Synthesis of prebiotic galactooligosaccharides from lactose using bifidobacterial β-galactosidase (BbgIV) immobilised on DEAE-Cellulose, Q-Sepharose and amino-ethyl agarose

The bifidobacterial β-galactosidase BbgIV was immobilised on DEAE-Cellulose and Q-Sepharose via ionic binding and on amino-ethyl- and glyoxal-agarose via covalent attachment, and was then used to catalyse the synthesis of galactooligosaccharides (GOS). The immobilisation yield exceeded 90 % using ionic binding, while it was low using aminoethyl agarose (25 – 28 %) and very low using glyoxal agarose (< 3 %). This was due to the mild conditions and absence of chemical reagents in ionic binding, compared to covalent attachment. The maximum GOS yield obtained using DEAE-Cellulose and Q-Sepharose was similar to that obtained using free BbgIV (49 – 53 %), indicating the absence of diffusion limitation and mass transfer issues. For amino-ethyl agarose, however, the GOS yield obtained was lower (42 – 44 %) compared to that obtained using free BbgIV. All the supports tried significantly (P < 0.05) increased the BbgIV operational stability and the GOS synthesis productivity up to 55 °C. Besides, six successive GOS synthesis batches were performed using BbgIV immobilised on Q-Sepharose; all resulted in similar GOS yields, indicating the possibility of developing a robust synthesis process. Overall, the GOS synthesis operation performance using BbgIV was improved by immobilising the enzyme onto solid supports, in particular on Q-Sepharose

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.

Modulation of the fecal microflora profile and immune function by a novel trans-galactooligosaccharide mixture (B-GOS) in healthy elderly volunteers

Background: Aging is associated with reduced numbers of beneficial colonic bifidobacteria and impaired immunity. Galactooligosaccharides (GOSs) stimulate the growth of bifidobacteria in younger adults, but little is known about their effects in the elderly and their immunomodulatory capacity. Objective: We assessed the effect of a prebiotic GOS mixture (B-GOS) on immune function and fecal microflora composition in healthy elderly subjects. Design: In a double-blind, placebo-controlled, crossover study, 44 elderly subjects were randomly assigned to receive either a placebo or the B-GOS treatment (5.5 g/d). Subjects consumed the treatments for 10 wk, and then went through a 4-wk washout period, before switching to the other treatment for the final 10 wk. Blood and fecal samples were collected at the beginning, middle (5 wk), and end of the test period. Predominant bacterial groups were quantified, and phagocytosis, natural killer (NK) cell activity, cytokine production, plasma cholesterol, and HDL cholesterol were measured. Results: B-GOS significantly increased the numbers of beneficial bacteria, especially bifidobacteria, at the expense of less beneficial groups compared with the baseline and placebo. Significant increases in phagocytosis, NK cell activity, and the production of antiinflammatory cytokine interleukin-10 (IL-10) and significant reduction in the production of proinflammatory cytokines (IL-6, IL-1 beta , and tumor necrosis factor-alpha) were also observed. B-GOS exerted no effects on total cholesterol or HDL-cholesterol production, however. Conclusions: B-GOS administration to healthy elderly persons resulted in positive effects on both the microflora composition and the immune response. Therefore, B-GOS may be a useful dietary candidate for the enhancement of gastrointestinal health and immune function in elderly persons. Am J Clin Nutr 2008; 88: 1438-46.

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.

Effect of pH and dose on the growth of gut bacteria on prebiotic carbohydrates in vitro

The effect of pH and substrate dose on the fermentation profile of a number of commercial prebiotics was analysed in triplicate using stirred, pH and temperature controlled anaerobic batch culture fermentations, inoculated with a fresh faecal slurry from one of three healthy volunteers. Bacterial numbers were enumerated using fluorescence in situ hybridisation. The commercial prebiotics investigated were fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), isomaltooligosaccharides (IMO) and lactulose. Two pH values were investigated, i.e. pH 6 and 6.8. Doses of 1% and 2% (w/v) were investigated, equivalent to approximately 4 and 8 g per day, respectively, in an adult diet. It was found that both pH and dose altered the bacterial composition. It was observed that FOS and inulin demonstrated the greatest bifidogenic effect at pH 6.8 and 1% (w/v) carbohydrate, whereas GOS, IMO and lactulose demonstrated their greatest bifidogenic effect at pH 6 and 2% (w/v) carbohydrate. From this we can conclude that various prebiotics demonstrate differing bifidogenic effects at different conditions in vitro. (C) 2003 Elsevier Science Ltd. All rights reserved.

