to : (i) enrichment or not with L-tryptophan and (ii) composition of the prepared fruit pulp. The responses to be evaluated will be defined between the physical-chemical (pH, rheology and colour attributes), microbiological, microstructure and the amounts of ellagic acid. Lactic beverages have shown low post-acidification without significant variations for beverages containing tryptophan. The improvement in the viability of the probiotic bacteria Lactobacillus acidophilus throughout the study period was achieved. The colour parameters were stable during cold storage. The rheological behaviour was similar and the addition of red fruit pulps provided a beneficial effect on the consistency and viscosity of the products. It was possible to estimate the optimum amount of ellagic acid throughout response surface methodology. Lactic beverages added with strawberry pulp have had higher amounts of ellagic acid, particularly when L-tryptophan was supplemented. The photomicrographs obtained by scanning electron microscopy allowed adequate assessing the interaction of the matrix components of symbiotic lactic beverages.

A ingestão de alimentos que, além de fornecerem a nutrição, promovem a saúde, como os alimentos funcionais adicionados de probióticos, que auxiliam no equilíbrio da microbiota intestinal, é de vital importância. Em virtude do crescente interesse da indústria e da comunidade científica por novos produtos probióticos, o desenvolvimento de uma sobremesa láctea que permita a adição de probióticos, como o manjar branco, é promissor. O presente trabalho visou desenvolver um manjar branco com a adição dos microrganismos potencialmente probióticos, Lactobacillus paracasei subsp. paracasei e/ou Bifidobacterium longum, adicionados isoladamente e em co-cultura, e avaliar o comportamento dos probióticos e as características do produto ao longo do seu armazenamento refrigerado. Quatro tratamentos de manjar branco foram produzidos (com 3 repetições cada): T1 (controle - sem a adição de culturas), T2 (com Bifidobacterium longum), T3 (com Lactobacillus paracasei) e T4 (com B. longum + L. paracasei). O produto foi avaliado sensorialmente (após 7, 14 e 21 dias de armazenamento a 4±1°C, através de escala hedônica estruturada, com 24 provadores não treinados em cada período) e, durante o seu armazenamento por até 28 dias, microbiologicamente quanto à viabilidade dos probióticos e à contagem de contaminantes (coliformes, Escherichia coli, Staphylococcus spp. e bolores e leveduras) e quanto às suas características físico químicas (umidade, pH) e de textura instrumental. Durante o armazenamento dos produtos por 28 dias, as populações médias de L. paracasei aumentaram de 6,6 para 8,6 log UFC/g e de 6,4 para 7,3 log UFC/g, para T3 e T4, respectivamente. B. longum revelou populações sempre entre 7,3 e 7,5 log UFC/g e entre 7,1 e 7,5 log UFC/g para T2 e T4, respectivamente. O manjar T3 resultou em uma redução mais acentuada do pH (p<0,05), mas a umidade não foi significativamente distinta para os diferentes produtos (p>0,05). A firmeza de todos os tratamentos de manjar branco estudados aumentou ao longo do armazenamento e o manjar T2 apresentou menor firmeza a partir de 14 dias de armazenamento. No entanto, essa diferença não foi observada durante a análise sensorial do manjar branco para nenhum dos tratamentos. De fato, a avaliação sensorial não detectou diferenças evidentes da adição de culturas isoladas ou em co-cultura de bifidobacteria e L. paracasei nas características sensoriais. Não foi observada nenhuma diferença significativa entre os tratamentos (p = 0,06) durante o período de vida-de-prateleira de 21 dias (p = 0,24). Entretanto, foi observada uma tendência de melhores notas para os manjares adicionados de microrganismos probióticos, comparados ao manjar branco controle e uma tendência de redução das notas quando ambos os microrganismos foram adicionados. A adição de Lactobacillus paracasei e Bifidobacterium longum ao manjar branco resultou em um produto potencialmente probiótico e com excelentes atributos sensoriais, particularmente quando a adição das culturas foi realizada individualmente. / The ingestion of foods that promote health in addition to providing basic nutrition, like foods supplemented with probiotics cultures, which may exert a positive effect on the gut indigenous microbiota is becoming increasingly important. The development of a non-fermented dairy dessert which allows the supplementation with probiotics, like coconut flan, appears to be a good option, due to the growing interest that food companies and the scientific community have demonstrated in new probiotic products. The present research aimed to study the viability of Lactobacillus paracasei subsp. paracasei and Bifidobacterium longum, added as single cultures or in co-culture, in coconut flan supplemented with these potentially probiotic cultures and to evaluate the behavior of the probiotics and the features of the product during refrigerated storage. Coconut