Tesi sul tema "Bifidobacterium infantis"

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.

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.

O objetivo deste trabalho foi estudar a viabilidade de Bifidobacterium animalis subsp. lactis HN019 em fórmulas infantis fermentadas ou não, probióticas durante armazenamento a 4°C. Três matrizes lácteas e três não lácteas (a base de soja) foram utilizadas para a elaboração de produtos fermentados ou não fermentados usando Bifidobacterium animalis subsp. lactis HN019, resultando em doze diferentes fórmulas probióticas para lactentes. O perfil de acidificação foi determinado a 42°C até pH 4,7. Determinações físico-químicas (sólidos totais, proteína, gordura, cinzas, carboidratos, calorias, densidade e pH) foram realizadas e foram focadas as contagens de bactérias viáveis durante o armazenamento refrigerado. A caracterização química dos produtos lácteos e a não lácteos apresentou resultados diferentes, à exceção FSL2, todos estavam de acordo com Codex Alimentarius. O perfil de acidificação de Bifidobacterium animalis subsp. lactis HN019 diferiu conforme a matriz. Durante o armazenamento dos produtos a 4°C, a contagem de bactérias viáveis de acordo com o preconizado, bem como a pós-acidificação, estando em conformidade com as recomendações da legislação brasileira. Processo (fermentação ou adição) e tipo de matriz (lácteos e não lácteos) influenciaram a pós-acidificação e a viabilidade de Bifidobacterium animalis subsp. lactis HN019. As fórmulas para lactentes podem ser considerados bons veículos de Bifidobacterium animalis subsp. lactis HN019.
This study proposed to study infant formulas as vehicles for Bifidobacterium animalis ssp.lactis HNOI9. Three dairy and three non-dairy matrices were employed for the preparation of fermented or unfermented products using Bifidobacterium animalis ssp. lactis HN019 resulting in twelve different probiotic infant formulas. Acidification profile of the probiotic was determined at 42°C until pH 4.7. Physicochemical determination (total solids, protein, fat, ash, carbohydrates and calories, density and pH) was conducted, and counts viable bacteria (in dairy and non dairy infant formulas fermented and unfermented) during cold storage was focused on. The chemical characterization of the dairy and non-dairy matrix showed different results, the exception FSL2, all were in accordance to the Codex Alimentarius. The acidification profile of B. animalis ssp. lactis HN019 differed according to the matrix. During storage of products at 4°C counts of viable bacteria were stable as well as post-acidification, and were in accordance with the recommendations of the Brazilian legislation. Process (fermentation or addition) and matrix type (dairy and non-dairy) influenced post-acidification and viability of B. animalis ssp. lactis BN019 . Infant formulas could be considered good vehicles for Bifidobacterium animalis ssp. lactis HN019.

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)

