Fermented Dairy Foods: Impact on Intestinal Microbiota and Health-Linked Biomarkers.

The intake of fermented foods is gaining increasing interest due to their health-promoting benefits. Among them, fermented dairy foods have been associated with obesity prevention, and reduction of the risk of metabolic disorders and immune-related pathologies. Fermented foods could lead to these health benefits by providing the consumer with both easily metabolizable nutrients and beneficial microorganisms. Our aim was to evaluate the relationship between the consumption of fermented dairy products and the intestinal microbiota, serum lipid profile, and the pro-oxidant/inflammatory status. 130 healthy adults were evaluated. Dietary fermented food intake was assessed by an annual food frequency questionnaire (FFQ), including 26 fermented dairy products. Levels of the major phylogenetic types of the intestinal microbiota were determined by qPCR, and concentration of fecal short chain fatty acids were assessed by gas chromatography. Serum glucose and lipid profile, as well as serum malondialdehyde (MDA), C-reactive protein (CRP), and leptin levels were determined by standardized protocols. Among fermented dairy foods, natural yogurt, sweetened yogurt and matured/semi-matured cheese were the most consumed. While natural yogurt consumers showed increased fecal levels of Akkermansia with respect to non-consumers, sweetened yogurt intake was associated to lower levels of Bacteroides. Serum levels of CRP were also significantly reduced in yogurt consumers. Our results underline the interest in exploring the potential effects of the different yogurt types and the role the microbiota may play in such effects.

Impact of probiotics on development and behaviour in Drosophila melanogaster - a potential in vivo model to assess probiotics.

In vitro models are frequently used in probiotic research. However, such models often fail to predict in vivo functionality and efficacy. This fact complicates the screening process for selecting the most suitable strains, prior to accomplish expensive animal studies and clinical intervention trials. Therefore, additional sensitive, discriminating and cost-effective models are needed to conduct preliminary assays before undertaking human intervention studies definitely proving efficacy. With this purpose in mind, we explored the potential of axenic Drosophila melanogaster populations as well as of these axenic flies treated with probiotic microbial strains as a model to test the effects of probiotics on a subset of developmental and behavioural traits. An axenic D. melanogaster progeny from the wild-type Canton S strain was obtained and its eggs were further developed until pupae eclosion occurred in growth medium containing either of two probiotic strains: Bifidobacterium animalis subsp. lactis Bb12 or Lactobacillus rhamnosus GG. Whereas B. animalis Bb12 colonised the flies, the capacity of L. rhamnosus LGG to colonise was considerably lower in our experimental conditions. Regarding the influence of microbial load on the flies' development, the axenic condition caused a decrease in egg survival, and lowered adults' average weight with respect to wild-type flies. Both probiotics were able to counteract these effects. An earlier emergence of adults was observed from eggs treated with L. rhamnosus GG in comparison to the other fly populations. The axenic condition did not influence negative geotaxis behaviour in Drosophila; however, flies mono-associated with B. animalis Bb12 moved faster than wild-type. Our results suggest that the use of axenic/probiotic-treated D. melanogaster populations may be an affordable model for preliminary testing of the effects of probiotics on developmental or behavioural aspects.

Real-time monitoring of HT29 epithelial cells as an in vitro model for assessing functional differences among intestinal microbiotas from different human population groups.

Several in vitro screening tests have been used for selecting probiotic strains; however they often show low predictive value and only a limited number of strains have demonstrated functionality in vivo. The most used in vitro tests represent a very simplified version of the gut environment, especially since they do not consider the accompanying microbiota. Therefore, there is a need to develop sensitive and discriminating in vitro models including the microbiota. Here we developed an in vitro model to discriminate among microbiotas/fecal waters from different population groups. To this end samples were obtained from seven healthy adults, five IBD-patients, ten full-term and ten preterm newborns. Fecal microbiotas were purified and their impact, as well as that of the fecal waters, on HT29 cells was continuously monitored for 22 h using a real-time cell analyzer (RTCA). The composition of the purified microbiotas was assessed by 16S rRNA gene profiling and qPCR and the levels of short chain fatty acids (SCFA) determined by gas chromatography. The microbiota fractions and SCFA concentrations obtained from IBD-patients, full-term and preterm babies, showed clear differences with regard to those of the control group (healthy adults). Moreover, the purified intestinal microbiotas and fecal waters also differed from the control group in the response induced on the HT29 cells assay developed. In short, we have developed a real-time, impedance-based in vitro model for assessing the functional response induced by purified microbiotas and fecal waters upon intestinal epithelial cells. The capability of the assay for discriminating the functional responses induced, by microbiotas or fecal waters from different human groups, promises to be of help on the search for compounds/strains to restore the functionality of the microbiota-host's interaction.