flans were produced with no addition of cultures (T1 - control), or supplemented with Bifidobacterium longum (T2), Lactobacillus paracasei subsp. paracasei (T3) and B. longum + L. paracasei (T4). The products were sensory evaluated (after 7, 14 and 21 days of storage, at 4±1°C, employing a structured hedonic scales, with 24 untrained panelists at each period) and monitored microbiologically for the viability of the probiotics and the counts of contaminants (coliforms, Escherichia coli, Staphylococcus spp., yeasts and moulds), as well as for their instrumental texture profile and their physicochemical features (pH, moisture) through the 28 days of storage at 4±1ºC. During storage of the products for 28 days, the mean populations of L. paracasei increased from 6.6 to 8.6 log CFU/g and from 6.4 to 7.3 log CFU/g, for T3 and T4, respectively. B. longum presented populations always between 7.3 and 7.5 log CFU/g, and between 7.1 and 7.5 log CFU/g for T2 and T4, respectively. Coconut flan T3 underwent higher decreases in pH values (P<0,05). On the other hand, moisture showed no significant differences (P>0,05) between trials and during the shelf life period. The firmness of all coconut flans studied increased during storage, and coconut flan T2 presented lower firmness after 14 days of storage. Nevertheless, this difference was not detected during the sensory evaluation of coconut flans. In fact, the sensory analyses did not shown marked differences from the addition of probiotic cultures of bifidobacteria and L. paracasei as single cultures or in co-culture, on sensory features. No significant differences between trials (P=0,06) and during the 21 days of shelf life period (P=0,24) were detected. However, a tendency of better scores for the coconut flans supplemented with the probiotic microorganisms, compared with the control product, and a tendency of reduction of scores when both microorganisms were presented in the coconut flan formulation were observed. The supplementation of coconut flans with Lactobacillus paracasei and Bifidobacterium longum resulted in great potential as a functional food, and with high sensory acceptability, particularly when single cultures were employed.

O objetivo deste trabalho foi estudar a viabilidade de Bifidobacterium animalis subsp. lactis HN019 em fórmulas infantis fermentadas ou não, probióticas durante armazenamento a 4°C. Três matrizes lácteas e três não lácteas (a base de soja) foram utilizadas para a elaboração de produtos fermentados ou não fermentados usando Bifidobacterium animalis subsp. lactis HN019, resultando em doze diferentes fórmulas probióticas para lactentes. O perfil de acidificação foi determinado a 42°C até pH 4,7. Determinações físico-químicas (sólidos totais, proteína, gordura, cinzas, carboidratos, calorias, densidade e pH) foram realizadas e foram focadas as contagens de bactérias viáveis durante o armazenamento refrigerado. A caracterização química dos produtos lácteos e a não lácteos apresentou resultados diferentes, à exceção FSL2, todos estavam de acordo com Codex Alimentarius. O perfil de acidificação de Bifidobacterium animalis subsp. lactis HN019 diferiu conforme a matriz. Durante o armazenamento dos produtos a 4°C, a contagem de bactérias viáveis de acordo com o preconizado, bem como a pós-acidificação, estando em conformidade com as recomendações da legislação brasileira. Processo (fermentação ou adição) e tipo de matriz (lácteos e não lácteos) influenciaram a pós-acidificação e a viabilidade de Bifidobacterium animalis subsp. lactis HN019. As fórmulas para lactentes podem ser considerados bons veículos de Bifidobacterium animalis subsp. lactis HN019. / This study proposed to study infant formulas as vehicles for Bifidobacterium animalis ssp.lactis HNOI9. Three dairy and three non-dairy matrices were employed for the preparation of fermented or unfermented products using Bifidobacterium animalis ssp. lactis HN019 resulting in twelve different probiotic infant formulas. Acidification profile of the probiotic was determined at 42°C until pH 4.7. Physicochemical determination (total solids, protein, fat, ash, carbohydrates and calories, density and pH) was conducted, and counts viable bacteria (in dairy and non dairy infant formulas fermented and unfermented) during cold storage was focused on. The chemical characterization of the dairy and non-dairy matrix showed different results, the exception FSL2, all were in accordance to the Codex Alimentarius. The acidification profile of B. animalis ssp. lactis HN019 differed according to the matrix. During storage of products at 4°C counts of viable bacteria were stable as well as post-acidification, and were in accordance with the recommendations of the Brazilian legislation. Process (fermentation or addition) and matrix type (dairy and non-dairy) influenced post-acidification and viability of B. animalis ssp. lactis BN019 . Infant formulas could be considered good vehicles for Bifidobacterium animalis ssp. lactis HN019.