IBDs are chronic inflammatory conditions, characterized by remissions and relapses, whose main manifestations are ulcerative colitis and Crohn's disease. Ulcerative colitis, one of the main forms of IBDS has as standard treatment the use of corticosteroids and anti-inflammatory drugs. The use of antibiotics has also been reported, but the possible adverse effects must be taken into consideration and thus the use of probiotics emerges as a real possibility. In this study, we evaluated the possibility of using Bifidobacterium longum subsp. infantis CHCC2228 as treatment for ulcerative colitis in a murine model. For induction of colitis in female BALB/c mice, we replace the water by a 3.5% DSS (dextran sodium sulphate) solution for 7 days. During this period, the animals were evaluated for weight variation, fecal consistency and presence of bleeding in feces. On the seventh day, the animals were euthanized to collect the organs to the histological analysis of the liver, small intestine and colon. Other experiments were done, as: dosage of sIgA in the small intestine; evaluation of the intestinal permeability; indirect evaluation of the infiltration of eosinophils, neutrophils and macrophages by measuring their specific enzymes EPO (eosinophil peroxidase), MPO (myeloperoxidase) and NAG (N-acetyl-glucosaminidase); dosage of the cytokines KC and Eotaxin-1; evaluation of the permeability and oxidative stress in the intestine. Treatment with the probiotic resulted in clinical improvement of animals. The histological and morphometric analyzes showed a reduction of lesions and edema in the animals, but there was no increase in the production of mucin. The dosage of sIgA was significantly higher in the colitis group and reduced in the group treated with the probiotic. There was also a reduction in the inflammation of the colon, as indicated by the reduction of EPO and MPO activity, but no change in the activity of NAG. The intestinal permeability, which is typically increased during the onset of IBD, was reduced after treatment with bifidobacteria. There were no differences on rates of reactive oxygen species (ROS) generation. Based on these data it can be concluded that the bacterium B. longum subsp. infantis CHCC2228 has probiotic potential for the treatment of ulcerative colitis, but further studies should be conducted in order to verify the mechanisms of action of the bacterium.
Doenças inflamatórias intestinais (IBDs) são condições inflamatórias crônicas, marcadas por remissões e recidivas, de origem idiopática, mas que possuem mediação imunológica. A colite ulcerativa (UC), uma das principais formas de IBDs, tem como tratamento padrão o uso de anti-inflamatórios e corticosteróides. O uso de antibióticos também tem sido relatado, mas aqui devem ser considerados os efeitos colaterais associados. Nos últimos anos, a utilização de probióticos no tratamento de IBDs vem ganhando atenção na comunidade médica. Este trabalho objetivou avaliar os efeitos de Bifidobacterium longum subsp. infantis CHCC2228 no tratamento de UC em um modelo murino. Para a indução da colite em camundongos BALB/c fêmeas, a água foi suplementada com 3,5% de DSS (sulfato sódico de dextrana) a 3,5% por 7 dias. Durante este período os animais foram avaliados quanto à variação de peso, consistência fecal e presença de sangue nas fezes. No sétimo dia os animais foram eutanasiados para coleta dos órgãos para realização de análises histológica do fígado, do intestino delgado e do cólon. Foram, ainda, realizados: dosagem de imunoglobulina secretada (sIgA) no intestino delgado; avaliação da permeabilidade intestinal; avaliação indireta dos infiltrados de neutrófilos pela enzima mieloperoxidase (MPO), eosinófilos pela peroxidase eosinofílica (EPO) e macrófagos pela N-acetil-glicosaminidase (NAG); dosagem das citocinas KC e eotaxina-1; avaliação da permeabilidade e do estresse oxidativo no intestino. O tratamento com o probiótico melhorou o quadro clínico provocado pelo DSS nos animais. As análises histológicas e morfométricas mostraram que houve uma tendência à redução de áreas de lesão e edema nos animais, mas não houve aumento na produção de mucina. A dosagem de sIgA mostrou-se maior no grupo com colite e reduzido no grupo com colite e tratado com o probiótico. Houve, ainda, uma redução no quadro inflamatório do cólon, com redução das atividades enzimáticas de EPO e MPO, mas sem alteração na atividade de NAG. A permeabilidade intestinal, que está tipicamente aumentada durante o acometimento das IBD, mostrou-se reduzida após o tratamento com a bifidobactéria. Não foram observadas diferenças nas taxas de geração de espécies reativas de oxigênio (ROS). Baseado nestes dados pode-se concluir que a bactéria B. Longum subsp. infantis CHCC2228 possui potencial probiótico no modelo estudado e é um bom candidato para o tratamento para o tratamento da colite ulcerativa.

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.

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.

