Survivability of a probiotic Lactobacillus casei in the gastrointestinal tract of healthy human volunteers and its impact on the faecal microflora

Aim: The aim of this study was to measure the gastrointestinal survival of Lactobacillus casei and its impact on the gut microflora in healthy human volunteers. Methods and Results: Twenty healthy volunteers took part in a double-blind placebo-controlled probiotic feeding study (10 fed probiotic, 10 fed placebo). The probiotic was delivered in two 65 ml aliquots of fermented milk drink (FMD) daily for 21 days at a dose of 8.6 +/- 0.1 Log(10)Lact. casei CFU ml(-1) FMD. Faecal samples were collected before, during and after FMD or placebo consumption, and important groups of faecal bacteria enumerated by fluorescent in situ hybridization (FISH) using oligonucleotide probes targeting the 16S rRNA. The fed Lact. casei was enumerated using selective nutrient agar and colony identity confirmed by pulsed field gel electrophoresis. Seven days after ingestion of FMD, the Lact. casei was recovered from faecal samples taken from the active treatment group at 7.1 +/- 0.4 Log(10) CFU g(-1) faeces (mean +/- SD, n = 9) and numbers were maintained at this level until day 21. Lact. casei persisted in six volunteers until day 28 at 5.0 +/- 0.9 Log(10) CFU g(-1) faeces (mean +/- SD, n = 6). Numbers of faecal lactobacilli increased significantly upon FMD ingestion. In addition, the numbers of bifidobacteria were higher on days 7 and 21 than on days 0 and 28 in both FMD fed and placebo fed groups. Consumption of Lact. casei had little discernible effect on other bacterial groups enumerated. Conclusions: Daily consumption of FMD enabled a probiotic Lact. casei strain to be maintained in the gastrointestinal tract of volunteers at a stable relatively high population level during the probiotic feeding period. Significance and Impact of the Study: The study has confirmed that this probiotic version of Lact. casei survives well within the human gastrointestinal tract.

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.

An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties

This study evaluated the use of a bile-salt-hydrolyzing Lactobacillus fermentum strain as a probiotic with potential hypocholesterolemic properties. The effect of L. fermentum on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system. Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the Lactobacillus strain was evaluated. Administration of L. fermentum resulted in a decrease in the overall bifidobacterial population (ca. 1 log unit). In the in vitro system, no significant changes were observed in the total bacterial, Lactobacillus, Bacteroides, and clostridial populations through L. fermentum supplementation. Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively). A general, although lesser, increase in the production of lactate was observed with the administration of the L. fermentum strain. Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%). Results from this in vitro study suggest that L.Fermentum KC5b is a candidate probiotic which may affect cholesterol metabolism. The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain.

In vitro studies on colonization resistance of the human gut microbiota to Candida albicans and the effects of tetracycline and Lactobacillus plantarum LPK

An anaerobic three-vessel continuous-flow culture system, which models the three major anatomical regions of the human colon, was used to study the persistence of Candida albicans in the presence of a faecal microbiota. During steady state conditions, overgrowth of C. albicans was prevented by commensal bacteria indigenous to the system. However antibiotics, such as tetracycline have the ability to disrupt the bacterial populations within the gut. Thus, colonization resistance can be compromised and overgrowth of undesirable microorganisms like C. albicans can then occur. In this study, growth of C. albicans was not observed in the presence of an established faecal microbiota. However, following the addition of tetracycline to the growth medium, significant growth of C. albicans occurred. A probiotic Lactobacillus plantarum LPK culture was added to the system to investigate whether this organism had any effects upon the Candida populations. Although C. albicans was not completely eradicated in the presence of this bacterium, cell counts were markedly reduced, indicating a compromised physiological function. This study shows that the normal gut flora can exert 'natural' resistance to C. albicans, however this may be diminished during antibiotic intake. The use of probiotics can help fortify natural resistance.

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.

In vitro fermentation of rice bran combined with Lactobacillus acidophilus 14 150B or Bifidobacterium longum 05 by the canine faecal microbiota

The fermentability of rice bran (RB), alone or in combination with one of two probiotics, by canine faecal microbiota was evaluated in stirred, pH-controlled, anaerobic batch cultures. RB enhanced the levels of bacteria detected by probes Bif164 (bifidobacteria) and Lab158 (lactic acid bacteria); however, addition of the probiotics did not have a significant effect on the predominant microbial counts compared with RB alone. RB sustained levels of Bifidobacterium longum 05 throughout the fermentation; in contrast, Lactobacillus acidophilus 14 150B levels decreased significantly after 5-h fermentation. RB fermentation induced changes in the short-chain fatty acid (SCFA) profile. However, RB combined with probiotics did not alter the SCFA levels compared with RB alone. Denaturing gradient gel electrophoresis analysis of samples obtained at 24 h showed a treatment effect with RB, which was not observed in the RB plus probiotic systems. Overall, the negative controls displayed lower species richness than the treatment systems and their banding profiles were distinct. This study illustrates the ability of a common ingredient found in pet food to modulate the canine faecal microbiota and highlights that RB may be an economical alternative to prebiotics for use in dog food.

