C.G. de los Reyes-Gavilán

Establishment and development of lactic acid bacteria and bifidobacteria microbiota in breast-milk and the infant gut.

The initial establishment of lactic acid bacteria (LAB) and bifidobacteria in the newborn and the role of breast-milk as a source of these microorganisms are not yet well understood. The establishment of these microorganisms during the first 3 months of life in 20 vaginally delivered breast-fed full-term infants, and the presence of viable Bifidobacterium in the corresponding breast-milk samples was evaluated. In 1 day-old newborns Enterococcus and Streptococcus were the microorganisms most frequently isolated, from 10 days of age until 3 months bifidobacteria become the predominant group. In breast-milk, Streptococcus was the genus most frequently isolated and Lactobacillus and Bifidobacterium were also obtained. Breast-milk contains viable lactobacilli and bifidobacteria that might contribute to the initial establishment of the microbiota in the newborn.

Production of exopolysaccharides by Lactobacillus and Bifidobacterium strains of human origin, and metabolic activity of the producing bacteria in milk

This work reports on the physicochemical characterization of 21 exopolysaccharides (EPS) produced by Lactobacillus and Bifidobacterium strains isolated from human intestinal microbiota, as well as the growth and metabolic activity of the EPS-producing strains in milk. The strains belong to the species Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus vaginalis, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. The molar mass distribution of EPS fractions showed 2 peaks of different sizes, which is a feature shared with some EPS from bacteria of food origin. In general, we detected an association between the EPS size distribution and the EPS-producing species, although because of the low numbers of human bacterial EPS tested, we could not conclusively establish a correlation. The main monosaccharide components of the EPS under study were glucose, galactose, and rhamnose, which are the same as those found in food polymers; however, the rhamnose and glucose ratios was generally higher than the galactose ratio in our human bacterial EPS. All EPS-producing strains were able to grow and acidify milk; most lactobacilli produced lactic acid as the main metabolite. The lactic acid-to-acetic acid ratio in bifidobacteria was 0.7, close to the theoretical ratio, indicating that the EPS-producing strains did not produce an excessive amount of acetic acid, which could adversely affect the sensory properties of fermented milks. With respect to their viscosity-intensifying ability, L. plantarum H2 and L. rhamnosus E41 and E43R were able to increase the viscosity of stirred, fermented milks to a similar extent as the EPS-producing Streptococcus thermophilus strain used as a positive control. Therefore, these human EPS-producing bacteria could be used as adjuncts in mixed cultures for the formulation of functional foods if probiotic characteristics could be demonstrated. This is the first article reporting the physicochemical characteristics of EPS isolated from human intestinal microbiota.

Exopolysaccharides produced by Lactobacillus and Bifidobacterium strains abrogate in vitro the cytotoxic effect of bacterial toxins on eukaryotic cells

Aims:  To evaluate the capability of the exopolysaccharides (EPS) produced by lactobacilli and bifidobacteria from human and dairy origin to antagonize the cytotoxic effect of bacterial toxins.

Preservation of the microbiological and biochemical quality of raw milk by carbon dioxide addition: a pilot-scale study

Carbon dioxide treatment of refrigerated raw milk was evaluated as a method for extending storage life by inhibiting growth of psychrotrophic bacteria and other bacterial groups in raw milk. The effect of CO2 acidification followed by degasification and pasteurization on biochemical and microbiological properties of cold stored milk was studied on a pilot scale, Two CO2 treatments (acidification to pH 6.2 and to pH 6.0) were compared with a control (untreated) milk during 4 days of storage at 4°C. Total bacterial counts in the categories of milk established in this study were mainly determined by the proteolytic psychrotroph levels. The inhibitory capability of CO2 was greater in the low-quality than in the high-quality milk category. Acidification at pH 6.0 was more inhibitory than that at pH 6.2, especially against proteolytic psychrotrophs. Neither caseins nor whey proteins were affected by CO2 treatment and pasteurization. Organic acid (orotic, citric, uric, formic, acetic, propionic, and hippuric) concentrations did not change after CO2 treatment, cold storage, or the pasteurization process; the lactic acid content of CO2-treated milks remained constant during the refrigeration time but increased slightly in the control. In general, lower amounts of volatile compounds were produced in CO2-treated milks during refrigeration than in control milk. Ethanol and 2-propanol levels were most affected by degasification and pasteurization. Sensory evaluation revealed no significant differences between CO2-treated milk after degasification and pasteurization and the untreated milk used as control. It was concluded that degasification and pasteurization on a pilot scale eliminated CO2 from milk with minimum detrimental effects on its biochemical and sensory properties, making this process acceptable for milk preservation.