En français : 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
En anglais : Inflammatory bowel diseases are characterized by abnormal inflammation of digestive tract. Several studies have shown positive effects of anti-inflammatory probiotic (native or recombinant) or commensal bacterial strains. The first part of this PhD thesis describes different optimization strategies of the use of lactic acid bacteria as proteins delivery vector. We have shown that the modification of peptidoglycan of Lactococcus lactis influencing lysis rate does not confer any advantage in both persistence in gastrointestinal tract and proteins delivery vector abilities. We showed that nature of bacterial vector is an essential parameter to deliver protein of health interest: Bifidobacterium infantis could increase higher immune response against E7 antigen than the ones obtained with L. Lactis and Lactobacillus plantarum. In the second part of this PhD thesis, we analized anti-inflammatory effects of recombinant probiotic or commensal bacteria. We showed that Lb. Casei BL23 producing superoxide dismutase could significantly protect mice from dextran sodium sulfate induced colitis damages. Finally, we observed anti-inflammatory properties on cellulars and animals models of Faecalibacterium prausnitzii, the first commensal anti-inflammatory bacterium identified by analysis of human clinical data

Selected bacterial groups within the infant gut micro biota were investigated in relation to mode of delivery, initial diet (i.e. breast-fed versus formula-fed) and weaning. Colonic bacteria from 50 faecal samples (obtained from 30 infants during the milk-fed and, post-weaning phases) were enumerated by fluorescence in situ hybridisation. The probes used were as follows: Bac 303 (bacteroides), Bif 164 (bifidobacteria), Chis 150 (Clostridium clusters I and II), Erec 482 (Clostridium cluster XIVa), EC 1531 (Escherichia coli), Lab 158 (lactobacilli/enterococci group) and the DAPI nucleic acid stain (total cell count). In all cases bifidobacteria were identified as the major group. The mode of delivery did not influence the composition of the gut microbiota. Exclusively breast-fed infants had significantly lower Clostridium cluster XIVa counts than those exclusively formula-fed. Infants fed predominantly breast-milk had significantly lower Clostridium cluster XIVa, and higher total and bifidobacterial counts than predominantly formula-fed infants. Amongst the samples obtained following the introduction of solid food, no differences were observed following segregation into original cohorts (of delivery mode and initial diet). Furthermore, such faecal samples had significantly lower E. coli, total bacterial and bifidobacterial counts than those obtained during the milk-fed phase. Subsequently, the bifidobacterial component was further examined in relation to diversity and anti-microbial activity. Predominant biotypes (as determined by genetic fingerprinting of isolates) were identified by partial16S rRNA gene sequencing as five recognised bifidobacterial species, novel Bifidobacterium spp., Enterococcus faecalis and Lactobacillus plantarum. B. breve was the principle species isolated. However the bifidobacterial composition differed between infant cohorts.

Since the 1970s, the United States has encountered an increasing proportion of Cesarean deliveries (CS), surpassing the advised 10-15% maximum rate established in 1985 by the World Health Organization (World Health Organization, 1985). This increasing rate has fueled correlational and causational studies observing the impact of Cesarean delivery on several aspects of infant health. Previous studies on CS infants have observed a delay in gut colonization by beneficial bacteria – for instance Bifidobacteria – traditionally transmitted from the mother’s gut and vaginal microbiome as other environmental factors have influenced the initial microflora (Biasucci et al., 2010; Dong, Yang, & Wang, 2010; Penders et al., 2006). In addition, an increasing proportion of births are occurring within a home setting, providing an opportunity to study how these possible environmental factors may influence bacterial colonization. This initial gastrointestinal colonization is considered one of the most important factors towards immune system development and general health. This thesis proposes an examination of how the mode and setting of delivery influence the diversity of Bifidobacterium species in infants’ initial gut microbiomes. Additionally, while several studies have examined the impact of specific bacterial species on immune system development, this study will provide an approach to understanding how differences in the overall gastrointestinal (GI) ecologies of CS and vaginal delivery (VD) infants impact immune system development.

