La caractérisation phénotypique de l’espèce Torulaspora delbrueckii en conditions œnologiques, à partir de l’étude d’un grand nombre de souches, a permis de mettre en évidence une grande variabilité au sein de cette espèce. En effet, les souches de T.delbrueckii présentent des différences au niveau des durées de phase de latence et de fermentation, des capacités biotiques mais aussi des productions d’éthanol (maximum 12% vol.). Cette variabilité se retrouve également pour la production d’acidité volatile, de glycérol et de certains arômes. Ce travail confirme les faibles productions d’acidité volatile et de glycérol de cette espèce et met en évidence une réponse au stress osmotique différente de celle de l’espèce Saccharomyces cerevisiae. Au final, l’espèce T. delbrueckii présente une grande « pureté » de fermentation et produit peu de composés indésirables comme le sulfure d’hydrogène, les phénols volatils, l’acetoïne, l’acétaldéhyde et le diacétyle. La réalisation de co-inoculations T. delbrueckii / S. cerevisiae sur moûts liquoreux, permet une réduction systématique de l’acidité volatile des vins, en comparaison à une fermentation pure de l’espèce S. cerevisiae, quelque soit la souche de T. delbrueckii utilisée. De plus, la souche T. delbrueckii OXT1 1//2 a permis de complexifier la composition aromatique d’un moût sec issu du cépage Sauvignon blanc (esters fermentaires +25%, phényl-2-éthanol +51% et thiols volatils +31%). Enfin, la mise au point d’un fermenteur à double compartiment, avec une séparation physique des levures tout en conservant l’homogénéité du milieu de culture, a permis d’aborder l’étude des interactions entre ces 2 espèces. Des inhibitions de type « cell-cell contact » ont ainsi été mises en évidence. / The phenotypic characterization of Torulaspora delbrueckii species in enological conditions, using a large number of strains, showed up a wide variability within this species. Indeed, the strains of T. delbrueckii present different fermentative capacities for lag phase and fermentation durations, biotic capacities and ethanol production (up to 12 % vol.). This phenotypic variability is also obtained for the production of volatile acidity, glycerol and certain aroma compounds. This work confirms the low productions of volatile acidity and glycerol of this species and reveals an osmotic stress response distinct from that of Saccharomyces cerevisiae. Finally, the T. delbrueckii species presents a high fermentation purity and produces low levels of undesirable compounds like hydrogen sulfide, volatile phenols, acetoin, acetaldehyde and diacetyl. The realization of co-inoculations T. delbrueckii / S. cerevisiae on high sugar must, allows a systematic reduction of the volatile acidity of wines, in comparison to a pure fermentation of S. cerevisiae. Moreover, co-inoculation with the T. delbrueckii strain OXT1 1 // 2 allowed the increase of aromatic complexity of a Sauvignon blanc dry wine (esters + 25%, 2-phenylethanol +51 % and volatile thiols + 31 %). At last, the development of a fermentor with double compartment allowing a physical separation of yeasts and preserving the homogeneity of culture medium, allowed us to approach the study of yeast interactions. Inhibitions of type “cell-cell contact” have been demonstrated.

