Dissertations / Theses on the topic 'Bifidobacterium bifidum'

La production de beta-d-galactosidase (EC. 3. 2. 1. 23), de n-acetyl-beta-d-glucosaminidase (EC. 3. 2. 1. 30), de neuraminidase (EC. 3. 2. 1. 18) et d'alpha-l-fucosidase (EC. 3. 2. 1. 51) a été optimisée par fermentation. La bactérie anaérobie Bifidobacterium bifidum souche AA/22 a été cultivée en milieu liquide renfermant par litre : 37 g d'un extrait sec de cerveau-cœur, 10 g de glucose et des facteurs bifidigènes (1 g de gynolactose ou 66 ml de lait de femme). La fermentation est conduite sous N2/CO2 à 37°C et à ph 6,8. L'extraction des systèmes enzymatiques par une technique aux ultra-sons a été optimisée en tampon phosphate de sodium 20 mm ph 6,8 renfermant de l'EDTA 20 mm et 0,1 p. 100 de nonidet P 40. Après centrifugation d'une heure à 100 000 g la solution enzymatique obtenue renferme: 7 U de beta-d-galactosidase, 1,6 U de n-acetyl-beta-d-glucosaminidase, 90 mU de neuraminidase et 400 mU de fucosidase par mg de protéine. Une neuraminidase a été purifiée jusqu'à homogénéité en électrophorèse dénaturante (SDS). La masse moléculaire de l'enzyme native est de 75 kDa, elle est constituée par deux sous-unités identiques (35 kDa). Le phi, la température et le ph optimum d'action sont respectivement de 4,2, 50°C et 5,0. Cette neuraminidase est sensible à l'action de la température, elle n'est stable qu'en dessous de 48°C. L'activité est indépendante des métaux. Elle permet l'hydrolyse des liaisons alpha 2,3 plus rapidement que les alpha 2,6 et les alpha 2,8. L'enzyme agit aussi bien sur les glycopeptides que sur les glycoprotéines natives.

Nous avons etudie la capacite de bifidobacterium bifidum dsm 20082 a assimiler des saccharides. Cette etude a ete realisee en utilisant la technique de microcalorimetrie. Les resultats obtenus montrent que bifidobacterium bifidum est capable de croitre sur milieu riche (tpy) en presence ou en absence de saccharides. Sur milieu semi-synthetique (garches modifie), seules les n-acetylhexosamines permettent une croissance bacterienne, elles sont donc assimilees comme seule source de carbone. De plus, la quantite de chaleur degagee est proportionnelle a la consommation du saccharide. Les facteurs bifidogenes ne sont en realite que des nutriments preferentiels des bifidobacteries. Nous nous sommes interesses ensuite aux mecanismes d'incorporation de differents saccharides, glucose, fructose, galactose, n-acetylglucosamine, lactose et lacto-n-biose. La determination du mecanisme de transport de chaque saccharide a ete realisee a l'aide d'inhibiteur de translocation et de l'etude de la phosphorylation des saccharides. Les six mecanismes de transport se sont averes etre constitutifs. Le saccharide utilise lors de la culture n'entraine qu'une adaptation de la bacterie au saccharide, voire aucun changement dans la capacite d'incorporation. Les systemes de transport montrent une grande specificite vis-a-vis du saccharide qu'ils transportent. Le glucose est vehicule par un symport a cation regule par les ions potassium, avant d'etre phosphoryle par une glucokinase atp-independante. Le galactose traverserait la membrane par un mecanisme de diffusion. La galactokinase est activee en presence d'atp. Le lacto-n-biose et le lactose sont incorpores par des symports a proton. La -d-galactosidase est presente en grande quantite au niveau intracellulaire afin de liberer le glucose et le galactose. Le fructose est vehicule par un symport a proton. La fructokinase voit son activite diminuer de 60% en presence d'atp. Bifidobacterium bifidum aurait trouve a ce niveau un moyen de reguler le transport du fructose par un mecanisme de retro-inhibition.

Quatre activités exoglycosidasiques de bifidobacterium bifidum souche AA/22 ont été étudiées. Il s'agit des activités: béta-galactosidase, n-acétyl-beta-hexosaminidase, alpha-neuraminidase et alpha-fucosidase. Ces activités sont endo-cellulaires, leur extraction est réalisée par ultra-sons. Le schéma de purification suivi nous a permis d'obtenir six fractions. Trois fractions ne contenant qu'une seule activité : une alpha-neuraminidase et deux beta-galactosidases. Trois fractions possèdent chacune deux activités : une béta-galactosidase et une alpha-fucosidase ; une alpha-neuraminidase et une n-acétyl-béta-hexosaminidase ; une béta-galactosidase et une n-acétyl-béta-hexosaminidase. Toutes ces fractions ne révèlent qu'une seule bande en électrophorèse non dénaturante. Nous avons vérifié que celles-ci correspondaient bien aux activités suivies. Les deux activités béta-galactosidase purifiées séparément sont homogènes en électrophorèse dénaturante. Toutes ces activités ont des températures optimales comprises entre 40 et 48°C. Les pH optimaux sont compris entre 5 et 6. 5 et les masses moléculaires relatives déterminées en gel filtration sur Superose 6 varient de 97000 à 420000. Les pHi sont acides (4 a 5. 1). Seule une activité béta-galactosidase est sensible à l'EDTA et à l'EGTA, de plus Elle est activée par le calcium et le magnésium. Les paramètres enzymatiques des béta-galactosidases ont été réalisés sur le pNP-Gal et sur le lactose, ils mettent en évidence trois groupes de réactivité différente. Tous ces paramètres nous permettent de conclure que Bifido bacterium bifidum souche AA/22 possède au moins trois béta-galactosidases différentes. Nous n'avons pas prouvé que dans les fractions contenant deux activités, nous étions en présence d'un complexe enzymatique. Mais nous avons montré que la présence d'une activité influençait la réactivité de l'autre activité présente.

In this thesis, the field of the bifidobacteria! physiology in relation to β- and α-galactosidases is described and perspectives of them for oligosaccharides synthesis are discussed- The experimental work focused on the isolation and characterisation of these cell constituents from Bifidobacterium bifidum NCIMB41171. To generate a basis for subsequent work the construction of a genomic DNA library was carried out. This resulted in the isolation of four different β-galactosidases (bbgI, bbgII, bbgIII and bbgIV) and one α-galactosidase (melA) genes. Molecular characterisation of the gene products indicated their putative location, which was subsequently confirmed after expression in an E. coli host. Nucleotide and amino acid sequence comparisons with other known bifidobacterial enzymes revealed aspects of either their evolution or conservation among species.