No presente trabalho avaliou-se o efeito antiparasitário de uma preparação probiótica constituída por quatro espécies de lactobacilos, L. casei ATCC 7469, L. plantarum ATCC 8014, L. fermentum ATCC 9338 e L. acidophillus ATCC 4536, no tratamento da eimeriose aviária causada por Eimeria acervulina. Para tanto, foram utilizados 120 animais, da espécie Gallus gallus, machos, com 14 dias de idade, distribuídos em seis grupos, sendo o Grupo 1 constituído por animais tratados apenas com água e ração (controle negativo); Grupo 2 constituído por animais que receberam diariamente uma dose da preparação probiótica contendo 1x108 UFC/mL; Grupo 3 constituído por animais infectados experimentalmente com 2x105 oocistos esporulados de Eimeria acervulina (controle positivo) e Grupo 4 onde os animais receberam diariamente uma dose da preparação probiótica contendo 1x108 UFC/mL durante o período do experimento e após 7 dias do início do tratamento, foram infectados com 2x105 oocistos esporulados de E. acervulina; no Grupo 5 os animais foram infectados com 2x105 oocistos esporulados de E. acervulina e paralelamente receberam uma dose diária da preparação probiótica em estudo contendo 1x108 UFC/mL; no Grupo 6 os animais foram infectados com 2x105 oocistos esporulados de E. acervulina e após o surgimento dos sinais clínicos da infecção passaram a receber diariamente uma dose da preparação probiótica contendo 1x108 UFC/mL, até o final do experimento. Durante 28 dias, semanalmente, amostras de sangue e fezes foram coletadas para determinação de OoPG, testes bioquímicos e parâmetros zootécnicos. Exames de fezes foram realizados diariamente para se determinar os períodos pré-patente e patente do parasita, bem como o efeito da preparação probiótica no combate a infecção. Os resultados demonstraram que a preparação probiótica em estudo reduziu a infecção intestinal causada por Eimeria acervulina, contribuindo para o bem estar animal. Referente aos parâmetros bioquímicos verificou-se efeito significativo sobre o perfil lipídico no 4º dia pós infecção, não sendo observado diferenças significativas para os demais parâmetros. No tocante ao desempenho zootécnico, verificou-se que a preparação probiótica não resultou em diferenças significativas em relação ao ganho de peso, eficiência e conversão alimentar dos animais avaliados. / This study evaluated the antiparasitic effect of a probiotic preparation which consisted of four species of lactobacilli (Lactobacillus casei ATCC 7469, L. plantarum ATCC 8014, L. fermentum ATCC 9338 and L. acidophilus ATCC 4536) in the treatment of eimeriosis in broilers caused by Eimeria acervulina. Therefore, during 28 days, 120 animals of Gallus gallus species, males, 14 days old, were distributed in six groups, as follow: Group 1 the animals received only feed and water \"ad libidum\" (negative control); Group 2 consisted of animals that received a daily dose of probiotic preparation which contained 1x108 CFU/mL; Group 3 the animals were experimentally infected with 2x105 oocysts of Eimeria acervulina (positive control); Group 4 the birds received a daily dose of probiotic preparation containing 1x108 CFU / mL during the experiment and after 7 days of the treatment beginning, these animals were infected with 2x105 oocysts of E. acervulina; Group 5 was composed by animals that were infected with 2x105 oocysts of the parasite and simultaneously received a daily dose of probiotic preparation containing 1x108 CFU / ml, Group 6 the animals were infected with 2x105 oocysts of E. acervulina and after clinical signs of infection were noticed, the birds were treated daily with a dose of the probiotic preparation containing 1x108 CFU / mL until the end of the experiment. During 28 days six blood and feces samples were collected and parameters such as oocyst elimination, histopathological, and biochemical were evaluated. Stool examinations were performed daily to determine the parasite prepatent and patent periods, and the probiotic effect on the infection. The results showed that the probiotic preparation under study reduced the intestinal infection caused by Eimeria acervulina, contributing to animal welfare. Regarding the biochemical parameters there was a significant effect on the lipid profile on the 4th day after infection, and no significant differences were observed for the other parameters. With regard to zootechnical performance, it was found that the probiotic preparation resulted in no significant differences in weight gain, and in feed conversion efficiency.