The Diploma thesis deals with use of selected probiotic strains Lactobacillus acidophilus and Bifidobacterium breve in different forms in food for infants. The theoretical part is focused on describing probiotics, encapsulation methods and intestinal gut microbiota of infants. Further, characterization of individual periods of infant feeding and food for infants were introduced. In experimental part the possibilities of encapsulation and lyophilisation of probiotic cells were observed. Probiotic cells were encapsulated into alginate particles. The encapsulator was used for preparation of particles and the most appropriate particles were prepared by encapsulation nozzle with size of 300 µm. Moreover, probiotics viability was monitored by Flow Cytometry, Fluorescence Microscopy and by cultivation (CFU method). Viability of probiotics was monitored during long-term storage in selected food for infants. The appropriate shelf life of non-lyophilized alginate particles in real food have been set at 1 to 2 months. Lyophilized alginate particles could be stored for more than 3 months. Finally, the stability of the particles and viability of encapsulated and non-encapsulated cells in the gastrointestinal tract conditions were also examined. The viabilities of lyophilized cells and cells encapsulated in lyophilized particles were also compared. From the results obtained, non-encapsulated probiotic bacteria cells are more susceptible to negative effects of digestive juices, the percentage of dead probiotic cells after digestion was approximately 80 %. On the other hand, alginate particles showed cell protection from digestive juices, after incomplete cell releasing from particles the percentage of dead probiotic cells did not exceed 20 %. After adequate rehydration, similar results were gained with lyophilized alginate particles. Lyophilized alginate particles have been determined to be the most suitable application form for infants’ food.

The Diploma thesis deals with designing of probiotic dietary supplement for children with strains Lactobacillus acidophilus and Bifidobacterium breve and with prebiotics. Used prebiotics were Inulin, Chia fiber, Bamboo fiber, Chlorella + Spirulina and Yakon syrup. The theoretical part is focused on probiotics, prebiotics and their biological influence. In experimental part the possibilities of encapsulation into alginate particle and lyophilisation of probiotic cells were observed to find their good form to final nutritional product for children. Several types of probiotic with addition of prebiotics were tested in model conditions of gastrointestinal tract. It was found that addition of prebiotic highly increases viability of probiotic cells and their resistance to model conditions of gastrointestinal tract. In this case, the best prebiotic was found in Yakon syrup. The prebiotics were also characterised in terms of nutritional composition, amount of total and reducing sugars, oligosaccharides, proteins, lipids, polyphenols and chlorophyll were obtained. Finally, Chia fiber, Chlorella + Spirulina and Yakon syrup were chosen as prebiotics with best characterisation/properties. In conclusion, a dietary supplement with lyophilized alginate particles containing probiotic cells and with the most appropriate prebiotics were designed. Forms of the product were powder and gummy-bear.

Atopic allergy is the most common chronic disease among children in the developed world. This high prevalence could be associated with low microbial exposure. The early gut microbiota appears to be important for immune maturation. Immunomodulatory components in human milk might differ between mothers and could therefore explain the contradictory results seen regarding breastfeeding and allergy development. The aim of this thesis was to investigate whether early colonization with certain gut microbiota species influences childhood immune responses and allergy development up to age five. Also, as human milk oligosaccharides (HMOs) might stimulate the growth of certain gut microbiota species, the consumption of neutral colostrum HMOs was investigated for their role in allergy development up to 18 months. The concentrations of neutral colostrum HMOs varied considerably between women; however this variation could not be explained by their allergic status. Neither was the consumption of neutral colostrum HMOs related to allergy development in their children up to 18 months. Infants who harboured lactobacilli group I and Bifidobacterium adolescentis one week after birth developed allergic disease less frequently during their first five years than infants who did not harbour these bacteria at the same time. Also, colonization with several Bifidobacterium species was associated with higher levels of house dust endotoxin and larger family size. The early Bifidobacterium flora influenced levels of salivary secretory IgA at six and 12 months but not during later childhood. Moreover, the intensity of early Bacteroides fragilis colonization was inversely associated with spontaneous Toll-like receptor 4 mRNA expression in peripheral blood cells collected 12 months after birth. In conclusion, these results indicate that the early infant gut microbiota influences systemic and mucosal immune maturation during infancy, and that it might be altered in infants developing allergic disease.