Sorvetes são alimentos que apresentam grande potencial como veículo de espécies de micro-organismos probióticos, tendo em vista que apresentam elevada aceitabilidade em todas as faixas etárias. Os alimentos elaborados a base de soja se destacam no mercado devido às inúmeras evidências científicas sobre seus efeitos benéficos à saúde humana. Desta forma, no presente trabalho objetivou-se o desenvolvimento de sorvetes probióticos formulados com diferentes extratos solúveis de soja (in natura e fermentados), utilizando-se a cepa Lactobacillus delbrueckii UFV H2B20. Primeiramente, avaliou-se o desempenho fermentativo (pH e acidez titulável) de 5 cepas de Lactobacillus em meio formulado com os extratos solúveis de soja ESL e ES60, cujos resultados demonstraram que estes extratos são adequados para a formulação de meios de fermentação. Na sequência foram processados sorvetes probióticos formulados com os respectivos extratos in natura (ESL e ES60) e fermentado (ESL F e ES60 F) por Lactobacillus delbrueckii UFV H2B20. Nos sorvetes confeccionados com os extratos na forma in natura, uma suspensão de células da referida cepa foi adicionada à calda previamente ao processo de batimento e congelamento. Os sorvetes foram estocados a -23 ºC por 5 meses e avaliados periodicamente quanto às características físico-químicas (pH, acidez titulável, composição centesimal e overrun), microbiológica (viabilidade celular) e sensoriais. Os resultados demonstram que a cepa de Lactobacillus avaliada foi capaz de fermentar os açúcares presentes nos respectivos extratos de soja produzindo ácido láctico, atingindo a concentração de 0,90% no meio formulado com o extrato de soja desengordurado ES60. Os sorvetes confeccionados com os respectivos extratos fermentados apresentaram diferenças significativas (P<0,05) quanto ao pH e acidez titulável em relação aos sorvetes com extratos in natura, atingindo valores de pH inferiores a 4,0 com produção máxima de ácido láctico de 1,36%. A composição centesimal dos sorvetes desenvolvidos está de acordo com o padrão mínimo estabelecido pela ANVISA, apresentando uma composição média de 2,5% de proteínas, 5,0-6,0% de gordura e 36,0% de sólidos totais. As características probióticas dos sorvetes estudados foram preservadas, apresentando contagens de células viáveis superiores a 106 UFC/g, com redução do número de células inferior a um ciclo logarítmico ao longo de 150 dias de estocagem a -23ºC. A análise sensorial revelou que os sorvetes formulados com os respectivos extratos in natura não apresentaram diferenças significativas entre si (P>0,05) para os atributos avaliados e apresentaram maior aceitabilidade, sendo o sorvete formulado com o extrato ESL o que obteve maior aceitação, com médias acima de 6,0 correspondente a \"gostei ligeiramente\". Os resultados demonstraram ainda que a fermentação dos extratos interferiu negativamente nas características sensoriais dos respectivos sorvetes, apresentando diferenças significativas em relação aos extratos in natura, além de resultar nos menores escores de aceitação, tendo o sorvete fermentado ES60 F sido avaliado como \"desgostei moderadamente\". Estes resultados permitem concluir que o extrato solúvel de soja é um insumo adequado para a formulação de meio de cultivo de espécies de microorganismos probióticos, além de apresentar potencial para a confecção de sorvetes probióticos mediante adaptações da tecnologia de produção de gelados comestíveis. / Ice creams are foods that have great potential as a vehicle for species of probiotic microorganisms due to its high acceptability by all age people. Foods made with soybeans byproducts stand out in the market due to the numerous scientific evidences that show its beneficial effects on human health. Therefore, this study aimed to the development of probiotic ice cream formulated with different soybean soluble extracts (in natura and fermented), using the strain Lactobacillus delbrueckii UFV H2B20. Primarily, it was evaluated the fermentation performance (pH and titratable acidity) of 5 Lactobacillus strains in media formulated with soluble soybean extracts ESL and ES60 (Olvebra®), and the results showed that these soybean byproducts are suitable for the formulation of fermentation media. The probiotic ice cream were formulated with the soybean extracts in natura (ESL and ES60) and fermented (ESL F and ES60 F) by Lactobacillus delbrueckii UFV H2B20. For the manufacture of ice cream with the soybean extracts in natura, a cell suspension was added to the basic ice cream formulation before beating and freezing procedures. The ice creams were stored at -23 ° C for 5 months and evaluated periodically regarding the physico-chemical characteristics (pH, titratable acidity, chemical composition and overrun), microbiological (cell viability) and sensorial evaluation. The results shown that the Lactobacillus strain were capable to metabolize the sugars presented in the soluble soybean extracts producing lactic acid reaching a concentration of 0.90% in the media formulated with defatted soybean extract ES60. The ice creams made with fermented extracts showed significant differences (P <0.05) regarding pH (< 4,0) and titratable acidity (1,36%) in relation to the ice creams manufactured with extracts in natura. The results concerned to the chemical composition of the ice cream, showed that the values obtaneid are in accordance with those stablished by ANVISA, presenting an average composition of 2.5% protein, 5,0-6,0% fat and 36,0% total solids. The probiotic characteristics, of the formulated ice creams, were preserved presenting viable cell counts greater than 106 UFC/g, and a decreasing in the cell number lower than one log cycle after 150 days of storage at -23 ºC. Regarding the sensorial evaluation, the results revealed that the ice creams formulated with soybean extracts in natura showed no significant differences between them (P> 0.05) for the evaluated attributes and they showed higher acceptability. The ice cream formulated with ESL extract presented higher acceptance, with average notes greater than 6.0, corresponding to \"like slightly\". The results also showed that the fermentation of the extracts influenced negatively on the sensory properties of the ice cream, showing significant differences regarding the ice cream made with the extracts in natura, and lower acceptance scores, being the fermented ice cream ES60 F evaluated as \"moderately disliked\". These results indicate that the soluble soybean extracts evaluated in the present work are suitable for culture medium formulation for probiotic microorganisms, and also shown its potential for making probiotic ice cream by adaptation of the technology.