O aquarismo é uma atividade mundialmente difundida e um segmento extremamente grande da indústria de animais de estimação. O Brasil, em 2007, exportou o valor de US$ 5.871.576,73 em peixes. Devido à biologia dos peixes, todo seu manejo pode desencadear uma resposta fisiológica de estresse, que, dependendo da duração, tipo e espécie de manejo leva a uma resposta imunossupressora, que pode acarretar doença e morte aguda e consequentemente grande prejuízo à produção desses animais. O uso de imunoestimulantes, como suplementação dietética pode prover defesa inata e resistência a patógenos. O presente trabalho teve como objetivo avaliar a viabilidade da utilização de mananoligossacarídeo de levedo de cerveja (Saccharomyces cerevesiae), como imunoestimulante adicionado à ração oferecida a peixes da espécie Cyprinus carpio durante 45 dias. Para avaliar a imunidade dos peixes, foram feitas avaliações hematológicas periódicas e, no final do período determinado, os peixes foram desafiados imunologicamente com estresse e infectados com Aeromonas hydrophila. As carpas do grupo controle tiveram uma taxa de crescimento de 0,05 g/dia, conversão alimentar de 14,09 e eficiência protéica de 0,25, enquanto o grupo imunoestimulado obteve a taxa de crescimento de 0,11g/dia, conversão alimentar de 6,15 e eficiência protéica de 0,57. Não houve diferença estatística entre o resultado da hematologia dos animais do grupo controle e do grupo imunoestimulado. Dos animais infectados experimentalmente, 88% morreram em menos de 24 horas por choque endotóxico e, no exame post mortem, não houve diferença entre os grupos. A carpa que recebeu alimentação controle e foi infectada teve anemia macrocítica normocrômica, trombocitopenia, linfopenia, monocitose e aumento do número de CGE. A carpa que recebeu alimentação com MOS, foi infectada e sobreviveu, não apresentou alteração nos parâmetros hematológicos. A ração com MOS foi zootecnicamente melhor para a nutrição das carpas. Com base na taxa de sobrevivência e na avaliação hematológica, não há como responder se a ração suplementada com MOS foi imunologicamente melhor do que a ração controle. / The aquarium hobby is an activity wordwide spread and a segment extremely large of the pet industry. Brazil, in 2007, exported an amount of US$ 5.871.576,73 of fishes. Due to the biology of the fishes, all its management can trigger a physiological response leading to stress, that depending on the duration, type and specie, can conclude in immunosuppressive response, leading to disease and acute death, creating a great prejudice. The use of immunostimulants, as dietary supplementation may provide an innate defense against pathogens. This study aims to evaluate the feasibility of using mannan oligosaccharide of brewer yeast (Saccharomyces cerevesiae) as immunostimulants added to the feed for koi fishes (Cyprinus carpio) during 45 days. To evaluate the immunity of the fishes, periodic hematologic evaluations were made and at the end of the determined period, the fishes were immunologically challenged with stress and inoculation with Aeromonas hydrophila. The control group had a growth rate of 0,05g/day, feed conversion of 14,09 and protein efficiency ratio of 0,25, while the treatment group had growth rate of 0,11g/day, feed conversion of 6,15 and protein efficiency ratio of 0,57. The hematological results showed no statistical difference between the control group and immunostimulant group according. Analyzing the experimental infected animals, 88% died within 24 hours, due to endotoxic shock, and in the post mortem examination, there were no difference between groups. The koi that received control feed and it was infected had macrocytic normochromic anemia, thrombocytopenia, lymphopenia, monocytosis and increased of special granulocytic cells. The koi that received the MOS feed, was infected with A. hydrophila and survived, had no alteration on haematological parameters. The feed with MOS was zootechnical better than the control feed to carps. Based on the rate of survival and hematology, there are no possibility of answering if supplemented feed with MOS was immunologic better than control feed.