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A adição de culturas probióticas tem sido testada em vários produtos lácteos, e a maior parte dos microrganismos consegue permanecer viável, alcançando características desejáveis no produto final. O objetivo da pesquisa foi desenvolver requeijão cremoso com probióticos, Lactobacillus acidophilus e Bifidobacterium bifidum, a fim de se tornar uma alternativa saudável de um alimento de amplo consumo. Foram elaboradas 3 formulações: requeijão cremoso fresco padrão, requeijão cremoso fresco com probióticos, requeijão cremoso fresco probiótico com reduzido teor de gordura. Para a caracterização do produto foram realizadas análises para determinação da composição centesimal, em triplicata para determinação de umidade, cinzas, lipídios e proteínas, e carboidratos. A contagem de coliformes totais e Escherichia coli foram realizadas nas amostras para análise da qualidade higiênico-sanitária. Realizou-se a contagem de L. acidophilus e B. bifidum durante o período de armazenamento de 15 dias. A avaliação da resistência das culturas probióticas às condições digestivas foi realizada empregando-se um modelo in vitro, utilizando suco gástrico e entérico simulados e enzimas do trato gastrintestinal. A análise da cor foi realizada por colorimetria em escala CIELab. A análise sensorial foi aplicada pelo teste de aceitação por escala hedônica para avaliação dos atributos e avaliação da qualidade global e intenção de compra. A composição centesimal dos requeijões padrão e adicionado de probióticos atendeu aos requisitos da legislação para o produto. O requeijão proposto como light apresentou redução de 26% no valor calórico total e 43% no teor de lipídeos em relação ao padrão. Quanto a acidez titulável, observou-se que o armazenamento influenciou significativamente os seus valores nas preparações com probióticos, diferindo do padrão que não apresentou alterações nos valores durante o armazenamento. A qualidade higênico-sanitária foi satisfatória, visto que as amostras foram negativas para presença de coliformes totais e Escherichia coli. As contagens individuais de Lactobacillus acidophilus e Bifidobacterium bifidum, estiveram de acordo com as exigências da legislação para alimentos com alegações de propriedades funcionais. O requeijão cremoso fresco probiótico apresentou contagem de L. acidophilus de 4,0x107UFC/g e B. bifidum de 2,0x108UFC/g aos sete dias de armazenamento e o requeijão cremoso fresco probiótico com reduzido teor de gordura apresentou contagem de L. acidophilus de 5,0x109UFC/g e B. bifidum de 2,0x109UFC/g. A redução de gordura no produto até os níveis estudados não interferiu na viabilidade dos probióticos no requeijão cremoso durante o armazenamento de 15 dias. Após a simulação da digestão in vitro, houve redução da população total de probióticos em ambas as formulações com resultados mais expressivos para a formulação com reduzido teor de gordura afirmando o efeito protetor das gorduras para a sobrevivência dos probióticos ao trato gastrintestinal. A análise instrumental da cor dos requeijões cremosos indicou produtos com boa luminosidade, com acentuação da cor amarela ao longo do tempo de armazenamento. A avaliação sensorial demonstrou que a adição de probióticos resultaram em requeijões cremosos com melhor perfil de características e maior aceitabilidade e intenção de compra que a preparação padrão. Pode-se concluir que os probióticos melhoraram a qualidade sensorial do produto, sendo tais amostras preferidas pelos julgadores. O requeijão cremoso tradicional e com baixo teor de gordura são tecnologicamente viáveis para a incorporação das culturas probióticas de L. acidophilus e B. bifidum.

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Two experiments were conducted to evaluate the effect of using probiotics and symbiotic in piglets diets in the nursery phase. The first experiment was the performance of piglets from 21 to 35, from 36 to 49 days and in total period, being also held the count of lactic bacteria, fecal coliforms and clostridia in the piglets faeces at 35 and 49 days old. The economic viability of probiotics and symbiotic used in piglets diets was also evaluated. There were used 120 piglets, with 21 days old, with initial average weight of 5.80 ± 0.30 kg, distributed in a randomized blocks design, in a factorial scheme consisting of two levels of probiotics (0.30 and 0.60%) and three levels of inulin inclusion (0.00; 0.25 and 0.50%), totalizing six treatments with five replicates. In the metabolism trial were used 24 barrows, with average initial weight of 18.00 ± 0.38 kg, distributed individualy in metabolic cages, and the experimental design and treatments used were the same described in the performance experiment, with four replicates. In this trial were determined the values of digestible energy, metabolizable energy, the digestibility and metabolizability coefficients of gross energy and at the end of metabolism trial, were collected fecal samples, by means of retal massage to determine fecal pH. The inclusion of probiotic and inulin in the diets did not influence (P>0.05) the performance of piglets, from 21 to 35 and from 21 to 49 days of age, the cost index, the index of economic efficiency, the count of lactic bacteria, fecal coliforms and clostridia in the faeces. In the metabolism trial was observed that the inclusion of probiotics in the diets did not influence in the energy metabolization. Nevertheless, the levels of inclusion of inulin reduced (P<0.05) the digestible energy and the digestibility coefficient of gross energy, but did not influence in the coefficient of gross energy metabolization. The pH fecal values were not influenced by the inclusion of probiotic and symbiotic. The use of probiotic and inulin, and their association, did not change the performance, thefaeces microbial counts, the economic viability and gross energy metabolization of piglets from 21 to 49 days old
Foram realizados dois experimentos para avaliar o efeito do uso de probióticos e simbiótico em rações para leitões na fase de creche. O primeiro experimento foi o de desempenho dos leitões dos 21 aos 35, dos 36 aos 49 dias e no período total, sendo também realizada a contagem das bactérias lácticas, coliformes e clostrídios nas fezes dos leitões, aos 35 e 49 dias de idade. A viabilidade econômica da utilização dos probióticos e simbiótico nas rações dos leitões também foi avaliada. Foram utilizados 120 leitões, com 21 dias de idade, com peso médio inicial de 5,80 ± 0,30 kg, distribuídos em um delineamento experimental de blocos ao acaso, em um esquema fatorial constituído de dois níveis de probióticos (0,30 e 0,60%) e três níveis de inclusão de inulina (0,00; 0,25 e 0,50%), totalizando seis tratamentos com cinco repetições. No ensaio de metabolismo foram utilizados 24 suínos machos castrados, com peso vivo médio inicial de 18,00 ± 0,38 kg, distribuídos individualmente em gaiolas de metabolismo, e o delineamento experimental e tratamentos utilizados foram os mesmos descritos no experimento de desempenho, com quatro repetições. Foram determinados os valores de energia digestível, energia metabolizável, os coeficientes de digestibilidade e metabolizibilidade da energia bruta e no final do ensaio metabólico foram coletadas amostras de fezes, por meio de massagem retal, para determinar o pH fecal. A inclusão do probiótico e inulina nas rações não influenciou (P>0,05) o desempenho dos leitões, dos 21 aos 35 e dos 21 aos 49 dias de idade, o índice de custo, o índice de eficiência econômica, a contagem de bactérias acidoláticas, coliformes e clostrídios das fezes. No ensaio metabólico foi observado que a inclusão de probiótico nas rações não influenciou na metabolizibilidade da energia. No entanto, os níveis de inclusão de inulina reduziram (P<0,05) os valores de energia digestível e o coeficiente de digestibilidade da energia bruta, mas não influenciaram no coeficiente de metabolizibilidade da energia bruta. Os valores de pH fecal não foram influenciados pela inclusão de probiótico e simbiótico. O uso de probiótico e inulina, e sua associação, não alteraram o desempenho, a contagem de micro-organismos, a viabilidade econômica e a metabolizibilidade da energia bruta de leitões dos 21 aos 49 dias de idade

