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.

In vitro fermentation of mixed linkage glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

The aim of this study was to develop selectively fermented (prebiotic) carbohydrate molecules which would also result in the generation of butyric acid. Glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from various types of maltodextrins were evaluated for their fermentation by mixed cultures of human colonic microflora. The selectivity of growth of desirable bacteria (bifidobacteria, lactobacilli) was studied in stirred pH-controlled (6.8) batch cultures. Bacterial populations were enumerated using fluorescent in situ hybridization (FISH). Gluco-oligosaccharides resulted in significantly (P<0.05) increased numbers of bifidobacteria and lactobacilli within 24 hours. Bacteroides, clostridial and eubacterial populations were slightly decreased at 48 h. There was very little difference in selectivity between the maltodextrin substrates and the products, although maltodextrin displayed a slightly less selective fermentation than the gluco-oligosaccharide products, also stimulating the growth of bacteroides, clostridia and eubacteria. Gluco-oligosaccharides, produced from G19 maltodextrin, resulted in the best prebiotic effect with the highest prebiotic index (PI) of 5.90 at 48 hours. Acetate, propionate and butyrate were all produced from glucooligosaccharides, derived from G19 maltodextrin, at 48 hours but no lactate or formate were detected.

In vitro effects of phosphatidylcholine and transgalactooligosaccharides on the production of 1,2-sn-diacylglycerol by Bifidobacterium longum biovar infantis

Aims: To investigate the production of the tumour promoter 1,2-sn-diacylglycerol (DAG) by a human gut isolate of Bifidobacterium longum biovar infantis. Methods and Results: Bifidobacterium longum biovar infantis was grown in vitro using anaerobic static batch cultures in the presence of phosphatidylcholine (PC) and trans-galactooligosaccharides (TOS). Production of DAG was found to be dependent upon the presence of PC, while TOS had a reducing effect. Considerable differences in morphology, growth and metabolic end products from the micro-organism were observed under the different culture conditions. Conclusions: Our results have provided evidence that B. longum biovar infantis can produce DAG in vitro and that a prebiotic exerted a reducing effect upon this production. Significance and Impact of the Study: The results presented in this study demonstrate an ability of ostensibly beneficial member of the colonic environment to produce unwanted compounds under certain conditions. Therefore, it may be important that a combination of substrates and other factors are assessed when studying the behaviour of any bacterial group or species, especially when designing the dietary interventions.

In vitro fermentation of chitosan derivatives by mixed cultures of human faecal bacteria

Stirred, pH controlled batch cultures were carried out with faecal inocula and various chitosans to investigate the fermentation of chitosan derivatives by the human gut flora. Changes in bacterial levels and short chain fatty acids were measured over time. Low, medium and high molecular weight chitosan caused a decrease in bacteroides, bifidobacteria, clostridia and lactobacilli. A similar pattern was seen with chitosan oligosaccharide (COS). Butyrate levels also decreased. A three-stage fermentation model of the human colon was used for investigation of the metabolism of COS. In a region representing the proximal colon, clostridia decreased while lactobacilli increased. In the region representing the transverse colon, bacteroides and clostridia increased. Distally a small increase in bacteroides occurred. Butyrate levels increased. Under the highly competitive conditions of the human colon, many members of the microflora, are unable to compete for chitosans of low, medium or high molecular weight. COS were more easily utilised and when added to an in vitro colonic model led to increased production of butyrate, but some populations of potentially detrimental bacteria also increased. (c) 2005 Elsevier Ltd. All rights reserved.

In vitro evaluation of the fermentation properties of galactooligosaccharides synthesised by alpha-galactosidase from Lactobacillus reuteri

Stirred, pH-controlled anaerobic batch cultures were used to evaluate the in vitro utilisation by canine gut microflora of novel alpha-galactooligosaccharides synthesised with an enzyme extract from a canine Lactobacillus reuteri strain. Fructooligosaccharides (FOS), melibiose and raffinose were used as reference carbohydrates for the prebiotic properties of the synthesised oligosaccharide (galactosyl melibiose mixture-GMM). Addition of Lactobacillus acidophilus was used as control for the evaluation of the synbiotic properties of the oligosaccharide with L. reuteri. Populations of predominant gut bacterial groups were monitored over 48 h of batch culture by fluorescent in situ hybridisation, and short-chain fatty acid (SCFA) production was measured. GMM showed a higher increase in bifidobacteria and lactobacilli population number and size as well as a higher decrease in clostridia population number and size compared to the commercial prebiotics (FOS, melibiose, raffinose). This prebiotic effect was further increased by the addition of L. reuteri followed by a change in the SCFA production pattern compared to GMM alone or GMM with L. acidophilus. The observed change in SCFA production was in accordance with the fermentation properties of L. reuteri, suggesting that the novel synbiotic had a significant effect on the canine gut microflora fermentation.

In vitro effects of selected synbiotics on the human faecal microbiota composition

Synbiotics are recognized means of modulating gut microbiota composition and activities. However, whether synbiotics are superior to prebiotics and probiotics alone in moderating the gut microbiota towards a purportedly healthy composition has not been determined. Eight selected synbiotics (short-chain fructooligosaccharides or fructooligosaccharides, each combined with one of four probiotics, Lactobacillus fermentum ME-3, Lactobacillus plantarum WCFS1, Lactobacillus paracasei 8700:2 or Bifidobacterium longum 46) were added to 24-h pH-controlled anaerobic faecal batch cultures. The prebiotic and probiotic components were also tested alone to determine their respective role within the synbiotic for modulation of the faecal microbiota. Effects upon major groups of the microbiota were evaluated using FISH. Rifampicin variant probiotic strains were used to assess probiotic levels. Synbiotic and prebiotics increased bifidobacteria and the Eubacterium rectale-Clostridium coccoides group. Lower levels of Escherichia coli were retrieved with these combinations after 5 and 10 h of fermentation. Probiotics alone had little effect upon the groups, however. Multivariate analysis revealed that the effect of synbiotics differed from the prebiotics as higher levels of Lactobacillus-Enterococcus were observed when the probiotic was stimulated by the prebiotic component. Here, the synbiotic approach was more effective than prebiotic or probiotic alone to modulate the gut microbiota.