Nas últimas décadas, a incidência de infecções hospitalares causadas por bactérias Gram-negativas multirresistentes vem crescendo de maneira vertiginosa em todo o mundo, de modo que a Organização Mundial de Saúde (OMS) recentemente reconheceu essas infecções como uma preocupação mundial devido ao seu impacto negativo sobre as taxas de mortalidade intra-hospitalar e dos custos da assistência à saúde, afetando tanto os países desenvolvidos quanto os em desenvolvimento. Atualmente considera-se que o uso racional de antimicrobianos, a higienização das mãos e o isolamento de contato são as principais medidas disponíveis para contenção desse avanço. Porém, elas são apenas parcialmente efetivas e de implementação trabalhosa e onerosa. Assim, considera-se necessário o desenvolvimento de formas mais simples e eficientes para lidar com esse problema. No presente estudo, nos propusemos a avaliar o impacto da administração de um produto simbiótico a pacientes colonizados e/ou infectados por bactérias Gram-negativas multirresistentes sobre a incidência subsequente de infecções hospitalares relacionadas ao trato respiratório e urinário. Trata-se de um ensaio clínico randomizado, duplamente cego, controlado com placebo, cuja intervenção consistiu na administração oral ou enteral diária de 1010 unidades de Lactobacillus bulgaricus e 1010 unidades de Lactobacillus rhamnosus associados a fruto-oligosacarídeos durante 7 dias, a pacientes internados em um hospital terciário, com colonização prévia por bactérias Gram-negativas multirresistentes, demonstrada por meio de cultura seletiva de swab retal. O desfecho primário do estudo foi a incidência de infecção hospitalar posterior à intervenção, que, na análise do tipo intenção de tratar foi 18/48 (37,50%) no grupo experimental e 12/53 (22,64%) no grupo controle (odds ratio ajustado=1,95, IC95%=0,69-5,50, p=0,21). Os desfechos secundários principais, também de acordo com a análise intenção de tratar, foram: o tempo de internação hospitalar; sendo a mediana de 17 dias no grupo controle e 31 dias no grupo experimental (p= 0,07), taxas de óbito; com valores de 3,77% no grupo placebo e 8,33% no grupo simbiótico (odds ratio ajustado = 1,34, IC95%= 0,454,00, p= 0,61) e ocorrência de eventos adversos; 7,55% no grupo que utilizou placebo e 6,25% no grupo sob intervenção (p= 1,00). Os dados obtidos pelo estudo nos levam à conclusão de que o simbiótico estudado demonstrou-se inefetivo na prevenção de infecções hospitalares do trato respiratório e urinário em pacientes colonizados e/ou infectados por bactérias Gram-negativas multirresistentes. / In recent decades the incidence of multidrug resistant Gram-negative nosocomial infections has been dramatically raising in the whole world. The World Health Organization (WHO) recently recognized nosocomial infections as a global concern due to its negative impact on patients, health care workers and health care institutions, affecting developed countries as well as developing ones. They negatively impact in-hospital mortality and healthcare related costs. Antibiotic stewardship, hand hygiene promotion and contact precautions are the main available measures to control such multidrug resistant Gram-negative organisms in hospitals. However, they are only partially effective as well as difficult to be implemented and expensive. Therefore, simpler and more effective actions are thought to be helpful and urgent. In the main study, we propose to analyze the impact of the administration of a symbiotic product on patients colonized and/or infected by Gram-negative multidrug resistant bacteria upon the subsequent incidence of respiratory and urinary tract nosocomial infections. A randomized, double- blinded, placebo controlled, clinical trial was proposed in order to provide oral or enteral daily administration of 1010 units of Lactobacillus bulgaricus and 1010 units of L. rhamnosus associated with fructo-oligosacharide (FOS) during 7 days, to previously colonized patients with multi-resistant Gram-negative bacteria, identified through selective culture of rectal swab, hospitalized in a tertiary-care hospital. The primary outcome was the incidence of nosocomial infections after the intervention, which in the intention to treat analysis was 18/48 (37,50%) in the experimental group versus 12/53 (22,64%) in the control group (adjusted odds ratio= 1,95, IC95%= 0,69-5,50, p=0,21). Secondary outcomes, according to intention to treat analysis, were hospital length of stay: median of 17 days in the control group and 31 days in the symbiotic group (p= 0,07), mortality rates: 3,77% in the placebo group versus 8,33% in the experimental group (adjusted odds ratio = 1,34, IC95%= 0,45 4,00, p= 0,61) and adverse effects: 7,55% in the control group and 6,25% in the intervention group (p= 1,00). The results of this study leads to the conclusion that the studied symbiotic proved to be ineffective to prevent nosocomial respiratory and urinary tract infections in patients colonized and/or infected by Gram-negative multi-resistant bacteria.