Lactic acid bacteria have been reported to be used as a health adjunct in food for u many years. However, these health benefits have not been proven. and how these bacteria pass through the digestion process and remain viable in the human intestinal tract is still not clear. The aim of this work was to isolate mutants from Lactobacillus acidophilus or Bifidobacterium bifidum that could tolerate the conditions of the digestion process (low pH and bile conduction) and to characterize these isolated mutants. Acid- and bile-tolerant mutants of L. acidophilus were isolated from parental strains successfully using natural selection techniques. These mutants survived and grew at conditions of pH 3.5 with 0.2% mixed bile salts added. After the selection, phenotypic characterization was identified to further clarify desirable traits for use as probiotic adjuncts in foods. These phenotypic characteristics included protease, aminopeptidase, ß-galactosidase, and bile salt hydrolase activity. Based on different protease, aminopeptidase, and ß-galactosidase activity, selected acid- and bile-tolerant mutants contained different growth characteristics compared with their parents. All the isolates tested showed different bile salt hydrolase activity, and this activity was not strain and medium dependent. Plasmid profiles and fatty acid analysis were conducted to provide more information of these acid/bile tolerant isolates and whether or not they were mutants from their parent strains rather than only adapted variants. Results showed the acid-/bile-tolerant isolates contained different plasmid profiles and cell wall fatty acids compared with their parents, which indicated these isolates were mutants. Protein expression by two-dimensional gel electrophoresis showed different protein expression patterns between acid- and bile-tolerant mutants and their parents. fm1her suggesting these isolates were mutants. We observed the protein production in parent strains decreased as the pH decreased. and protein expression in mutants remained the same as pH decreased. Two of the proposed health benefits of probiotic bacteria are anticholesterol activity and antimicrobial activity. These were evaluated using selected acid- and bile-tolerant mutants. Results showed no decrease of cholesterol in the test medium during bacterial growth. The observed antimicrobial activity was due to the presence of active cells. and this may relate to the acid production during cell growth and not to the production of antimicrobial substances. We concluded that the acid-/bile-tolerant isolates were mutants, and they survived and grew better in harsh environments compared with their parent strains. These mutants may be useful as a food adjunct in the future, but further study is needed to establish their use and possible probiotic benefits.

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The objective of this work was evaluate the effect of different levels of inclusion of inulin (chicory root extract) in diets containing ingredients of plant and animal on performance, carcass characteristics (cuts and abdominal fat) and the parameters blood, for broiler chickens 10-40 days old. 1056 chicks were used from one day old, housed on reused litter in a completely randomized design with eight treatments and six replicates of 22 birds each, which were arranged in a factorial 2 x 4, where the first factor was the use of two diets (plant and animal) and the second factor represented by four levels of inulin (0.00, 0.25, 0.50, 0.75%). The probiotic used was composed of Lactobacillus acidophillus, Bifidobacterium bifidum and Streptococcus faecium. The characteristics evaluated were weight gain, final weight, average feed intake, feed conversion, viability 1-40 days of age. At 21 and 35 days of age were measured blood parameters (calcium, phosphorus, uric acid and total protein). At 40 days was carried out the slaughter of birds to assess the yield of carcass, breast, drumstick, thigh, wing and abdominal fat percentage. Within the analysis of economic viability was calculated the cost of the experimental diets, the index of economic efficiency and cost index. In the initial phase and growth was not found significant results for the performance variables. In the total periods of 1-40 days of age for the variable feed no significant effect (P <0.05) for levels of inclusion. Carcass yield was influenced (P <0.05) by food type regardless of the levels of inclusion of inulin. For the cuts, only the thigh showed a significant difference (P <0.05) for the type of diet. Was observed in abdominal fat interaction (P <0.05) between type of diet (plant and animal) and the levels of inclusion of inulin. For the blood parameters for 21 days was significantly different (P <0.05) between levels of inulin inclusion for the variables P and total protein, and 35 was again a significant difference (P <0.05) for phosphorus . The other variables were not affected with the use of inulin. The use of 2.5 g / kg inulin provides better feed for broilers at 1-40 days old. Inclusion of inulin reduces abdominal fat independent mind the type of diet. The use of feed containing animal ingredients provides improved higher carcass yield and higher thigh yield. The use of inulin affected the phosphorus stock you live at 21 and 35 days old. The use of inulin in diets of broiler feed cost rises mind independent of the use of ingredients of animal or vegetable
Objetivou-se com este trabalho avaliar o efeito de diferentes níveis de inclusão de inulina (extrato de raiz chicória) em rações contendo ingredientes de origem vegetal e animal, sobre o desempenho, as características de carcaça (cortes nobres e gordura abdominal) e os parâmetros sanguíneos, para frangos de corte de 1 a 40 dias de idade. Foram utilizados 1056 pintos de um dia de idade, alojados sobre cama reutilizada, em um delineamento experimental inteiramente casualizado, com oito tratamentos e seis repetições e 22 aves por unidade experimental, os quais foram arranjados em um esquema fatorial 2 x 4, onde o primeiro fator foi a utilização de duas dietas (vegetal e animal) e o segundo fator representado por quatro níveis de inclusão de inulina (0,00; 0,25; 0,50; 0,75%). O probiótico utilizado era composto por Lactobacillus acidophillus; Streptococcus faecium e Bifidobacterium bifidum. As características avaliadas foram ganho de peso, peso final, consumo médio de ração, conversão alimentar, viabilidade de 1 a 40 dias de idade. Aos 21 e 35 dias de idade foram mensurados os parâmetros sanguíneos (cálcio, fósforo, ácido úrico e proteínas totais). Aos 40 dias foi realizado o abate das aves para avaliar o rendimento de carcaça, peito, coxa, sobrecoxa, asa e percentual de gordura abdominal. Dentro da análise da viabilidade econômica foram calculados os custos das dietas experimentais, o índice de eficiência econômica e o índice de custo. Na fase inicial e de crescimento não foi encontrado resultado significativo para as variáveis de desempenho. No período total de criação de 1 a 40 dias de idade, para a variável conversão alimentar houve efeito significativo (P<0,05) para os níveis de inclusão. O rendimento de carcaça foi influenciado (P<0,05) pelo tipo de ração independente dos níveis de inclusão de inulina. Para os cortes, somente a coxa apresentou diferença significativa (P<0,05) para o tipo de ração. Na gordura abdominal foi observado interação (P<0,05) entre o tipo de ração (vegetal e animal) e os níveis de inclusão de inulina. Para os parâmetros sanguíneos aos 21 dias foi observada diferença significativa (P<0,05) entre os níveis de inclusão inulina para as variáveis fósforo e proteínas totais, e aos 35 novamente foi observada diferença significativa (P<0,05) para o fósforo. As demais variáveis não foram afetadas com a utilização da inulina. A utilização de 2,5g/kg de inulina proporciona melhor conversão alimentar para aves de 1 a 40 dias de idade. A inclusão de inulina reduz a deposição de gordura abdominal independentemente do tipo de ração. A utilização de ração com ingredientes de origem animal proporciona melhor rendimento de carcaça e maior rendimento de coxa. A utilização de inulina afetou os níveis de fósforo circulante aos 21 e 35 dias de idade. A utilização de inulina em rações de frango de corte eleva o custo da ração independente mente da utilização de ingredientes de origem animal ou vegetal

