Catabolic flexibility of mammalian-associated lactobacilli

Metabolic flexibility may be generally defined as “the capacity for the organism to adapt fuel oxidation to fuel availability”. The metabolic diversification strategies used by individual bacteria vary greatly from the use of novel or acquired enzymes to the use of plasmid-localised genes and transporters. In this review, we describe the ability of lactobacilli to utilise a variety of carbon sources from their current or new environments in order to grow and survive. The genus Lactobacillus now includes more than 150 species, many with adaptive capabilities, broad metabolic capacity and species/strain variance. They are therefore, an informative example of a cell factory capable of adapting to new niches with differing nutritional landscapes. Indeed, lactobacilli naturally colonise and grow in a wide variety of environmental niches which include the roots and foliage of plants, silage, various fermented foods and beverages, the human vagina and the mammalian gastrointestinal tract (GIT; including the mouth, stomach, small intestine and large intestine). Here we primarily describe the metabolic flexibility of some lactobacilli isolated from the mammalian gastrointestinal tract, and we also describe some of the food-associated species with a proven ability to adapt to the GIT. As examples this review concentrates on the following species - Lb. plantarum, Lb. acidophilus, Lb. ruminis, Lb. salivarius, Lb. reuteri and Lb. sakei, to highlight the diversity and inter-relationships between the catabolic nature of species within the genus.

The application of next generation sequencing to profile microbe related cheese quality defects

High throughput next generation sequencing, together with advanced molecular methods, has considerably enhanced the field of food microbiology. By overcoming biases associated with culture dependant approaches, it has become possible to achieve novel insights into the nature of food-borne microbial communities. In this thesis, several different sequencing-based approaches were applied with a view to better understanding microbe associated quality defects in cheese. Initially, a literature review provides an overview of microbe-associated cheese quality defects as well as molecular methods for profiling complex microbial communities. Following this, 16S rRNA sequencing revealed temporal and spatial differences in microbial composition due to the time during the production day that specific commercial cheeses were manufactured. A novel Ion PGM sequencing approach, focusing on decarboxylase genes rather than 16S rRNA genes, was then successfully employed to profile the biogenic amine producing cohort of a series of artisanal cheeses. Investigations into the phenomenon of cheese pinking formed the basis of a joint 16S rRNA and whole genome shotgun sequencing approach, leading to the identification of Thermus species and, more specifically, the pathway involved in production of lycopene, a red coloured carotenoid. Finally, using a more traditional approach, the effect of addition of a facultatively heterofermentative Lactobacillus (Lactobacillus casei) to a Swiss-type cheese, in which starter activity was compromised, was investigated from the perspective of its ability to promote gas defects and irregular eye formation. X-ray computed tomography was used to visualise, using a non-destructive method, the consequences of the undesirable gas formation that resulted. Ultimately this thesis has demonstrated that the application of molecular techniques, such as next generation sequencing, can provide a detailed insight into defect-causing microbial populations present and thereby may underpin approaches to optimise the quality and consistency of a wide variety of cheeses.

Étude du rôle pathogénique de la formiminotransférase-cyclodésaminase dans l'hépatite auto-immune de type 2

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Étude des autoanticorps marqueurs de l'hépatite autoimmune présents chez les patients infectés de façon chronique par le virus de l'hépatite C

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Enteric methane emissions and nitrogen utilisation efficiency for two genotype of hill hoggets offered fresh, ensiled and pelleted ryegrass

