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.

Metabolism of the soyabean isoflavone glycoside genistin in vitro by human gut bacteria and the effect of prebiotics

The isoflavone genistein is found predominantly in soyabeans and is thought to possess various potent biological properties, including anticarcinogenic effects. Studies have shown that genistein is extensively degraded by the human gut microflora, presumably with a loss of its anti-carcinogenic action. The aim of the present study was to investigate the potential of a prebiotic to divert bacterial metabolism away from genistein breakdown: this may be of benefit to the host. Faecal samples were obtained from healthy volunteers and fermented in the presence of a source of soyabean isoflavones (Novasoy(TM) (10 g/l); ADM Neutraceuticals, Erith, Kent, UK). Bacterial genera of the human gut were enumerated using selective agars and genistein was quantified by HPLC. The experiment was repeated with the addition of glucose (10 g/l) or fructo-oligosaccharide (10 g/l; FOS) to the fermentation medium. The results showed most notably that counts of Bifidobacterium spp. and Lactobacillus spp. were significantly increased (P<0.05 and P<0.01 respectively) under steady-state conditions in the presence of FOS. Counts of Bacteroides spp. and Clostridium spp. were, however, both significantly reduced (P<0.05) during the fermentation. A decline in genistein concentration by about 52 and 56% over the 120h culture period was observed with the addition of glucose or FOS to the basal medium (P<0.01), compared with about 91% loss of genistein in the vessels containing Novasoy(TM) (ADM Neutraceuticals) only. Similar trends were obtained using a three-stage chemostat (gut model), in which once again the degradation of genistein was about 22% in vessel one, about 24% in vessel two and about 26% in vessel three in the presence of FOS, compared with a degradation of genistein of about 67% in vessel one, about 95% in vessel two and about 93% in vessel three in the gut model containing Novasoy(TM) (ADM Neutraceuticals) only. The present study has shown that the addition of excess substrate appeared to preserve genistein in vitro. In particular, the use of FOS not only augmented this effect, but also conferred an additional benefit in selectively increasing numbers of purportedly beneficial bacteria such as bifidobacteria and lactobacilli.

Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC50 value of 5.521 mg/mL is high compared with 0.2811 mu g/mL (182.5 pM or 1.825 x 10(-10) M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E (R) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%. 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease. (C) 2009 Elsevier Ltd. All rights reserved

In vitro investigation into the potential prebiotic activity of honey oligosaccharides

The effect of honey oligosaccharides on the growth of fecal bacteria was studied using an in vitro fermentation system. Prior to treatment, glucose and fructose (31.73 and 21.41 g/100 g of product, respectively) present in honey, which would be digested in the upper gut, were removed to avoid any influence on bacterial populations in the fermentations. Nanofiltration, yeast (Saccharomyces cerevisiae) treatment, and adsorption onto activated charcoal were used to remove monosaccharides. Prebiotic (microbial fermentation) activities of the three honey oligosaccharide fractions and the honey sample were studied and compared with fructooligosaccharide (FOS), using 1% (w/v) fecal bacteria in an in vitro fermentation system (10 mg of carbohydrate, 1.0 mL of basal medium). A prebiotic index (PI) was calculated for each carbohydrate source. Honey oligosaccharides seem to present potential prebiotic activity (PI values between 3.38 and 4.24), increasing the populations of bifidobacteria and lactobacilli, although not to the levels of FOS (PI of 6.89).

Carbohydrate preferences of Bifidobacterium species isolated from the human gut

The growth of nine species of Bifidobacterium on media containing glucose, xylose, xylooligosaccharides (XOS), xylan or fructooligosaccharides (FOS) as the sole carbon source were compared in pure culture. The bifidobacteria differed in fermentation profiles when tested on different carbohydrates. All species grew to their highest final optical density (OD) on a glucose containing medium, with the exception of B. catenulatum which demonstrated a preference for xylose over glucose, and XOS over FOS. B. bifidum grew to the highest OD on XOS compared to xylose suggesting a specific transport system for the oligosaccharide over the monomer. This is consistent with a lack of β-xylosidase activity present in the culture medium. Lactate, formate and acetate levels were determined and the ratios of these metabolites altered between and within species growing on different carbohydrates. In general, high lactate production correlated with low formate production and low lactate concentrations were obtained at higher levels of formate. Bifidobacteria may alter their metabolic pathways based upon the carbohydrates that are available for their use.