Els efectes beneficiosos que els oligosacàrids de la llet materna (OLM) confereixen a la salut dels lactants s'ha estudiat durant anys. Aquests oligosacàrids proporcionen una barrera protectora i un suport nutritiu essencials, als quals no tenen accés els nens que no prenen llet materna. La llet humana és considerada única respecte a la resta de llets de mamífers pel que fa a la quantitat i la complexitat d'oligosacàrids. Actualment, s'han identificat més de 130 estructures químiques diferents de OLM, i no es disposa de cap recurs natural que proporcioni accés a aquestes estructures tan complexes i a bastament. De la mateixa manera, la síntesi química és complicada a causa de l'estructura tan complexa i diversa que presenten els OLM, i de moment, la síntesi en gran escala no ha estat possible. La síntesi enzimàtica, en canvi, es presenta com una eina alternativa de síntesi d'aquestes molècules complexes atès que, en la naturalesa els enzims són els responsables de formar enllaços glicosídics entre carbohidrats amb alta regio- i estereoselectivitat. L'objectiu d'aquesta tesi és avaluar l'ús de l'enzim Lacto-N-biosidasa de Bifidobacterium bifidum (LnbB) com un biocatalizador eficient des de dues perspectives diferents: i) l'estudi estructural-funcional de LnbB i ii) la generació de biocatalizadors capaços de sintetitzar l'oligosacàrid d'interès (lacto-N-tetraosa) mitjançant enginyeria de proteïnes en l'enzim LnbB. En aquesta tesi, hem analitzat l'organització dels dominis dels enzims de la família GH20, i, en conseqüència, hem definit dos models d'arquitectures. El Model A conté almenys dos dominis, un domini GH20b no catalític i el GH20 catalític, que sempre es presenta acompanyat d'una α-hèlix extra. En canvi, el Model B consisteix únicament en el domini catalític GH20. Mitjançant l'expressió de diferents formes truncades de LnbB, hem descrit els requeriments estructurals per a la funcionalitat dels enzims GH20, i en particular per LnbB, per tal d’obtenir la unitat funcional mínima que conservi l'activitat enzimàtica. Respecte a la síntesi de la lacto-N-tetraosa usant com a biocatalizador noves proteïnes de LnbB obtingudes mitjançant enginyeria de proteïnes, hem contemplat dues estratègies enzimàtiques diferents. En primer lloc, l'estratègia de glicosintasa, en la qual l'enzim (amb mutació en el residu assistent) és capaç de transferir el corresponent donador activat (sucre sintètic derivat d’oxazolina) a un acceptor, sense hidròlisi del producte. En segon lloc, l'estratègia de transglicosilació millorada, en la qual, una nova generació de mutants en els llocs d'unió a l'acceptor seran capaços d'acomodar de manera més favorable un sucre en lloc d'una molècula d'aigua, i d'aquesta manera, augmentar l'activitat de transglicosilació.
Los efectos beneficiosos que los oligosacáridos de la leche materna (OLM) confieren a la salud de los lactantes se han estudiado durante años. Estos oligosacáridos proporcionan una barrera protectora y un soporte nutritivo esenciales, a los que, los niños que no toman leche materna no tienen acceso. La leche humana se considerada única respecto al resto de leches de mamíferos en cuanto a cantidad y complejidad de oligosacáridos. Actualmente, se han identificado más de 130 estructuras químicas diferentes de OLM, y no se dispone de ningún recurso natural que proporcione acceso a estas estructuras tan complejas y en cantidad suficiente. Del mismo modo, la síntesis química es complicada debido a la estructura tan compleja y diversa que presentan los OLM, y por el momento, la síntesis en gran escala no ha sido posible. La síntesis enzimática, en cambio, se presenta como una herramienta alternativa de síntesis de éstas moléculas complejas dado que, en la naturaleza las enzimas son las responsables de formar enlaces glicosídicos entre carbohidratos con alta regio- y estereoselectividad. El objetivo de esta tesis es evaluar el uso del enzima Lacto-N-biosidase de Bifidobacterium bifidum (LnbB) como un biocatalizador eficiente desde dos perspectivas diferentes: i) el estudio estructural-funcional de LnbB y ii) la generación de biocatalizadores capaces de sintetizar el oligosacárido de interés (lacto-N-tetraosa) mediante ingeniería de proteínas en el enzima LnbB. En esta tesis, hemos analizado la organización de los dominios de enzimas GH20, y, en consecuencia, hemos definido dos modelos de arquitecturas de dominio. El Modelo A contiene al menos dos dominios, un dominio GH20b no catalítico y el GH20 catalítico, que siempre se presenta acompañado de una α-hélice extra. Por el contrario, el Modelo B consiste únicamente en el dominio catalítico GH20. Mediante la expresión de diferentes formas truncadas de LnbB, hemos descrito los requerimientos estructurales para la funcionalidad de las enzimas GH20, y en particular para LnbB, de modo que se obtenga la unidad funcional mínima que conserve la actividad enzimática. Respecto a la síntesis de la lacto-N-tetraosa usando como biocatalizador nuevas proteínas de LnbB obtenidas mediante ingeniería, hemos contemplado dos estrategias enzimáticas diferentes. En primer lugar, la estrategia de glicosintasa, en la que el enzima (un mutante en el residuo asistente) es capaz de transferir el correspondiente dador activado (azúcar sintético derivado de oxazolina) a un aceptor, sin hidrólisis del producto. En segundo lugar, la estrategia de transglicosilación mejorada, en la que, una nueva generación de mutantes en los sitios de unión al aceptor serán capaces de acomodar de manera más favorable un aceptor de azúcar en lugar de una molécula de agua, y de este modo, aumentar la actividad de transglicosilación.
The potential health benefits of human milk oligosaccharides (HMO) have been studied for many years. It is well known that these oligosaccharides provide a protective barrier and nutritive support that infants with poor access to breast milk do not acquire in the first years of life. Human milk is considered to be unique among mammals in terms of the quantity and complexity of its oligosaccharides. To date, 130 chemical structures within HMO have been identified. No other natural resources provide access to these complex oligosaccharides in such large amounts, and until now, large scale synthesis of HMO has not been possible by any traditional organic chemistry methodology. Enzymatic synthesis is an alternative synthetic tool since enzymes can form the new glycosidic linkage between carbohydrates with high regio- and stereoselectivity. The objective of this thesis is to evaluate the use of Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) as an efficient biocatalyst in the following two ways: i) the structural-functional study of LnbB and ii) protein engineering of LnbB to generate biocatalysts able to synthesize the target lacto-N-tetraose. Here, we have analysed the domain organization of GH20 enzymes, and accordingly, have defined two models of domain architectures. Model A, contains at least two domains, a non-catalytic GH20b domain, and the catalytic GH20 which is always accompanied with an extra α-helix. In contrast, Model B consists only of the catalytic GH20 domain. By expressing different truncated forms of LnbB, we have described the structural requirements for functionality of GH20 enzymes, and in particular for LnbB, to obtain a minimal functional unit that retains the enzymatic activity. With regard to the synthesis of lacto-N-tetraose using new engineered LnbB proteins as biocatalysts, we envisage two different enzymatic strategies. First, the glycosynthase strategy, in which the activated donor is the corresponding synthetic sugar oxazoline and the enzyme, a mutant on the assisting residue, is able to transfer the donor to an acceptor without hydrolysis of the product. Second, the enhanced transglycosidase strategy, in which, a new generation of mutants on the acceptor subsites of the enzyme will be able to more favourably accommodate a sugar acceptor instead of water, and thus, increase transglycosylation activity.