Profiling of composition and metabolic activities of the colonic microflora of growing pigs fed diets supplemented with prebiotic oligosaccharides

It is evident that quantitative information on different microbial groups and their contribution in terms of activity in the gastrointestinal (GI) tract of humans and animals is required in order to formulate functional diets targeting improved gut function and host health. In this work, quantitative information on levels and spatial distributions of Bacteroides spp, Eubacterium spp, Clostridium spp, Escherichia coli, Bifidobacterium spp and Lactobacillus/Enterococcus spp. along the porcine large intestine was investigated using 16S rRNA targeted probes and fluorescent in situ hybridisation (FISH). Caecum, ascending colon (AC) and rectum luminal digesta from three groups of individually housed growing pigs fed either a corn-soybean basal diet (CON diet) or a prebiotic diet containing 10 g/kg oligofructose (FOS diet) or trans-galactooligosaccharides (TOS diet) at the expense of cornstarch were analysed. DAPI staining was used to enumerate total number of cells in the samples. Populations of total cells, Bacteroides, Eubacterium, Clostridium and Bifidobacterium, declined significantly (P < 0.05) from caecum to rectum, and were not affected by dietary treatments. Populations of Lactobacillus/ Enterococcus and E coli did not differ throughout the large intestine. The relative percent (%) contribution of each bacterial group to the total cell count did not differ between caecum and rectum, with the exception of Eubacterium that was higher in the AC digesta. FISH analysis showed that the sum of all bacterial groups made up a small percentage of the total cells, which was 12.4%, 21.8% and 10.3% in caecum, AC and rectum, respectively. This supports the view that in swine, the diversity of GI microflora might be higher compared to other species. In terms of microflora metabolic activity, the substantially higher numerical trends seen in FOS and TOS treatments regarding total volatile fatty acid, acetate concentrations and glycolytic activities, it could be postulated that FOS and TOS promoted saccharolytic activities in the porcine colon. (c) 2006 Elsevier Ltd. All rights reserved.

Established and emerging prebiotics and their effects on the gut microflora

The use of probiotics combined with prebiotics (synbiotics) has been proved to be more and more interesting in the market of functional foods. The use of probiotics alone has a long history whereas the concept of prebiotics is rather new, introduced by Gibson & Roberfroid(1). Efficient prebiotics are considered the compounds that are not digested and selectively promote the growth of beneficial microorganisms (such as lactobacilli and bifidobacteria) in the colon. Some established prebiotics that are currently used in the European market are fructooligosaccharides (FOS), galactooligosaccharides (GOS) and inulin. However, there are more compounds considered as "emerging prebiotics" which have not been established yet, but there is a need of further investigation on them. Some of them are oligomers of soya & xylan, isomalto-oligosaccharides (IMO), polydextrose and possibly some oligosaccharides in honey. There is still an incomplete picture of their fermentation properties but according to the studies performed till now, it is quite possible that these molecules might have the same or more desirable properties than the established ones. In this review, the effects of the established and emerging prebiotics on the gut microflora are presented, based on in vitro and in vivo studies (healthy volunteers).

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.

Effects of a novel galactooligosaccharide on the faecal microbiota of healthy and inflammatory bowel disease cats during a randomized, double-blind, cross-over feeding study

Inflammatory bowel disease (IBD) is a common gastrointestinal disorder of cats with no known aetiological agent. Previous work has suggested that the faecal microbiota of IBD cats is significantly different from that of healthy cats, including significantly lower bifidobacteria, bacteroides and total counts in IBD cats and significantly lower levels of sulfate-reducing bacteria in healthy cats. Prebiotics, including galactooligosaccharides (GOS), have been shown to elicit a bifidogenic effect in humans and other animals. The purpose of the current study was to examine the impact of a novel GOS supplementation on the faecal microbiota of healthy and IBD cats during a randomized, double-blind, cross-over feeding study. Eight oligonucleotide probes targeting specific bacterial populations and DAPI stain (total bacteria) were used to monitor the feline faecal microbiota. Overall, inter-animal variation was high; while a trend of increased bifidobacterial levels was seen with GOS supplementation it was not statistically significant in either healthy or IBD cats. No significant differences were observed in the faecal microbiota of IBD cats and healthy cats fed the same diet. Members of the family Coriobacteriaceae (Atopobium cluster) were found to be the most abundant bacteria in the feline microbiota.