Sorvetes são alimentos que apresentam grande potencial como veículo de espécies de micro-organismos probióticos, tendo em vista que apresentam elevada aceitabilidade em todas as faixas etárias. Os alimentos elaborados a base de soja se destacam no mercado devido às inúmeras evidências científicas sobre seus efeitos benéficos à saúde humana. Desta forma, no presente trabalho objetivou-se o desenvolvimento de sorvetes probióticos formulados com diferentes extratos solúveis de soja (in natura e fermentados), utilizando-se a cepa Lactobacillus delbrueckii UFV H2B20. Primeiramente, avaliou-se o desempenho fermentativo (pH e acidez titulável) de 5 cepas de Lactobacillus em meio formulado com os extratos solúveis de soja ESL e ES60, cujos resultados demonstraram que estes extratos são adequados para a formulação de meios de fermentação. Na sequência foram processados sorvetes probióticos formulados com os respectivos extratos in natura (ESL e ES60) e fermentado (ESL F e ES60 F) por Lactobacillus delbrueckii UFV H2B20. Nos sorvetes confeccionados com os extratos na forma in natura, uma suspensão de células da referida cepa foi adicionada à calda previamente ao processo de batimento e congelamento. Os sorvetes foram estocados a -23 ºC por 5 meses e avaliados periodicamente quanto às características físico-químicas (pH, acidez titulável, composição centesimal e overrun), microbiológica (viabilidade celular) e sensoriais. Os resultados demonstram que a cepa de Lactobacillus avaliada foi capaz de fermentar os açúcares presentes nos respectivos extratos de soja produzindo ácido láctico, atingindo a concentração de 0,90% no meio formulado com o extrato de soja desengordurado ES60. Os sorvetes confeccionados com os respectivos extratos fermentados apresentaram diferenças significativas (P<0,05) quanto ao pH e acidez titulável em relação aos sorvetes com extratos in natura, atingindo valores de pH inferiores a 4,0 com produção máxima de ácido láctico de 1,36%. A composição centesimal dos sorvetes desenvolvidos está de acordo com o padrão mínimo estabelecido pela ANVISA, apresentando uma composição média de 2,5% de proteínas, 5,0-6,0% de gordura e 36,0% de sólidos totais. As características probióticas dos sorvetes estudados foram preservadas, apresentando contagens de células viáveis superiores a 106 UFC/g, com redução do número de células inferior a um ciclo logarítmico ao longo de 150 dias de estocagem a -23ºC. A análise sensorial revelou que os sorvetes formulados com os respectivos extratos in natura não apresentaram diferenças significativas entre si (P>0,05) para os atributos avaliados e apresentaram maior aceitabilidade, sendo o sorvete formulado com o extrato ESL o que obteve maior aceitação, com médias acima de 6,0 correspondente a \"gostei ligeiramente\". Os resultados demonstraram ainda que a fermentação dos extratos interferiu negativamente nas características sensoriais dos respectivos sorvetes, apresentando diferenças significativas em relação aos extratos in natura, além de resultar nos menores escores de aceitação, tendo o sorvete fermentado ES60 F sido avaliado como \"desgostei moderadamente\". Estes resultados permitem concluir que o extrato solúvel de soja é um insumo adequado para a formulação de meio de cultivo de espécies de microorganismos probióticos, além de apresentar potencial para a confecção de sorvetes probióticos mediante adaptações da tecnologia de produção de gelados comestíveis. / Ice creams are foods that have great potential as a vehicle for species of probiotic microorganisms due to its high acceptability by all age people. Foods made with soybeans byproducts stand out in the market due to the numerous scientific evidences that show its beneficial effects on human health. Therefore, this study aimed to the development of probiotic ice cream formulated with different soybean soluble extracts (in natura and fermented), using the strain Lactobacillus delbrueckii UFV H2B20. Primarily, it was evaluated the fermentation performance (pH and titratable acidity) of 5 Lactobacillus strains in media formulated with soluble soybean extracts ESL and ES60 (Olvebra®), and the results showed that these soybean byproducts are suitable for the formulation of fermentation media. The probiotic ice cream were formulated with the soybean extracts in natura (ESL and ES60) and fermented (ESL F and ES60 F) by Lactobacillus delbrueckii UFV H2B20. For the manufacture of ice cream with the soybean extracts in natura, a cell suspension was added to the basic ice cream formulation before beating and freezing procedures. The ice creams were stored at -23 ° C for 5 months and