En milieu acide, l'hydrolyse du lactose par B. Bifidum entraîne la formation d'oligosaccharides. Les produits néosynthétisés (depuis les disaccharides jusqu'aux nonasaccharides) représentent 30% (p/p) des saccharides totaux. Ils se caractérisent par un regreffage spécifique du galactose par des liaisons beta (1-3) glycosidiques. L'enzyme responsable de cette activité de transgalactosylation a été purifiée. C'est une beta-d-galactosidase membranaire qui ne représente que 0,8% de l'activité hydrolytique initiale. Elle résulte d'un assemblage de plusieurs sous-unités de masse moléculaire apparente de l'ordre de 362 kDa. Son point isoionique est de 5,25. L'étude des propriétés enzymatiques a montré que les conditions de transfert (pH 4,8 et température de 45°C, Km 800 mM) sont très différentes des conditions d'hydrolyse (pH 6,5 et 37°C, Km 13 mM). La réaction est sensible au calcium qui est un activateur de l'activité hydrolytique au détriment de la réaction de transfert. La transgalactosylation ne peut s'effectuer que sur des accepteurs glycosylés possédant une structure parfaitement définie (fonction hémiacétalique bloquée, structure pyranique, fonction hydroxyle du C2 en position équatoriale). L'ensemble des résultats nous amène à proposer deux modèles pour expliquer la synthèse par mécanisme intermoléculaire des oligosaccharides. Le rôle biologique des produits de transgalactosylation à partir de lactose a été abordé.

The presented diploma thesis is focused on the preparation and the monitoring of the biological stability of beer enriched with probiotics. Probiotic bacterial strains of Lactobacillus acidophilus, Bifidobacterium breve and Bifidobacterium bifidum were used in this study. The theoretical part is divided into two sections. In the first section, probiotics are generally characterized, and their role within a gut microbiota is described. Next, the microbiology of particular probiotic microorganisms including the genera of Lactobacillus and Bifidobacterium is described. Then, some factors influencing the viability and the growth of probiotics are stated. In this section, biological effects of probiotics on the human organism and their potential clinical applications are described. The second section of the theoretical part deals with the technology of brewing, the chemical composition of beer and particular beer styles. In the experimental part, the methods of probiotic bacterial cell concentration and viability determination were optimized. Several techniques for determination of these parameters were selected, particularly the cultivation method, flow cytometry and the spectrophotometric measurement of turbidity. Then, the growth curves of the probiotic strains were measured in MRS medium. Probiotic bacteria were cultivated in model beer samples, i.e. in MRS media with several different concentrations of ethanol. It is possible to say that ethanol did not have significant effect on probiotics growth. Next, experimental cultivations of individual probiotic bacteria and their mixtures in nine real beer samples were conducted. No increase of viable cells concentrations was detected in the samples. On the contrary, a decrease of the concentrations was observed, mainly in the samples with individual bacterial strains. However, certain values of viable cells concentrations were determined at the end of the cultivations in all cases. A pale, top-fermented beer was brewed and supplemented with probiotics, and the concentrations of viable probiotic cells were monitored during 37 days of fermentation. A decrease of concentrations by two orders of magnitude of CFU/ml was observed in almost all samples. Yet, viable cells of probiotic bacteria were detected in all samples of beer at the end of the fermentation. Maximal concentration of viable probiotic cells in the brewed beer was determined with the cultivation method at (3,80 ± 0,14)10^5 CFU/ml. Chosen samples were analyzed with HPLC-RI method that quantified the common beer concentrations of ethanol in all chosen samples, lactic acid was not detected. Sensory analysis was conducted as well. Based on the results of the experimental part and the bibliography, an optimal technology of the preparation of beer enriched with probiotics is discussed in this study.

L'objectif de ce travail était de sélectionner de manière rationnelle une bactérie probiotique par la mise en place d'un crible ainsi que d'étudier et de comprendre l'impact du potentiel d'oxydoréduction (Eh) et du bullage de gaz sur la survie de Bifidobacterium bifidum dans un produit laitier fermenté. Nous avons tout d'abord développé des techniques de sélection des bactéries sur des critères de viabilité / vitalité (analyse par cytométrie en flux) ainsi que sur des critères de fonctionnalité (pouvoir antioxydant). Nous avons pu sélectionner des souches d'intérêt industriel ainsi qu'une souche d'étude académique, Bifidobacterium bifidum.Nous avons ensuite étudié l'effet de la modification du Eh par bullage de gaz sur la survie de B. bifidum dans un produit laitier fermenté. Les laits fermentés conditionnés sous atmosphère anaérobie (Azote) et/ou réductrice (Azote-Hydrogène) permettent une meilleure survie de la souche au cours du stockage (28 jours - 4 °C) par rapport au Contrôle. Puis, nous avons analysé l'effet d'une croissance sous différents Eh sur la viabilité en milieu modèle et la fonctionnalité de B. bifidum. Une croissance en condition anaérobie et/ou réductrice permet d'améliorer à la fois la résistance aux stress (stress oxydant d'ordre physiologique, stress aux sels biliaires et stress côlon), le pouvoir réducteur (sels de tétrazolium), le pouvoir antioxydant (test KRL et test des comètes) et l'adhérence par rapport au Contrôle. Une modulation des propriétés biochimiques membranaires sous Azote et sous Azote-Hydrogène pourraient expliquer ces phénomènes. L'augmentation des composés thiols exofaciaux et l'augmentation des acides gras insaturés à longues chaines est observée pour des cellules produites sous conditions Azote et Azote-Hydrogène. Ainsi ces conditions de croissance présentent une amélioration majeure de l'effet probiotique de B. bifidum, à la fois d'un point de vue de sa résistance aux stress que d'un point de vue de sa fonctionnalité