Probiotics are marketed throughout the world to promote the health of the consumer by improving the microorganisms that normally occur in the intestinal tract (Tannock, 1997). It has also been suggested that probiotics can prevent pathogen infections by adhering to the intestinal mucosa (Lee, Lim, Teng, Ouwehand, Tuomola, & Salminen, 2000). While probiotics can be delivered to the infected areas in multiple fashions, microencapsulation is a newer form of delivering probiotics straight to the infected area. A whey protein microcapsule is thought to protect the probiotics from stomach acid and delivers the treatment to the affected area. To ensure this microencapsulation treatment is affective, the microcapsules will be stained and imaged to see if the microcapsules are constructed in a way which is consistent with the theory: a whey protein microcapsule surrounding bacteria and fat droplets. Through these experiments, it was shown that the microcapsule was not constructed as previously thought. Instead of a thin layer of protein surrounding the bacteria, it more closely resembled a solid ball of protein with bacteria and fat trapped inside. The bacteria are able to survive stomach like conditions (0.1M HCl for 8 hours) due to other forms of microencapsulation.

Bacteria communicate with their hosts in part via surface, secreted and transmembrane proteins (collectively the secretome) resulting in probiotic (beneficial) or pathogenic (harmful) outcomes to the host. Therapeutic benefits of probiotic bacteria have been shown previously, but the molecular mechanisms and the health-promoting effector components involved are still being elucidated. Some evidence suggests that probiotic bacteria can competitively adhere to intestinal mucus and displace pathogens. The adherence of probiotic bacteria to human intestinal mucus and cells appears to be mediated, at least in part, by secretome proteins. Secretome proteins-encoding open reading frames can be identified in bacterial genome sequences using bioinformatics. However, functional analysis of the translated secretome is possible only if many secretome proteins are expressed and purified individually. Phage display technology offers a very efficient way to purify and functionally characterise proteins by displaying them on the surface of the bacteriophage. While a phage display system for cloning secretome proteins has been previously reported it is not efficient for enrichment and display of Gram-positive secretome proteins. In this study a new phage display system has been developed and applied in direct selection, identification, expression and purification of Gram-positive Lactobacillus rhamnosus strain HN001 secretome proteins. The new phage display system is based on the requirement of a signal sequence for assembly of sarcosyl-resistant filamentous phage virions. Using this system 89 secretome open reading frames were identified from a library of only 106 clones, performing at least 20-fold more efficiently than the previously reported enrichment method. Seven of the identified secretome proteins are unique for L. rhamnosus HN001. A L. rhamnosus HN001 shot-gun phage display library was also constructed to capture proteins that mediate adhesion or aggregation, initial steps in establishing host-microbe contact or forming multicellular aggregates, both of which may lead to beneficial effects – colonisation of the gastro-intestinal tract and exclusion of pathogens. In search for proteins involved in adhesion, a L. rhamnosus HN001 shot-gun phage display library was screened against the human extracellular matrix component fibronectin commonly used as binding target by bacteria that colonise diverse tissues. This screen selected, instead of a fibronectin-binding protein, a protein that binds to avidin, used to immobilise biotinylated fibronectin. Affinity screening of the shot-gun library for binding to L. rhamnosus HN001 cells identified a secretome protein, Lrh33, as an HN001-cell surface binding protein. This protein contains two bacterial immunoglobulin-like domains type 3. Analysis of phage-displayed nested deletions of Lrh33 determined that the proximal (N-terminal) immunoglobulin-like domain is not sufficient for binding; only the constructs displaying both domains demonstrated binding to HN001. Lrh33 does not have any similarity to previously identified Lactobacillus-binding proteins and no match in the NCBI database (at a cutoff value of > e-13), hence it represents potentially a new type of bacterial auto-aggregation protein.

