Metabolism of Oligosaccharides and Starch in Lactobacilli: A Review

Oligosaccharides, compounds that are composed of 2–10 monosaccharide residues, are major carbohydrate sources in habitats populated by lactobacilli. Moreover, oligosaccharide metabolism is essential for ecological fitness of lactobacilli. Disaccharide metabolism by lactobacilli is well understood; however, few data on the metabolism of higher oligosaccharides are available. Research on the ecology of intestinal microbiota as well as the commercial application of prebiotics has shifted the interest from (digestible) disaccharides to (indigestible) higher oligosaccharides. This review provides an overview on oligosaccharide metabolism in lactobacilli. Emphasis is placed on maltodextrins, isomalto-oligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides, and raffinose-family oligosaccharides. Starch is also considered. Metabolism is discussed on the basis of metabolic studies related to oligosaccharide metabolism, information on the cellular location and substrate specificity of carbohydrate transport systems, glycosyl hydrolases and phosphorylases, and the presence of metabolic genes in genomes of 38 strains of lactobacilli. Metabolic pathways for disaccharide metabolism often also enable the metabolism of tri- and tetrasaccharides. However, with the exception of amylase and levansucrase, metabolic enzymes for oligosaccharide conversion are intracellular and oligosaccharide metabolism is limited by transport. This general restriction to intracellular glycosyl hydrolases differentiates lactobacilli from other bacteria that adapted to intestinal habitats, particularly Bifidobacterium spp.


INTRODUCTION
Lactobacilli have complex nutritional requirements for fermentable carbohydrates, amino acids, nucleic acids and other substrates, and derive metabolic energy from homofermentative or heterofermentative carbohydrate fermentation (Hammes and Hertel, 2006). Habitats of lactobacilli are nutrient-rich and often acidic and include plants, milk and meat, and mucosal surfaces of humans and animals (Hammes and Hertel, 2006). Intestinal microbiota are characterized by a high proportion of lactobacilli particularly in animals harboring non-secretory epithelia in the upper intestinal tract, including the crop of poultry, the pars esophagus of swine, and the forestomach or rodents and members of the Equidae family (Walter, 2008). Lactobacilli are also dominant in fermentation microbiota of a majority of food fermentations, and are applied as probiotic cultures to benefit host health (Hammes and Hertel, 2006). Owing to their association with humans, food animals, and food as well as their economic importance, they have been studied for more than a century and the physiology and genetics of their monosaccharide metabolism is well understood (Orla-Jensen, 1919;Kandler, 1983;de Vos and Vaughan, 1994;Axelsson, 2004;Makarova et al., 2006;Gänzle et al., 2007).
Oligosaccharides are defined as compounds that are composed of few (2-10) monosaccharide residues (Anonymous, 1982). The use of the term "oligosaccharides" in the current scientific literature, however, differs from this definition in a number of cases. For example, the term "fructo-oligosaccharides" is generally used to include β-(1 → 2) linked fructo-oligosaccharides, excluding the digestible disaccharide sucrose; the term "galactooligosaccharides" generally includes the (indigestible) β-(1 → 3 or 6) linked disaccharides and α-galactosyllactose but excludes the disaccharide lactose, which is also indigestible in a majority of humans, and melibiose; the term "isomalto-oligosaccharides" generally includes the digestible disaccharide isomaltose (Roberfroid et al., 1998;MacFarlane et al., 2008;Seibel and Buchholz, 2010). This paper will use the IUPAC definition of oligosaccharides to include the corresponding disaccharides.
Oligosaccharide metabolism is essential for ecological fitness of lactobacilli in most of their food-related and intestinal habitats (de Vos and Vaughan, 1994;Bron et al., 2004;Gänzle et al., 2007;Walter, 2008;Tannock et al., 2012). Oligosaccharides are the major carbohydrate sources in cereals, milk, fruits, and the upper intestine of animals. The metabolism of mono-and disaccharides is well understood; however, few data are available on the metabolism of higher oligosaccharides which are equally abundant in many habitats. Moreover, interest in the intestinal microbial ecology as well as the widespread commercial application of prebiotic oligosaccharide preparations has shifted the research interest from www.frontiersin.org (digestible) disaccharides to (indigestible) higher oligosaccharides (Barrangou et al., 2003;Kaplan and Hutkins, 2003;Walter, 2008;Seibel and Buchholz, 2010). However, the sound description of the metabolism of higher oligosaccharides is challenging. First, the in silico assignment of the specificity of carbohydrate transport systems or glycosyl hydrolases is unreliable (see e.g., Thompson et al., 2008;Francl et al., 2010) and often results in questionable assignments of gene function. Second, few of the relevant higher oligosaccharides are available in purified form for use as substrate. However, the determination of the growth of lactobacilli on poorly described substrates provided little relevant information on the capacity of lactobacilli to utilize oligosaccharides as carbon source. The lack of reference compounds also impedes identification and quantification of individual compounds in a mixture of oligosaccharides with chromatographic methods. Only few studies characterized oligosaccharide preparations with regards to composition, linkage type, and degree of polymerization, or monitored the metabolism of individual compounds during growth of lactobacilli (Gopal et al., 2001;Kaplan and Hutkins, 2003;Saulnier et al., 2007;Ketabi et al., 2011;Teixeira et al., 2012). Despite the importance of oligosaccharide metabolism for the performance of lactobacilli in food fermentations and in intestinal habitats, our understanding of oligosaccharide metabolism in lactobacilli remains thus limited. Particularly the delineation of metabolism of disaccharides and higher oligosaccharides is unclear, and a comprehensive description of metabolic pathways is currently not available.
This review aims to provide an overview on oligosaccharide metabolism by lactobacilli. Hydrolysis of starch, the only polysaccharide hydrolyzed by extracellular enzymes of lactobacilli, is also discussed. Oligosaccharide fermentation is discussed on the basis of metabolic studies, the cellular location, and substrate specificity of carbohydrate transport systems, glycosyl hydrolases and phosphorylases, and the distribution of the genes coding for metabolic enzymes in selected genomes of lactobacilli. Oligosaccharide metabolism is discussed in detail for four major groups of compounds (i) starch, maltodextrins, and isomaltooligosaccharides (IMO); (ii) fructo-oligosaccharides (FOS); (iii) β-galacto-oligosaccharides (βGOS); (iv) raffinose-family oligosaccharides (RFO) as well as α-galacto-oligosaccharides (RFO and αGOS, respectively).

