Effects of probiotic or prebiotic supplemented milk formulas on fecal microbiota composition of infants

The aim of the study was to evaluate whether supplementation of milk-formulas with prebiotic fructooligosaccharides or a probiotic, Lactobacillus johnsonii La1 (La1), could modulate the composition of the fecal microbiota of formula-fed infants, compared to breastfed (BF) infants. Ninety infants close to 4 months of age were randomized into one of three groups to be blindly assigned to receive for 13 weeks: a) an infant formula (Control), b) the same formula with fructo-oligosaccharides (Prebio), or c) with La1 (Probio). At the end of this period, all infants received the control formula for 2 additional weeks. Twenty-six infants, breastfed throughout the study, were recruited to form group BF. Fecal samples were obtained upon enrolment and after 7 and 15 weeks. Bacterial populations were assessed with classical culture techniques and fluorescent in situ hybridisation (FISH). Seventy-six infants completed the study. On enrolment, higher counts of Bifidobacterium and Lactobacillus and lower counts of enterobacteria were observed in BF compared to the formula-fed infants; these differences tended to disappear at weeks 7 and 15. No major differences for Clostridium, Bacteroides or Enterococcus were observed between the groups or along the follow up. Probio increased fecal Lactobacillus counts (P<0.001); 88% of the infants in this group excreted live La1 in their stools at week 7 but only 17% at week 15. Increased Bifidobacterium counts were observed at week 7 in the 3 formula groups, similar to BF infants. These results confirm the presence of higher counts of bifidobacteria and lactobacilli in the microbiota of BF infants compared to formula-fed infants before dietary diversification, and that La1 survives in the infant digestive tract.

In vitro effects of synbiotic fermentation on the canine faecal microbiota.

Stirred, pH-controlled anaerobic batch cultures were used to investigate the in vitro effects of galacto-oligosaccharides (GOS) alone or combined with the probiotic Bifidobacterium bifidum 02 450B on the canine faecal microbiota of three different donors. GOS supported the growth of B. bifidum 02 450B throughout the fermentation. Quantitative analysis of bacterial populations by FISH revealed significant increases in Bifidobacterium spp. counts (Bif164) and a concomitant decrease in Clostridium histolyticum counts (Chis150) in the synbiotic-containing vessels compared with the controls and GOS vessels. Vessels containing probiotic alone displayed a transient increase in Bifidobacterium spp. and a transient decrease in Bacteroides spp. Denaturing gradient gel electrophoresis analysis showed that GOS elicited similar alterations in the microbial profiles of the three in vitro runs. However, the synbiotic did not alter the microbial diversity of the three runs to the same extent as GOS alone. Nested PCR using universal primers, followed by bifidobacterial-specific primers illustrated low bifidobacterial diversity in dogs, which did not change drastically during the in vitro fermentation. This study illustrates that the canine faecal microbiota can be modulated in vitro by GOS supplementation and that GOS can sustain the growth of B. bifidum 02 450B in a synbiotic combination.

Investigation of the faecal microbiota associated with canine chronic diarrhoea.

Diarrhoea is a common problem in dogs and can result in disturbance of the normal intestinal microbiota. However, little is known about the gastrointestinal microbiota of dogs with chronic diarrhoea and controlled canine studies of dietary management are scarce. The aims of this study were to investigate the predominant faecal microbiota of chronic diarrhoea dogs and to examine the effect(s) of a fibre blend on the canine faecal microbiota. A 3-week fibre supplementation feeding study was performed in nine chronic diarrhoea and eight control dogs. Atopobium cluster, Lactobacillus-Enterococcus group and Clostridium cluster XIV were the predominant bacterial groups in all dogs. Chronic diarrhoea dogs had significantly higher Bacteroides counts at baseline and significantly lower Atopobium cluster counts following fibre supplementation compared with control dogs. Atopobium cluster levels increased significantly in control dogs, while counts of sulphate-reducing bacteria decreased significantly and Clostridium clusters I and II counts increased significantly in chronic diarrhoea dogs during fibre supplementation. Microbial profiles (detected by denaturing gradient gel electrophoresis) demonstrated interindividual variation, with greater similarity seen between the chronic diarrhoea and control dogs' profiles after fibre supplementation compared with baseline. In conclusion, fibre supplementation induced changes in the canine faecal microbiota, with greater resemblance between the microbiota of chronic diarrhoea and control dogs after this dietary modulation.

