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.

Development of a fermentation system to model sessile bacterial populations in the human colon

A fermentation system was designed to model the human colonic microflora in vitro. The system provided a framework of mucin beads to encourage the adhesion of bacteria, which was encased within a dialysis membrane. The void between the beads was inoculated with faeces from human donors. Water and metabolites were removed from the fermentation by osmosis using a solution of polyethylene glycol (PEG). The system was concomitantly inoculated alongside a conventional single-stage chemostat. Three fermentations were carried out using inocula from three healthy human donors. Bacterial populations from the chemostat and biofilm system were enumerated using fluorescence in situ hybridization. The culture fluid was also analysed for its short-chain fatty acid (SCFA) content. A higher cell density was achieved in the biofilm fermentation system (taking into account the contribution made by the bead-associated bacteria) as compared with the chemostat, owing to the removal of water and metabolites. Evaluation of the bacterial populations revealed that the biofilm system was able to support two distinct groups of bacteria: bacteria growing in association with the mucin beads and planktonic bacteria in the culture fluid. Furthermore, distinct differences were observed between populations in the biofilm fermenter system and the chemostat, with the former supporting higher populations of clostridia and Escherichia coli. SCFA levels were lower in the biofilm system than in the chemostat, as in the former they were removed via the osmotic effect of the PEG. These experiments demonstrated the potential usefulness of the biofilm system for investigating the complexity of the human colonic microflora and the contribution made by sessile bacterial populations.

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.

In vitro determination of prebiotic properties of oligosaccharides derived from an orange juice manufacturing by-product stream

Fermentation properties of oligosaccharides derived from orange peel pectin were assessed in mixed fecal bacterial culture. The orange peel oligosaccharide fraction contained glucose in addition to rhamnogalacturonan and xylogalacturonan pectic oligosaccharides. Twenty-four-hour, temperature- and pH-controlled, stirred anaerobic fecal batch cultures were used to determine the effects that oligosaccharides derived from orange products had on the composition of the fecal microbiota. The effects were measured through fluorescent in situ hybridization to determine changes in bacterial populations, fermentation end products were analyzed by high-performance liquid chromatography to assess short-chain fatty acid concentrations, and subsequently, a prebiotic index (PI) was determined. Pectic oligosaccharides (POS) were able to increase the bifidobacterial and Eubacterium rectale numbers, albeit resulting in a lower prebiotic index than that from fructo-oligosaccharide metabolism. Orange albedo maintained the growth of most bacterial populations and gave a PI similar to that of soluble starch. Fermentation of POS resulted in an increase in the Eubacterium rectale numbers and concomitantly increased butyrate production. In conclusion, this study has shown that POS can have a beneficial effect on the fecal microflora; however, a classical prebiotic effect was not found. An increase in the Eubacterium rectale population was found, and butyrate levels increased, which is of potential benefit to the host.

A double-blind placebo-controlled study to establish the bifidogenic dose of inulin in healthy humans

Objective: To evaluate the bifidogenic efficacy of two inulin doses in healthy human adults. Design: A double-blind, placebo-controlled, crossover human study. Setting: Food Microbial Sciences Unit, The University of Reading, Reading, UK. Subjects: Thirty healthy volunteers, 15 men, 15 women ( age range 19-35). Interventions: Subjects consumed a chocolate drink containing placebo ( maltodextrin, 8 g/day), 5 g/day inulin and 8 g/day inulin for a 2-week treatment period. Each treatment was followed by a 1-week washout at the end of which volunteers progressed to the next treatment. Faecal samples were obtained at the start of the study ( baseline) and at the end of each treatment and washout period. Fluorescent in situ hybridization was used to monitor populations of Bifidobacterium genus, Bacteroides - Prevotella, Lactobacillus - Enterococcus and Clostridium perfringens - histolyticum subgroup. Results: Bifidobacterial levels increased significantly upon ingestion of both the low ( 9.78 +/- 0.29 log(10) cells/g faeces, P < 0.05) and the high inulin dose ( 9.79 +/- 0.38 log(10) cells/g faeces, P < 0.05) compared to placebo ( 9.64 +/- 0.23 log(10) cells/g faeces). Conclusions: Both inulin doses exhibited a bifidogenic effect but a higher volunteer percentage responded to the high dose. A dose response effect was not observed but the magnitude of increase in bifidobacteria levels depended on their initial numbers. The higher the initial concentrations the smaller was the increase upon ingestion of the active treatments. Sponsorship: Financial support for the completion of this project was provided by Sensus ( Roosendaal, The Netherlands).

Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

In vitro fermentation by human fecal microflora of wheat arabinoxylans

The fermentation of three arabinoxylan (AX) fractions from wheat by the human fecal microflora was investigated in vitro. Three AX fractions, with average molecular masses of 354, 278, and 66 kDa, were incorporated into miniature-scale batch cultures (with inulin as a positive prebiotic control) with feces from three healthy donors, aged 23-29. Microflora changes were monitored by the culture-independent technique, fluorescent in situ hybridization, and short chain fatty acid (SCFA) and lactic acid production were measured by high-performance liquid chromatography. Total cell numbers increased significantly in all treated cultures, and the fermentation of AX was associated with a proliferation of the bifidobacteria, lactobacilli, and eubacteria groups. Smaller but statistically significant increases in bacteroides and clostridia groups were also observed. All AX fractions had comparable bifidogenic impacts on the microflora at 5 and 12 h, but the 66 kDa AX was particularly selective for lactobacilli. Eubacteria increased significantly on all AX fractions, particularly on 66 kDa AX. As previously reported, inulin gave a selective increase in bifidobacteria. All supplemented cultures showed significant rises in total SCFA production, with a particularly high proportion of butyric acid being produced from AX fermentation. The prebiotic effect, that is, the selectivity of AX for bifidobacteria and lactobacilli groups, increased as the molecular mass of the AX decreased. This suggests that molecular mass may influence the fermentation of AX in the colon.

In vitro fermentation of oat and barley derived beta-glucans by human faecal microbiota

Fermentation of beta-glucan fractions from barley [average molecular mass (MM), of 243, 172, and 137 kDa] and oats (average MM of 230 and 150 kDa) by the human faecal microbiota was investigated. Fractions were supplemented to pH-controlled anaerobic batch culture fermenters inoculated with human faecal samples from three donors, in triplicate, for each substrate. Microbiota changes were monitored by fluorescent in situ hybridization; groups enumerated were: Bifidobacterium genus, Bacteroides and Prevotella group, Clostridium histolyticum subgroup, Ruminococcus-Eubacterium-Clostridium (REC) cluster, Lactobacillus-Enterococcus group, Atopobium cluster, and clostridial cluster IX. Short-chain fatty acids and lactic acid were measured by HPLC. The C. histolyticum subgroup increased significantly in all vessels and clostridial cluster IX maintained high populations with all fractions. The Bacteroides-Prevotella group increased with all but the 243-kDa barley and 230-kDa oat substrates. In general beta-glucans displayed no apparent prebiotic potential. The SCFA profile (51 : 32 : 17; acetate : propionate : butyrate) was considered propionate-rich. In a further study a beta-glucan oligosaccharide fraction was produced with a degree of polymerization of 3-4. This fraction was supplemented to small-scale faecal batch cultures and gave significant increases in the Lactobacillus-Enterococcus group; however, the prebiotic potential of this fraction was marginal compared with that of inulin.

In vitro evaluation of the fermentation properties and potential prebiotic activity of Agave fructans

Aims: This study was carried out to evaluate in vitro the fermentation properties and the potential prebiotic activity of Agave-fructans extracted from Agave tequilana (Predilife). Methods and Results: Five different commercial prebiotics were compared using 24-h pH-controlled anaerobic batch cultures inoculated with human faecal slurries. Measurement of prebiotic efficacy was obtained by comparing bacterial changes, and the production of short-chain fatty acids (SCFA) was also determined. Effects upon major groups of the microbiota were monitored over 24 h incubations by fluorescence in situ hybridization. SCFA were measured by HPLC. Fermentation of the Agave fructans (Predilife) resulted in a large increase in numbers of bifidobacteria and lactobacilli. Conclusions: Under the in vitro conditions used, this study has shown the differential impact of Predilife on the microbial ecology of the human gut. Significance and Impact of the Study: This is the first study reporting of a potential prebiotic mode of activity for Agave fructans investigated which significantly increased populations of bifidobacteria and lactobacilli compared to cellulose used as a control.

