Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays

Short-chain fructooligosaccharides (scFOS) and other prebiotics are used to selectively stimulate the growth and activity of lactobacilli and bifidobacteria in the colon. However, there is little information on the mechanisms whereby prebiotics exert their specific effects upon such microorganisms. To study the genomic basis of scFOS metabolism in Lactobacillus plantarum WCFS1, two-color microarrays were used to screen for differentially expressed genes when grown on scFOS compared to glucose (control). A significant up-regulation (8- to 60-fold) was observed with a set of only five genes located in a single locus and predicted to encode a sucrose phosphoenolpyruvate transport system (PTS), a beta-fructofuranosidase, a fructokinase, an alpha-glucosidase, and a sucrose operon repressor. Several other genes were slightly overexpressed, including pyruvate dehydrogenase. For the latter, no detectable activity in L. plantarum under various growth conditions has been previously reported. A mannose-PTS likely to encode glucose uptake was 50-fold down-regulated as well as, to a lower extent, other PTSs. Chemical analysis of the different moieties of scFOS that were depleted in the growth medium revealed that the trisaccharide 1-kestose present in scFOS was preferentially utilized, in comparison with the tetrasaccharide nystose and the pentasaccharide fructofuranosylnystose. The main end products of scFOS fermentation were lactate and acetate. This is the first example in lactobacilli of the association of a sucrose PTS and a beta-fructofuranosidase that could be used for scFOS degradation.

Volatile compounds produced by the probiotic strain Lactobacillus plantarum NCIMB 8826 in cereal-based substrates

The production of volatile compounds by the probiotic strain, Lactobacillus plantarum NCIMB 8826, in cereal-based media (oat, wheat. barley and malt) was investigated. Sixty compounds, including fatty acids and their esters, amides, alcohols, aldehydes, aromatic hydrocarbons, furans, ketones, peroxides and pyrans, were identified. L. plantarum significantly changed the aroma profile of the four cereal broths. and each substrate showed a specific volatiles profile. Oat and barley media were the substrates more influenced by the fermentation process. The most abundant volatiles detected in oat, wheat, barley and malt were oleic acid, linoleic acid. acetic acid, and 5-hydroxymethylfurfural, respectively. Analysis of these products confirmed the heterofermentative pathway of L plantarum. Maillard compounds were not detected during sterilisation and fermentation. This study is the first to report the volatile composition of probiotic drinks produce with non-supplemented cereal-based media and the results obtained could contribute to the development of new non-dairy probiotic formulations. (C) 2009 Elsevier Ltd. All rights reserved.

Modelling and validation of Lactobacillus plantarum fermentations in cereal-based media with different sugar concentrations and buffering capacities

An unstructured mathematical model is proposed to describe the fermentation kinetics of growth, lactic acid production, pH and sugar consumption by Lactobacillus plantarum as a function of the buffering capacity and initial glucose concentration of the culture media. Initially the experimental data of L plantarum fermentations in synthetic media with different buffering capacity and glucose were fitted to a set of primary models. Later the parameters obtained from these models were used to establish mathematical relationships with the independent variables tested. The models were validated with 6 fermentations of L. plantarum in different cereal-based media. In most cases the proposed models adequately describe the biochemical changes taking place during fermentation and are a promising approach for the formulation of cereal-based probiotic foods. (C) 2008 Elsevier B.V. All rights reserved.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Influence of encapsulation and coating materials on the survival of Lactobacillus plantarum and Bifidobacterium longum in fruit juices

The aim of this work was to compare alginate and pectin beads for improving the survival of Lactobacillus plantarum and Bifidobacterium longum during storage in pomegranate and cranberry juice, and to evaluate the influence of various coating materials, including chitosan, gelatin and glucomannan on cell survival and on the size and hardness of the beads. In pomegranate juice, free cells of L. plantarum died within 4 weeks of storage and those of B. longum within 1 week; in cranberry juice both types of cells died within one week. Encapsulation within either alginate or pectin beads improved cell survival considerably, but coating of the beads with chitosan or gelatin improved it even further; coating with glucomannan did not have any positive effect. The double gelatin coated pectin beads gave the highest protection among all types of beads, as a final concentration of approximately 108 CFU/mL and 106 CFU/mL for both L. plantarum and B. longum was obtained after 6 weeks of storage in pomegranate and cranberry juice, respectively. The good protection could be attributed to the very strong interaction between the two polymers, as measured by turbidity experiments, leading to the formation of a polyelectrolyte complex. It was also shown that the coating was able to inhibit the penetration of gallic acid within the beads, which was used in this study as a model phenolic compound with antimicrobial activity; this is a likely mechanism through which the beads were able to protect the cells from the antimicrobial activity of phenolic compounds present in both types of juices. Despite their good protective effect, the pectin beads were considerably softer than the alginate beads, an issue that should be addressed in order to increase their mechanical stability.