Foram conduzidos três experimentos no confinamento experimental do Departamento de Zootecnia da ESALQ/USP com o objetivo de estudar os efeitos de diferentes aditivos em rações para bovinos terminados em confinamento. No experimento 1 foram utilizados 100 bovinos machos Nelore castrados (392 kg), distribuídos em 20 baias, por 60 dias. As rações continham 41% de sorgo moído, 40% de polpa cítrica peletizada e 15% de silagem de cana-de-açúcar. Os tratamentos foram: (1) controle, (2) monesina sódica Rumensin (MON1), (3) monensina sódica Rumenfort (MON2), levedura Saccharomyces cerevisiae Yea-Sacc 1026 (LEV1) e levedura Saccharomyces cerevisiae Biosaf SC47 (LEV2). A IMS foi reduzida pelo tratamento MON1 (P<0,05), quando comparada ao tratamento controle. O GPD dos animais não foi afetado pelos tratamentos (P>0,05). A EA dos animais não foi afetada pelos tratamentos (P>0,05). O tratamento MON2 apresentou um menor rendimento de carcaça (P<0,05) e o tratamento MON 1 apresentou uma maior AOL (P<0,05). No experimento 2 foram utilizados 96 tourinhos Nelore não castrados (396 kg), distribuídos em 16 baias por 95 dias. Os tratamentos foram: (1) Controle, (2) levedura Saccharomyces cerevisiae Biosaf SC47 (LEV), (3) combinação de levedura Saccharomyces cerevisiae e bactérias probióticas na dose de 1g /bovino/dia (PROB1) e (4) combinação de levedura Saccharomyces cerevisiae e bactérias probióticas na dose de 3g/bovino/dia (PROB2). As rações continham 59% de polpa cítrica peletizada, 35% de farelo de glúten de milho úmido e 5% de feno de tifton 65. A adição dos aditivos levedura (Saccharomyces cerevisae) e a combinação de leveduras e bactérias probióticas não afetou a IMS o GPD e a EA dos animais (P>0,05). A energia líquida de manutenção e de ganho das rações também não foi afetada pelos tratamentos (P>0,05), assim como os dados de carcaça. No experimento 3, que avaliou a digestibilidade das rações, foram utilizados 20 tourinhos Nelore, alocados em 20 baias individuais durante 15 dias, sendo 10 dias de adaptação ao marcador e 5 dias de coleta de fezes. A ração foi a mesma utilizada no experimento 2, e o marcador utilizado foi o óxido de cromo. Os tramentos utilizados foram: (1) Controle, (2) monensina sódica Rumenfort (MON), (3) levedura Saccharomyces cerevisiae Biosaf SC47 (LEV), (4) combinação de levedura Saccharomyces cerevisiae e bactérias probióticas na dose de 1g /bovino/dia (PROB1) e (5) combinação de levedura Saccharomyces cerevisiae e bactérias probióticas na dose de 3g/bovino/dia (PROB2). Os aditivos testados não afetaram as digestibilidades da MS, da MO, da FDN e da PB das rações (P>0,05). Com os resultados obtidos é possível afirmar que bovinos Nelore, castrados ou não, confinados com rações com altos teores de concentrado ricos em co-produtos como polpa cítrica e farelo de glúten de milho úmido não apresentam melhor desempenho nem melhor digestibilidade dos nutrientes quando suplementados com monensina sódica ou com micorganismos probióticos.
Three trials were conducted at the ESALQ/USP Animal Sciences Department experimental feedlot to evaluate the effects of different feed additives in feedlot finished cattle. On trial 1 100 Nellore steers (392kg) were allocated to 20 pens for 60 days. Experimental rations had 41% ground milo, 40% dried citrus pulp and 15% sugarcane silage. Treatments were: (1) control, (2) sodium monensin Rumensin (MON1), (3) sodium monensin Rumenfort (MON2), (4) yeast culture Saccharomyces cerevisiae Yea-Sacc 1026 (LEV1) and (5) yeast culture Saccharomyces cerevisiae Biosaf SC47 (LEV2). DMI was reduced by MON1 trestment (P<0,05) in relation to control. WDG was not affected by treatments (P>0,05). Treatments did not affect FE (P>0,05). Animals on treatment MON2 showed the lowest dressing percentage (DP) and those on MON1 showed the highest rib eye area (REA) (P<0,05). Trial 2 used 96 young Nellore bulls (396kg), allocated to 16 pens for 95 days. Treatments were: (1) control, (2) Saccharomyces cerevisiae Biosaf SC47 (LEV), (3) Saccharomyces cerevisiae and probiotic bacteria mix at 1g /animal/day dose (PROB1) and (4) Saccharomyces cerevisiae probiotic bacteria mix at 3g/animal/day dose (PROB2). Rations contained 59% dried citrus pulp, 35% wet corn gluten feed and 5% Tifton 65 hay. Treatments didnt affect DMI, WDG and FE (P>0,05). Rations net energy for maintenance and gain were also not affect by treatments (P>0,05), well as carcass data. Trial 3 utilized 20 young Nellore bulls allocated to individual pens for 15 days (10 days for adaptation to marker and 5 days for data collection) to evaluate rations digestibility. Experimental ration was the same utilized on trial 2, with chromium oxide as an external marker. Treatments were (1) control, (2) sodium monensin Rumenfort (MON1), (3) yeast culture Saccharomyces cerevisiae Biosaf SC47 (LEV), (4) Saccharomyces cerevisiae and probiotic bacteria mix at 1g /animal/day dose (PROB1) and (5) Saccharomyces cerevisiae probiotic bacteria mix at 3g/animal/day dose (PROB2). Treatments did not affect rations DM, OM, NDF and CP digestibilities (P>0,05). Results show that Nellore cattle, castrated or not, feedlot finished receiving rations with high levels of byproducts such as dried citrus pulp and wet corn gluten feed dont have higher performance nor better nutrients digestibility when supplemented with sodium monensin or probiotic microorganisms.