Pectins and pectic-oligosaccharides inhibit Escherichia coli O157 : H7 Shiga toxin as directed towards the human colonic cell line HT29

Pectins and pectic-oligosaccharides, as derived by controlled enzymatic hydrolysis, were evaluated for their ability to interfere with the toxicity of Shiga-like toxins from Escherichia coli O157:H7. Both types of material resulted in some degree of protection but this was significantly higher (P > 0.01) with the oligosaccharide fractions (giving 90-100% cell survival, compared to 70-80% with the polymer). An effect of methylation on the protective effect was detected with lower degrees being more active. The pectic-oligosaccharides and galabiose, the minimum toxin receptor analogue, were shown to inhibit toxicity and were both protective at 10 mg ml(-1), but not at lower concentrations. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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.

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using alpha-mannosidases from Penicillium citrinum, Aspergillus phoenicis and almond

Recombinant Penicillium citrinum alpha-1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of alpha-D-mannopyranosyl-(1-->2)-D-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with alpha1-->2 linkages present at 98.5% of the total linkages formed. Non-specific alpha-mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45-50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 1-->2, 1-->3 and 1-->6 isomers. alpha-1,2-linkage specific mannosidase from P. citrinum and alpha-1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in alpha-D-mannopyranosyl-(1-->3)-D-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as 'decoys' of receptors of type-1 fimbriae carried by enterobacteria.

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.

The use of dead-end and cross-flow nanofiltration to purify prebiotic oligosaccharides from reaction mixtures

Nanofiltration (NF) of model sugar solutions and commercial oligosaccharide mixtures were studied in both dead-end and cross-flow modes. Preliminary trials, with a dead-end filtration cell, demonstrated the feasibility of fractionating monosaccharides from disaccharides and oligosaccharides in mixtures, using loose nanofiltration (NF-CA-50, NF-TFC-50) membranes. During the nanofiltration purification of a commercial oligosaccharide mixture, yields of 19% (w w-1) for the monosaccharides and 88% (w w-1) for di, and oligosaccharides were obtained for the NF-TFC-50 membrane after four filtration steps, indicating that removal of the monosaccharides is possible, with only minor losses of the oligosaccharide content of the mixture. The effects of pressure, feed concentration, and filtration temperature were studied in similar experiments carried out in a cross-flow system, in full recycle mode of operation. The rejection rates of the sugar components increased with increasing pressure, and decreased with both increasing total sugar concentration in the feed and increasing temperature. Continuous diafiltration (CD) purification of model sugar solutions and commercial oligosaccharide mixtures using NF-CA-50 (at 25oC) and DS-5-DL (at 60oC) membranes, gave yield values of 14 to 18% for the monosaccharide, 59 to 89% for the disaccharide and 81 to 98% for the trisaccharide present in the feed. The study clearly demonstrates the potential of cross flow nanofiltration in the purification of oligosaccharide mixtures from the contaminant monosaccharides.

Fractionation of oligosaccharides by nanofiltration

Two loose nanofiltration membranes (NF-CA-50 and NF-TFC-50) and one dense ultrafiltration membrane (UF-CA-1) were used to fractionate commercial oligosaccharide mixtures by applying diafiltration in a 'dead-end' filtration cell at 40bar constant pressure with a maximum volume concentration ratio (VCR) of 6 at each fractionation. The rejections of a monosaccharide (glucose) and a disaccharide (lactose) were determined for each membrane; the results indicated that fractionation between these two sugars was possible using the two nanofiltration membranes. During the nanofiltration purification of a commercial oligosaccharide mixture, yields of 19% (w/w) for monosaccharides and 88% (w/w) for di- and oligosaccharides were obtained with the NF-TFC-50 membrane after four filtration steps, indicating that removal of the monosaccharides is possible with only minor losses of the oligosaccharide content of the mixture. The ultrafiltration membrane, at the same time, gave purification levels similar to the NF-TFC-50 membrane with fewer diafiltration steps but with higher losses of di- and oligosaccharides (12% (w/w) for monosaccharides and 53% (w/w) for di- and oligosaccharides on the third run). (C) 2003 Society of Chemical Industry.