evaluated periodically regarding the physico-chemical characteristics (pH, titratable acidity, chemical composition and overrun), microbiological (cell viability) and sensorial evaluation. The results shown that the Lactobacillus strain were capable to metabolize the sugars presented in the soluble soybean extracts producing lactic acid reaching a concentration of 0.90% in the media formulated with defatted soybean extract ES60. The ice creams made with fermented extracts showed significant differences (P <0.05) regarding pH (< 4,0) and titratable acidity (1,36%) in relation to the ice creams manufactured with extracts in natura. The results concerned to the chemical composition of the ice cream, showed that the values obtaneid are in accordance with those stablished by ANVISA, presenting an average composition of 2.5% protein, 5,0-6,0% fat and 36,0% total solids. The probiotic characteristics, of the formulated ice creams, were preserved presenting viable cell counts greater than 106 UFC/g, and a decreasing in the cell number lower than one log cycle after 150 days of storage at -23 ºC. Regarding the sensorial evaluation, the results revealed that the ice creams formulated with soybean extracts in natura showed no significant differences between them (P> 0.05) for the evaluated attributes and they showed higher acceptability. The ice cream formulated with ESL extract presented higher acceptance, with average notes greater than 6.0, corresponding to \"like slightly\". The results also showed that the fermentation of the extracts influenced negatively on the sensory properties of the ice cream, showing significant differences regarding the ice cream made with the extracts in natura, and lower acceptance scores, being the fermented ice cream ES60 F evaluated as \"moderately disliked\". These results indicate that the soluble soybean extracts evaluated in the present work are suitable for culture medium formulation for probiotic microorganisms, and also shown its potential for making probiotic ice cream by adaptation of the technology.

Made available in DSpace on 2015-03-26T13:52:02Z (GMT). No. of bitstreams: 1 texto completo.pdf: 207338 bytes, checksum: e8ae32d5d73829fe54385cc857a2a2cd (MD5) Previous issue date: 2006-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The effects of heat shock, acid treatment, exposure to bile salts and the presence of H2O2 on antimicrobial resistance in Lactobacillus delbrueckii UFV H2b20 were determined. Agar diffusion assays results indicated that this bacterium is resistant to vancomycin, sulphamethoxazole, colistin, some [Beta]-lactams, aminoglycosides, nitrofurans and quinolones. It is moderately susceptible to choramphenicol, tetracycline, cephalothin, ampicillin and erythromycin. L. delbrueckii UFV H2b20 can grow on high concentrations of H2O2 and complete inhibition will be observed only at 70 µg mL-1. Diferent effects on the Minimal Inhibitory Concentration (MIC) of the antimicrobials were observed when the bacterial cells were exposed to several stress conditions. Heat shock affected resistance to different aminoglycosides and [Beta]-lactams in various ways; it diminished resistance to nitrofurans and to tetracycline; however, it had no effect over resistance to choramphenicol and espiramycin. The same results were observed for acid pre-treatment at pH 3,5 for 30 min, except that it resulted in enhancement of espiramycin resistance. Cell exposure to 0,5% bile salts resulted in diminuished MICs for almost all tested antibiotics, except, apparently, furazolidone, sulphametoxol and nalidix acid, which maintained resistance to the highest concentrations tested. The presence of H2O2, 20 µg mL-1, had various effects on aminoglycosides, diminuished resistance to nitrofurans, [Beta]-lactams, tetracycline and espiramycin; however, it enhanced resistance to choramphenicol. These results demonstrate an ample diversity of responses to heat shock, acid shock, presence of H2O2, and also a general response when cells of L. delbrueckii UFV H2b20 are exposed to bile salts. / A resistência a antimicrobianos e o efeito de peróxido de hidrogênio sobre o crescimento em Lactobacillus delbrueckii UFV H2b20 foram estudados. Os efeitos de choque térmico, choque ácido, exposição a sais biliares e presença de peróxido de hidrogênio sobre a resistência a antimicrobianos nesta bactéria também foram investigados. A determinação do modelo de resistência, pelo método de difusão em meio sólido, indicou que L. delbrueckii UFV H2b20 apresenta resistência a vancomicina, a alguns [Beta]-lactâmicos, a sulfametoxol, a aminoglicosídeos, a nitrofuranos, a