Mutagenesis of asp-569 of glucosyltransferase I glucansucrase modulates glucan and oligosaccharide synthesis

Glucansucrases of oral streptococci and Leuconostoc mesenteroides are enzymes of medical and biotechnological interest that synthesize alpha-glucans. They can also synthesize oligosaccharides in the presence of a sugar acceptor. Previous reports have identified an amino acid residue that may affect the structure of the glucan product; therefore, random mutagenesis of the corresponding Asp-569 of Streptococcus downei glucosyltransferase I (GTF-I) was used to further understanding of its involvement in the catalytic mechanism and to evaluate how different amino acids can modulate glucan and oligosaccharide synthesis. GTF-I variants were obtained where Asp-569 was replaced by each of the different possible classes of amino acids. These were expressed in Escherichia coli and purified by means of a His(6) tag. The results showed that the amino acid in position 569 influences the structure of the glucan and the size of the oligosaccharides produced by GTF-I. The results suggest that the amino acid occupying this position is more likely to interact with the acceptor molecules (oligosaccharides or elongating glucan chain) than to be directly involved in glucosyl transfer from sucrose. Engineering of the equivalent position in glucansucrases thus appears to be a good target to expand the range of oligosaccharides synthesized.     

Bifidogenic effect and in vitro immunomodulatory roles of melibiose-derived oligosaccharides produced by the acceptor reaction of glucansucrase E81

Glucansucrases are responsible for the production of α-glucans using sucrose as the substrate. Glucansucrases may also produce different oligosaccharides by transferring the glucose moiety from sucrose to a variety carbohydrates acting as acceptor nucleophile. In this study, melibiose-derived oligosaccharides were produced by the glucansucrase from Lactobacillus reuteri E81 expressed without the N-terminal region (GtfA-ΔN). The reaction products were characterized by TLC, LC-MS and NMR analysis and it was found that GtfA-ΔN synthesized melibiose-derived oligosaccharides (DP3 and DP4) by adding glucose units through alpha 1->3 or 1->6 glycosidic bond. The functional characteristics of these melibiose-derived oligosaccharides were determined by testing the immune-modulatory functions in HT-29 cells and testing their growth promoting effects for important probiotic and pathogenic strains. The melibiose-derived oligosaccharides triggered the production of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokine TNF-α depending on their concentrations. Finally, melibiose-derived oligosaccharides showed bifidogenic effect as potential prebiotics.     

Biochemical and crystallographic characterization of a glucansucrase from Lactobacillus reuteri 180

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass α-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no three-dimensional structure has been reported for any. With the aim of solving the first structure of a GH70 glucansucrase, purification and crystallization experiments were performed with a fully active, 117 kDa N-terminally truncated fragment of glucansucrase GTF180 from Lactobacillus reuteri 180 (residues 742–1772). Crystallization experiments yielded crystals that belong to two different triclinic crystal forms (space group P1) and one orthorhombic crystal form (space group P2₁2₁2₁). Native data sets for both triclinic and the orthorhombic crystals were collected at 1.7 and 2.0 Å resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.     

Characterization of a glucansucrase from Lactobacillus reuteri E81 and production of malto-oligosaccharides

Abstract

Glucansucrases, which can be produced by different Lactic Acid Bacteria (LAB), catalyze the synthesis of α-glucans with different structures and properties using sucrose as substrate. In this study, a novel glucansucrase (GTFA) from Lactobacillus reuteri E81 was identified and heterologously expressed. Alignments of GTFA with other glucansucrases revealed its novelty and a putative 3D model structure was obtained. The biochemical properties of the truncated enzyme without the N-terminal variable region, GTFA-ΔN, was characterized. The K_m and V_max were found to be 7.5?mM and 1.49?IU/mg, respectively, and it showed optimum activities at pH 7 and at 50?°C. The GTFA-ΔN produced in vitro an α-glucan with (α1 → 3) and (α1 → 6) glycosidic linkages using sucrose as the substrate. Importantly, GTFA-ΔN synthesized DP = 9 oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively, as detected by TLC, HPLC, LC-MS and NMR analysis.     

Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose

Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical properties are produced, which have found diverse (potential) commercial applications, e.g. in food, health and as biomaterials. Originally, the GH70 family was established only for glucansucrase enzymes of lactic acid bacteria that catalyze the synthesis of α-glucan polymers from sucrose. In recent years, we have identified 3 novel subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD), inactive on sucrose but converting starch/maltodextrin substrates into novel α-glucans. These novel starch-acting enzymes considerably enlarge the panel of α-glucans that can be produced. They also represent very interesting evolutionary intermediates between sucrose-acting GH70 glucansucrases and starch-acting GH13 α-amylases. Here we provide an overview of the repertoire of GH70 enzymes currently available with focus on these novel starch-acting GH70 enzymes and their biotechnological potential. Moreover, we discuss key developments in the understanding of structure-function relationships of GH70 enzymes in the light of available three-dimensional structures, and the protein engineering strategies that were recently applied to expand their natural product specificities.     

Molecular characterization and expression analysis of the glucansucrase DSRWC from Weissella cibaria synthesizing a α(1→6) glucan

Weissella cibaria isolated from human saliva produces a soluble glucan that predominantly has α-1,6-glucosidic type linkages. Using degenerated primers that were selected based on the amino acid sequences of conserved regions from known glucansucrases, a single 2.7-kb fragment was isolated. In subsequent steps, a 4969-bp product was obtained using inverse PCR. The coding region for the glucansucrase gene ( dsrWC ) consisted of a 4419-bp ORF that encoded a 1472-amino acid protein with a calculated molecular mass of 161.998 Da. The produced DSRWC glucansucrases exhibited similarity with the enzymes of the glucosylhydrolase family 70, which includes the Lactobacillus fermentum glucansucrase. The expressed recombinant DSRWC (rDSRWC) synthesized oligosaccharides in the presence of maltose or isomaltose as an acceptor and the synthesized products included α-1,6-linked glucosyl residues in addition to the maltosyl or isomaltosyl residue. rDSRWC synthesized water-soluble polymers using sucrose as substrate. According to the ¹³C-nuclear magnetic resonance analysis, the polymer that was synthesized by rDSRWC was a linear dextran, which formed predominately α-1,6-glucosidic linkages. This is the first report on the molecular characterization of glucansucrase from a W. cibaria strain.     

Screening and Application of Microbial Esterases for the Enantioselective Synthesis of Chiral Glycerol Derivatives

Glucansucrases from family 70 of glycoside-hydrolases catalyse the synthesis of α-glucans with various types of osidic linkages from sucrose. Among these enzymes, alternansucrase (ASR) and dextransucrase E (DSR-E) catalyse the formation of unusual α-glucans. ASR catalyses the synthesis of linear glucan with α-1,3 and α-1,6 alternating linkages and DSR-E synthesizes a glucan containing α-1,6 linkages in the linear chain and α-1,2 branches. The sequence analysis of these enzymes enabled the identification of structural elements suspected to be involved in the enzyme specificities. Biochemical characterization of ASR and DSR-E variants obtained from gene truncations or site-directed mutagenesis experiments showed that the specificity of these enzymes to form different types of osidic linkage is controlled by two different approaches. For ASR, the double specificity is controlled by only one catalytic domain where important amino acids involved in the enzyme specificity have been identified. In the case of DSR-E, the double specificity is controlled by two different catalytic domains both belonging to family 70, each domain being specific of one type of linkage.     

Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu⁹⁴⁰ in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu⁹⁴⁰ of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67–100% of (α1→6) linkages) were produced by GTF180 and its Leu⁹⁴⁰ mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu⁹⁴⁰ in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.     