Thirty-six 12-month-old hill hoggets were used in a 2 genotype (18 Scottish Blackface vs. 18 Swaledale×Scottish Blackface)×3 diet (fresh vs. ensiled vs. pelleted ryegrass) factorial design experiment to evaluate the effects of hogget genotype and forage type on enteric methane (CH4) emissions and nitrogen (N) utilisation. The hoggets were offered 3 diets ad libitum with no concentrate supplementation in a single period study with 6 hoggets for each of the 6 genotype×diet combinations (n=6). Fresh ryegrass was harvested daily in the morning. Pelleted ryegrass was sourced from a commercial supplier (Aylescott Driers & Feeds, Burrington, UK) and the ryegrass silage was ensiled with Ecosyl (Lactobacillus plantarum, Volac International Limited, Hertfordshire, UK) as an additive. The hoggets were housed in individual pens for at least 14 d before being transferred to individual respiration chambers for a further 4 d with feed intake, faeces and urine outputs and CH4 emissions measured. There was no significant interaction between genotype and forage type on any parameter evaluated. Sheep offered pelleted grass had greater feed intake (e.g. DM, energy and N) but less energy and nutrient apparent digestibility (e.g. DM, N and neutral detergent fibre (NDF)) than those given fresh grass or grass silage (P<0.001). Feeding pelleted grass, rather than fresh grass or grass silage, reduced enteric CH4 emissions as a proportion of DM intake and gross energy (GE) intake (P<0.01). Sheep offered fresh grass had a significantly lower acid detergent fibre (ADF) apparent digestibility, and CH4 energy output (CH4-E) as a proportion of GE intake than those offered grass silage (P<0.001). There was no significant difference, in CH4 emission rate or N utilisation efficiency when compared between Scottish Blackface and Swaledale × Scottish Blackface. Linear and multiple regression techniques were used to develop relationships between CH4 emissions or N excretion and dietary and animal variables using data from sheep offered fresh ryegrass and grass silage. The equation relating CH4-E (MJ/d) to GE intake (GEI, MJ/d), energy apparent digestibility (DE/GE) and metabolisability (ME/GE) resulted in a high r2 (CH4-E=0.074 GEI+9.2 DE/GE−10.2 ME/GE−0.37, r2=0.93). N intake (NI) was the best predictor for manure N excretion (Manure N=0.66 NI+0.96, r2=0.85). The use of these relationships can potentially improve the precision and decrease the uncertainty in predicting CH4 emissions and N excretion for sheep production systems managed under the current feeding conditions.

Caratterizzazione della popolazione microbica di salami fermentati spontaneamente

Le fermentazioni spontanee nei salami sono un fenomeno molto complesso in cui si assiste ad una successione di diverse popolazioni microbiche (micrococchi, stafilococchi e batteri lattici), avente ognuna un ruolo importante nell’ottenimento delle qualità desiderate del prodotto finale. Questo susseguirsi vorticoso di popolazioni microbiche nelle fermentazioni spontanee viene ormai regolamentato dall’uso di colture starter che, aggiunte in quantità idonee immediatamente prima dell’insaccamento, contribuiscono in maniera essenziale ad un rapido, corretto (e soprattutto sicuro) processo di fermentazione prima e di disidratazione poi. Poiché però l’impiego di starter segue spesso protocolli indifferenziati, è importante la selezione di colture starter “taylor made”, in grado di differenziare i prodotti in maniera riconoscibile con l’apporto di specifici tratti organolettici e sensoriali. Dunque, con questo elaborato di tesi si è voluto procedere alla raccolta di potenziali colture starter provenienti da diversi salami della tradizione mediterranea (salame di tipo bresciano, romagnolo e lucano), fermentati spontaneamente. Questi prodotti sono stati dapprima caratterizzati per quanto riguarda le loro caratteristiche microbiologiche, chimico-fisiche, profilo aromatico e il loro contenuto in ammine biogene. Successivamente, in particolare per il salame di tipo bresciano, sono stati isolati ceppi di interesse dalla popolazione lattica, poi identificati e testati per alcune caratteristiche di interesse tecnologico (capacità di produrre ammine biogene e profilo aromatico). I ceppi riscontrati, appartenenti alle specie Lactobacillus sakei e Lactobacillus curvatus, hanno mostrato una notevole biodiversità, soprattutto per quanto riguarda la produzione di molecole aromatiche, e quindi una notevole capacità, anche nell’ambito della stessa specie, di modulare la produzione di composti molto importanti dal punto di vista organolettico.