In this study, strains of probiotic bacteria have been selected for tolerance to low pH, bile, sucrose, oxygen in media and low storage temperatures. Lactobacillus acidophilus 2401 and Bifidobacterium infantis 1912 were selected as strains able to survive in these conditions. These two strains were then offered further protection from the adverse conditions of food processing and storage by microencapsulation in a calcium alginate and starch gel matrix. Encapsulation in calcium alginate increases survival in yoghurt. In cheddar cheese the free L. acidophilus 2401 and B. infantis 1912 cells survived better than the encapsulated cells, probably due to the dense nature of the cheddar cheese matrix combined with the encapsulation restricting the flow of the nutrients and metabolites between the outside environment and the cells. In ice cream survival was high, probably due to the high fat and solids nature of the ice cream combined with the low storage temperature. The trial results of the laboratory scale production was consistent with the survival results for yoghurt and cheddar cheese. Incorporation of encapsulated probiotic bacteria into ice cream and cheddar cheese was acceptable by sensory standards and largely unnoticeable in comparison with the same foods without capsules. The capsules were visible and able to be felt on the tongue when eaten in yoghurt causing the product to be disliked by the panellists. / Master of Science (Hons)

Background: The World Health Organization defines probiotics as ‘live microorganisms which, when administered in adequate amounts, confer a health benefit to the host’. Traditionally, probiotic microorganisms have been used to prevent or treat gastrointestinal tract diseases. In the last 15 years, there has been increasing interest of a possible probiotic impact on the oral microbiota and dental caries. Dental caries is a multifactorial disease, and the causative factor in the oral microbiota includes a shift from a balanced microflora to a microflora that includes more aciduric species such as mutans streptococci (MS), non-mutans streptococci, and Actinomyces. MS is considered an opportunistic pathogen although several other bacteria also contribute to the disease. Early acquisition of MS is associated with early development of caries; therefore a desirable complement to other prophylactic measures would be a MS colonization inhibitor. Objective: To better understand how selected strains of probiotic lactobacilli interact with MS in vitro and in vivo and to study the impact of probiotic lactobacilli on caries development during childhood. Material and methods: The in vitro properties of probiotic lactobacilli were studied with regard to (i) acid production from sugars and sugar alcohols, (ii) growth inhibition capacity on clinical isolates and reference strains of MS as well as Candida albicans and (iii) the capacity to co-aggregate with MS. A randomized controlled trial (RCT) tested the short-term effect of intervention with two Lactobacillus reuteri strains on MS, which was evaluated after treatment with chlorhexidine. The re-growth patterns of MS and 19 other selected strains were also evaluated. In the second clinical study  we investigated the long-term effect on MS prevalence and dental caries after an intervention with Lactobacillus paracasei ssp. paracasei F19 (LF19) between 4 and 13 months of age. Results: The results from the in vitro testing showed that strains of probiotic lactobacilli differed in their fermentation patterns, inhibition capacity and their capacity to co-aggregate, which should be kept in mind in the translation to clinical research. The clinical study on short-term effects of two L. reuteri strains on MS and other oral strains showed no effect on re-growth patterns after intervention. The clinical study on long-term effects of LF19 showed no effect on the prevalence of MS. Furthermore, the clinical follow-up at 9 years of age showed no differences in either decayed, missing, and filled surface (dmfs) or DMFS between the probiotic and placebo groups. Evaluation of saliva samples showed no signs of oral colonization with LF19 in the study group. Conclusion: The in vitro testing showed potentials of the selected probiotic Lactobacillus strains for interference with MS and C. albicans. The results from the clinical studies showed no such effect on MS or dental caries. Evidence regarding the effectiveness of specific probiotic applications in the prevention of dental caries is limited and does not allow for conclusions concerning the use of probiotic bacteria as a preventive measure.