C-section and the Neonatal Gut Microbiome Acquisition: Consequences for Future Health.

BACKGROUND: The human gut microbiota is assembled during infancy with an increase in diversity and stability. The correct colonization and the establishment of this microbiome are linked to the early and future health status of the individual. It is known that caesarean delivery alters this optimal microbial foundation. C-section (CS) is a common obstetrician surgery; however, it is not without risk for the mother/infant dyad. The World Health Organization recommends not exceeding 10-15% of the total deliveries; nevertheless, this rate has been increasing rapidly worldwide in the last decades. SUMMARY: This review discloses the clinical parameters for correct CS recommendation. Moreover, the major microbial changes in the infant gut microbiome acquisition as a consequence of delivery mode and medical practices surrounding it, as well as, the early and long-lasting effects for both mother and babies are discussed. In addition, some strategies for the gut microbiota restoration are analysed. The aim of this review is to show the need for the development of strategies for minimizing or limiting the impact of caesarean on the microbiome development, favouring future health.

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.

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.

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. METHODS AND RESULTS: The cytotoxicity of Bacillus cereus extracellular factors on Caco-2 colonocytes in the presence/absence of the EPS was determined by measuring the integrity of the tissue monolayer and the damage to the cell membrane (extracellular lactate dehydrogenase activity). Additionally, the protective effect of EPS against the haemolytic activity of the streptolysin-O was evaluated on rabbit erythrocytes. The EPS produced by Bifidobacterium animalis ssp. lactis A1 and IPLA-R1, Bifidobacterium longum NB667 and Lactobacillus rhamnosus GG were able to counteract the toxic effect of bacterial toxins on the eukaryotic cells at 1mg ml(-1) EPS concentration. The EPS A1 was the most effective in counteracting the effect of B. cereus toxins on colonocytes, even at lower doses (0·5mg ml(-1) ), whereas EPS NB667 elicited the highest haemolysis reduction on erythrocytes. CONCLUSIONS: The production of EPS by lactobacilli and bifidobacteria could antagonize the toxicity of bacterial pathogens, this effect being EPS and biological marker dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: This work allows gaining insight about the mechanisms that probiotics could exert to improve the host health.

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.

Short communication: effect of exopolysaccharide isolated from "viili" on the adhesion of probiotics and pathogens to intestinal mucus.

The strong ropy character of the Scandinavian fermented milk viili is conferred by the exopolysaccharides (EPS) produced by lactococcal strains. These biopolymers can be responsible for some health benefits. We have assessed the influence of the EPS fraction isolated from commercial viili on the adhesion of some probiotics and pathogens to human intestinal mucus. Concentrations of viili EPS greater than 0.1 mg/mL promoted a decrease in adherence of Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus GG and this effect was dose-dependent. However, no modifications were detected on the adhesion levels of the pathogenic strains tested at a concentration of 1 mg/mL of EPS. Results obtained in the present work should be considered in the design of new probiotic products.

Invited review: methods for the screening, isolation, and characterization of exopolysaccharides produced by lactic acid bacteria.

The ability to produce exopolysaccharides (EPS) is widespread among lactic acid bacteria (LAB), although the physiological role of these molecules has not been clearly established yet. Some EPS confer on LAB a "ropy" character that can be detected in cultures that form long strands when extended with an inoculation loop. When EPS are produced in situ during milk fermentation they can act as natural biothickeners, giving the product a suitable consistency, improving viscosity, and reducing syneresis. In addition, some of these EPS may have beneficial effects on human health. The increasing demand by consumers of novel dairy products requires a better understanding of the effect of EPS on existing products and, at the same time, the search for new EPS-producing strains with desirable properties. The use of genetically modified organisms capable of producing high levels of EPS or newly designed biopolymers is still very limited. Therefore, exploration of the biodiversity of wild LAB strains from natural ecological environments is currently the most suitable approach to search for the desired EPS-phenotype. The screening of ropy strains and the isolation and characterization of EPS responsible for this characteristic have led to the application over the past years of a wide variety of techniques. This review summarizes the available information on methods and procedures used for research on this topic. The information provided deals with methods for screening of EPS-producing LAB, detection of the ropy phenotype, and the physicochemical and structural characterization of these molecules, including parameters related to their viscosifying properties. To our knowledge, this is the first compilation of methods available for the study of EPS produced by LAB.

