George Tzortzis

Clinical trial: the effects of a trans‐galactooligosaccharide prebiotic on faecal microbiota and symptoms in irritable bowel syndrome

Background  Gut microflora‐mucosal interactions may be involved in the pathogenesis of irritable bowel syndrome (IBS).

Microencapsulation of probiotics for gastrointestinal delivery

The administration of probiotic bacteria as nutraceuticals is an area that has rapidly expanded in recent years, with a global market worth

A Mixture of trans-Galactooligosaccharides Reduces Markers of Metabolic Syndrome and Modulates the Fecal Microbiota and Immune Function of Overweight Adults

32.6 billion predicted by 2014. Many of the health promoting claims attributed to these bacteria are dependent on the cells being both viable and sufficiently numerous in the intestinal tract. The oral administration of most bacteria results in a large loss of viability associated with passage through the stomach, which is attributed to the high acid and bile salt concentrations present. This loss of viability effectively lowers the efficacy of the administered supplement. The formulation of these probiotics into microcapsules is an emerging method to reduce cell death during GI passage, as well as an opportunity to control release of these cells across the intestinal tract. The majority of this technology is based on the immobilization of bacteria into a polymer matrix, which retains its structure in the stomach before degrading and dissolving in the intestine, unlike the diffusion based unloading of most controlled release devices for small molecules. This review shall provide an overview of progress in this field as well as draw attention to areas where studies have fallen short. This will be followed by a discussion of emerging trends in the field, highlighting key areas in which further research is necessary.

Production and evaluation of dry alginate-chitosan microcapsules as an enteric delivery vehicle for probiotic bacteria.

Metabolic syndrome is a set of disorders that increases the risk of developing cardiovascular disease. The gut microbiota is altered toward a less beneficial composition in overweight adults and this change can be accompanied by inflammation. Prebiotics such as galactooligosaccharides can positively modify the gut microbiota and immune system; some may also reduce blood lipids. We assessed the effect of a galactooligosaccharide mixture [Bi2muno (B-GOS)] on markers of metabolic syndrome, gut microbiota, and immune function in 45 overweight adults with ≥3 risk factors associated with metabolic syndrome in a double-blind, randomized, placebo (maltodextrin)-controlled, crossover study (with a 4-wk wash-out period between interventions). Whole blood, saliva, feces, and anthropometric measurements were taken at the beginning, wk 6, and end of each 12-wk intervention period. Predominant groups of fecal bacteria were quantified and full blood count, markers of inflammation and lipid metabolism, insulin, and glucose were measured. B-GOS increased the number of fecal bifidobacteria at the expense of less desirable groups of bacteria. Increases in fecal secretory IgA and decreases in fecal calprotectin, plasma C-reactive protein, insulin, total cholesterol (TC), TG, and the TC:HDL cholesterol ratio were also observed. Administration of B-GOS to overweight adults resulted in positive effects on the composition of the gut microbiota, the immune response, and insulin, TC, and TG concentrations. B-GOS may be a useful candidate for the enhancement of gastrointestinal health, immune function, and the reduction of metabolic syndrome risk factors in overweight adults.

A mixture containing galactooligosaccharide, produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium infection in mice

This study investigates the production of alginate microcapsules, which have been coated with the polysaccharide chitosan, and evaluates some of their properties with the intention of improving the gastrointestinal viability of a probiotic ( Bifidobacterium breve ) by encapsulation in this system. The microcapsules were dried by a variety of methods, and the most suitable was chosen. The work described in this Article is the first report detailing the effects of drying on the properties of these microcapsules and the viability of the bacteria within relative to wet microcapsules. The pH range over which chitosan and alginate form polyelectrolyte complexes was explored by spectrophotometry, and this extended into swelling studies on the microcapsules over a range of pHs associated with the gastrointestinal tract. It was shown that chitosan stabilizes the alginate microcapsules at pHs above 3, extending the stability of the capsules under these conditions. The effect of chitosan exposure time on the coating thickness was investigated for the first time by confocal laser scanning microscopy, and its penetration into the alginate matrix was shown to be particularly slow. Coating with chitosan was found to increase the survival of B. breve in simulated gastric fluid as well as prolong its release upon exposure to intestinal pH.

Influence of galacto-oligosaccharide mixture (B-GOS) on gut microbiota, immune parameters and metabonomics in elderly persons

The prebiotic Bimuno is a mixture containing galactooligosaccharide, produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41171 in the presence of lactose. Previous studies have implicated prebiotics in reducing infections by enteric pathogens, thus it was hypothesized that Bimuno may confer some protection in the murine host from Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. In this study, infection caused by S. Typhimurium SL1344nal(r) in the presence or absence of Bimuno was assessed using tissue culture assays, a murine ligated ileal gut loop model and a murine oral challenge model. In tissue culture adherence and invasion assays with HT-29-16E cells, the presence of approximately 2 mM Bimuno significantly reduced the invasion of S. Typhimurium SL1344nal(r) (P<0.0001). In the murine ligated ileal gut loops, the presence of Bimuno prevented colonization and the associated pathology of S. Typhimurium. In the BALB/c mouse model, the oral delivery of Bimuno prior to challenge with S. Typhimurium resulted in significant reductions in colonization in the five organs sampled, with highly significant reductions being observed in the spleen at 72 and 96 h post-challenge (P=0.0002, <0.0001, respectively). Collectively, the results indicate that Bimuno significantly reduced the colonization and pathology associated with S. Typhimurium infection in a murine model system, possibly by reducing the invasion of the pathogen into host cells.