BIOINFORMATIC ANALYSES OF OLIGOSACCHARIDE AND STARCH METABOLISM OF LACTOBACILLI
To assess the distribution of different oligosaccharide metabolic pathways in lactobacilli, this study identified genes related to oligosaccharide metabolism in lactobacilli by bioinformatic analyses. The analysis included 38 genomes of lactobacilli that were assembled to the chromosome level ( Table 1). The selection of genomes includes representatives of the six major phylogenetic groups in the genus Lactobacillus, the L. salivarius group, the l. delbrueckii group, the L. buchneri group, the L. plantarum group, the L. casei group, the L. reuteri group, as well as the representatives of L. brevis and L. sakei (Hammes and Hertel, 2006). The selection of organisms includes obligately homofermentative species, facultatively heterofermentative species, and obligately heterofermentative species; which were isolates from milk, meat, cereal fermentations, vegetable fermentations, and intestinal habitats. Data were obtained from NCBI GenBank (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/) on 9 September 2011 ( Table 1). For each species and its associated plasmids, coding sequences were extracted, translated, and a BLAST database was built using "makeblastdb" of the NCBI Standalone BLAST+ software package (version 2.2.25;Camacho et al., 2009). The query sequences were searched in each database using "blastp" of the BLAST+ software package with the standard settings and the best match was reported. Additionally, a Smith-Waterman alignment of the query sequence with the highest match was performed using a BLOSUM62 substitution matrix. The score of this alignment was divided by the score of the alignment of the query sequence to itself resulting in a score ratio. An enzyme was marked as present in a given genome or plasmid if the score ratio is above a threshold of 0.3-0.4.
The bioinformatic analysis of genes contributing to oligosaccharide metabolism allows an assessment of the frequency of alternative pathways for oligosaccharide metabolism, identifies genes that occur together to form a functional metabolic pathway, and delineates major and convergent or divergent metabolic strategies of lactobacilli for niche adaptation by specialized oligosaccharide metabolism. However, it does not account for silent genes or orphan genes that are not expressed or not functional (Obst et al., 1992). Moreover, carbohydrate fermentation is highly variable within strains of the same species due to the loss of plasmid encoded traits (de Vos and Vaughan, 1994) and gene acquisition by lateral gene transfer (Barrangou et al., 2003). For example, gene cassettes coding for carbohydrate utilization in L. plantarum are highly variable and were designated as "lifestyle cassettes" that may be added or deleted according to the requirements of specific ecological niches (Siezen and van Hylckama Vlieg, 2011).
Comparable to amylolytic enzymes in bifidobacteria, extracellular amylases of lactobacilli are endoamylases hydrolyzing α-(1 → 6) as well as α-(1 → 4) glucosidic bonds in amylose, amylopectin, or pullulan. The activity increases with increasing degree of polymerization of the substrate (Talamond et al., 2006;Kim et al., 2008). Amylases activity on raw starch was dependent on the sequence of starch binding domains exhibiting significant sequence diversity (Rodriguez-Sanoja et al., 2005). Oligosaccharides with a degree of polymerization of 3 and 4 are the major products of catalysis. Amylase activity in lactobacilli is strain-specific. This study identified an extracellular amylase only in L. acidophilus and L. amylovorus