Effect of inclusion of lactulose and Lactobacillus plantarum on the intestinal environment and performance of piglets at weaning

The prebiotic lactulose, a probiotic strain of Lactobacillus plantarum (L. plantarum) and a synbiotic combination of these two agents were evaluated as growth promoters in 25–39-day old commercial weaning pigs. Ninety-six weaning pigs were allocated into 32 pens, taking initial weight into account, and distributed into four groups as follows: a control diet (CTR), the same diet supplemented daily with L. plantarum (109 CFU/mL sprayed on top; 20 mL/pig) (LPN); 10 g/kg lactulose (LAC) or a combination of both treatments (SYN). At day 14, eight piglets from each group were euthanized and proximal colon digesta was sampled for luminal pH, short-chain fatty acids (SCFA) and lactic acid concentrations. Deoxyribonucleic acid was extracted from colonic digesta and the microbial community was profiled by terminal restriction fragment length polymorphism analysis (T-RFLP) and qPCR. Blood urea nitrogen (BUN) and acute-phase proteins (Pig-MAP) were measured. Lactulose treatment (LAC) improved feed intake (P<0.05), average daily gain (P<0.01), feed:gain ratio (P<0.05) and reduced BUN (P<0.01). Both, LAC and LPN treatment, decreased the Enterobacteriaceae:Lactobacillus spp. ratio in the colonic luminal contents (P<0.05). Moreover LPN treatment promoted a decrease in the percentage of branched fatty acids (P<0.01) suggesting a reduction in proteolytic microbial activity. Microbial profiling of colonic luminal contents by T-RFLP revealed changes in some microbial species. Terminal restriction fragments (TRFs) compatible with Bifidobacterium thermoacidophilum were more frequently detected in experimental diets compared to CTR (P<0.05). Pigs receiving SYN diet demonstrated the combined positive effects of individual LAC and LPN treatment although we were not able to show a specific increase in the probiotic strain with the inclusion of lactulose. Collectively, these data suggest the combination of lactulose and L. plantarum acts as a complementary synbiotic, but not as a synergistic combination.

Lactulose and Lactobacillus plantarum, a potential complementary synbiotic to control postweaning colibacillosis in piglets

The potential of a prebiotic oligosaccharide lactulose, a probiotic strain of Lactobacillus plantarum, or their synbiotic combination to control postweaning colibacillosis in pigs was evaluated using an enterotoxigenic Escherichia coli (ETEC) K88 oral challenge. Seventy-two weanlings were fed four diets: a control diet (CTR), that diet supplemented with L. plantarum (2 × 10(10) CFU · day(-1)) (LPN), that diet supplemented with 10 g · kg(-1) lactulose (LAC), or a combination of the two treatments (SYN). After 7 days, the pigs were orally challenged. Six pigs per treatment were euthanized on days 6 and 10 postchallenge (PC). Inclusion of lactulose improved the average daily gain (ADG) (P < 0.05) and increased lactobacilli (P < 0.05) and the percentage of butyric acid (P < 0.02) in the colon. An increase in the ileum villous height (P < 0.05) and a reduction of the pig major acute-phase protein (Pig-MAP) in serum (P < 0.01) were observed also. The inclusion of the probiotic increased numbers of L. plantarum bacteria in the ileum and colon (P < 0.05) and in the total lactobacilli in the colon and showed a trend to reduce diarrhea (P = 0.09). The concentrations of ammonia in ileal and colonic digesta were decreased (P < 0.05), and the villous height (P < 0.01) and number of ileal goblet cells (P < 0.05) increased, at day 10 PC. A decrease in plasmatic tumor necrosis factor alpha (TNF-α) (P < 0.01) was also seen. The positive effects of the two additives were combined in the SYN treatment, resulting in a complementary synbiotic with potential to be used to control postweaning colibacillosis.