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.

It is recognised that ageing induces various changes to the human colonic microbiota. Most relevant is a reduction in bifidobacteria, which is a health-positive genus. Prebiotics, such as galacto-oligosaccharides (GOS), are dietary ingredients that selectively fortify beneficial gut microbial groups. Therefore, they have the potential to reverse the age-related decline in bifidobacteria and modulate associated health parameters. We assessed the effect of GOS mixture (Bimuno (B-GOS)) on gut microbiota, markers of immune function and metabolites in forty elderly (age 65-80 years) volunteers in a randomised, double-blind, placebo (maltodextrin)-controlled, cross-over study. The intervention periods consisted of 10 weeks with daily doses of 5·5 g/d with a 4-week washout period in between. Blood and faecal samples were collected for the analyses of faecal bacterial populations and immune and metabolic biomarkers. B-GOS consumption led to significant increases in bacteroides and bifidobacteria, the latter correlating with increased lactic acid in faecal waters. Higher IL-10, IL-8, natural killer cell activity and C-reactive protein and lower IL-1β were also observed. Administration of B-GOS to elderly volunteers may be useful in positively affecting the microbiota and some markers of immune function associated with ageing.

Layer-by-layer coating of alginate matrices with chitosan-alginate for the improved survival and targeted delivery of probiotic bacteria after oral administration

The prebiotic Bimuno(®) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41171 using lactose as the substrate. Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno(®) 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(®), may confer the direct anti-invasive and protective effects of Bimuno(®). In this study the efficacy of Bimuno(®), a basal solution of Bimuno(®) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno(®)] 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 ∼ 5 mg Bimuno(®) ml(-1) or ∼ 2.5 mg GOS ml(-1) significantly reduced the invasion of S. Typhimurium (SL1344nal(r)) (P<0.0001). Furthermore, ∼ 2.5 mg GOS ml(-1) significantly reduced the adherence of S. Typhimurium (SL1344nal(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 ileal gut loops, the presence of Bimuno(®) 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(®) 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(®) can be attributed to GOS.

Molecular cloning and comparative analysis of four β-galactosidase genes from Bifidobacterium bifidum NCIMB41171

The oral administration of probiotic bacteria has shown potential in clinical trials for the alleviation of specific disorders of the gastrointestinal tract. However, cells must be alive in order to exert these benefits. The low pH of the stomach can greatly reduce the number of viable microorganisms that reach the intestine, thereby reducing the efficacy of the administration. Herein, a model probiotic, Bifidobacterium breve, has been encapsulated into an alginate matrix before coating in multilayers of alternating alginate and chitosan. The intention of this formulation was to improve the survival of B. breve during exposure to low pH and to target the delivery of the cells to the intestine. The material properties were first characterized before in vitro testing. Biacore™ experiments allowed for the polymer interactions to be confirmed; additionally, the stability of these multilayers to buffers simulating the pH of the gastrointestinal tract was demonstrated. Texture analysis was used to monitor changes in the gel strength during preparation, showing a weakening of the matrices during coating as a result of calcium ion sequestration. The build-up of multilayers was confirmed by confocal laser-scanning microscopy, which also showed the increase in the thickness of coat over time. During exposure to in vitro gastric conditions, an increase in viability from <3 log(CFU) per mL, seen in free cells, up to a maximum of 8.84 ± 0.17 log(CFU) per mL was noted in a 3-layer coated matrix. Multilayer-coated alginate matrices also showed a targeting of delivery to the intestine, with a gradual release of their loads over 240 min.

A comprehensive investigation of the synthesis of prebiotic galactooligosaccharides by whole cells of Bifidobacterium bifidum NCIMB 41171

Bifidobacterium bifidum NCIMB41171 carries four genes encoding different β-galactosidases. One of them, named bbgIII, consisted of an open reading frame of 1,935 amino acid (a.a.) residues encoding a protein with a multidomain structure, commonly identified on cell wall bound enzymes, having a signal peptide, a membrane anchor, FIVAR domains, immunoglobulin Ig-like and discoidin-like domains. The other three genes, termed bbgI, bbgII and bbgIV, encoded proteins of 1,291, 689 and 1,052 a.a. residues, respectively, which were most probably intracellularly located. Two cases of protein evolution between strains of the same species were identified when the a.a. sequences of the BbgI and BbgIII were compared with homologous proteins from B. bifidum DSM20215. The homologous proteins were found to be differentiated at the C-terminal a.a. part either due to a single nucleotide insertion or to a whole DNA sequence insertion, respectively. The bbgIV gene was located in a gene organisation surrounded by divergently transcribed genes putatively for sugar transport (galactoside-symporter) and gene regulation (LacI-transcriptional regulator), a structure that was found to be highly conserved in B. longum, B. adolescentis and B. infantis, suggesting optimal organisation shared amongst those species.