Enzymatic synthesis and anti-coagulant effect of salicin analogs by using the Leuconostoc mesenteroides glucansucrase acceptor reaction

Glucansucrases from Leuconostoc mesenteroides catalyze the transfer of glucosyl units from sucrose to other carbohydrates by acceptor reaction. We modified salicyl alcohol, phenol and salicin by using various glucansucrases and with sucrose as a donor of glucosyl residues. Salicin, phenyl glucose, isosalicin, isomaltosyl salicyl alcohol, and a homologous series of oligosaccharides, connected to the acceptors and differing from one another by one or more glucose residues, were produced as major reaction products. By using salicin and salicyl alcohol as acceptors, B-1355C2 and B-1299CB-BF563 dextransucrases synthesized most widely diverse products, producing more than 12 and 9 different kinds of saccharides, respectively. With phenol, two acceptor products and oligosaccharides were synthesized by using the B-1299CB-BF563 dextransucrase. Salicyl derivatives, as acceptor products, showed higher anti-coagulation activity compared with that of salicin or salicyl alcohol that were used as acceptors.     

Glycosidic bond specificity of glucansucrases: on the role of acceptor substrate binding residues

Many lactic acid bacteria produce extracellular α-glucan polysaccharides using a glucansucrase and sucrose as glucose donor. The structure and the physicochemical properties of the α-glucans produced are determined by the nature of the glucansucrase. Typically, the α-glucans contain two types of α-glycosidic linkages, for example, (α1-2), (α1-3), (α1-4) or (α1-6), which may be randomly or regularly distributed. Usually, the α-glucan chains are also branched, which gives rise to an additional level of complexity. Even though the first crystal structure was reported in 2010, our current understanding of the structure–function relationships of glucansucrases is not advanced enough to predict the α-glucan specificity from the sequence alone. Nevertheless, based on sequence alignments and site-directed mutagenesis, a few amino acid residues have been identified as being important for the glycosidic bond specificity of glucansucrases. A new development in GH70 research was the identification of a cluster of α-glucan disproportionating enzymes. Here, we discuss the current insights into the structure–function relationships of GH70 enzymes in the light of the recently determined crystal structure of glucansucrases.     

Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity: toward tailor-made polymer and oligosaccharide products

Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly alpha-1,6-glucosidic linkages to a mutan type polymer with predominantly alpha-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency.     

Enzymatic synthesis of l-DOPA α-glycosides by reaction with sucrose catalyzed by four different glucansucrases from four strains of Leuconostoc mesenteroides

l-DOPA α-glycosides were synthesized by reaction of l-DOPA with sucrose, catalyzed by four different glucansucrases from Leuconostoc mesenteroides B-512FMC, B-742CB, B-1299A, and B-1355C. The glucansucrases catalyzed the transfer of d-glucose from sucrose to the phenolic hydroxyl position-3 and -4 of l-DOPA. The glycosides were fractionated and purified by Bio-Gel P-2 column chromatography, and the structures were determined by ¹H NMR spectroscopy. The major glycoside was 4-O-α-d-glucopyranosyl l-DOPA, and the minor glycoside was 3-O-α-d-glucopyranosyl l-DOPA. The two glycosides were formed by all four of the glucansucrases. The ratio of the 4-O-α-glycoside to the 3-O-α-glycoside produced by the B-512FMC dextransucrase was higher than that for the other three glucansucrases. The glycosylation of l-DOPA significantly reduced the oxidation of the phenolic hydroxyl groups, which prevents their methylation, potentially increasing the use of l-DOPA in the treatment of Parkinson’s disease. The use of one enzyme, glucansucrase, and sucrose as the d-glucosyl donor makes the synthesis considerably simpler and cheaper than the formerly published procedure using cyclomaltodextrin and cyclomaltodextrin glucanyltransferase, followed by glucoamylase, and β-amylase hydrolysis.     

