This study aimed to isolate and characterize in-vitro and under simulated colonic conditions, probiotic candidates isolated from food environment producing γ-aminobutyric acid (GABA), a major inhibitory neuromediator of the enteric nervous system with a potential role in modulating the immune system in many health disorders. Several lactic acid bacteria were isolated and detected for the presence of the gadB gene using PCR and GAD enzymatic assay. The most active strains with high and fast production kinetics were identified, characterized, and included Streptococcus thermophilus, Lactiplantibacillus plantarum, and Lactobacillus delbrueckii subsp. bulgaricus. The biological safety (i.e., sensitivity to antibiotics and the presence of virulence factors) and probiotic potential (i.e., resistance to gastrointestinal conditions and whole-genome sequencing) of identified bioactive strains was also confirmed in vitro. The growth, GABA production, and competitiveness of selected probiotic candidates (B. animalis, S. thermophilus, and L. bulgaricus) were investigated in the presence of human gut microbiota ex vivo in a model of a proximal colon mimicking physiological and microbiological conditions of the human large intestine. Supplementation with GABA-producing probiotic candidates did not affect the overall gut microbiota diversity over 48 h of treatment. However, we observed modulation of the microbiome composition, especially change of Bacteroides population, a key gut microbe associated with anti-depressive and anti-inflammatory activities. The level of microbiota-generated butyrate within 12 h of treatment was significantly increased compared to control. Results from this study demonstrated the probiotic potential of tested GABA-producing bacteria and their impact on gut microbiota structure and metabolism, suggesting their suitability for gut health-promoting application.

Akamu is a lactic acid bacteria fermented cereal-based food that complements infant diets in most African countries. Uncontrolled fermentation increases the variability in quality and safety of akamu. This study was aimed at the controlled fermentation of akamu with selected lactic acid bacteria (LAB), investigation of the probiotic potential of the LAB and the effect of variation in production method on the product quality and sensory properties. PCR-DGGE analysis of traditional akamu samples revealed LAB community dominated by Lactobacillus fermentum, L. plantarum, L. delbrueckii subsp. bulgaricus and L. helveticus. Isolated yeasts were Candida tropicalis, C. albicans, Clavispora lusitaniae and Saccharomyces paradoxus. The isolated Lactobacillus plantarum strains (NGL5 and NGL7) fermented irradiated ground maize slurries and produced significant levels of lactic acid (>73 mmol L-1) and low pH ≤3.63 displaying inhibitory activity against Salmonella enterica serovar Enteritidis NCTC 5188, Escherichia coli 1077 (NCTC 11560), Bacillus cereus NCIMB 11925, Staphylococcus aureus NCTC 3750 and Listeria monocytogenes NCTC 7973 in MRS agar and E. coli 1077 in maize slurry fermentation. Viability of both strains of L. plantarum at pH 2 after 3 h was reduced from ≥8.26±0.05 to ≤4.94±0.49 Log10 CFU mL-1 while incubation in 0.3% bile allowed growth to 5.73±0.13 and 7.93±0.12 Log10 CFU mL-1 after 6 h for NGL5 and NGL7 respectively. Auto-aggregation of the L. plantarum strains at 37oC (≥25 after 5 h) correlated with adhesion to hydrocarbons (<15, 26, 33 and 64% for Hexane, Hexadecane, Ethyl acetate and Chloroform respectively). The strains failed to exhibit gelatinase or haemolytic activity but adhered to porcine mucin (OD403 nm ≥0.63 with viability ≥6.52 Log10 CFU mL-1) and Caco-2 cells (≥5.13 Log10 CFU mL-1). The ash, mineral (Ca, K, Mg, Na, S and Zn), IDF, SDFP and TDF content of the L. plantarum fermented ground maize slurries were significantly (p≤0.05) higher than that of the traditional akamu but the peak and final viscosities (139.5 and 68.5 cP respectively) were significantly (p≤0.05) the least. The aroma, appearance, colour, flavour and texture of the resultant porridges were liked moderately by 75% of the assessors. This study demonstrated that fermentation with the L. plantarum strains would contribute towards product safety and the L. plantarum strains possessed some probiotic potential that could be beneficial to the consumers particularly in those developing countries were the main staple foods are fermented cereals.