Effetto di batteri lattici probiotici microincapsulati sulle proprietà chimico-fisiche e microbiologiche di Squacquerone funzionale

In questo studio sono state prodotte 5 diverse tipologie di Squacquerone inoculate con St. thermophilus e con batteri lattici funzionali in forma planctonica o microincapsulata al fine di garantire la loro sopravvivenza, in ambienti caratterizzati da condizioni chimico-fisiche proibitive. Sono stati utilizzati Lb. crispatus BC4 e Lb. paracasei A13 caratterizzati da comprovata attività anti-Candida e battericida o probiotica, rispettivamente. I campioni sono stati caratterizzati a livello microbiologico, chimico-fisico, reologico, proteolitico ed organolettico durante la maturazione/conservazione a 4°C. I dati hanno dimostrato che l’incapsulazione non ha inciso sulla sopravvivenza dei ceppi funzionali in quanto la loro concentrazione è sempre stata costante. La proteolisi ha dimostrato un’evidente relazione tra il ceppo e le modalità della sua immissione nel prodotto: in presenza dei co-starter non incapsulati i processi litici sono accelerati rispetto al campione di controllo, mentre la cinetica di proteolisi viene influenzata negativamente dalle microcapsule. I campioni funzionali, dopo 4 giorni, sono risultati nettamente più apprezzati a livello sensoriale, indipendentemente dal tipo di inclusione dei microrganismi. A fine shelf-life i 5 Squacqueroni sono invece molto più simili tra loro. Di conseguenza, la microincapsulazione può essere vista come uno strumento per modulare le cinetiche di maturazione dello Squacquerone e il momento di immissione di questo sul mercato, per estendere la sua shelf-life ed allargare i mercati. In conclusione sia l’addizione dei co-starter in forma planctonica, che in forma microincapsulata con St. thermophilus, hanno permesso di produrre quattro diversi Squacqueroni identificabili come alimenti funzionali caratterizzati da specifici patterns proteolitici e organolettici. La microincapsulazione e la selezione dei ceppi sono da considerarsi strumenti utili alla innovazione e alla differenziazione dei prodotti lattiero-caseari.

Diet and Probiotics during Pregnancy - Endorsing developmental Programming

Maternal obesity has been shown to increase the risk for adverse reproductive health outcomes such as gestational diabetes, hypertension, and preeclampsia. Moreover, several studies have indicated that overnutrition and maternal obesity adversely program the development of offspring by predisposing them to obesity and other chronic diseases later in life. The exact molecular mechanisms leading to developmental programming are not known, but it has recently been suggested that obesity-related low-grade inflammation, gut microbiota and epigenetic gene regulation (in particularly DNA methylation) participate in the developmental programming phenomenon. The aim of this thesis was to evaluate the effect of diet, dietary counseling and probiotic intervention during pregnancy in endorsing favorable developmental programming. The study population consisted of 256 mother-child pairs participating in a prospective, double-blinded dietary counselling and probiotic intervention (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12) NAMI (Nutrition, Allergy, Mucosal immunology and Intestinal microbiota) study. Further overweight women were recruited from maternal welfare clinics in the area of Southwest Finland and from the prenatal outpatient clinic at Turku University Hospital. Dietary counseling was aimed to modify women’s dietary intake to comply with the recommended intake for pregnant women. Specifically, counseling aimed to affect the type of fat consumed and to increase the amount of fiber in the women’s diets. Leptin concentration was used as a marker for obesity-related low-grade inflammation, antioxidant vitamin status as an efficiency marker for dietary counselling and epigenetic DNA methylation of obesity related genes as a marker for probiotics influence. Results revealed that dietary intake may modify obesity-associated low-grade inflammation as measured by serum leptin concentration. Specifically, dietary fiber intake may lower leptin concentration in women, whereas the intakes of saturated fatty acids and sucrose have an opposite effect. Neither dietary counselling nor probiotic intervention modified leptin concentration in women, but probiotics tended to increase children’s leptin concentration. Dietary counseling was an efficient tool for improving antioxidant vitamin intake in women, which was reflected in the breast milk vitamin concentration. Probiotic intervention affected DNA methylation of dozens of obesity and weight gain related genes both in women and their children. Altogether these results indicate that dietary components, dietary counseling and probiotic supplementation during pregnancy may modify the intrauterine environment towards favorable developmental programming.