This Diploma thesis deals with preparation of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium breve enriched with prebiotics meant for application in baby food products. Natural extracts from matcha, moringa, young beat, young barley, chlorella and spirulina were selected as prebiotics. The theoretical part is focused on probiotic bacteria, their biological effects and their effects on the child´s body. The experimental part deals with the cultivation of probiotic bacteria with plant extracts, monitoring their viability and stabilization in an encapsulated form. Mixtures of probiotic cells with prebiotics were encapsulated into alginate particles to increase stability. Some of the alginate particles were processed by freeze drying. Mixtures of probiotic cultures with plant extracts were subjected to model human digestion by the action of model digestive juices in unencapsulated, encapsulated and lyophilized form. Selected extracts of plant materials were characterized in terms of amount of total and reducing sugars, total phenolic substances, individual phenolic substances and antioxidant activity. Further, two baby commercial dietary supplements containing probiotics were selected, which were characterized in terms of cell number and viability. Probiotic products were also subjected to model digestion.

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
國立中興大學
食品暨應用生物科技學系所
99
Over the years, lactic acid bacteria have long been regarded as the effectiveness of probiotics, which can maintain of intestinal microflora, reduce lactose intolerance, blood pressure, blood fats, anti-cancer, anti-inflammation and regulate the immune system. It is kind of functional food is widely liked by people. Bifidobacterium, is belong to lacitic acid bacteria family and the greatest microflora in the infant gut. In the studies found that the population of Bifidobacterium in the gut is associated with the life span of the host, and the more longevity, the higher proportion of this bacteria.The main function of Bifidobacterium is to treat diarrhea, maintain intestinal microbial homeostasis and protect against harmful intestinal bacteria colonizing. Although there have confirmed their effectiveness in the immune experiments , but generally found that their ability to regulate immunity is not particularly powerful.The previous experiments has been shown that Bifidobacterium longum supsp. infants. GB-1496 had best capacity to modulate immunity in vitro. Therefore, further investigations on regulation of immune responses in murine model in vivo. In the present studies, we fed the Der p-sensitized BALB/c mice on high (3.0*107 cfu/per mice) , medium (9*107 cfu/per mice) and low (3.0*106 cfu/per mice) dose. The proliferation of splenocyte, cytokines secreted by splenocyte (IFN-γ, IL-4, IL-10 and IL-17) , and specific immunoglobulin in serum were measured. The results showed that only medium dose group stimulated by ConA had significant enhance proliferation of splenocyte; high and mediumn dose group can significantly increase the production of IFN-γ, reduced the secretion of IL-4 and no effect of IL-10 and IL-17; IFN-γ/IL-4 ratio is also significantly improved, so that the immune response directed Th1; specific immunoglobulin trial, three doses were significantly lower specific IgE, and High dose can down regular specific IgG1. These results showed that Bifidobacterium longum subsp. infants GB-1640. had ability to modulate in mite allergen-sensitize mice.

碩士
國立交通大學
生物科技學系
103
Carbohydrates are distributed widely in nature. They are important nutrients for the human beings. It can be divided into four different categories: monosaccharides, disaccharides, oligosaccharides and polysaccharides. It plays a quite important role for lives. Polysaccharides can storage as energy substances, like starch and glycogen, or as animals’ exoskeleton and plants’ cell wall, like chitin and cellulose. In addition, the five-carbon aldose ribose is the material of diverse cofactor, including ATP, FAD and NAD. It is also related some genetic molecular materials. According to some literatures previously published, many sugars derivatives showed great relevance to the immune system, fertilization, disease prevention, blood clotting and growth. There are many limitation and barriers about glycochemistry development. Glycorandomization, it provides a new approach to select sugars as possible candidates easily and quickly. It acceletate the speed of synthesis of new carbohydrates. Base on this concept, in this study, we selected two of the potential anomeric kinase genes, named NahK and GalK, respectively. NahK, its full name is N-acetylhexosamine 1-kinase, comes from the species Bifidobacterium infantis. GalK, galactokinase, comes from Escherichia coli. Our monosaccharide libraries are composed of 23 diverse monosaccharides, as the testifying substrates. In our study, two kinases were successfully cloned, functionally expressed in Eschericia coli system and sequently purified using Ni-NTA resin. Then, the enzymes were combined with different monosaccharides to do enzymatic activity assay. We checked the experimental results firstly by TLC analysis. After we isolated the products, the products was applied to ESI-MS to confirm the molecular weight and characterization. We hope that the selected monosaccharides can be potential candidates to new drug industry.

