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Among 29 strains of bifidobacteria studied as sources of beta-galactosidase enzyme, Bifidobacterium infantis HL96 showed the highest hydrolytic and transgalactosylic activities. This strain grew well in a MRS medium containing various sugars including lactose, and produced three beta-galactosidases (termed beta-Gal I, II, III). / Two genes, beta-galI and beta-galIII, located on 4.6 and 4.4 kb DNA fragments respectively, were cloned into E. coli, and the nucleotide sequences were determined. The 3,069 by-long beta-galI, encoded a polypeptide with a Mr of 113 kDa. A putative ribosome-binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. A partial sequence of an ORF transcribing divergently from beta-galI resembled a lactose permease gene. The beta-galIII gene, which is 2,076 bp long, encoded a polypeptide with a Mr of 76 kDa. A rho-independent, transcription terminator-like sequence was found 25 bp downstream of the termination codon. / The amino acid sequences of beta-GalI and beta-GalIII were homologous to those in the LacZ and LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, containing a glutamate at residue 160. beta-GalI and beta-GalIII were over-expressed 35 and 96 times respectively in E. coli by using a pET expression system. / Both beta-GalI and beta-GalIII were specific for beta-D -anomeric linked galactosides, but beta-GalI showed more hydrolytic and synthetic activities toward lactose than beta-GalIII. The galacto-oligosaccharides (GaOS) production mediated by beta-GalI at 37°C in 20% (w/v) lactose was 130 mg/ml, which is six times higher than that of beta-GalIII. The yield of GaOS further increased to 190 mg/ml in 30% (w/v) lactose. A major tri-saccharide produced by beta-GalI was characterized as O-beta- D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)- D-glucopyranose. / beta-GalI was purified by ammonium sulphate precipitation, and anion-exchange (Mono-Q) and gel filtration (Superose 12) chromatographic steps. The enzyme appeared to be a tetramer, with a Mr of 470 kDa as estimated by native PAGE and gel-filtration chromatography. The optimum temperature and pH for ONPG and lactose as substrates were 60°C, pH 7.5, and 50°C, pH 7.5, respectively. The enzyme was stable over the pH range of 5~8.5, and was particularly active at 50°C for more than 80 min. The enzyme was significantly activated by reducing agents, especially glutathione, as well as by Na+ and K+ cations. Maximal activity required both Na+ and K+ at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, and by most bivalent metal ions. Hydrolytic activity using 20 mM lactose as substrate was significantly inhibited by 10 mM galactose. The Km and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. / The objectives of this research were to characterize beta-galactosidases of B. infantis HL96 at the molecular and biochemical levels, and to over-express the enzymes in Escherichia coli. Two beta-galactosidase isoenzymes with unique properties were genetically characterized for the first time. beta-GalI properties included a neutral pH optimum, relatively higher temperature stability and a high transgalactosylic activity that makes it very competitive for GaOS synthesis. The results were also important for the advancement of knowledge on the catalytic mechanism and the evolutionary aspect of this enzyme.

Investigations of the molecular binding partners of the probiotic bacterium Bifidobacterium longum subspecies infantis (B. infantis) and the pathogen Salmonella enterica subspecies enterica serovar Typhimurium LT2 (Salmonella ser. Typhimurium) found that these two very different bacteria bind gangliosides. However, these organisms lead to completely different host health outcomes when present in the gut. B. infantis is the founding microbial population in the intestinal tract of breast-fed infants. S. typhimurium is the most important food-borne pathogen that results in humans. This study used an in vitro gut epithelial cell model to examine the host cellular response to adhesion of B. infantis, which led to an increase in intestinal epithelium survival. This observation led to a series of experiments to elucidate the pathway for host signaling initiated by adherence of B. infantis to the host membrane to explain the increase in host cell survival. B. infantis adhesion induced significant (q≤0.05) differential expression of 208 host genes. These genes were associated with increased broad mechanisms of cell survival that included BIRC3, TNFAIP3, and SERPINB9. We hypothesized that a biochemical link existed between the host membrane adhesion protein and the increase in cell survival, mediated via AKT. We tested this hypothesis to demonstrate that B. infantis interaction initiated signal transduction using G-proteins via phosphorylation of AKT and induced production of the BIRC3, TNFAIP3, and SERPINB9. This study discovered adhesion of B. infantis initiated activation of AKT via phosphorylation of both Ser473 and Thr308, which results in increased cell survival.

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

Self-injurious behavior (SIB) is a behavioral pathology seen in a small percentage of humans and non-human primates. In one previous study, macaques with SIB had more sleep disruption than controls, but observations were limited. Two studies were conducted: a baseline study to investigate nighttime activity in rhesus macaques (Macaca mulatta) displaying SIB and controls, and a probiotic study to assess probiotic Bifidobacterium infantis 35624 for high nighttime activity. Subjects were 13 rhesus macaques, 5 with SIB (3 females; 1 SIB). Videocapture of Nighttime Activity (VNRA) was developed to record in complete darkness. IR-receptive webcams were connected to a laptop running ISPYCONNECT, software which recorded movement. Subjects were observed during the entire lights-off period (8pm-7am). Measures included total movement time (TMT), movement in hour 1 (HR1) and hour 11 (HR11), and number of videos. In the baseline, SIB subjects had higher TMT (pBifidobacterium infantis 35624 had no effect on sleep disruption, and also that increased nighttime activity seems to be a persistent characteristic of SIB subjects. It is unknown if increased nighttime activity affects SIB subjects; it may result in elevated SIB, or the SIB pathology could result in sleep disruption.

Les maladies inflammatoires chroniques de l'intestin sont caractérisées par une inflammation anormale et récurrente du tractus digestif. De nombreuses études ont démontré des effets bénéfiques de souches probiotiques anti-inflammatoires recombinantes ou non. La première partie de cette thèse décrit différentes stratégies d'optimisation de souches de bactéries lactiques en tant que vecteurs de protéines d'intérêt santé. Nous avons ainsi démontré qu'une modification du peptidoglycane de la paroie de Lactococcus lactis influençant la lyse bactérienne ne permettait pas de moduler l'immunogénicité de l'antigène E7 délivré par L. lactis. Nous avons également démontré que la nature du vecteur bactérien était un paramètre essentiel dans la vectorisation de la protéine délivrée : ainsi l'espèce Bifidobacterium infantis induit une réponse immunitaire spécifique à l'antigène E7 supérieure à celle obtenue avec les vecteurs L. lactis et Lactobacillus plantarum. La deuxième partie de cette thèse porte sur l'étude des effets anti-inflammatoires de bactéries recombinantes ou non. Nous avons ainsi démontré que la souche Lb. casei BL23 produisant une superoxyde dismutase à manganèse permettait de diminuer significativement des colites murines induites par administration de dextran sodium sulfate. Enfin, nous avons mis en évidence des propriétés anti-inflammatoires sur divers modèles d'inflammation in vitro / in vivo de Faecalibacterium prausnitzii, première bactérie commensale anti-inflammatoire identifiée sur la base de données cliniques humaines.