Ice cream mix (12% fat, 11% milk solids nonfat, 12.5% sugar, and 4.5% corn syrup solids) was fermented with Lactobacillus acidophilus and Bifidobacterium bifidum. Half of the mix was heat treated at 82°C for 30 minutes and cooled to 40-41°C. The other half was warmed to 40-41°C and inoculated with the starter cultures. Both were made into ice cream and stored at -29°C. Survival of L. acidophilus and B. bifidum and of β-galactosidase activity were monitored during 17 weeks of frozen storage. Reinforced clostridial medium was used to enumerate culture bacteria. Colony counts, after fermentation, for both L. acidophilus and B. bifidum were about 5 x 108. The population of cultures decreased less than one log cycle after initial freezing. After 17 weeks storage the bacterial counts were 1 x 107 for B. bifidum and were 4 x 106 for L. acidophilus. During the same period, β-galactosidase activity decreased only 31%. Therefore, frozen fermented dairy products provide a good vehicle to supply β-galactosidase enzymes to people who are lactose maldigestors. Frozen fermented ice cream was prepared at four different pH's (5.0, 5.5, 6.0, 6.5) by blending fermented mix with unfermented mix and then was frozen to produce samples for sensory evaluation. All samples were strawberry flavored. These were then evaluated by 88 judges. The preferred pH, based on overall acceptance, was 5.5. A second sensory evaluation was conducted to compare heat-treated with non-heat-treated ice cream. There were no significant differences in appearance, texture, flavor, and overall acceptance between the two samples. Our study shows that ice cream is a suitable vehicle for delivering these beneficial microorganisms and enzymes to consumers.

Certaines souches de bifidobactéries entrent dans la composition de laits infantiles. Leurs propriétés (modulation du microbiome, régulation de la translocation bactérienne, maturation de cellules dendritiques) sont liées à la capacité de sécrétion de composés de haut poids moléculaire lors de la fermentation bactérienne. Les objectifs de ce travail sont, dans un premier temps, de caractériser les molécules issues de la fermentation de la souche de référence B.breve C50, et de déterminer si d’autres bifidobactéries peuvent sécréter des molécules de propriétés similaires. Les analyses associant chromatographie gazeuse (GC), spectrométrie de masse (MS), électrophorèse, séquençage protéique montrent que les composés fermentaires de B.breve C50 sont des agrégats (>600kDa) associant des unités lipoprotéiques de la paroi cellulaire contenant un domaine CHAP et des sucres, en majorité du glucose. Ces agrégats sont reconnus par le TLR6, indiquant une structure lipoprotéique di-acylée. Ils sont également ligand de la galectine 1, ce qui suggère que les hexosamines et le galactose détectés par GC sont exposés à l’extérieur des agrégats. L’analyse in silico des homologies avec le gène codant pour la séquence protéique révèle la proximité du gène de B.longum avec B.breve. Par contre, il est peu probable que B.bifidum sécrète des agrégats semblables, la séquence du gène homologue étant dépourvu de lipobox. Les agrégats (>600 kDa), isolés après fermentation de la souche B.longum CBi0703 dans le milieu lacté de référence, montrent une composition similaire (unités lipoprotéique associées à des sucres, en majorité du glucose et du mannose), une reconnaissance par la Galectine 1 mais non par le TLR6. La différence de composition en lipide et l’hydrophobicité de la séquence protéique semble prévenir la reconnaissance de la structure lipoprotéique par le récepteur TLR6. Les agrégats de B.longum ayant montré des propriétés anti-inflammatoires, l’hypothèse d’une phagocytose a été explorée dans un deuxième temps. Les agrégats marqués par des marqueurs fluorescents ne sont pas détectés dans les cellules après contact direct (ex-vivo) ou gavage des animaux (in vivo). Une capture des agrégats par les cellules présentatrices d’antigènes est donc peu probable. La reconnaissance par la galectine 1 des deux types d’agrégats bifides nous fait privilégier la piste d’un mécanisme de régulation de la translocation du microbiome par l’intermédiaire des structures hexosamines et galactose exposées à la surface des agrégats. Dans un troisième temps, l’implication des agrégats bifides dans la prévention du diabète de type 1 a été explorée. En effet, le lait de femme prévient la survenue du diabète chez les souris NOD. La recherche par PCR du gène codant pour la lipoprotéine d’intérêt a permis la détection de B.longum dans 21 échantillons de lait maternel sur 33 (soit 12 mamans parmi 16). A l’inverse, B.breve est rarement isolé (2 mamans parmi 16). Comme l’analyse transcriptomique indique que les lipoprotéines sont synthétisées de façon continue, elles peuvent donc être sécrétées par les bifidobactéries dans le lait de femme. Aussi le choix s’est porté sur les agrégats de B.longum, testés à une dose anti-inflammatoire en prévention anti-diabétique. Si l’administration de lait maternel réduit l’incidence du DT1 chez des souris NOD âgées de plus de 18 semaines (p < 0.001), c’est au contraire une protection précoce mais qui ne persiste pas qui est observée sous agrégats bifides. L’effet protecteur est observé en absence de bifidobactéries intestinales. Les agrégats bifides à la dose anti-inflammatoire ne modulent pas à la même vitesse les bactéries intestinales sensibles au lait de femme, ce qui pourrait expliquer le retard d’apparition des premiers diabètes lors du traitement
Some bifidobacterial strains are used to ferment infant formulas. Their properties (modulation of microbiome, regulation of bacterial translocation, dendritic cells maturation) are related to their ability to secrete high molecular weight compounds during the bacterial fermentation. The first objective of the study was to characterize the molecules secreted by the strain B.breve C50 used as a reference, and to determine whether other bifidobacteria can secrete molecules with similar properties. Analysis using gas chromatography (GC), mass spectrometry (MS), electrophoresis, protein sequencing showed that the B.breve C50 fermentation compounds are constituted of aggregates (>600kDa) combining units of a cell wall lipoprotein with a CHAP domain and sugars moities, mostly glucose. The aggregates are recognized by TLR6, indicating that the protein was diacetylated. They are also ligand of the galectin 1, suggesting that the hexosamine and galactose moieties detected by GC surrounded the aggregates. In silico analysis showed that a B.longum gene exhibiting a high homology with the B.breve C50 gene, coded for a lipoprotein, which was secreted during fermentation, and formed aggregates with sugars. B.bifidum species likely does not secrete similar aggregates since the sequence of the homologous gene is deprived of lipobox. B.longum CBi0703 and B.breve C50 aggregates shared the same global structure (lipoproteins with CHAP domain bordered by sugars primarily constituted of glucose and mannose). Remarkably, the CBi0703 aggregates were also able to bind Gal-1 but were lacking binding capacities to TLR6. It is likely that the hydrophobicity of the protein sequence, as well as the lipid and sugar compositions prevented the recognition of the lipoprotein structure by the TLR6 receptor. Secondly, a putative phagocytosis of aggregates was investigated. Fluorescent-labeled aggregates are not detected within cells after direct contact (ex-vivo) or oral challenge in animals (in vivo). Capture of the aggregates by antigen presenting cells seemed improbable. The two types of aggregates being recognized by galectin-1, regulation of the intestinal bacterial translocation by the aggregates likely involves the hexosamines and galactose surrounding their surface. In a third step, the possible involvement of the bifidobacterial aggregates in the prevention of type 1 diabetes was investigated. Actually, breast milk was previously shown to prevent diabetes onset in old NOD mice. Detection of bifidobacteria using amplification of the gene encoding the B.longum lipoprotein was positive in 21 human milk samples out of 31 (i.e. 12 mothers out of 16). Conversely, B.breve is rarely isolated (2/16 mothers). Since transcriptomic analysis showed that the lipoproteins were continuously synthesized, we hypothesized that the bifidobacterial aggregates were secreted by the bifidobacteria harbored in human milk. To ensure that B.longum aggregates play a role in the protection induced by human milk, they were assayed at an anti-inflammatory dose. Contrary to breast milk which reduced the incidence of T1D in NOD mice older than 18 weeks (p <0.001), only early but not persistent protection is observed during bifidobacterial aggregates intake. The protective effect was observed in the absence of intestinal bifidobacteria. Variation in intestinal bacterial colonization did not match in groups drinking human milk or bifidobacterial aggregates at an inflammatory dose. The difference in kinetics could support the delay in diabetes onset induced by the bifidobacterial aggregates