Bifidobacterium infantis (BI) and other probiotics are non-pathogenic living organisms that have recently gained attention for their possible therapeutic implications on the health of the digestive tract. The mechanisms by which probiotics exert their effects are largely unknown. This study explored the protective and regulatory effect of oral BI on the enteric nervous system (ENS) in the TNBS-induced colitis rats. Electrical field stimulation and chemical stimulation by serotonin (5-HT) were used to elicit changes in the short-circuit current (Isc) response of the colonic rat tissue. BI-fed colitis rats expressed trends of higher secretomotor activity and revealed signs of decreased macroscopic inflammatory damage when compared to sham-fed colitis rats, suggesting a protective and preventative role of oral BI. These findings may provide additional insights for understanding the prophylactic and therapeutic value of specific probiotics in intestinal inflammatory disorders, offering the possibility of a noninvasive alternative to toxic and immune-compromising drugs.
Access to thesis permanently restricted to Ball State community only
Department of Physiology and Health Science

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.

Inflammation has modulatory effects on the brain, particularly during development. These plastic changes can hold severe functional consequences. Perinatal hypoxia-ischemia (HI)-induced inflammation can result in cerebral palsy and cognitive impairment. In an attempt to reduce inflammation in the brain, we assessed the probiotic Bifidobacterium (B.) infantis as an HI intervention, using a rat model. Rat pups, developmentally equivalent to preterm infants, were exposed to chronic hypoxia from postnatal (PND) 3 –PND 10. Inflammation was assessed through hippocampal concentrations of the cytokine interleukin-6 (IL-6). Tissue was collected from pups on PND 10 and analyzed via enzyme-linked immunosorbent assay (ELISA). Results showed lower IL-6 concentrations in hypoxic groups , regardless of B. infantis administration. Qualitative observations suggested poor gut health in association with hypoxia and probiotic exposure. These preliminary findings support the chronic hypoxia exposure model of HI and suggest the association with IL-6 and HI events is less straightforward than expected.
October 2016

