Prebiotics are nondigestible food ingredients that encourage proliferation of selected groups of the colonic microflora, thereby altering the composition toward a more beneficial community. In the present study, the prebiotic potential of a novel galactooligosaccharide (GOS) mixture, produced by the activity of galactosyltransferases from Bifidobacterium bifidum 41171 on lactose, was assessed in vitro and in a parallel continuous randomized pig trial. In situ fluorescent hybridization with 16S rRNA-targeted probes was used to investigate changes in total bacteria, bifidobacteria, lactobacilli, bacteroides, and Clostridium histolyticum group in response to supplementing the novel GOS mixture. In a 3-stage continuous culture system, the bifidobacterial numbers for the first 2 vessels, which represented the proximal and traverse colon, increased (P < 0.05) after the addition of the oligosaccharide mixture. In addition, the oligosaccharide mixture strongly inhibited the attachment of enterohepatic Escherichia coli (P < 0.01) and Salmonella enterica serotype Typhimurium (P < 0.01) to HT29 cells. Addition of the novel mixture at 4% (wt:wt) to a commercial diet increased the density of bifidobacteria (P < 0.001) and the acetate concentration (P < 0.001), and decreased the pH (P < 0.001) compared with the control diet and the control diet supplemented with inulin, suggesting a great prebiotic potential for the novel oligosaccharide mixture.

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A novel strain of Bifidobacterium bifidum NCIMB 41171, isolated from a faecal sample from a healthy human volunteer and able to express beta-galactosidase activity, was used in synthesis reactions for the production of galactooligosaccharide from lactose. The beta-galactosidase activity of whole bifidobacterial cells showed an optimum activity at pH 6.8-7.0 and 40 degrees C. The transgalactosylation activity of the B. bifidum cells from 50% (w/w) lactose resulted in a galactooligosaccharide mixture (20% w/w) comprising (w/w): 25% disaccharides, 35% trisaccharides, 25% tetrasaccharides and 15% pentasaccharides. Using different initial lactose concentrations, the conversion rate to galactooligosaccharides was maximum (35%) when 55% (w/w) lactose was used. In fermentation experiments, B. bifidum showed an increased preference towards the produced galactooligosaccharide mixture, displaying higher growth rate and short-chain fatty acid production when compared with commercially available oligosaccharides.

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Four different beta-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4-5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4-6.8). Na(+) and/or K(+) ions were prerequisite for BbgI and BbgIV activity in Bis-Tris-buffered solutions, whereas Mg(++) was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg(++), Mn(++) and Ca(++) ions exerting the most positive effect. Determination of the specificity constants (k(cat)/K(m)) clearly indicated that BbgI (6.11 x 10(4) s(-1) M(-1)), BbgIII (2.36 x 10(4) s(-1) M(-1)) and especially BbgIV (4.01 x 10(5) s(-1) M(-1)) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for beta-D-(1-->6) galactobiose (5.59 x 10(4) s(-1) M(-1)) than lactose (1.48 x 10(3) s(-1) M(-1)). Activity measurements towards other substrates (e.g. beta-D-(1-->6) galactobiose, beta-D-(1-->4) galactobiose, beta-D-(1-->4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the beta-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.

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 40 bar 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). INTRODUCTIONThe food industry has recently shown increased interest in oligosaccharides as functional food ingredients owing to their proposed beneficial effects on human health. 1 Oligosaccharides are carbohydrates consisting of 2-10 linked monomer sugars and either occur naturally or are produced chemically or by enzymatic transglycosylation or controlled degradation reactions. 2 Oligosaccharides are increasingly included in foods for their prebiotic properties. A prebiotic is defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or more of a limited number of bacteria in the colon (ie Bifidobacterium spp and Lactobacillus spp). 2 Certain oligosaccharides have also been reported to act as soluble dietary fibres and as anticarcinogenic agents. 2 Food-grade oligosaccharides are mixtures containing oligosaccharides of different degrees of polymerisation and monosaccharides. Not all of these sugars possess prebiotic activity and it would be useful to be able to separate the active oligosaccharides from the contaminants. This is particularly important when the materials are to be tested using in vitro models of the human gut, as these models test the fermentation of all carbohydrate material present, whereas in the human situation some components would not reach the colon.

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.

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