Polymorphism of Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses.

Thirty isolates of Listeria monocytogenes and 18 of L. innocua obtained from different short-ripened cheeses manufactured in Asturias (northern Spain), were compared with each other and with reference strains using serotype, phage type and pulsed-field restriction endonuclease digestion profiles analysis of the total DNA. Restriction enzymes ApaI and SmaI defined five clusters in L. monocytogenes (m1 to m5) and two main clusters in L. innocua (i1 and i2). Cluster i2 was further arranged into three subclusters (i2a, i2b and i2c) based on the different Eco52I (XmaIII) and Crf42I (SacII) patterns of its isolates. Clusters of L. innocua were clearly different whereas those of L. monocytogenes were more closely related to each other. In this latter species, serotype 4b isolates (m4 and m5) constituted a more homogeneous group than serogroup I isolates (m1, m2 and m3). Cluster m3 contained two strains of serotype 1/2a whereas m1 and m2 harboured strains of both serotypes, 1/2a and 1/2b. Therefore, the combined use of restriction patterns and serotype may be useful to differentiate L. monocytogenes strains showing identical restriction profiles but differing in serotype. The cheese source of Listeria strains proved that isolates from cluster m1 were repeatedly detected as a contaminant in the same type of cheese. Comparison of L. monocytogenes ApaI profiles showed a genetic proximity of m4 and m5 to the recognized pathogenic strains ATCC 13932 and NCTC 11994, responsible for meningitis cases in other countries. Finally, bacteriophage typing data indicated that m4, the sole phage typable group, had a phage type resembling that of strains causing the Auckland (New Zealand) outbreak of listeriosis in 1969. These data suggest a wide distribution of closely related types which might cause, under several circumstances, sporadic cases of listeriosis.

Characterization of plasmids from Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses.

The plasmid content of 30 isolates of Listeria monocytogenes and 18 isolates of Listeria innocua obtained from short-ripened cheeses was analysed. The isolates of L. monocytogenes serogroup 1 harboured a single plasmid, pLM33 (33.2 kbp), whereas the serogroup 4 isolates did not contain plasmids. One group of L. innocua strains harboured the plasmid pLI71 (71 kbp) and another one contained two plasmids: pLI59 (59.5 kbp) and pLI56 (56.5 kbp). These plasmid groups were in accordance with clusters previously defined by pulsed-field gel electrophoresis analysis of the chromosomal DNA of Listeria isolates. Plasmids pLM33, pLI71 and pLI59 shared homology regions of at least 20 kbp. Plasmid pLI56 did not encode genes for any known character (such as carbohydrate fermentation, resistance to antibiotics, heavy metals or disinfectants, growth at low pH, NaCl tolerance or thermal inactivation by pasteurisation) and displayed different characteristics to the other three plasmids. It was also the only one cured from the parent strain and the sole plasmid not digested by the restriction enzyme PstI. In addition, its lack of homology with pLM33, pLI71 and pLI59 enhanced the possibility of a different origin for plasmid pLI56.

A novel exocytoplasmic endonuclease from Streptomyces antibioticus.

A new exocytoplasmic, nutritionally controlled endodeoxyribonuclease (EC 3.1.21.-) was purified to homogeneity from Streptomyces antibioticus. The enzyme showed an apparent molecular mass of 29 kDa (being active in the monomeric form) and a pI of approximately 7.8. The nuclease hydrolysed endonucleolytically double-stranded circular and linear DNA. The enzyme makes nicks in one strand of the DNA in G-rich regions, leaving either 5' or 3' short, single-stranded overhangs with 3'-hydroxy and 5'-phosphate termini. Breaks in the DNA occur when two nicks in opposite strands are close together. The enzyme had an optimum pH of 7.5 and an absolute requirement for bivalent cations and > or = 100 mM NaCl in the reaction buffer. Activity was greatly diminished in the presence of phosphate, Hg2+ or iodoacetate and was stimulated by dimethyl sulphoxide. Single-stranded DNA was a much poorer substrate than double-stranded DNA. The nuclease hydrolyses sequences of three or preferably more (dG).(dC) tracts in the DNA. The initial specificity shifts to other sequences (including sequences shorter than those initially hydrolysed) during the course of the reaction, giving the changing pattern of bands observed in agarose gels. 5-Methylcytosine-hemimethylated DNA is not hydrolysed by the nuclease. The properties of this novel enzyme suggest a relationship with class II restriction endonucleases and also with some eukaryotic nucleases.