Prebiotic evaluation of a novel galactooligosaccharide mixture produced by the enzymatic activity of Bifidobacterium bifidum NCIMB 41171, in healthy humans: a randomized, double-blind, crossover, placebo-controlled intervention study

Background: Galactooligosaccharides are selectively fermented by the beneficial member of the colonic microflora contributing to the health of the host. Objective: We assessed the prebiotic potential of a novel galactooligosaccharide produced through the action of beta-galactosidases, originating from a probiotic Bifidobacterium bifidum strain, against a galactooligosaccharide produced through the action of an industrial P-galactosidase and a placebo. Design: Fifty-nine healthy human volunteers participated in this study. Initially, the effect of the matrix on the prebiotic properties of a commercially available galactooligosaccharide (7 g/d) was assessed during 7-d treatment periods with a 7-d washout period in between. During the second phase, 30 volunteers were assigned to a sequence of treatments (7 d) differing in the amount of the novel galactooligosaccharide (0, 3.6, or 7 g/d). Stools were recovered before and after each intervention, and bacteria numbers were determined by fluorescent in situ hybridization. Results: Addition of the novel galactooligosaccharide mixture significantly increased the bifidobacterial population ratio compared with the placebo (P < 0.05), whereas 7 g/d of the novel galactooligosaccharide significantly increased the bifidobacterial ratio compared with the commercial galactooligosaccharide (P < 0.05). Moreover, a significant relation (P < 0.001) between the bifidobacteria proportion and the novel galactooligosaccharide dose (0, 3.6, and 7 g/d) was observed. This relation was similar to the effect of the novel galactooligosaccharide on the prebiotic index of each dose. Conclusions: This study showed that galactooligosaccharide mixtures produced with different beta-galactosidases show different prebiotic properties and that, by using enzymes originating from bifidobacterial species, an increase in the bifidogenic properties of the prebiotic product is achievable.

Safety evaluation of oligofructose: 13 Week rat study and in vitro mutagenicity

Oligofructose (OF), comprised of fructose oligomers with a terminal glucose unit, is a family Of oligosaccharides derived from the hydrolysis of inulin. Consumption of OF in animals and humans increases colonic bifidobacteria levels. The present study evaluates the safety of OF in both a 13 week rat feeding Study and Using in Vitro mutagenicity tests. Fecal bifidobacteria levels were also determined by in situ hybridization to assess a biological function of OF. Rats received either a control diet OF diets containing one of four doses of OF. Total, HDL, and LDL-cholesterol levels were significantly lower at several time points during the study in groups receiving OF compared to controls with the largest effects Occurring in the high dose male animals. Weight gain in the male high dose group was significantly lower at early time points compared to controls but]lot Significantly different at the end of study. As expected, cecal weights increased in a dose-related manner and fecal bifidobacteria levels also demonstrated a dose-related increase. There were no consistent differences in gross pathology or histopathology related to dietary OF. OF did not induce a positive response in the Ames test or chromosomal aberration test with CHO cells. These results demonstrate no adverse effects of OF. (C) 2008 Elsevier Ltd. All rights reserved.

In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora

Aims: To determine the fermentation profiles by human gut bacteria of arabino-oligosaccharides of varying degree of polymerization. Materials and Methods: Sugar beet arabinan was hydrolyzed with a commercial pectinase and eight fractions, of varying molecular weight, were isolated by gel-filtration chromatography. Hydrolysis fractions, arabinose, arabinan and fructo-oligosaccharides were fermented anaerobically by gut bacteria. Total bacteria, bifidobacteria, bacteroides, lactobacilli and the Clostridium perfringens/histolyticum sub. grp. were enumerated using fluorescent in situ hybridization. Results: Bifidobacteria were stimulated to different extents depending on molecular weight, i.e. maximum increase in bifidobacteria after 48 h was seen on the lower molecular weight fractions. Lactobacilli fluctuated depending on the initial inoculum levels. Bacteroides numbers varied according to fraction; arabinan, arabinose and higher oligosaccharides (degree of polymerization, dp > 8) resulted in significant increases at 24 h. Only carbohydrate mixtures with dp of 1-2 resulted in significant increases at 48 h (log 8.77 +/- 0.23). Clostridia decreased on all substrates. Conclusions: Arabino-oligosaccharides can be considered as potential prebiotics. Significance and Impact of the Study: Arabinan is widely available as it is a component of sugar beet pulp, a co-product from the sugar beet industry. Generation of prebiotic functionality from arabinan would represent significant added value to a renewable resource.