Physiological and bifidogenic effects of prebiotic supplements in infant formulae

This randomized controlled trial involving 110 healthy neonates studied physiological and bifidogenic effects of galactooligosaccharides (GOS), oligofructose and long-chain inulin (FOS) in formula. Subjects were randomized to Orafti Synergy1 (50 oligofructose: 50 FOS) 0.4g/dl or 0.8g/dl, GOS:FOS (90:10) 0.8g/dl or a standard formula according to Good Clinical Practise (GCP) guidelines. A breast-fed group was included for comparison. Outcome parameters were weight, length, intake, stool characteristics, crying, regurgitation, vomiting, adverse events and fecal bacterial population counts. Statistical analyses used non-parametric tests. During the first month of life weight, length, intake and crying increased significantly in all groups. Regurgitation and vomiting scores were low and similar. Stool frequency decreased significantly and similarly in all formula groups but was lower than in the breast-fed. All prebiotic groups maintained soft stools, only slightly harder than those of breast-fed infants. The standard group had significantly harder stools at wks 2 and 4 compared to 1 (P<0.001 & P=0.0279). The total number of fecal bacteria increased in all prebiotic groups (9.82, 9.73 and 9.91 to 10.34, 10.38 and 10.37, respectively, log10 cells/g feces, P=0.2298) and resembled more the breast-fed pattern. Numbers of lactic acid bacteria, bacteroides and clostridia were comparable. In the SYN1 0.8 g/dl and GOS:FOS groups Bifidobacterium counts were significantly higher at D14 & 28 compared to D3 and comparable to the breast-fed group. Tolerance and growth were normal. In conclusion, stool consistency and bacterial composition of infants taking SYN1 0.8 g/dl or GOS:FOS supplemented formula was closer to the breast-fed pattern. There was no risk for dehydration.

Effect of prebiotics on the human gut microbiota of elderly persons

The colonic microbiota undergoes certain age related changes that may affect health. For example, above the age of 55–65 y, populations of bifidobacteria are known to decrease markedly. Bifidobacteria are known inhibitors of pathogenic microbes and a decrease in their activities may increase susceptibility to infections. There is therefore interest in trying to reverse their decline in aged persons. As the gut microbiota responds to dietary intervention, both probiotics and prebiotics have been tested in this regard. Probiotics are live microbes in the diet, whereas prebiotics are fermentable ingredients that specifically target components of the indigenous microbiota seen to be beneficial. We have published a recent paper demonstrating that prebiotic galactooligosaccharides can exert power effects upon bifidobacteria in the gut flora of elderly persons (both in vivo and in vitro). This addendum summarizes research that led up to this study and discusses the possible impact of prebiotics in impacting upon the gut health of aged persons.

Purified galactooligosaccharide, derived from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium adhesion and invasion in vitro and in vivo

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 411 71 using lactose as the substrate Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno (R) in reducing Salmonella enterica serovar Typhimurium (S Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS Here we wished to test the hypothesis that GOS, derived from Bimuno (R) may confer the direct anti-invasive and protective effects of Bimuno (R) In this study the efficacy of Bimuno (R), a basal solution of Bimuno (R) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno (R)] and purified GOS to reduce S Typhimurium adhesion and invasion was assessed using a series of in vitro and in vivo models The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics in vitro demonstrated that the presence of similar to 5 mg Bimuno (R) ml(-1) or similar to 2 5 mg GOS ml(-1) significantly reduced the invasion of S Typhimurium (SL1344nal(r)) (P<0 0001) Furthermore, similar to 2 5 mg GOS ml(-1) significantly reduced the adherence of S Typhimurium (SU 344nal(r)) (P<0 0001) It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions In the murine ligated deal gut loops, the presence of Bimuno (R) or GOS prevented the adherence or invasion of S Typhimurium to enterocytes, and thus reduced its associated pathology This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen In all assays, Bimuno (R) without GOS conferred no such protection, indicating that the basal solution confers no protective effects against S Typhimurium Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno (R) can be attributed to GOS