quinolonas e a colistina e susceptibilidade moderada a cloranfenicol, a tetraciclina, a cefalotina, a ampicilina e a eritromicina. L. delbrueckii UFV H2b20 é capaz de crescer em altas concentrações de peróxido de hidrogênio e inibição completa do crescimento só foi observada com 70 µg mL-1. Os efeitos de condições de estresse sobre a resistência a antimicrobianos foram determinados pela comparação da Concentração Inibitória Mínima (CIM), realizada pelo método de microdiluição em meio líquido, de células submetidas e de não submetidas às condições de estresse citadas. O choque térmico provocou efeitos diversos sobre a resistência a aminoglicosídeos e a [Beta]-lactâmicos, diminuiu a resistência a nitrofuranos e a tetraciclina e não alterou a resistência a cloranfenicol e a espiramicina. O choque ácido também provocou efeitos diferentes sobre a resistência a aminoglicosídeos e a [Beta]-lactâmicos, diminuiu a resistência a nitrofuranos e a tetraciclina, não alterou a CIM de cloranfenicol e aumentou a resistência a espiramicina. A exposição das células de L. delbrueckii UFV H2b20 a sais biliares provocou diminuição da CIM de quase todos os antimicrobianos testados, exceto furazolidona, sulfametoxol e ácido nalidíxico, que não tiveram a resistência alterada até a concentração máxima testada. A presença de peróxido de hidrôgenio provocou efeitos diferentes sobre a resistência a aminoglicosídeos, diminuiu a resistência a nitrofuranos, a [Beta]-lactâmicos, a tetraciclina e a espiramicina e aumentou a resistência a cloranfenicol. Os resultados demonstram ampla diversidade nas respostas ao choque térmico, ao choque ácido e ao peróxido de hidrogênio e similaridade na resposta à exposição das células de L. delbrueckii UFV H2b20 a sais biliares.

Lactobacillus delbrueckii est une bactérie laitière non commensale qui transite dans notre tractus gastro-intestinal du fait de son utilisation dans les produits laitiers fermentés, notamment les yaourts. Nous avons cherché à déterminer les profils immuno-modulateurs de 59 souches de L. delbrueckii subsp. lactis et subsp. bulgaricus. Nous avons d'abord criblé notre panel de souches sur leur capacités à inhiber l'activation de NF-kB par le TNF-α dans des cellules HT-29. Nous avons ainsi pu mettre en évidence une variabilité des capacités immuno-modulatrices chez ces souches en repérant des souches aux capacités anti- ou pro-inflammatoires. Nous avons retenu les deux souches les plus anti-inflammatoires (L. delbrueckii subsp. lactis CNRZ327 et 333) et avons observé qu'elles perdaient leurs effets après traitement à la trypsine suggérant que les effecteurs inhibant l'activation de NF-kB soient de nature protéique. Nous avons pu aussi déterminer que cette inhibition passait par la réduction de la phosphorylation d'IkB affectant la translocation de NF-kB dans le noyau. Nous avons ensuite testé ces deux souches dans un modèle murin de colite induite au DSS. La souche CNRZ327 a amélioré de façon significative les dommages induits par le DSS confirmant ainsi ses effets anti-inflammatoires in vivo. Nous avons ensuite tenté de déterminer les mécanismes des effets anti-inflammatoires de la CNRZ327. Cette souche i) augmente les taux d'IgA sécrétoires; ii) module la production de TGF-β dans le colon et de l'IL-6 et de l'IL-10 dans la rate; iii) augmente les cellules T régultrices CD4+Foxp3+ dans la rate et les ganglions cécaux et iv) module la fréquence des cellules dendritiques TLR2+2 and TLR4+. Nos travaux constituent la première démonstration d'effets immuno-modulateurs d'une souche de lactobacille laitier. Les lactobacilles laitiers très présents dans nos aliments laitiers fermentés peuvent ainsi moduler notre réponse immune et influencer ainsi notre santé. / The work presented in this thesis aims to review the present knowledge on bacteria-host interactions in the GI tract, and to demonstrate the immune modulatory effects of Lactobacillus delbrueckii, a non-commensal dairy bacterium that is in constant transit in our GI tract through the ingestion of fermented food products, like milk and cheese.The main results presented in this thesis can be divided into two parts. In the first part we revisited the anti-inflammatory properties of L. delbrueckii by screening a collection of different strains for their ability to inhibit TNF-α-induced NF-kB activation in an IEC cell line. Our results demonstrated the existence of inter strain variation for immune modulation properties in the L. delbrueckii species. Two of the most effective strains completely lost their ability to suppress NF-kB after trypsin treatment, indicating that the bacterial effectors involved