Synthesis of isomaltooligosaccharides and oligodextrans by the combined use of dextransucrase and dextranase

Batch syntheses of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase were performed with the aim of understanding the reaction mechanism and the parameters which affect product characteristics and molecular size. Both activities described for dextransucrase (dextran formation and acceptor reaction) achieved synthesis whilst the hydrolytic activity of dextranase regulated the product molecular size and acceptor availability. Depending on the reaction conditions, the product oligosaccharide mixtures contained mainly sugars (up to 36%) with degrees of polymerization (DP) varying between 10 and 60 together with lower concentrations of both lower and higher molecular weight sugars. Alterations in substrate and dextranase concentrations (50-400 mg ml(-1) and 2.5-46 U ml(-1), respectively) affected the molecular weight of IMO, the reaction rate and the formation of leucrose. This permitted manipulation of the product characteristics. It was found that higher substrate and dextranase concentrations gave rise to products with lower molecular sizes and a dextransucrase:dextranase ratio of 1: 1 or 1:2 appeared to produce a polymer with a molecular weight which is desirable for prebiotic use. (C) 2004 Elsevier Inc. All rights reserved.

Development of a process for the production and purification of alpha- and beta-galactooligosaccharides from Bifobacterium bifidum NCIMB 41171

Synthesis of prebiotic alpha- and beta-galactooligosaccharides (GOS) using the whole cells of Bifidobacterium bifidum NCIMB 41171 was investigated. Determination of alpha- and beta-galactosidase activities showed them to be at 3 and 205 g(-1) of freeze dried biomass, respectively, and they increased to 5 and 344 U g(-1), respectively, when cells were treated with toluene. Starting with 450-500 mg mL(-1) lactose, maximum GOS concentrations were observed at 80-85% lactose conversions and the mixtures contained oligosaccharides (with a degree of polymerisation >= 3) at 77-109 mg mL(-1) and trans-galactosylated disaccharides between 85-115 mg mL(-1). The GOS yield values varied between 36% and 43%. An alpha-linked disaccharide was detected and its presence was confirmed by gas chromatography mass spectroscopy. Cells were re-used up to 8 times without changes in reaction times or the substrate conversions to GOS. Oligosaccharide synthesis was not inhibited by the presence of glucose or galactose. The mixtures were successfully purified from glucose (92% of glucose removed) by fermentation with Saccharomyces cerevisiae with no losses in the oligosaccharide content and only a small decrease on the galactose. (c) 2006 Elsevier Ltd. All rights reserved.

Prebiotics and resistance to gastrointestinal infections

Acute gut disorder is a cause for significant medicinal and economic concern. Certain individual pathogens of the gut, often transmitted in food or water, have the ability to cause severe discomfort. There is a need to manage such conditions more effectively. The route of reducing the risk of intestinal infections through diet remains largely unexplored. Antibiotics are effective at inhibiting pathogens; however, these should not be prescribed in the absence of disease and therefore cannot be used prophylactically. Moreover, their indiscriminate use has reduced effectiveness. Evidence has accumulated to suggest that some of the health-promoting bacteria in the gut (probiotics) can elicit a multiplicity of inhibitory effects against pathogens. Hence, an increase in their numbers should prove effective at repressing pathogen colonisation if/when infectious agents enter the gut. As such, fortification of indigenous bifidobacteria/lactobacilli by using prebiotics should improve protection. There are a number of potential mechanisms for lactic acid bacteria to reduce intestinal infections. Firstly, metabolic endproducts such as acids excreted by these micro-organisms may lower the gut pH to levels below those at which pathogens are able to effectively compete. Also, many lactobacilli and bifidobacteria species are able to excrete natural antibiotics, which can have a broad spectrum of activity. Other mechanisms include an improved immune stimulation, competition for nutrients and blocking of pathogen adhesion sites in the gut. Many intestinal pathogens like type 1 fimbriated Escherichia coli, salmonellae and campylobacters utilise oligosaccharide receptor sites in the gut. Once established, they can then cause gastroenteritis through invasive and/or toxin forming properties. One extrapolation of the prebiotic concept is to simulate such receptor sites in the gut lumen. Hence, the pathogen is 'decoyed' into not binding at the host mucosal interface. The combined effects of prebiotics upon the lactic acid flora and anti-adhesive strategies may lead towards new dietary interventions against food safety agents.