Biological significance of short-chain fatty acid metabolism by the intestinal microbiome

Purpose of review Evidence suggests that short-chain fatty acids (SCFAs) derived from microbial metabolism in the gut play a central role in host homeostasis. The present review describes the current understanding and physiological implications of SCFAs derived from microbial metabolism of nondigestible carbohydrates. Recent findings Recent studies indicate a role for SCFAs, in particular propionate and butyrate, in the metabolic and inflammatory disorders such as obesity, diabetes and inflammatory bowel diseases, through the activation of specific G-protein-coupled receptors and modification of transcription factors. Established prebiotics, such as fructooligosaccharides and galactooligosaccharides, which support the growth of Bifidobacteria, mainly mediate acetate production. Thus, recent identification of prebiotics which are able to stimulate the production of propionate and butyrate by benign saccharolytic populations in the colon is of interest. Summary Manipulation of saccharolytic fermentation by prebiotic substrates is beginning to provide information on structure–function relationships relating to the production of SCFAs, which have multiple roles in host homeostasis.

Effects of orange juice formulation on prebiotic functionality using an in vitro colonic model sytem

A three-stage continuous fermentative colonic model system was used to monitor in vitro the effect of different orange juice formulations on prebiotic activity. Three different juices with and without Bimuno, a GOS mixture containing galactooligosaccharides (B-GOS) were assessed in terms of their ability to induce a bifidogenic microbiota. The recipe development was based on incorporating 2.75g B-GOS into a 250 ml serving of juice (65°Brix of concentrate juice). Alongside the production of B-GOS juice, a control juice - orange juice without any additional Bimuno and a positive control juice, containing all the components of Bimuno (glucose, galactose and lactose) in the same relative proportions with the exception of B-GOS were developed. Ion Exchange Chromotography analysis was used to test the maintenance of bimuno components after the production process. Data showed that sterilisation had no significant effect on concentration of B-GOS and simple sugars. The three juice formulations were digested under conditions resembling the gastric and small intestinal environments. Main bacterial groups of the faecal microbiota were evaluated throughout the colonic model study using 16S rRNA-based fluorescence in situ hybridization (FISH). Potential effects of supplementation of the juices on microbial metabolism were studied measuring short chain fatty acids (SCFAs) using gas chromatography. Furthermore, B-GOS juices showed positive modulations of the microbiota composition and metabolic activity. In particular, numbers of faecal bifidobacteria and lactobacilli were significantly higher when B-GOS juice was fermented compared to controls. Furthermore, fermentation of B-GOS juice resulted in an increase in Roseburia subcluster and concomitantly increased butyrate production, which is of potential benefit to the host. In conclusion, this study has shown B-GOS within orange juice can have a beneficial effect on the fecal microbiota.

Effect of prebiotics on the fecal microbiota of elderly volunteers after dietary supplementation of Bacillus coagulans GBI-30, 6086

In advancing age, gut populations of beneficial microbes, notably Bifidobacterium spp., show a marked decline. This contributes to an environment less capable of maintaining homoeostasis. This in vitro investigation studied the possible synergistic effects of probiotic supplementation in modulating the gut microbiota enabling prebiotic therapy to in elderly persons. Single stage batch culture anaerobic fermenters were used and inoculated with fecal microbiota obtained from volunteers after taking a 28 day treatment of Bacillus coagulans GBI-30, 6086 (GanedenBC30 (BC30)) or a placebo. The response to prebiotic supplements fructooligosaccharides (FOS) and galactooligosaccharides (GOS) in the fermenters was assessed. Bacterial enumeration was carried out using fluorescent in situ hybridisation and organic acids measured by gas chromatography. Baseline populations of Faecalibacterium prausnitzii, Clostridium lituseburense and Bacillus spp. were significantly higher in those having consumed BC30 compared to the placebo. Both prebiotics increased populations of several purportedly beneficial bacterial groups in both sets of volunteers. Samples from volunteers having ingested the BC30 also increased populations of C. lituseburense, Eubacterium rectale and F. prausnitzii more so than in persons who had consumed the placebo, this also resulted in significantly higher concentrations of butyrate, acetate and propionate. This shows that consumption of BC30 and subsequent use of prebiotics resulted in elevated populations of beneficial genres of bacteria as well as organic acid production