Characterization of ø393-A2, a bacteriophage that infects Lactobacillus casei

Bacteriophage o393-A2, isolated from an artisanal cheese whey sample, is a temperate phage able to generate stable lysogens through integration of its DNA into the bacterial genome. One-step growth kinetics of its lytic development revealed eclipse and latent periods of 100 and 140 min, respectively, with a burst size of about 200 p.f.u. per infected cell. ϕ393-A2 virions have an isometric head and a long, non-contractile tail terminating in a baseplate. The capsid is composed of two major and at least nine minor structural polypeptides. The phage genome consists of a double-stranded DNA molecule of 44 kbp bearing 3′-protruding cohesive ends. A physical map of the phage DNA has been constructed for six restriction enzymes. The whole ϕ393-A2 genome has been cloned in Escherichia coli using plasmid- and phage-derived cloning vectors.

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.

Selection of potential probiotic bifidobacteria and prebiotics for elderly by using in vitro faecal batch cultures

The gut microbiota plays an important role in host health. The ageing process affects this microbial community, and therefore, the use of functional foods to restore the microbiota of elderly constitutes an interesting strategy. To this end, probiotics and prebiotics targeted at correcting the specific microbiota alterations occurring during senescence are needed. We performed an in vitro selection of bifidobacterial strains and prebiotic substrates on the basis of their ability to counterbalance the specific microbiota aberrancies previously identified in the elderly population. Batch cultures of faeces from elderly were carried out adding different strains of Bifidobacterium or prebiotics. The effects of these strains/prebiotics upon gut microbiota were assessed by quantitative PCR and the concentrations of short chain fatty acids determined by gas chromatography-FID/MS. The target-specific selection process applied in this study allowed the preliminary selection of two Bifidobacterium strains and a prebiotic fructooligosaccharide on the basis of their specific properties for the modulation of the microbiota of elders. Overall, this study identifies potentially probiotic strains and prebiotic substrates for the development of functional foods specifically targeted to the senior population.

Production of immune response mediators by HT-29 intestinal cell-lines in the presence of Bifidobacterium-treated infant microbiota

The colonisation and establishment of the intestinal microbiota starts immediately at birth and is essential for the development of the intestine and the immune system. This microbial community gradually increases in number and diversity until the age of two or three years when it becomes a stable ecosystem resembling that of adults. This period constitutes a unique window of opportunity to modulate it through probiotic action, with a potential impact in later health. In the present work we have investigated how putative bifidobacterial probiotics modify the metabolic profiles and immune-modulatory properties of faecal microbiotas. An in vitro pH-controlled single-stage continuous-culture system (CCS) inoculated with infant faeces was employed to characterise the effects of two Bifidobacterium species on the intestinal microbiotas in three children, together with the effects of these modified microbiotas on cytokine production by HT-29 cells. Intestinal bacterial communities, production of short-chain fatty acids and lactate were determined by quantitative PCR and gas chromatography, respectively. Cytokines production by HT-29 cells was measured by ELISA. The combination of CCS with infant faeces and human intestinal cells provided a suitable model to evaluate the specific modulation of the intestinal microbiota and immune system by probiotics. In the CCS, infant faecal microbiotas were influenced by the addition of bifidobacteria, resulting in changes in their ability to induce the production of immune mediators by HT-29 cells. The different metabolic and immunological responses induced by the bifidobacterial species tested indicate the need to assess potential probiotics in model systems including complex intestinal microbiotas. Potential probiotic bifidobacteria can modulate the infant microbiota and its ability to induce the production of mediators of the immune response by intestinal cells.

Glucolytic fingerprinting reveals metabolic groups within the genus Bifidobacterium: an exploratory study.