USE OF STARTER CULTURES TO IMPROVE PORTUGUESE TRADITIONAL SAUSAGES

In Mediterranean countries, such as Portugal, traditional dry-fermented sausages are highly appreciated. They are often still being manufactured in small processing units, according to traditional procedures. The aims of the present study were to evaluate the effect of different starter cultures and their optimal concentration, to reduce the microbial load in end-products, with the purpose to improve the sausages’ safety, without deteriorating sensory acceptability. pH, aw, colour, texture and microbiological profile were assessed. On the other hand, a sensory panel evaluated the products. Based on the first results, S. xylosus and L. sakei were chosen to be inoculated together with a yeast strain. In the mixed starter culture experiment, a food safety issue arose probably related to the higher aw value (0.91). The presence of Salmonella spp. detected in a few end-products sausages did not allow a full sensory evaluation in the mixed starter culture experiment. However, in the two preliminary experiments, the use of starter cultures did not depreciate the panellists’ overall appreciation and products acceptability.

Conversão da lactose e síntese de galacto-oligossacarídeos: uma abordagem experimental e teórica

O presente trabalho avaliou, na etapa experimental, um processo simultâneo de catálise e fermentação láctica visando obter um iogurte com potenciais características nutracêuticas e, na sua etapa teórica, estabeleceu uma interlocução entre a vivência experimentalista e a teoria da cinética enzimática, no que se refere à conversão da lactose e à síntese de galactooligossacarídeos (GOS). Na abordagem experimental, para um substrato específico, avaliouse biocatálise conduzida simultaneamente à fermentação, defasando a adição da enzima em relação ao início do processo fermentativo. A fermentação foi realizada a partir de cultura láctica liofilizada comercial contendo dois micro-organismos probióticos, Bifidobacterium animalis e Lactobacillus acidophilus, associados aos micro-organismos característicos do iogurte, Lactobacillus bulgaricus e Streptococcus thermophilus. Foi utilizado um preparado enzimático contendo -galactosidases obtidas de duas origens distintas: Kluyveromyces lactis e Aspergillus niger. Foram avaliados os efeitos da concentração da enzima e do tempo de adição da enzima em um planejamento experimental 2 2 . As respostas foram às concentrações, ao final do processo, de lactose, de GOS, de glicose e de galactose e a hidrólise dos galactooligossacarídeos ao longo do tempo. No que se refere à abordagem teórica, o presente trabalho considerou modelos matemáticos de hidrólise de dissacarídeos e conversão da lactose, em que a inibição foi representada a partir do incremento da concentração dos produtos da reação. No que se refere à conversão da lactose e síntese de GOS, o presente trabalho buscou estabelecer um modelo matemático em que a inibição ocorreu por efeito do incremento das concentrações de glicose e de galactose, comparando-o com os modelos conhecidos na literatura. Verificou-se que o desempenho do modelo obtido no presente trabalho foi robusto em relação às premissas estabelecidas. Na comparação com resultados experimentais de conversão enzimática, o modelo mostrou-se capaz de minimizar o erro e de ajustar-se aos dados experimentais.

Aditivos químicos e biológicos na ensilagem de cana-de-açúcar: 1. composição química das silagens, ingestão, digestibilidade e comportamento ingestivo

Avaliou-se o efeito da inclusão de aditivos na ensilagem de cana-de-açúcar (Saccharum officinarum L.) sobre a composição químico-bromatológica das silagens, o comportamento ingestivo, o consumo voluntário e a digestibilidade em bovinos de corte. Utilizaram-se cinco novilhos da raça Nelore providos de cânula ruminal, alocados em delineamento quadrado latino 5 ´ 5 e alimentados com dietas com 65% de volumoso na MS. Foram avaliadas cinco silagens (base úmida): controle - cana-de-açúcar sem aditivos; uréia - cana-de-açúcar + 0,5% uréia; benzoato - cana-de-açúcar + 0,1% de benzoato de sódio; LP - cana-de-açúcar inoculada com Lactobacillus plantarum (1 ´ 10(6) ufc/g MV); LB - cana-de-açúcar inoculada com L. buchneri (3,6 ´ 10(5) ufc/g forragem). A forragem foi armazenada em silos do tipo poço por 90 dias antes do fornecimento aos animais. A composição químico-bromatológica da cana-de-açúcar foi alterada após a ensilagem, em relação à cana-de-açúcar original, com redução no teor de carboidratos solúveis e na digestibilidade in vitro e elevação relativa nos teores de FDN e FDA. Os teores de etanol (0,30% da MS) e ácidos orgânicos (0,99% de ácido lático e 2,31% de acético) foram baixos e semelhantes entre as silagens. Os aditivos aplicados na ensilagem não promoveram alterações no consumo e na digestibilidade aparente da MS (7,2 kg/dia e 63,6%, respectivamente). O comportamento ingestivo dos animais também não foi alterado, com tempos médios de 230,6; 519,6 e 672,8 minutos/dia despendidos com ingestão de ração, ruminação e ócio, respectivamente. Os aditivos acrescidos à cana-de-açúcar promoveram pequenas alterações na maioria das variáveis avaliadas.