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No. of bitstreams: 1 Dissertação Charlene - VERSÂO Final CD.docx: 1141880 bytes, checksum: 525cb42fb5cb9562edb967f1bbd44c49 (MD5) Previous issue date: 2017-12-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A procura por bactérias ácido-láticas (BAL) com propriedades benéficas a saúde, melhoria nas características sensoriais, digestibilidade, qualidade nutricional, bem como na preservação dos alimentos tem sido o foco de intensas pesquisas na última década.O isolamento e caracterização de novas espécies são vantajosos para obtenção de linhagens com características taxonômicas, tecnológicas e funcionais diferenciadas. O objetivo deste trabalho foi avaliar fatores de virulência e atividade probiótica do isoladoEnterococcus durans(LAB18s), proveniente de queijo minas frescal, bem como isolar, identificar, caracterizar, avaliar o potencial probiótico e os aspectos de segurança de isolados de BAL, provenientes de queijo ricota, para possível aplicação como cultura probiótica. Verificou-se que o isolado LAB18séhomofermentativo, lipase positiva e gelatinase negativa, possui atividade antagonista contra Salmonella Enteritidis, Listeria monocytogenes, Staphylococcus aureus eEscherichia coli. Não foi detectada a presença do operon icaADBC, os quais são relacionados à formação de biofilme,indicando segurança em relação a um possível uso em alimentos. Além disso, o isolado LAB18scarreia dois genes de enterocinas, o gene L50A/B e o gene P. O isolado R7, obtido de queijo ricota, foi identificado molecularmente como Lactococcus lactis subsp lactise apresentou característicasprobióticas nasanálises de tolerância ao pH ácido, sais biliares, suco gástrico simulado e suco intestinal simulado. Além disso, foi sensível aos seis antimicrobianos testados, não forma biofilme, é homofermentativo, bem como não apresentou atividade hemolítica, nem das enzimas DNAse, gelatinase e lipase. O isolado R7 apresentou ainda, capacidade de autoagregação, coagregação e hidrofobicidade, indicando potencial para ser utilizado como probiótico. Da mesma forma, apresentou atividade antagonista contra Salmonella Enteritidis,Listeria monocytogenes, Staphylococcus aureus eEscherichia coli e atividade antioxidante. Por meio dos resultados obtidos conclui-se que, tanto o LAB18s isolado de queijo minas frescal quanto o R7 isolado de queijo ricotaapresentaram características probióticas e aspectos de segurança compatíveis para futura utilização como culturas iniciadoras em alimentos. São resultados promissores, no entanto, há necessidade de pesquisa de genes codificadores de virulência e toxicidade para possível aplicação. / The search for new lactic-acid bacteria (LAB) with beneficial properties has been the focus of intensive research in the current years. The isolation and characterization of new species are advantageous to obtain lineages with different taxonomic, technological and functional properties. The am of this study was to evaluate the virulence and antimicrobial activity of Enterococcus durans (LAB18s) isolated from cheese "minas frescal", as well as to isolate, identify, characterize and evaluate the probiotic potential and safety aspects of a new strain of BAL from Cheese "ricotta type" for the possible application as a probiotic culture. In the results of isolateLAB18s, gelatinase negative, lipase positive, homofermentative activity, antagonistic activity against the Salmonella Enteritidis, Listeria monocytogenes and Staphylococcus aureus were observed in the well diffusion assay by well, and activity against E. coli, S. Enteritidis, L. monocytogenes by the "Spot on the lawn" method. The presence of the icaADBC operon was not detected indicating safety in relation to a possible alimentary use; the presence of two enterocin genes, the L50A/B gene and the P gene werepronounced. The R7 strain isolated from ricotta cheese, identified as Lactococcus lactis subsp lactis, presented probiotic characteristics against tolerance analysis at acid pH, bile salts, simulated gastric juice and simulated intestinal juice. It was classified as sensitive against all antibiotics tested, non-biofilm forming, homofermentative, presented negative activity for analysis of hemolysis, DNAse, gelatinase and lipase. The isolate R7 also presented self-aggregation, coagregation and hydrophobicity capacity indicating potential to be used as probiotic. Likewise, it presented antagonistic activity against Salmonella Enteritidis, Listeria monocytogenes, Staphylococcus aureus eEscherichia coli, and antioxidant activity both by the TBARS and DPPH methods. By means of the results obtained, it was concluded that both LAB18s isolated from fresh minas cheese and R7 isolated from ricotta cheese presented probiotic characteristics and safety aspects compatible for future use as starter cultures in foods. There are promising results, however, there is a need for research on genes coding for virulence and toxicity for possible application.