In vitro evaluation of the fermentation properties and potential probiotic activity of Lactobacillus plantarum C4 in batch culture systems

Lactobacillus plantarum C4 has been tested in in vitro pH-controlled anaerobic faecal batch cultures as compared to Lactobacillus rhamnosus GG to determine changes caused to the composition of faecal bacteria. Effects upon major groups of the microbiota and levels of short-chain fatty acids (SCFA) were assessed over 24 h. Concomitantly, hydrophobic character and ability of both bacterial cells to adhere in vitro to Caco-2 cells were investigated. Quantitative analysis of bacterial populations revealed that there was a significant increase in Lactobacillus/Enterococcus numbers in vessels with probiotic supplemented with fructooligosaccharides (FOS), compared to the negative control. L. plantarum C4 showed to have more hydrophilic behaviour and fulfilled better adhesive properties, compared to L. rhamnosus GG. Thus, L. plantarum C4 can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of SCFA.

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.

Comparative effects of six probiotic strains on immune function in vitro

There is considerable interest in the strain specificity of immune modulation by probiotics. The present study compared the immunomodulatory properties of six probiotic strains of different species and two genera in a human peripheral blood mononuclear cell (PBMC) model in vitro. Live cells of lactobacilli (Lactobacillus casei Shirota, L. rhamnosus GG, L. plantarum NCIMB 8826 and L. reuteri NCIMB 11951) and bifidobacteria (Bifidobacterium longum SP 07/3 and B. bifidum MF 20/5) were individually incubated with PBMC from seven healthy subjects for 24 h. Probiotic strains increased the proportion of CD69+ on lymphocytes, T cells, T cell subsets and natural killer (NK) cells, and increased the proportion of CD25+, mainly on lymphocytes and NK cells. The effects on activation marker expression did not appear to be strain specific. NK cell activity was significantly increased by all six strains, without any significant difference between strains. Probiotic strains increased production of IL-1β, IL-6, IL-10, TNF-α, granulocyte-macrophage colony-stimulating factor and macrophage inflammatory protein 1α to different extents, but had no effect on the production of IL-2, IL-4, IL-5 or TNF-β. The cytokines that showed strain-specific modulation included IL-10, interferon-γ, TNF-α, IL-12p70, IL-6 and monocyte chemotactic protein-1. The Lactobacillus strains tended to promote T helper 1 cytokines, whereas bifidobacterial strains tended to produce a more anti-inflammatory profile. The results suggest that there was limited evidence of strain-specific effects of probiotics with respect to T cell and NK cell activation or NK cell activity, whereas production of some cytokines was differentially influenced by probiotic strains.

New insights into the impact of Lactobacillus population on host-bacteria metabolic interplay

We aimed at evaluating the association between intestinal Lactobacillus sp. composition and their metabolic activity with the host metabolism in adult and elderly individuals. Faecal and plasma metabolites were measured and correlated to the Lactobacillus species distribution in healthy Estonian cohorts of adult (n=16; <48 y) and elderly (n=33; >65 y). Total cholesterol, LDL, C-reactive protein and glycated hemoglobin were statistically higher in elderly, while platelets, white blood cells and urinary creatinine were higher in adults. Aging was associated with the presence of L. paracasei and L. plantarum and the absence of L. salivarius and L. helveticus. High levels of intestinal Lactobacillus sp. were positively associated with increased concentrations of faecal short chain fatty acids, lactate and essential amino acids. In adults, high red blood cell distribution width was positively associated with presence of L. helveticus and absence of L. ruminis. L. helveticus was correlated to lactate and butyrate in faecal waters. This indicates a strong relationship between the composition of the gut Lactobacillus sp. and host metabolism. Our results confirm that aging is associated with modulations of blood biomarkers and intestinal Lactobacillus species composition. We identified specific Lactobacillus contributions to gut metabolic environment and related those to blood biomarkers. Such associations may prove useful to decipher the biological mechanisms underlying host-gut microbial metabolic interactions in an ageing population.