A method for estimating the turbulent kinetic energy dissipation rate from a vertically pointing Doppler lidar, and independent evaluation from balloon-borne in situ measurements

A method of estimating dissipation rates from a vertically pointing Doppler lidar with high temporal and spatial resolution has been evaluated by comparison with independent measurements derived from a balloon-borne sonic anemometer. This method utilizes the variance of the mean Doppler velocity from a number of sequential samples and requires an estimate of the horizontal wind speed. The noise contribution to the variance can be estimated from the observed signal-to-noise ratio and removed where appropriate. The relative size of the noise variance to the observed variance provides a measure of the confidence in the retrieval. Comparison with in situ dissipation rates derived from the balloon-borne sonic anemometer reveal that this particular Doppler lidar is capable of retrieving dissipation rates over a range of at least three orders of magnitude. This method is most suitable for retrieval of dissipation rates within the convective well-mixed boundary layer where the scales of motion that the Doppler lidar probes remain well within the inertial subrange. Caution must be applied when estimating dissipation rates in more quiescent conditions. For the particular Doppler lidar described here, the selection of suitably short integration times will permit this method to be applicable in such situations but at the expense of accuracy in the Doppler velocity estimates. The two case studies presented here suggest that, with profiles every 4 s, reliable estimates of ϵ can be derived to within at least an order of magnitude throughout almost all of the lowest 2 km and, in the convective boundary layer, to within 50%. Increasing the integration time for individual profiles to 30 s can improve the accuracy substantially but potentially confines retrievals to within the convective boundary layer. Therefore, optimization of certain instrument parameters may be required for specific implementations.

Structure of single-wall peptide nanotubes: in situ flow aligning X-ray diffraction

The structure of single wall peptide nanotubes is presented for the model surfactant-like peptide A6K. Capillary flow alignment of a sample in the nematic phase at high concentration in water leads to oriented X-ray diffraction patterns. Analysis of these, accompanied by molecular dynamics simulations, suggests the favourable self-assembly of antiparallel peptide dimers into beta-sheet ribbons that wrap helically to form the nanotube wall.

The effect of condensed tannins in fresh sainfoin (Onobrychis viciifolia) on in vivo and in situ digestion in sheep

This study focused on effects of structure, content and biological activity of condensed tannins (CT) in leaves, stems and whole plant of sainfoin (Onobrychis viciifolia) on its in vivo and in situ digestive characteristics in sheep. Sainfoin was studied as fresh forage during the first vegetation cycle at two phenological stages (i.e., end of flowering and green seeds) and during the second vegetation cycle (i.e., start of flowering). The feeding experiment used 12 sheep; with six dosed, through the rumen cannula, with polyethylene glycol (PEG) to neutralise CT effects. Organic matter digestibility (OMD), total tract N disappearance and N balance were measured in sheep fed the whole plant. The residues of dry matter (DM) and N from nylon bags suspended in the rumen were determined on leaves and stems. Intestinal digestibility was measured using other, intestinally fistulated sheep. PEG addition and vegetation cycle increased total tract N digestibility (P<0.001) but PEG affected OMD only at the end of flowering. PEG inactivated the CT and increased urinary N excretion (P<0.05) but this was offset by lower faecal N excretion (P<0.001). Feeding sainfoin can be used to alter the form of excreted N (i.e., urine vs faeces) and thus potentially reduce environmental N pollution without affecting body N retention. Kinetic studies of total N, ammonia N (NH3-N) and volatile fatty acids (VFA) in rumen fluid were made before and 1.5, 3 and 6 h after feeding. Sainfoin CT decreased rumen fluid soluble N (P<0.05) and NH3-N (P<0.01). Ruminal N disappearance (DisN) of leaves or stems was lower in the presence of active CT compared to PEG-inactivated CT (P<0.001) for both vegetation cycles. PEG also increased intestinal digestibility (P<0.05) of leaves and stems. Leaves had lower ruminal DisN, but higher N disappearing from intestine than stems. The biological activity and content of CT in the whole plant decreased as phenological stage increased. Prodelphinidin:procyanidin (PD:PC) ratios of leaves varied with vegetation cycle and phenological stage. The molecular size of CT in the whole plant, as indicated by their mean degree of polymerisation (mDP), was lowest at the start of flowering and coincided with the higher biological activity and content of CT.