The consumption of functional foods has increased over the years, the awareness of the population to purchase foods that benefit health. Among the main and most functional foods are consumed fermented milk. The kefir is fermented milk that has stood out for its natural functional origin, with significant effects on health when consumed regularly, and despite not being marketed in Brazil is easily found by hand. Thus, the study of this important source functional food has gained ground among researchers, and the aim of this study was to evaluate the physicochemical and microbiological characteristics of the craft of kefir northwest region of Rio Grande do Sul State and the characteristics of probiotic lactic acid bacteria in the product. Eight kefir samples were analyzed produced by hand in the northwest region of of Rio Grande do Sul. The samples were cultured in the laboratory and fermented milk subjected to physical, chemical and microbiological analyzes. The physicochemical analyzes the moisture, ash, fat, total and degreased dry extract, protein, acidity, titratable acidity, pH, water activity and color. In the microbiological evaluation, the samples were subjected to counting of yeasts and molds and analysis the most probable numbers of total and fecal coliforms. We also assessed the potential of probiotic the isolated by BALs antagonistic activity against pathogenic bacteria strains of reference and resistance tests to acidic conditions and bile salts. In this work, we can to observe that the handmade kefir of the study area has similar characteristics between the different samples, both in physicochemical as microbiological parameters, and some isolates have important features in regard to the probiotic potential. However, we suggest further studies, especially in relation to microbiological behavior to assess more parameters to classify this as a functional food. / O consumo de alimentos funcionais tem aumentando no decorrer dos anos, pela conscientização da população em adquirir alimentos que tragam benefícios à saúde. Dentre os principais e mais consumidos alimentos funcionais estão os leites fermentados. O kefir é um leite fermentado que tem se destacado pela sua origem funcional natural, com efeitos significativos sobre a saúde quando consumido regularmente, e apesar de não ser comercializado no Brasil é encontrado facilmente de forma artesanal. Dessa forma, o estudo sobre essa fonte importante de alimentação funcional tem ganhado espaço entre pesquisadores, sendo o objetivo desse trabalho avaliar as características físico-químicas e microbiológicas do kefir artesanal da região noroeste do Estado do Rio Grande do Sul, bem como as características probióticas das bactérias ácido láticas presentes no produto. Foram analisadas oito amostras de kefir produzidos artesanalmente na região noroeste do Estado do Rio Grande do Sul. As amostras coletadas foram cultivadas em laboratório e os leites fermentados submetidos às analises físico-químicas e microbiológicas. Nas análises físico-químicas foram avaliados os teores de umidade, cinzas, gordura, extrato seco total e desengordurado, proteína, acidez titulável, acidez potenciométrica, pH, atividade de água e cor. Na avaliação microbiológica, as amostras foram submetidas a contagem de bolores e leveduras, análise do número mais provável de coliformes totais e termotolerantes e contagem de bactérias láticas (BALs). Também foi avaliado o potencial probiótico dos isolados através da atividade antagônica de BALs em relação a cepas de bactérias patogênicas de referência, além de testes de resistência à condições ácidas e sais biliares. Com a realização deste trabalho, é possível observar que o kefir artesanal da região de estudo tem características semelhantes entre as diferentes amostras, tanto nos parâmetros físico-químicos quanto microbiológicos, além de alguns isolados apresentarem características importantes no que diz respeito ao potencial probiótico. No entanto, sugerem-se mais estudos, especialmente em relação ao comportamento microbiológico para avaliar mais parâmetros que classifique esse alimento como funcional.