Microorganisms of the genus Bifidobacterium are inhabitants of diverse niches including the digestive tract of humans and animals. The species Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum have qualified presumption of safety status granted by EFSA and several strains are considered probiotic, and are being included in functional dairy fermented products. In the present work we carried out a preliminary exploration of general metabolic characteristics and organic acid production profiles of a reduced number of strains selected from these and other species of the genus Bifidobacterium. The use of resting cells allowed obtaining metabolic fingerprints without interference of metabolites accumulated during growth in culture media. Acetic acid was the most abundant organic acid formed per mol of glucose consumed (from 1.07 ± 0.03 to 1.71 ± 0.22 mol) followed by lactic acid (from 0.34 ± 0.06 to 0.90 ± 0.12 mol), with moderate differences in production among strains; pyruvic, succinic and formic acids were also produced at considerably lower proportions, with variability among strains. The acetic to lactic acid ratio showed lower values in stationary phase as regard to the exponential phase for most, but not all, the microorganisms; this was due to a decrease in acetic acid molar proportions together with increases of lactic acid proportions in stationary phase. A linear discriminant analysis allowed to cluster strains into species with 51-100% probability, evidencing different metabolic profiles, according to the relative production of organic acids from glucose by resting cells, of microorganisms collected at the exponential phase of growth. Looking for a single metabolic marker that could adequately discriminate metabolic groups, we found that groups established by the acetic to lactic acid ratio fit well with differences previously evidenced by the discriminant analysis. The proper establishment of metabolic groups within the genus Bifidobacterium could help to select the best suited probiotic strains for specific applications.

P180: Intérêt de la modulation du microbiote intestinal par les oligosaccharides non digestibles dans le contrôle de la leucémie et de la cachexie cancéreuse

Introduction et but de l’etude Il est a present clairement etabli que l’ensemble des bacteries presentes dans l’intestin (le microbiote intestinal) est capable d’influencer l’homeostasie energetique et immunitaire de son hote. Nous avons teste l’hypothese selon laquelle une modulation du microbiote intestinal par des oligosaccharides issus de la pectine (POS) ou de l’inuline (INU) permet d’interferer avec la progression de la leucemie et des desordres metaboliques associes. Materiel et methodes Des souris ont ete transplantees avec des lymphocytes proB murins (lignee BaF3) transfectees avec la proteine de fusion Bcr-Abl. Ce modele mime une leucemie aigue. Pendant deux semaines, les souris ont recu une diete enrichie ou non en INU ou en POS (5 %). La composition du microbiote intestinal a ete analyse par pyrosequencage, reaction en chaine par polymerase (RCP) quantitative et RCP-EGGD (electrophorese sur gel en gradient denaturant) de l’ARNr 16S. Resultats et Analyse statistique En accord avec nos travaux precedents, la supplementation en INU augmente les acides car-boxyliques a chaine courte au niveau de la veine porte et diminue l’accumulation de cellules leucemiques au niveau du foie. Le traitement par les POS n’affecte pas l’accumulation des cellules cancereuses au niveau hepatique, mais est plus efficace que l’INU en termes de regulation des desordres metaboliques. En effet, les POS retardent l’anorexie liee au cancer. De plus, les POS augmentent les taux d’acetate dans le contenu caecal, changent le profil des acides gras dans le tissu adipeux et contrent l’induction de marqueurs de la (β-oxydation, empechant par-la la perte de masse grasse. Une analyse de l’ARNr 16S par pyrosequencage et par RCP quantitative a mis en evidence que les POS diminuent la diversite et la richesse specifique du microbiote caecal alors qu’ils augmentent le nombre total de bacteries, les bifidobacteries, les roseburia et les bacteroides , de facon plus importante que l’INU. L’usage combine du pyrosequencage, de la RCP-EGGD et de la RCP quantitative a permis de mettre en avant une augmentation specifique, de l’ordre de 40 fois, de Bacteroides dorei par les POS. Conclusion Les oligosaccharides non digestibles, de par leurs proprietes prebiotiques, pourraient constituer une strategie nutritionnelle nouvelle et complementaire a l’approche therapeutique classique de certains cancers et de la cachexie qui y est associee.