Production of mannose-containing oligosaccharides by glucansucrase E81 and determination of their functional characteristics

Abstract

Oligosaccharides are one of the functional ingredients to be used in food technology. In this study, by using an active glucansucrase GTFA-ΔN E81 in the acceptor reaction of mannose, mannose-containing oligosaccharides were produced and their functionality was tested. The formation of the oligosaccharides were visualised by TLC analysis and mannose-containing oligosaccharides up to DP 7 were determined by LC-MS analysis. The presence of the (1,6)Glc and (1,3)Glc units within the oligosaccharides were determined by NMR analysis. The in vitro immune-modulatory functions of the mannose-containing oligosaccharides were determined but no induction in IL-4, IL-10, IL-12 and TNF-α cytokine levels were detected. Importantly this oligosaccharide mixture showed prebiotic effect by triggering the growth of tested probiotics and not affecting the growth of pathogen strains. Our findings reveals the potential of the role of glucansucrases for the production of functional oligosaccharides.     

Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.     

Rational transformation of Lactobacillus reuteri 121 reuteransucrase into a dextransucrase

Glucansucrase or glucosyltransferase (GTF) enzymes of lactic acid bacteria display high sequence similarity but catalyze synthesis of different alpha-glucans (e.g., dextran, mutan, alternan, and reuteran) from sucrose. The variations in glucosidic linkage specificity observed in products of different glucansucrase enzymes appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. This notion was derived from mutagenesis of amino acids of GTFA (reuteransucrase) from Lactobacillus reuteri strain 121 putatively involved in acceptor substrate binding. A triple amino acid mutation (N1134S:N1135E:S1136V) in a region immediately next to the catalytic Asp1133 (putative transition state stabilizing residue) converted GTFA from a mainly alpha-(1-->4) ( approximately 45%, reuteran) to a mainly alpha-(1-->6) ( approximately 80%, dextran) synthesizing enzyme. The subsequent introduction of mutation P1026V:I1029V, involving two residues located in a region next to the catalytic Asp1024 (nucleophile), resulted in synthesis of an alpha-glucan containing only a very small percentage of alpha-(1-->4) glucosidic linkages ( approximately 5%) and a further increased percentage of alpha-(1-->6) glucosidic linkages ( approximately 85%). This changed glucosidic linkage specificity was also observed in the oligosaccharide products synthesized by the different mutant GTFA enzymes from (iso)maltose and sucrose. Amino acids crucial for glucosidic linkage type specificity of reuteransucrase have been identified in this report. The data show that a combination of mutations in different regions of GTF enzymes influences the nature of both the glucan and oligosaccharide products. The amino acids involved most likely contribute to sugar-binding acceptor subsites in glucansucrase enzymes.     

Molecular characterization of DSR-E,an alpha-1,2 linkage-synthesizing dextransucrase with two catalytic domains

A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of alpha-1,6 and alpha-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and alpha-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.     

Gluco-oligosaccharides synthesized by glucosyltransferases from constitutive mutants of Leuconostoc mesenteroides strain Lm 28

Aims:  To find different types of glucosyltransferases (GTFs) produced by Leuconostoc mesenteroides strain Lm 28 and its mutant forms, and to check the effectiveness of gluco-oligosaccharide synthesis using maltose as the acceptor.
Methods and Results:  Constitutive mutants were obtained after chemical mutagenesis by ethyl methane sulfonate. Lm M281 produced more active GTFs than that obtained by the parental strain cultivated on sucrose. GTF from Lm M286 produced a resistant glucan, based on endo-dextranase and amyloglucosidase hydrolysis. The extracellular enzymes from Lm M286 catalyse acceptor reactions and transfer the glucose unit from sucrose to maltose to produce gluco-oligosaccharides (GOS). By increasing the sucrose/maltose ratio, it was possible to catalyse the synthesis of oligosaccharides of increasing degree of polymerization (DP).
Conclusions:  Different types of GTFs (dextransucrase, alternansucrase and levansucrase) were produced from new constitutive mutants of Leuc. mesenteroides . GTFs from Lm M286 can catalyse the acceptor reaction in the presence of maltose, leading to the synthesis of branched oligosaccharides.
Significance and Impact of the Study:  Conditions were optimized to synthesize GOS by using GTFs from Lm M286, with the aim of producing maximum quantities of branched-chain oligosaccharides with DP 3–5. This would allow the use of the latter as prebiotics.     

Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I

Related streptococcal and Leuconostoc mesenteroides glucansucrases are enzymes of medical and biotechnological interest. Molecular modelling has suggested that the catalytic domain contains a circularly permuted version of the (β/α)₈ barrel structure found in the amylase superfamily, and site-directed mutagenesis has identified critical amino acids in this region. In this study, sequential N-terminal truncations of Streptococcus downei GTF-I showed that key amino acids are also present in the first one-third of the core domain. Mutations were introduced at Trp-344, Glu-349 and His-355, residues that are conserved in all glucansucrases and lie within a region which is a target for inhibitory antibodies. W344L, E349L and H355V substitutions were assayed for their effect on mutan synthesis and also on oligosaccharide synthesis with various acceptors. It appeared that Trp-344 and His-355 are involved in the action mechanism of GTF-I; His-355 may also play a role in a binding subsite necessary for oligosaccharide and glucan elongation. Received: 14 May 1999 / Received revision: 8 July 1999 / Accepted: 9 July 1999     

Determining the optimum model parameters for oligosaccharide production efficiency using response surface integrated particle swarm optimization method: an experimental validation study

Abstract

Glucansucrases (GTFs) catalyzes the synthesis of α-glucans from sucrose and oligosaccharides in the presence of an acceptor sugar by transferring glucosyl units to the acceptor molecule with different linkages. The acceptor reactions can be affected by several parameters and this study aimed to determine the optimal reaction parameters for the production of glucansucrase-based oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively via a hybrid technique of Response Surface Method (RSM) and Particle Swarm Optimization (PSO). The experimental design was performed using Central Composite Design and the tested parameters were enzyme concentration, acceptor:donor ratio and the reaction period. The optimization studies showed that enzyme concentration was the most effective parameter for the final oligosaccharides yields. The optimal values of the significant parameters determined for enzyme concentration and acceptor:donor ratio were 3.45?U and 0.62, respectively. Even the response surface plots for input parameters verified the PSO results, an experimental validation study was performed for the reverification. The experimental verification results obtained were also consistent with the PSO results. These findings will help our understanding in the role of different parameters for the production of oligosaccharides in the acceptor reactions of GTFs.     

Non-digestible oligosaccharides used as prebiotic agents: mode of production and beneficial effects on animal and human health

Prebiotic agents are food ingredients that are potentially beneficial to the health of consumers. The main commercial prebiotic agents consist of oligosaccharides and dietary fibres (mainly inulin). They are essentially obtained by one of three processes: 1) the direct extraction of natural polysaccharides from plants; 2) the controlled hydrolysis of such natural polysaccharides; 3) enzymatic synthesis, using hydrolases and/or glycosyl transferases. Both of these enzyme types catalyse transglycosylation reactions, allowing synthesis of small molecular weight synthetic oligosaccharides from mono- and disaccharides. Presently, in Europe, inulin-type fructans, characterised by the presence of fructosyl units bound to the beta-2,1 position of sucrose, are considered as one of the carbohydrate prebiotic references. Prebiotics escape enzymatic digestion in the upper gastrointestinal tract and enter the caecum without change to their structure. None are excreted in the stools, indicating that they are fermented by colonic flora so as to give a mixture of short-chain fatty acids (acetate, propionate and butyrate), L-lactate, carbon dioxide and hydrogen. By stimulating bifidobacteria, they may have the following implications for health: 1) potential protective effects against colorectal cancer and infectious bowel diseases by inhibiting putrefactive bacteria (Clostridium perfringens ) and pathogen bacteria (Escherichia coli, Salmonella, Listeria and Shigella ), respectively; 2) improvement of glucid and lipid metabolisms; 3) fibre-like properties by decreasing the renal nitrogen excretion; 4) improvement in the bioavailability of essential minerals; and 5) low cariogenic factor. These potential beneficial effects have been largely studied in animals but have not really been proven in humans. The development of a second generation of oligosaccharides and the putative implication of a complex bacterial trophic chain in the intestinal prebiotic fermentation process are also discussed.