In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk

Commensal bacteria, including some species of lactobacilli commonly present in human breast milk, appear to colonize the neonatal gut and contribute to protection against infant infections, suggesting that lactobacilli could potentially modulate immunity. In this study, we evaluated the potential of two Lactobacillus strains isolated from human milk to modulate the activation and cytokine profile of peripheral blood mononuclear cell (PBMC) subsets in vitro. Moreover, these effects were compared to the same probiotic species of non-milk origin. Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 at 105, 106 and 107 bacteria/mL were co-cultured with PBMC (106/mL) from 8 healthy donors for 24 h. Activation status (CD69 and CD25 expressions) of natural killer (NK) cells (CD56+), total T cells (CD3+), cytotoxic T cells (CD8+) and CD4+ T cells was determined by flow cytometry. Regulatory T cells (Treg) were also quantified by intracellular Foxp3 evaluation. Regarding innate immunity, NK cells were activated by addition of both Lactobacillus strains, and in particular, the CD8+ NK subset was preferentially induced to highly express CD69 (90%, p<0.05). With respect to acquired immunity, approximately 9% of CD8+ T cells became activated after co-cultivation with L. fermentum or L salivarius. Although CD4+ T cells demonstrated a weaker response, there was a preferential activation of Treg cells (CD4+CD25+Foxp3+) after exposure to both milk probiotic bacteria (p<0.05). Both strains significantly induced the production of a number of cytokines and chemokines, including TNFα, IL-1β, IL-8, MIP-1α, MIP-1β, and GM-CSF, but some strain-specific effects were apparent. This work demonstrates that L salivarius CECT5713 and L. fermentum CECT5716 enhanced both natural and acquired immune responses, as evidenced by the activation of NK and T cell subsets and the expansion of Treg cells, as well as the induction of a broad array of cytokines.

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.

Effect of food models and low-temperature storage on the adhesion of Lactobacillus rhamnosus GG to Caco-2 cells

This study evaluated the effects of fat and sugar levels on the surface properties of Lactobacillus rhamnosus GG during storage in food model systems, simulating yogurt and ice cream, and related them with the ability of the bacterial cells to adhere to Caco-2 cells. Freeze-dried L. rhamnosus GG cells were added to the model food systems and stored for 7 days. The bacterial cells were analyzed for cell viability, hydrophobicity, ζ potential, and their ability to adhere to Caco-2 cells. The results indicated that the food type and its composition affected the surface and adhesion properties of the bacterial cells during storage, with yogurt being a better delivery vehicle than ice cream in terms of bacterial adhesion to Caco-2 cells. The most important factor influencing bacterial adhesion was the storage time rather than the levels of fats and sugars, indicating that conformational changes were taking place on the surface of the bacterial cells during storage.

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.

Influence of fermentation conditions on the surface properties and adhesion of Lactobacillus rhamnosus GG

Background: The surface properties of probiotic bacteria influence to a large extent their interactions within the gut ecosystem. There is limited amount of information on the effect of the production process on the surface properties of probiotic lactobacilli in relation to the mechanisms of their adhesion to the gastrointestinal mucosa. The aim of this work was to investigate the effect of the fermentation pH and temperature on the surface properties and adhesion ability to Caco-2 cells of the probiotic strain Lactobacillus rhamnosus GG. Results: The cells were grown at pH 5, 5.5, 6 (temperature 37 °C) and at pH 6.5 (temperature 25 °C, 30 °C and 37 °C), and their surfaces analysed by X-ray photoelectron spectrometry (XPS), Fourier transform infrared spectroscopy (FT-IR) and gel-based proteomics. The results indicated that for all the fermentation conditions, with the exception of pH 5, a higher nitrogen to carbon ratio and a lower phosphate content was observed at the surface of the bacteria, which resulted in a lower surface hydrophobicity and reduced adhesion levels to Caco-2 cells as compared to the control fermentation (pH 6.5, 37 oC). A number of adhesive proteins, which have been suggested in previous published works to take part in the adhesion of bacteria to the human gastrointestinal tract, were identified by proteomic analysis, with no significant differences between samples however. Conclusions: The temperature and the pH of the fermentation influenced the surface composition, hydrophobicity and the levels of adhesion of L. rhamnosus GG to Caco-2 cells. It was deduced from the data that a protein rich surface reduced the adhesion ability of the cells.