An in vitro assessment of the effects of broad-spectrum antibiotics on the human gut microflora and concomitant isolation of a Lactobacillus plantarum with anti-Candida activities

Chemostat culture was used to determine the effects of the antimicrobial agents tetracycline and nystatin on predominant components of the human gut microflora. Their addition to mixed culture systems caused a non-specific, and variable, decrease in microbial populations, although tetracycline allowed an increase in numbers of yeasts. Both had a profound inhibitory effect upon populations seen as important for gut health (bifidobacteria, lactobacilli). However, a tetracycline resistant Lactobacillus was enriched from the experiments. A combination of genotypic and phenotypic characterisations confirmed its identity as Lactobacillus plantarum. This strain exerted powerful inhibitory effects against Candida albicans. Because of its ability to resist the effects of tetracycline, this organism may be useful as a probiotic for the improved management of yeast related conditions such as thrush and irritable bowel syndrome. (C) 2004 Elsevier Ltd. All rights reserved.

Survival of Lactobacillus plantarum in model solutions and fruit juices

The aim of the work was to study the survival of Lactobacillus plantarum NCIMB 8826 in model solutions and develop a mathematical model describing its dependence on pH, citric acid and ascorbic acid. A Central Composite Design (CCD) was developed studying each of the three factors at five levels within the following ranges, i.e., pH (3.0-4.2), citric acid (6-40 g/L), and ascorbic acid (100-1000 mg/L). In total, 17 experimental runs were carried out. The initial cell concentration in the model solutions was approximately 1 × 10(8)CFU/mL; the solutions were stored at 4°C for 6 weeks. Analysis of variance (ANOVA) of the stepwise regression demonstrated that a second order polynomial model fits well the data. The results demonstrated that high pH and citric acid concentration enhanced cell survival; one the other hand, ascorbic acid did not have an effect. Cell survival during storage was also investigated in various types of juices, including orange, grapefruit, blackcurrant, pineapple, pomegranate, cranberry and lemon juice. The model predicted well the cell survival in orange, blackcurrant and pineapple, however it failed to predict cell survival in grapefruit and pomegranate, indicating the influence of additional factors, besides pH and citric acid, on cell survival. Very good cell survival (less than 0.4 log decrease) was observed after 6 weeks of storage in orange, blackcurrant and pineapple juice, all of which had a pH of about 3.8. Cell survival in cranberry and pomegranate decreased very quickly, whereas in the case of lemon juice, the cell concentration decreased approximately 1.1 logs after 6 weeks of storage, albeit the fact that lemon juice had the lowest pH (pH~2.5) among all the juices tested. Taking into account the results from the compositional analysis of the juices and the model, it was deduced that in certain juices, other compounds seemed to protect the cells during storage; these were likely to be proteins and dietary fibre In contrast, in certain juices, such as pomegranate, cell survival was much lower than expected; this could be due to the presence of antimicrobial compounds, such as phenolic compounds.

Investigation of the impact of feeding Lactobacillus plantarum CRL 1815 encapsulated in microbially derived polymers on the rat faecal microbiota

AIMS: The aim of this study was to evaluate the impact of the administration of microencapsulated Lactobacillus plantarum CRL 1815 with two combinations of microbially derived polysaccharides, xanthan : gellan gum (1%:0·75%) and jamilan : gellan gum (1%:1%), on the rat faecal microbiota. METHODS AND RESULTS: A 10-day feeding study was performed for each polymer combination in groups of 16 rats fed either with placebo capsules, free or encapsulated Lact. plantarum or water. The composition of the faecal microbiota was analysed by fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Degradation of placebo capsules was detected, with increased levels of polysaccharide-degrading bacteria. Xanthan : gellan gum capsules were shown to reduce the Bifidobacterium population and increase the Clostridium histolyticum group levels, but not jamilan : gellan gum capsules. Only after administration of jamilan : gellan gum-probiotic capsules was detected a significant increase in Lactobacillus-Enterococcus group levels compared to controls (capsules and probiotic) as well as two bands were identified as Lact. plantarum in two profiles of ileum samples. CONCLUSIONS: Exopolysaccharides constitute an interesting approach for colon-targeted delivery of probiotics, where jamilan : gellan gum capsules present better biocompatibility and promising results as a probiotic carrier. SIGNIFICANCE AND IMPACT OF STUDY: This study introduces and highlights the importance of biological compatibility in the encapsulating material election, as they can modulate the gut microbiota by themselves, and the use of bacterial exopolysaccharides as a powerful source of new targeted-delivery coating material.