in the NF-kB modulation are proteinaceous in nature. We also showed that L. delbrueckii inhibits NF-kB activation by reducing the phosphorylation of IkB, which would affect translocation of NF-kB to the nucleus. Based on our in vitro results, we selected three strains for tests in a DSS model of experimental colitis. One of the strains tested, L. delbrueckii subsp.Lactis CNRZ327 (Lb CNRZ327) consistently improved the DSS-induced damage, thus confirming its anti-inflammatory properties in vivo. The second part of my thesis was dedicated to the study of the mechanisms involved in the anti-inflammatory effects of L. delbrueckii in the DSS-model of colitis. Lb CNRZ327 showed a tendency to increases s-IgA levels, modulates the production of TGF-β in colonic tissue, and of IL-6 and IL-10 in the spleen, expands the frequency of CD4+Foxp3+ regulatory T cells in the spleen and CLN during colitis and modulates the frequencies of TLR2 and TLR4 expressing dendritic cells in the cecal lymph nodes. TLR2 appears to be involved in Lb CNRZ327 recognition and affect IL-12 production in BMDM. This is the first demonstration of systemic immune modulation effects exerted by a dairy Lactobacillus. The results of this study show that dairy lactobacilli that often are part of a regular diet can modulate innate immune responses and may thus affect health more than generally thought.

Made available in DSpace on 2015-03-26T13:51:56Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1119528 bytes, checksum: 3f39721f8aa2a5aa0f53cfd21af511b6 (MD5) Previous issue date: 2012-07-03 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Bacteria of the genus Lactobacillus are inhabit the human gastrointestinal tract (GHT) and species such as Lactobacillus delbrueckii UFV H2B20, isolated from feces of infant exclusively fed breast milk, have physiological characteristics, technology, immunological and genetic desirable and necessary to probiose. The objective of this work was to study the tolerance of L. delbrueckii UFV H2b20 to gastric juice and simulated intestinal when incorporated in a food matrix, fermented milk, and also the expression of the gene encoding the fbpA, binding protein fibronectin, involved in adhesion to the human gastrointestinal tract. The results showed that L. delbrueckii UFV H2b20 is able to maintain its viability at levels above 108 UFC.mL-1 after 28 days of storage. When subjected to the conditions prevailing in TGH found that the food matrix evaluated, the fermented milk, confer protection. After the fourteenth day of the experiment, no significant survival in simulated intestinal juice. The fbpA gene was identified by PCR and sequencing. This gene is clustered with the orthologs of probiotic bacteria. However, exposure of cells of L. delbrueckii UFV H2B20 incorporated in fermented milk, for 240 minutes to the conditions prevailing in TGH caused reduced expression of this gene, detected by RT-PCR. Confirmation of the involvement of this gene under stress conditions, in adherence to the TGH compartments remains dependent on studies with mutant bacteria. / Bactérias do gênero Lactobacillus são habitam do trato gastrointestinal humano (TGH) e espécies como Lactobacillus delbrueckii UFV H2b20, isolado a partir de fezes de criança alimentada exclusivamente com leite materno, apresentam características fisiológicas, tecnológicas, imunológicas e genéticas desejáveis e necessárias à probiose. O objetivo deste trabalho foi estudar a tolerância de L. delbrueckii UFV H2b20 ao suco gástrico e intestinal simulado quando incorporado a uma matriz alimentar, leite fermentado, e também a expressão do gene fbpA que codifica a proteína de ligação a fibronectina, envolvida na adesão ao trato gastrointestinal humano. Os resultados mostraram que L. delbrueckii UFV H2b20 é capaz de manter sua viabilidade em níveis superiores 108 UFC.mL-1 após 28 dias de estocagem refrigerada. Quando submetida às condições prevalecentes no TGH detectou-se que a matriz alimentar avaliada, o leite fermentado, conferiu proteção.. Após o décimo quarto dia de experimento, não houve sobrevivência expressiva ao suco intestinal simulado. O gene fbpA foi identificado por PCR e sequenciamento. Este gene se agrupou com os ortólogos de bactérias probióticas.No entanto, a exposição das células de L. delbrueckii UFV H2b20 incorporadas em leite fermentado, por 240 minutos às condições prevalecentes no TGH provocou redução da expressão desse gene, detectado por RT-PCR. A confirmação do envolvimento desse gene sob condições de estresse, na adesão a compartimentos do TGH continua dependente de estudos com mutantes da bactéria.