Aditivos químicos ou biológicos na ensilagem de cana-de-açúcar: 2. parâmetros ruminais e degradabilidade da matéria seca e das frações fibrosas

Objetivou-se avaliar o efeito da inclusão de aditivos na ensilagem de cana-de-açúcar (Saccharum officinarum L.) sobre a degradação de MS e de componentes da parede celular e sobre os parâmetros de fermentação ruminal em bovinos alimentados com dietas contendo essas silagens. Utilizaram-se cinco novilhos da raça Nelore providos de cânula ruminal, alocados em delineamento quadrado latino 5 ´ 5 e alimentados com dietas com 65% de volumoso (%) MS. Foram avaliadas cinco silagens (base úmida): controle - cana-de-açúcar, sem aditivos; uréia - cana-de-açúcar + 0,5% ureia; benzoato - cana-de-açúcar + 0,1% de benzoato de sódio; LP - cana-de-açúcar inoculada com Lactobacillus plantarum (1 ´ 10(6) ufc/g MV); LB - cana-de-açúcar inoculada com L. buchneri (3,6 ´ 10(5) ufc/g forragem). A forragem foi armazenada em silos do tipo poço por 90 dias antes do fornecimento aos animais. Os parâmetros ruminais foram afetados de forma moderada pelas silagens e tiveram forte efeito do horário de coleta de amostras. As concentrações molares médias dos ácidos acético, propiônico e butírico foram de 60,9; 19,3 e 10,2 mM, respectivamente. O ambiente ruminal proporcionado por dietas formuladas com silagens de cana-de-açúcar foi satisfatório e similar ao tradicionalmente observado em dietas contendo cana. O uso de aditivos na ensilagem influenciou, de forma não-significativa, a degradabilidade ruminal da MS e da MO, mas não alterou a degradabilidade ruminal da fração fibrosa. Os aditivos aplicados à cana-de-açúcar resultaram em pequenas alterações na maior parte das variáveis avaliadas. Apesar de a degradabilidade ruminal das silagens ter sido pouco afetada pelo uso de aditivos, os valores observados foram próximos aos observados para a cana-de-açúcar in natura.