碩士
朝陽科技大學
應用化學系
103
In this study, astragalus and ginger extract was added to the medium for the lactic acid bacteria Lactobacillus acidophilus, Bifidobacterium bifidum, Lactobacillus sporogenes to observe their influence on cell growth. Changes in the respective viability were observed during refrigeration. Astragalus extract was added to the medium, and results showed that for B. bifidum grew best at 1500ppm, the viability increased from 7.82 108 CFU / mL to 2.03 109 CFU / mL. When aged ginger extract was added to the culture, at 3% addition, the viability of B. bifidum increased from 2.86 x109 CFU / mL to 3.44 x1010 CFU / mL. The effect of aged ginger extract on the other two species was not significant. The optimum concentration of 1% of the dried ginger extract added to medium for B. bifidum, cell concentration increased from 3.59 x109 CFU / mL to 6.23 x109 CFU / mL. When raw ginger extract was used at 5%, the growth enhancement for L. acidophilus and B. bifidum were respectively, from 8.35 109 CFU / mL to 1.45 x1010 CFU / mL, and from 1.74 x109 CFU / mL to 1.26 1010 CFU / mL. The extracts were added to the refrigerated liquid cultures to observe the viability variations during refrigeration. The viability of L. acidophilus in the control group decreased. With added extracts, all species studied were still growing during refrigerated storage for 9 days.

博士
國立屏東科技大學
食品生技碩士學位學程在職專班
105
Ice cream is an ideal material for delivery of probiotics to the human body compared to fermented dairy products due to the pH of ice cream is closer to neutral, whereas that the fermented dairy products could be much lower, and lower pH may affect the survival and metabolic activity of probiotics. Due to the lipid content will influence the survival of probiotics, the survival of Lactobacillus acidophilus and Bifidobacterium bifidum in three ice cream formulations (low fat 8.0%, middle fat 12.0% and high fat 15.0%) were evaluated after the processing and storage at -18℃. As to the emulsification to the survival of Lactobacillus acidophilus and Bifidobacterium bifidum , we adjust the emulsification time to the ice cream for 1, 5, 15, 30 minutes, respectively. The results showed that the survival of both Lactobacillus acidophilus and Bifidobacterium bifidum are best in the 30 minutes-treatment after emulsification. The survival of Lactobacillus acidophilus and Bifidobacterium bifidum was not significantly affected among three ice cream formulations after processing. Resistance to the production of ice cream will also be showed in vitro by the ability of the cells incorporated in ice cream to survive under acid stress and maintain growth capacity in the presence of bile salts. At the last sensory science test, not only the sensory score, the total score with the majority likes shows that the ice cream is a successful products.

Bifidobacterium bifidum MIMBb75 is a novel strain whose behavior within the intestinal tract is not known. This work attempts to determine the survival and molecular determinant responsible for its colonization through two studies involving administration to mice of 1) B. bifidum MIMBb75 or 2) engineered B. longum NCC2705 expressing the B. bifidum-specific surface protein BopA. MIMBb75 was able to transiently colonize the murine intestinal tract. As a result, endogenous bifidobacteria increased significantly in the proximal colon whereas Clostridia clusters were differentially affected in a region-dependent fashion. Genetically engineered B. longum NCC2705, expressing the BopA gene, did not impact endogenous bacterial groups in the proximal colon or feces, albeit, it down-regulated the gene expression of KC and mucin 4 in the proximal colon. These outcomes indicate that MIMBb75 encompasses characteristics of a probiotic, but further studies are required to assess the role of BopA as the molecular determinant of its action.

碩士
中華科技大學
健康科技研究所
102
New cosmetic ingredients have been developed in recent years. As the Chinese herbal medicines derived from natural plants, they are safe and low side effect to human. Thus, the use of natural, organic and non-toxic ingredients extracted from Chinese herbal medicines is widely accepted by people. The past studies have found that Angelicae, Rhodiola, and Terminalia possessed the physiological activities of whitening and antioxidant. Therefore, the purpose of this research is to understand the physiological properties of the extracted compounds of Chinese herbal medicines by different solvents. In addition, the physiological properties (anti-tyrosinase and antioxidant) of the extracted compounds fermented by the Lactobacillus inhibit are also evaluated. HPLC is applied to analyze the active components of the fermented compounds. Experimental results showed that the active compounds were found at these operating conditions: 1) Terminalia first extracted by 50% ethyl acetate and then fermented by Lactobacillus acidophilus for 8 hours; 2) Terminalia first extracted by water and then fermented by Bifidobacterium bifidum for 16 hours. The active compounds achieved 100% anti-tyrosinase activity. Other extracted and fermented compounds also achieved not bad activity of anti-tyrosinase. In all operating conditions, the highest antioxidant efficiency (95%) was found under the operating condition (Terminalia extracted by 50% ethyl acetate and fermented by L.acidophilus for 8 hours). The highest reducing power was found in the operating condition (Angelicae extracted by 50% alcohol and fermented by B. bifidum for 8 hours). In conclusion, Terminalia first extracted and then fermented by L. acillusis has potential as whitening agent and antioxidants.

碩士
國立中正大學
化學工程所
95
beta-D-Galactosidase (EC 3.2.1.23) catalyzes not only the hydrolysis of lactose into glucose and galactose, but also transgalactosylation reactions to form galacto-oligosaccharides (GOSs). A high transgalactosylation beta-galactosidase gene was isolated from chromosomal DNA of Bifidobacterium bifidum and amplified by polymerase chain reaction (PCR) with designed primers. The recombinate plasmid was constructed by inserting PCR fragment into plasmid pET-32a. The the recombinate plasmid was then transformed into Escherichia coli BL21(DE3) and overexpressed soluble target fusion protein. The overexpressed recombinant enzyme was characterized and used for the production of galacto-oligosaccharides. The best transgalactosylation activity was at 37℃ and it was still good at 50℃. The highest galacto-oligosaccharide purity could be achieved about 65% when the lactose initial concentration of lactose was 60%. The result was better than the enzyme from Bacillus sp. Low-content galacto-oligosaccharide syrups was converted into high-content galactooligosaccharides by yeast Kluyveromyces marxianus fermentation. The digestable sugars such as glucose, galactose, lactose and some other nondigestable disaccharides were removed. The final galacto-oligosaccharide purity up to 95% was achieved.