This work describes the development, validation and application of sensor systems to monitor phase transition events of cryoprotectant mixtures in samples and cryopreservation profiles and post-thaw recovery of Lactobacillus delbrueckii subsp. bulgaricus CFL1. Ice nucleation and glass transition (Tg) temperatures influence cell viability during cryopreservation. Knowledge of these phase changes for cryoprotectant mixtures is an essential step in optimising cryopreservation protocols for cell survival. Differential scanning calorimetry (DSC) is used to determine Tg, but the expensive nature of such instrumentation limits its widespread use. Cost-effective sensor systems have been designed to monitor ice-initiation and Tg events in small volume samples of cryoprotectants solutions. Tg values were measured for glycerol, sucrose and Me2SO (with and without NaCl supplement and ice-nucleators) in cryotubes and cryostraws, using temperature and screen-printed impedance sensors. The effect of changes to ice-initiation temperature on Tg was also investigated at different cooling and warming rates by using a Grant Asymptote (EF600) controlled rate freezer. The resulting Tg values obtained by single-channel transition monitoring system (TMS 1) were not significantly different from the values obtained by DSC reported in the literature. However multiple channelled transition monitoring system (TMS 2) requires further circuit modification and multiple screen-printed temperature probes to study the phase-change temperatures and to determine transition events in more than one sample at a time. The lactic acid bacterium (LAB) Lactobacillus delbrueckii was investigated as a model system to monitor the effect of different cryopreservation protocols on post-thaw cell metabolic activity. An important parameter for monitoring the post-thaw quality of LAB for starter culture preparation is the change in pH of the culture medium during incubation at 40 oC. Glass pH combination electrodes are the most common and widely used sensors. However, they are fragile, must be conditioned before use and are not disposable. An alternative to conventional glass electrodes are screen-printed carbon-metal electrodes. Different percentage mixtures of ruthenium and antimony pastes were tested and 54.5% carbon-antimony electrodes gave the best sensitivity and consistency in potentials at fixed pH with a screen-printed salt-bridged Ag/AgCl reference. LAB cultures were cryo-preserved at very rapid, moderate and very slow cooling rates and their post-thaw metabolic activity after overnight incubation in MRS broth was determined using screen-printed pH electrodes. Back to back testing with conventional glass pH sensors was performed to compare responses. Results indicated that early ice-initiation (by means of nucleators) prevents the cells from extensive dehydration (during cooling) and enables maximum post-thaw recovery after incubation (due to equilibrium ice formation and ice melting). In future, screen-printed pH sensors require development with integrated salt-bridged Ag/AgCl reference to make it robust in signalling response. The availability of low cost, disposable, non-fragile sensors and sensor systems to monitor transition events allows the determination of Tg of cryopreservation media during both cooling and warming cycles. A combined screen-printed (impedance + temperature) sensor is proposed for this purpose. A combined screen-printed (pH + reference) sensor would allow the monitoring of metabolic activity in post-thaw and fresh starter cultures of LAB. At present the salt-bridged pH reference is manually attached to the screen-printed pH working electrode but it requires further modifications to the method of attachment. The two sensor systems would enable optimisation of cryopreservation protocols for LAB and could enable such measurements to become routine at commercial scale.