Use of a continuous culture fermentation system to investigate the effect of GanedenBC30 (Bacillus coagulans GBI-30, 6086) supplementation on pathogen survival in the human gut microbiota

Single-stage continuous fermentation systems were employed to examine the effects of GanedenBC30 supplementation on the human gastrointestinal microbiota in relation to pathogen challenge in vitro. Denaturing gradient gel electrophoresis analysis demonstrated that GanedenBC30 supplementation modified the microbial profiles in the fermentation systems compared with controls, with profiles clustering according to treatment. Overall, GanedenBC30 supplementation did not elicit major changes in bacterial population counts in vitro, although notably higher Bcoa191 counts were seen following probiotic supplementation (compared to the controls). Pathogen challenge did not elicit significant modification of the microbial counts in vitro, although notably higher Clit135 counts were seen in the control system post-Clostridium difficile challenge than in the corresponding GanedenBC30-supplemented systems. Sporulation appears to be associated with the anti-microbial activity of GanedenBC30, suggesting that a bi-modal lifecycle of GanedenBC30 in vivo may lead to anti-microbial activity in distal regions of the gastrointestinal tract.

A double-blind, placebo-controlled, crossover-designed probiotic feeding study in children diagnosed with autistic spectrum disorders

There is growing interest in the role of gastrointestinal (GI) pathology and clinical expression of autism. Recent studies have demonstrated differences in the faecal clostridial populations harboured by autistic and non-autistic children. The potential of Lactobacillus plantarum WCSF1 (a probiotic) to modulate the gut microbiota of autistic subjects was investigated during a double-blind, placebo-controlled, crossover-designed feeding study. The faecal microbiota, gut function and behaviour scores of subjects were examined throughout the 12-week study. Lactobacillus plantarum WCFS1 feeding significantly increased Lab158 counts (lactobacilli and enterococci group) and significantly reduced Erec482 counts (Clostridium cluster XIVa) compared to placebo. Probiotic feeding also resulted in significant differences in the stool consistency compared to placebo and behaviour scores (total score and scores for some subscales) compared to baseline. The major finding of this work was the importance of study protocol in relation to the specific considerations of this subject population, with an extremely high dropout rate seen (predominantly during the baseline period). Furthermore, the relatively high inter-individual variability observed suggests that subsequent studies should use defined subgroups of autistic spectrum disorders, such as regressive or late-onset autism. In summary, the current study has highlighted the potential benefit of L. plantarum WCFS1 probiotic feeding in autistic individuals.

The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota.

We have examined the gut bacterial metabolism of pomegranate by-product (POMx) and major pomegranate polyphenols, punicalagins, using pH-controlled, stirred, batch culture fermentation systems reflective of the distal region of the human large intestine. Incubation of POMx or punicalagins with faecal bacteria resulted in formation of the dibenzopyranone-type urolithins. The time course profile confirmed the tetrahydroxylated urolithin D as the first product of microbial transformation, followed by compounds with decreasing number of phenolic hydroxy groups: the trihydroxy analogue urolithin C and dihydroxylated urolithin A. POMx exposure enhanced the growth of total bacteria, Bifidobacterium spp. and Lactobacillus spp., without influencing the Clostridium coccoides–Eubacterium rectale group and the C. histolyticum group. In addition, POMx increased concentrations of short chain fatty acids (SCFA) viz. acetate, propionate and butyrate in the fermentation medium. Punicalagins did not affect the growth of bacteria or production of SCFA. The results suggest that POMx oligomers, composed of gallic acid, ellagic acid and glucose units, may account for the enhanced growth of probiotic bacteria.

Current level of consensus on probiotic science: report of an expert meeting, London, 23 November 2009

The present paper summarizes  the consensus views of a group of 9 European clinicians and scientists on the current state of scientific knowledge on probiotics, covering those areas where there is substantial evidence for beneficial effects and those  where the evidence base is poor or inconsistent. There was general agreement that probiotic effects were species and often strain specific. The experts agreed  that some probiotics were effective in reducing the incidence and duration of rotavirus diarrhoea in infants, antibiotic-associated diarrhoea in adults and, for certain probiotics, Clostridium difficile infections. Some probiotics are associated with symptomatic improvements in irritable bowel syndrome and alleviation of digestive discomfort. Probiotics can reduce the frequency and severity of necrotizing enterocolitis in premature infants and have been shown to regulate intestinal immunity.  Several other clinical effects of probiotics, including their role in inflammatory bowel disease, atopic dermatitis, respiratory or genito-urinary infections or H.pylori adjuvant treatment were thought promising but inconsistent.