Characterization and in vitro digestibility of bovine beta-lactoglobulin glycated with galactooligosaccharides

Galactooligosaccharides (GOS) are well-known prebiotic ingredients which can form the basis of new functional dairy products. In this work, the production and characterization of glycated beta-lactoglobulin beta-LG) with prebiotic GOS through the Maillard reaction under controlled conditions (a(w) = 0.44, 40 degrees C for 23 days) have been studied. The extent of glycation of beta-LG was evaluated by formation of furosine which progressively increased with storage for up to 16 days, suggesting that the formation of Amadori compounds prevailed over their degradation. RP-HPLC-UV, SIDS-PAGE, and IEF profiles of beta-LG were modified as a consequence of its glycation. MALDI-ToF mass spectra of glycated beta-LG showed an increase of up to similar to 21% in its average molecular mass after storage for 23 days. Moreover, a decrease in unconjugated GOS (one tri-, two tetra-, and one pentasaccharide) was observed by HPAEC-PAD upon glycation. These results were confirmed by ESI MS. The stability of the glycated beta-LG to in vitro simulated gastrointestinal digestion was also described and compared with that of the unglycated protein. The yield of digestion products of glycated beta-LG was lower than that observed for the unglycated protein. The conjugation of prebiotic carbohydrates to stable proteins and peptides could open new routes of research in the study of functional ingredients.

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 fermentation of carbohydrates by porcine faecal inocula and their influence on Salmonella Typhimurium growth in batch culture systems

The aim of this study was to evaluate in vitro the influence of fermentable carbohydrates on the activity of porcine microbiota and survival of Salmonella Typhimurium in a batch culture system simulating the porcine hindgut. The carbohydrates tested were xylooligosaccharides, a mixture of fructooligosaccharides/inulin (FIN), fructooligosaccharides (FOS), gentiooligosaccharides (GEO) and lactulose (LAC). These ingredients stimulated the growth of selected Bifidobacterium and Lactobacillus species in pure cultures. In batch cultures, the carbohydrates influenced some fermentation parameters. For example, GEO and FIN significantly increased lactic acids compared with the control (no added carbohydrate). With the exception of LAC, the test carbohydrates increased the production of short-chain fatty acid (SCFA) and modified SCFA profiles. Quantitative analysis of bacterial populations by FISH revealed increased counts of the Bifidobacterium group compared with control and, with exception of FOS, increased Lactobacillus, Leuconostoc and Weissella spp. counts. Salmonella numbers were the lowest during the fermentation of LAC. This work has looked at carbohydrate metabolism by porcine microbiota in a pH-controlled batch fermentation system. It provides an initial model to analyse interactions with pathogens.

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.

In vitro evaluation of the prebiotic activity of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).

In vitro fermentation of oat bran obtained by debranning with a mixed culture of human fecal bacteria

The prebiotic potential of oat samples was investigated by in vitro shaker-flask anaerobic fermentations with human fecal cultures. The oat bran fraction was obtained by debranning and was compared with other carbon sources such as whole oat flour, glucose, and fructo-oligosaccharide. The oat bran fraction showed a decrease in culturable anaerobes and clostridia and an increase in bifidobacteria and lactobacilli populations. A similar pattern was observed in fructo-oligosaccharide. Butyrate production was higher in oat bran compared to glucose and similar to that in fructo-oligosaccharide. Production of propionate was higher in the two oat media than in fructo-oligosaccharide and glucose, which can be used as energy source by the liver. This study suggests that the oat bran fraction obtained by debranning is digested by the gut ecosystem and increases the population of beneficial bacteria in the indigenous gut microbiota. This medium also provides an energy source preferred by colonocytes when it is metabolized by the gut flora.

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.

Effect of fecal water on an in vitro model of colonic mucosal barrier function

Fecal water (FW) has been shown to exert, in cultured cells, cytotoxic and genotoxic effects that have implications for colorectal cancer (CRC) risk. We have investigated a further biological activity of FW, namely, the ability to affect gap junctions in CACO2 cell monolayers as an index of mucosal barrier function, which is known to be disrupted in cancer. FW samples fi-om healthy, free-living, European subjects that were divided into two broad age groups, adult (40 +/- 9.7 yr; n = 53) and elderly (76 +/- 7.5 yr; n = 55) were tested for effects on gap junction using the transepithelial resistance (TER) assay. Overall, treatment of CACO2 cells with FW samples fi-om adults increased TER (+ 4 %), whereas FW from elderly subjects decreased TER (-5%); the difference between the two groups was significant (P < 0.05). We also measured several components of FW potentially associated with modulation of TER, namely, short-chain fatty acid (SCFA) and ammonia. SCFAs (propionic, acetic, and n-butyric) were significantly lower in the elderly population (-30%, -35%, and -21%, respectively, all P pound 0.01). We consider that FW modulation of in vitro epithelial barrier function is a potentially useful noninvasive biomarker, but it requires further validation to establish its relationship to CRC risk.