Thesis (MTech (Chemistry))--Cape Peninsula University of Technology, 2016. / Wines contain a number of phenolic compounds, belonging to non-flavonoid and flavonoid complexes. Phenolic compounds in wine are responsible for wine colour, astringency, and bitterness. Saccharomyces cerevisiae yeast is normally used in winemaking but it has been proved to decrease the phenolic content in wines. Current research on the use of non-Saccharomyces yeast in winemaking has produced better quality wines than S. cerevisiae yeast therefore improving the sensory profile of wine. This study evaluated effect of Torulaspora delbrueckii yeast on the phenolic content of experimental wines derived from Chenin blanc grapes. A reversed phase high-performance liquid chromatographic (RP-HPLC) method was used for the identification and quantitation of the phenolic compounds. The difference test method was used to determine the sensory attributes of wines. The data was subjected to analysis of variance to compare treatment differences between the wines and principal component analysis to establish possible correlation between the data sets. Furthermore, a gas chromatographic-flame ionization detection method (GC-FID) was used for the quantification of volatile compounds in the wines. In this work, wines made with T. delbrueckii strain M2/1 had high concentration of (+)-catechin, caffeic acid, ferulic acid and p-coumaric acid in all studied vintages. Wines made with VIN13 had higher concentrations of flavan-3-ols, compared to wines made with M2/1 and 654. In sensory evaluation, M2/1 wines were prominent in astringency and complexity. Yeast strain M2/1, also attributed to body and complexity of the wine. However, in this study no correlations were observed between the phenolic content and sensory attributes and vice versa. The quality of wine cannot be concluded by chemical or sensory analysis alone, but the data sets are complementary. Although the phenolic concentration of wines made with S. cerevisiae strain (VIN 13) and T. delbrueckii (M2/1) were similar in measured phenolic concentrations, they had different sensory attributes. Wines made during the 2013 vintage indicated the importance of the use of a strain with higher enzyme activity and high fermentation rate. There is minimal to no skin contact in white winemaking. Therefore, the use of a yeast strain with an increased enzyme activity can facilitate the extraction of phenolics from grape, resulting in wine with improved quality.

S. thermophilus et L. bulgaricus sont les bactéries lactiques utilisées dans la fabrication du yaourt. En milieu laitier, cette association est appelée proto-coopération lorsqu'elle est bénéfique au développement de chacune des espèces. Même si quelques facteurs nutritionnels impliqués dans cette coopération ont été identifiés, ce phénomène reste encore peu connu au niveau moléculaire. Notre objectif est d'identifier plus amplement les bases moléculaires de cette association, d'évaluer la nature et l'étendue des échanges bactériens et d'estimer si des phénomènes de régulation interviennent entre les deux espèces. Nous avons sélectionné le couple de souches, S. thermophilus LMG18311 et L. bulgaricus ATCC11842, dont les génomes sont séquencés, pour lequel nous avons observé un effet proto-coopératif. En utilisant des analyses protéomique et transcriptomique, nous avons comparé la croissance en lait de S. thermophilus en présence (co-culture) ou en absence (monoculture) de L. bulgaricus et à deux stades de croissance différents. Nous avons obtenu la variation de 88 différents gènes ou protéines (4.6% des CDS) de S. thermophilus qui variaient en présence de L. bulgaricus. Notamment des variations concernant les métabolismes azoté, des purines et du fer. De plus, la co-culture conduit à des changements de régulation chez S. thermophilus avec la variation d'un régulateur de réponse, homologue au régulateur de réponse CovR des streptocoques. Cette étude révèle des échanges nutritionnels inattendus et des relations de régulation entre St. thermophilus et Lb. bulgaricus offrant de nouvelles pistes pour la compréhension du comportement proto-coopératif entre ces deux bactéries.

Lactic acid bacteria are associated with preservation of foods, including milk, meat and vegetables. Yoghurt is produced by the cooperative action of two starter bacteria / S. thermophilus and L. delbrueckii subsp. bulgaricus. In this study, identification and typing of yoghurt starter bacteria were aimed. Traditional home made yoghurts were collected from different areas of Turkey, identification of those isolates at species and subspecies level and typing at strain level were achieved using PCR based methods. In our study, identification of yogurt starter bacteria was studied using species specific primers and ARDRA. These methods were inefficient in identification of yoghurt starter bacteria, at species and subspecies level. Consequently, a reliable and quick method for accurate identification of yoghurt starter bacteria was developed. The new method focuses on amplification of methionine biosynthesis genes, for selective identification of yoghurt starter bacteria together with some cheese starters. Further discrimination by ARDRA enabled differentiation of yoghurt starter bacteria from cheese starters. Confirmation of the proposed method has been accomplished by partial sequencing of the 16S rRNA gene. After correct identification of starter bacteria had been achieved, the strains were typed at strain level using RAPD-PCR and MLST. RAPD-PCR with primer 1254 resulted better fingerprints, compared to primer M13 at strain level. Comparisons of the two typing methods showed that RAPD-PCR revealed strain diversity better than MLST, however MLST was a more robust and reliable method and resulted in clustering of the strains depending on the isolation source.