Investigation of the faecal microbiota of geriatric cats

Aims: To investigate the faecal microbiota of geriatric cats, as aging affects the nutrient digestibility and metabolic function of the feline intestine. Methods and results: 20 geriatric cats were randomly assigned to two groups that were fed different foods. Coriobacteriaceae, Clostridium cluster XIV, bifidobacteria, and lactic acid bacteria were the dominant faecal bacterial groups, accounting for ∼40% of total bacteria. Clostridium cluster IX was less predominant (0.5% of total bacteria), while the remaining bacterial populations enumerated only accounted for 0.2% of total bacteria. Highly diverse microbial profiles were demonstrated for geriatric cats with denaturing gradient gel electrophoresis, although a few common bands were evident. Some differences were seen in the feline faecal microbiota between animal groups at the same time or over time for individual animals. However, no obvious clustering based on animal group or sample time was indicated. Conclusions: geriatric cats harboured a complex faecal microbiota and ∼41% of total bacteria have been detected with the probes employed. Significance and impact of study: First molecular-based study examining faecal microbiota of geriatric felines. Knowledge of the microbiota associated with ageing in cats may allow improved development of foods specific for the needs of senior cats.

Investigation of the faecal microbiota of kittens: monitoring bacterial succession and effect of diet

Weaning is a stressful process for kittens, and is often associated with diarrhoea and the onset of infectious diseases. The gastrointestinal microbiota plays an essential role in host well-being, including improving homeostasis. Composition of the gastrointestinal microbiota of young cats is poorly understood, and the impact of diet on the kitten microbiota unknown. The aims of this study were to monitor the faecal microbiota of kittens and determine the effect(s) of diet on its composition. Bacterial succession was monitored in two groups of kittens (at 4 and 6 weeks, and 4 and 9 months of age) fed different foods. Age-related microbial changes revealed significantly different counts of total bacteria, lactic acid bacteria, Desulfovibrionales, Clostridium cluster IX and Bacteroidetes between 4-week- and 9-month-old kittens. Diet-associated differences in the faecal microbiota of the two feeding groups were evident. In general, fluorescence in situ hybridization analysis demonstrated bifidobacteria, Atopobium group, Clostridium cluster XIV and lactic acid bacteria were dominant in kittens. Denaturing gradient gel electrophoresis profiling showed highly complex and diverse faecal microbiotas for kittens, with age- and/or food-related changes seen in relation to species richness and similarity indices. Four-week-old kittens harboured more diverse and variable profiles than those of weaned kittens.

Impact of polydextrose on the faecal microbiota: a double-blind, crossover, placebo-controlled feeding study in healthy human subjects

In this placebo-controlled, double-blind, crossover human feeding study, the effects of polydextrose (PDX; 8 g/d) on the colonic microbial composition, immune parameters, bowel habits and quality of life were investigated. PDX is a complex glucose oligomer used as a sugar replacer. The main goal of the present study was to identify the microbial groups affected by PDX fermentation in the colon. PDX was shown to significantly increase the known butyrate producer Ruminococcus intestinalis and bacteria of the Clostridium clusters I, II and IV. Of the other microbial groups investigated, decreases in the faecal Lactobacillus–Enterococcus group were demonstrated. Denaturing gel gradient electrophoresis analysis showed that bacterial profiles between PDX and placebo treatments were significantly different. PDX was shown to be slowly degraded in the colon, and the fermentation significantly reduced the genotoxicity of the faecal water. PDX also affected bowel habits of the subjects, as less abdominal discomfort was recorded and there was a trend for less hard and more formed stools during PDX consumption. Furthermore, reduced snacking was observed upon PDX consumption. This study demonstrated the impact of PDX on the

In vitro fermentation and prebiotic potential of novel low molecular weight polysaccharides derived from agar and alginate seaweeds