A Lactobacillus helveticus-Specific DNA Probe Detects Restriction Fragment Length Polymorphisms in This Species.

A cloned 2-kb EcoRI fragment (fragment f) from a 34-kb plasmid of Lactobacillus helveticus CNRZ 1094 was shown by dot blot to specifically hybridize to total DNAs of 75 L. helveticus strains. No hybridization was found with L. acidophilus, L. crispatus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. gasseri, or L. intestinalis strains. When Southern blots of EcoRI digests of L. helveticus strains were probed with fragment f, these strains displayed restriction fragment length polymorphisms on the basis of which they could be grouped into several clusters.

Evidence for a Plasmid-Linked Restriction-Modification System in Lactobacillus helveticus.

The presence of a restriction-modification (R/M) system against two bacteriophages, 328-B1 and hv, was demonstrated in three Lactobacillus helveticus strains, CNRZ 1094, CNRZ 1095, and CNRZ 1096. In addition, the burst size of phage 328-B1 in the three restrictive strains CNRZ 1094, CNRZ 1095, and CNRZ 1096 was reduced with respect to the values obtained in its propagating strain, CNRZ 328. Heating at 60 degrees C did not inactivate the R/M system. Nonrestrictive variants from CNRZ 1094 were easily obtained under several culture conditions, but treatment with novobiocin at 42 degrees C followed by storage at -20 degrees C resulted in drastic elimination of the R/M phenotype from all clones tested. Electrophoretic analysis of CNRZ 1094 nonrestrictive variants revealed the concomitant loss of a 34-kb plasmid. Four EcoRI fragments from the 34-kb plasmid were cloned in the Escherichia coli vector pACYC184. The use of one or several of these fragments as probes confirmed the plasmidic location of the genes responsible for the R/M system. These probes also showed the presence of R/M plasmids in the two other restrictive strains, CNRZ 1095 and CNRZ 1096. Lactose-fermenting ability and/or proteolytic capacity was not linked to the 34-kb plasmid.

A non-specific deoxyribonuclease with restriction function in Streptomyces glaucescens.

A non-specific deoxyribonuclease with a possible role in the restriction of some actinophages was detected in Streptomyces glaucescens ETHZ 22794. Production of this enzyme activity was influenced by the medium composition, indicating nutritional control of enzyme synthesis. Restriction was confirmed when phage adsorption and efficiency of plating in nuclease-productive and non-productive media were investigated, and also by analysis of a mutant which lacked exonucleolytic activity. In vivo escape from restriction in nuclease-productive media is mainly related to the ability of phages to adsorb in a growth phase earlier than that in which enzyme synthesis occurs.

An exocytoplasmic endonuclease with restriction function in Streptomyces antibioticus.

Streptomyces antibioticus produces a strong endo-DNase which is located between the cytoplasmic membrane and the cell wall. All DNA substrates assayed, including the chromosomal DNA of this species and several bacteriophage DNAs, were completely degraded in vitro by the enzyme. The rate of synthesis of the nuclease depended on the growth medium. In NBG medium, in which the enzyme is not produced, the size of lytic plaques of several actinophages was larger than that in GYM or GAE medium, in which synthesis of the nuclease takes place late in growth. In addition, one of the phages assayed, phi A6, showed a diminution of its efficiency of plating in GYM medium with respect to that in NBG medium; another phage, phi A9, grew in NBG medium but not in the other two media. It is postulated that the presence of the host nuclease, together with the capability of the particular phage to absorb on S. antibioticus of different growth phases, determines the efficiency of growth and the plaque size of the phages on productive media. This hypothesis was confirmed when the growth of phi A6 and phi A9 in a mutant of S. antibioticus lacking the endonuclease activity was analyzed. It is concluded that the enzyme can assume, under some circumstances, a role in in vivo restriction.