MICROBIOLOGICAL ANALYSIS OF TRADITIONALLY FERMENTED MILK SOLD IN KINIGI SECTOR OF MUSANZE DISTRICT IN RWANDA

A research work entitled: “Microbiological analysis of traditionally fermented milk (Ikivuguto) sold in Kinigi Sector of Musanze District,” was carried out at Higher Learning Institution of Applied Sciences (INES-Ruhengeri) Laboratory of Microbiology located near Volcanoes in the Northern Province of Rwanda. The main objective of this work was to determine the microbiological quality of traditionally fermented milk, which is consumed by Kinigi Center local people. The hypothesis was to analyze if traditionally fermented milk commercialized in Kinigi restaurants contained pathogenic bacteria such as fecal coliforms and Escherichia coli , in addition to staphylococci and yeasts. Milk samples were collected from Kinigi sector and examined in the microbiology laboratory in order to assess the microbiological quality and safety of traditionally fermented milk in rural areas. The samples were analyzed qualitatively and quantitatively for the microbes found in fermented milk sold in Kinigi Center, and the results were as follows: 7.21x107 CFU/ml for total counts; 3.89x107 CFU/ml for Lactobacillus ; 2.77x107 CFU/ml for yeasts; 1.196x105 CFU/ml for total coliforms; 9.63x104 CFU/ml for fecal coliforms and 8.92x103 CFU/ml for staphylococci. Biochemical tests were carried out and the results showed that identified pathogens were E. coli, Providencia alcalifaciens , and the staphylococci group. It was found that fermented milk contained genera and species of Staphylococcus haemolyticus , Staphylococcus aureus , Staphylococcus intermedius , Staphylococcus xylosus and Staphylococcus saprophyticus . Findings showed that the commercial milk samples were cross-contaminated by different pathogens from environment. These contaminations could have been due to improper handling, presence of flies, soil erosion, dust from atmosphere, as well as contaminated milk vessels or pots, stirrers and unpasteurized water. It was concluded that local farmers and milk retailers did not adhere to required hygienic conditions for milk safety. In this regard, the sold traditional fermented milk does not meet health and safety standards because people did not respect good manufacturing practices. The hypothesis and main objective were confirmed, because traditionally fermented milk of Kinigi was cross-contaminated before consumption. Thus, it would be better to train farmers in the areas of product hygiene, sanitation and safety during milking, processing and marketing.

Amino acid composition of processed fish silage using different raw materials

The objective was to evaluate amino acid composition of silages produced from three raw materials. Commercial marine fish waste, commercial freshwater fish waste, and tilapia filleting residue were used to produce fish silage by acid digestion (20 ml/kg formic acid and 20 ml/kg sulfuric acid) and anaerobic fermentation (50 g/kg Lactobacillus plantarum, 150 g/kg sugar cane molasses). Protein content and amino acid composition were determined for raw materials and silage. Marine fish waste had higher crude protein content (776.7 g/kg) compared to freshwater fish waste (496.2 g/kg) and tilapia filleting residue (429.9 g/kg). All silages lacked up to three amino acids for each product according to FAO standards for essential amino acids. However, considering as the limiting factor only the amino acids below the 30% minimum requirement for fish in general, all products were satisfactory with respect to essential amino acids. Therefore, the results suggest that all products investigated are appropriate for use in balanced fish diets. (C) 2003 Elsevier B.V. B.V. All rights reserved.

Production of bioactive metabolites by intestinal bacteria

The adult intestinal microbiota comprises a microbial ecosystem of approximately 100 trillion microorganisms, with specific bacterial communities holding distinct metabolic capabilities. Bacteria produce a range of bioactive compounds to survive unfavourable stimuli and to interact with other organisms, and generate several bioactive products during degradation of dietary constituents the host is not capable of digesting. This thesis addressed the impact of feeding potential probiotic bacteria and other dietary strategies such as pure fatty acids and prebiotics, on gut microbiota composition, short chain fatty acid (SCFA) production and modulation of metabolism in animal models. In the first experimental chapter (Chapter 2) a gas chromatography method for the quantification of SCFA was optimized and applied in the analysis of caecal samples obtained in animal studies described in other chapters of this thesis. In Chapter 3, t10, c12 CLA supplementation was shown to significantly alter murine gut microbiota composition and SCFA production rather than no supplementation. These changes were suggested to be extra factors affecting host lipid metabolism. Chapter 4 described the contrasting effects of CLA-producing strains, Bifidobacterium breve DPC 6330 and B. breve NCIMB 702258, on murine fat distribution/composition and gut microbiota composition, suggesting that these changes were most likely strain-dependent. In Chapter 5, dietary GABA-producing strain Lactobacillus brevis DPC 6108 was shown to significantly increase (p<0.05) serum insulin in healthy rats, leading to a second experiment using a type 1 diabetes rat model. Lb. brevis DPC 6108 administration did not change insulin levels in diabetic rats, but attenuated high levels of glucose when compared to diabetic control. However, an auto-immune-induced diabetes model was suggested as a better model to study GABA-related effects on diabetes. In Chapter 6 bovine milk oligosaccharides, 6’sialyllactose and Beneo Orafti P95 oligofructose supplementations were associated with depletion or reduction of less favourable bacteria, demonstrating that ingestion of these oligosaccharides might be a safe and effective approach to modulate populations of the intestinal microbiota. In Chapter 7 (General discussion) the major findings of all studies were reviewed and discussed.