Fermentation properties and prebiotic potential of novel low molecular weight polysaccharides (LMWPs) derived from agar and alginate bearing seaweeds was investigated. Ten LMWPs were supplemented to pH, temperature controlled anaerobic batch cultures inoculated with human feces from three donors, in triplicate. Microbiota changes were monitored using Fluorescent in-situ hybridization and short chain fatty acids, the fermentation end products were analysed using gas chromatography. Of the ten LMWPs tested, Gelidium seaweed CC2253 of molecular weight 64.64 KDa showed a significant increase in bifidobacterial populations from log(10) 8.06 at 0 h to log(10) 8.55 at 24 h (p = 0.018). For total bacterial populations, alginate powder CC2238 produced a significant increase from log(10) 9.01 at 0 h to log(10) 9.58 at 24 h (p = 0.032). No changes were observed in the other bacterial groups tested viz. Bacteroides, Lactobacilli/Enterococci, Eubacterium rectale/Clostridium coccoides and Clostridium histolyticum. The polysaccharides also showed significant increases in total SCFA production, particularly acetic and propionic acids, indicating that they were readily fermented. In conclusion, some LMWPs derived from agar and alginate bearing seaweeds were fermented by gut bacteria and exhibited potential to be used a novel source of prebiotics.

In vitro fermentation by human gut bacteria of proteolytically digested caseinomacropeptide nonenzymatically glycosylated with prebiotic carbohydrates

The in vitro fermentation selectivity of hydrolyzed caseinomacropeptide (CMP) glycosylated, via Maillard reaction (MR), with lactulose, galacto-oligosaccharides from lactose (GOSLa), and galacto-oligosaccharides from lactulose (GOSLu) was evaluated, using pH-controlled small-scale batch cultures at 37 °C under anaerobic conditions with human feces. After 10 and 24 h of fermentation, neoglyconjugates exerted a bifidogenic activity, similar to those of the corresponding prebiotic carbohydrates. No significant differences were found in Bacteroides, Lactobacillus�Enterococcus, Clostridium histolyticum subgroup, Atopobium and Clostridium coccoides�Eubacterium rectale populations. Concentrations of lactic acid and short-chain fatty acids (SCFA) produced during the fermentation of prebiotic carbohydrates were similar to those produced for their respective neoglycoconjugates at both fermentation times. These findings, joined with the functional properties attributed to CMP, could open up new applications of MR products involving prebiotics as novel multiple-functional ingredients with potential beneficial effects on human health.

In vitro fermentation of alternansucrase raffinose-derived oligosaccharides by human gut bacteria

In this work, in vitro fermentation of alternansucrase raffinose-derived oligosaccharides, previously fractionated according to their degree of polymerization (DP; from DP4 to DP10), was carried out using small-scale pH-controlled batch cultures at 37 °C under anaerobic conditions with human feces. Bifidogenic activity of oligosaccharides with DP4�6 similar to that of lactulose was observed; however, in general, a significant growth of lactic acid bacteria Bacteroides, Atopobium cluster, and Clostridium histolyticum group was not shown during incubation. Acetic acid was the main short chain fatty acid (SCFA) produced during the fermentation process; the highest levels of this acid were shown by alternansucrase raffinose acceptor pentasaccharides at 10 h (63.11 mM) and heptasaccharides at 24 h (54.71 mM). No significant differences between the gas volume produced by the mixture of raffinose-based oligosaccharides (DP5�DP10) and inulin after 24 h of incubation were detected, whereas lower gas volume was generated by DP4 oligosaccharides. These findings indicate that novel raffinose-derived oligosaccharides (DP4�DP10) could be a new source of prebiotic carbohydrates.

In vitro fermentation of a red wine extract by human gut microbiota: changes in microbial groups and formation of phenolic metabolites

An in vitro batch culture fermentation experiment was conducted with fecal inocula from three healthy volunteers in the presence and absence of a red wine extract. Changes in main bacterial groups were determined by FISH during a 48 h fermentation period. The catabolism of main flavonoids (i.e., flavan-3-ols and anthocyanins) and the formation of a wide a range of phenolic microbial metabolites were determined by a targeted UPLC-PAD-ESI-TQ MS method. Statistical analysis revealed that catechol/pyrocatechol, as well as 4-hydroxy-5-(phenyl)-valeric, 3- and 4-hydroxyphenylacetic, phenylacetic, phenylpropionic, and benzoic acids, showed the greatest increases in concentration during fermentation, whereas 5-(3′-hydroxyphenyl)-γ-valerolactone, its open form 4-hydroxy-5-(3′-hydroxyphenyl)-valeric acid, and 3,4-dihydroxyphenylacetic acid represented the largest interindividual variations in the catabolism of red wine polyphenols. Despite these changes, microbial catabolism did not produce significant changes in the main bacterial groups detected, although a slight inhibition of the Clostridium histolyticum group was observed.

In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria. In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism. Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied. The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.