The fecal flora of healthy bottle or breast-fed infants was examined for the presence of Bifidobacterium. Identification was based on the presence of fructose-6-phosphate phosphoketolase, which is found only in these bacteria. No bifidobacteria were recovered from bottle-fed infants. However, bifidobacteria were readily isolated from 15 day to 3 month old breast-fed infants. Further characterization revealed B. breve and B. longum were the dominant species in feces of breast-fed infants, but atypical strains also were found. A whey-based medium (7% sweet whey, 0.05% cysteine and 0.3% yeast extract, WCY-0.3) was developed to grow Bifidobacterium species without use of anaerobic incubation conditions. Freshly pasteurized WCY-0.3 was inoculated with 0.2% (10⁶ to 10⁷ CFU/ml) of the following active cultures of bifidobacteria: B. bifidum 15696, B. breve 15700, B. longum 15707, B. breve 15698, B. longum L10, B. longum L12, and B. longum 3j. Following incubation for 12 hours, most strains reached cell densities of 10⁹ to 5 x 10⁹ CFU/ml, except B. bifidum 15696 and B. longum 3j. Addition of Oxyrase to the WCY (WC with any level of yeast extract) at 0.03 unit/ml (WCYO) reduced the lag phase of all strains, allowing maximum populations to be reached more quickly. A higher population density (2 to 7 times) could be achieved in the WCOY-0.3 medium with strains 15696, 15700, 15707, and L10 by incorporating 1.9% sodium glycerophosphate or trimagnesium phosphate with incubation for 12 hours at 37°C. Also, viability of these strains was retained throughout a 24-hour incubation period, in contrast to rapid death of cells grown without the neutralizing agents. Inoculation of WCY-0.3 or WCOY-0.3 medium with frozen concentrates (10⁷ to 10⁸ CFU/ml) of bifidobacteria allowed equal growth of all species, except B. bifidum 15696, which grew much better in WCOY-0.3 than in WCY- 0.3. Survival stability of whey-based medium-grown bifidobacteria when resuspended in pasteurized skim milk and refrigerated at 4°C was strain dependent and enhanced by the presence of 0.05% cysteine; generally ATCC strains were more stable than strains freshly isolated from baby feces. In this regard, B. breve 15700, B. longum 15707, and B. breve 15698 did not lose viability in 11% skim milk with 0.05% cysteine within 10 days of storage. Stability of whey-based medium-grown bifidobacteria in WCY with 15% glycerol during six months storage at -40°C was strain dependent. Bifidobacterium bifidum 15696, B. breve 15700, B. longum 15707, B. breve 15698, and B. longum L12 did not lose viability; however B. bifidum L6 lost about 50% viability, while B. longum L10, B. breve T10, and B. breve T2 lost about one log population density. The plasmid profiles of 35 strains of bifidobacteria from human sources were examined. Only one strain, B. breve 15698, harbored a 5.8Kb plasmid. A curing process using UV-light treatment to remove the plasmid was carried out but characterictics of the cured strain were identical to those of the parent strain, indicating the plasmid is cryptic.
Graduation date: 1991

碩士
國立中興大學
食品暨應用生物科技學系所
99
Bifidobacterium strains were well known as a predominant component of the intestinal flora in breast-fed infant feces and breast milk. This bacterial group plays a vital role in modulating host immunity and maintaining the gut health. In this study, the eighteen strains of Bifidobacterium strains which were isolated from infant feces and breast milk were screened for their ability to induce cytokines production by peripheral blood mononuclear cells (hPBMCs). The results indicated that not only different strains but also different species have different effect on the stimulation of all tested cytokines production. Moreover, the results also revealed that capacity of cytokine induction was dose-dependent. B. adolescentis DB-2458 and B. longum subsp. infantis GB-1496 at a concentration ratio 1:30 (hPBMCs: bacterial cells) which were potent inducers for IFN-γ secretion (Th1 cytokine) and induced low level of IL-5 and IL-13 (Th2 cytokines) were viable choices to down regulate Th2 cytokines. B. adolescentis DZN092, B. adolescentis DZN365 (concentration ratio 1:3, 1:0.3), B. adolescentis DB-2458 (concentration ratio 1:30, 1:3) and B. longum HB-762 (concentration ratio 1:30) which strongly induced TNF-α and IL-6 were promoted for the inflammation to initiate the healing process due to the infection. While B. adolescentis DZN092 (concentration ratio 1:30, 1:3, 1:0.3), B. adolescentis DZN365 (concentration ratio 1:30, 1:0.3), B. longum GK - 22 (concentration ratio 1:30) and B. longum GL-78 (concentration ratio 1:0.3) which highly induced both of IL-10 and TGF-β (anti-inflammatory cytokines) releases and induced low level of TNF-α and IL-6 (pro-inflammatory cytokines) could exert immune balance toward anti-inflammatory response. Based on comparison of immunomodulating activity and general criteria tests for probiotic, B. adolescentis DZN092, B. adolescentis DB-2458, B. longum subsp. infantis GB-1496, B. longum HB-762, B. longum GK-22 and B. longum GL-78 were acid and bile resistant strains. They could survive under acidic condition at pH 2-4 and bile condition (0.3% bile salts). For adhesion ability, all of test strains were adhesive strains. In conclusion, the selected strains were viable choices for therapeutic use and could be applied in desirable functional food due to their specific health benefits.