Aci1
Aci2 Figure 1 for metabolic pathways; genome accession numbers are provided in Table 1.
Gray Background: Presence of Gene, white Background: Absence of Gene, * query sequence.
( Table 2). The infrequent occurrence of amylase genes corresponds to the observation that the majority of lactobacilli are not amylolytic. However, amylolytic lactobacilli are more frequently isolated from cereal fermentations in tropical climates, possibly reflecting the lower β-amylase activity in C4 plants when compared to wheat or rye (Gänzle and Schwab, 2009;Turpin et al., 2011). Maltose transport by the ABC-transporter MalEFG-MsmK was characterized in L. casei and l. acidophilus (Monedero et al., 2008;Nakai et al., 2009). This ABC-transporter is homologous to maltodextrin transport proteins of Bacillus subtilis and has a low affinity for maltose transport. Moreover, several intracellular glucanases are co-transcribed with MalEFG-MsmK, indicating the transport system functions as oligosaccharide transporter (Monedero et al., 2008;Nakai et al., 2009). The MalEFG/MsmK transport system is widespread in lactobacilli but noticeably absent in many lactobacilli that grow rapidly with maltose as the sole source of carbon ( Table 2). Maltose transport in L. sanfranciscensis was attributed to a maltose-H + symport system that was not characterized on the genetic level (Neubauer et al., 1994). Maltose phosphotransferase systems were characterized in other lactic acid bacteria (Le Breton et al., 2005) but are absent in lactobacilli ( Table 2).

METABOLISM OF SUCROSE AND FRUCTO-OLIGOSACCHARIDES
FOS consist of β-(2 → 1) or β-(2 → 6)-linked d-fructose units linked to a terminal d-glucose or d-fructose. Sucrose is widespread in plants and is the most abundant sugar in many fruits, grain legumes, and ungerminated cereal grains. The inulin-type β-(2 → 1) linked FOS 1-kestose, nystose, and 1fructofuranosylnystose are also widespread in nature, although they typically occur at lower concentrations than sucrose. High concentrations are found in Jerusalem artichoke, onions, and chicory root. Wheat, rye, and barley contain 0.15-0.4% of the FOS with a degree of polymerization of 3-5 (Campbell et al., 1997). 1-Kestose is more abundant than nystose and fructofuranosylnystose in cereal grains whereas the tri-, tetra-, and pentasaccharides are approximately equally abundant in onions and chicory roots (Campbell et al., 1997). Levan-type β-(2 → 6) linked FOS, 6-kestose, and higher oligosaccharides, are less abundant in nature but are formed by degradation of levan-type fructans, or by bacterial levansucrases (Praznik et al., 1992;van Hijum et al., 2006). Inulin-type FOS are commercially applied as prebiotic food ingredients (Roberfroid et al., 1998). Commercial production of FOS relies on inulin hydrolysis or synthesis from sucrose by fructansucrases (Yun, 1996;Seibel and Buchholz, 2010).
Three pathways for sucrose metabolism exist in lactobacilli; (i) extracellular hydrolysis by glucansucrases or fructansucrases; (ii) transport and concomitant phosphorylation of sucrose by the Pts1BCA phosphotransferase system, and hydrolysis by the (phospho-)fructo-furanosidase SacA/ScrB; and (iii) transport, followed by phosphorolysis by sucrose phosphorylase or hydrolysis by the (phospho)-fructo-furanosidases BfrA or SacA/ScrB (Figure 2; for review, see Reid and Abratt, 2005). The (phospho)-αglucosidase MalL of B. subtilis also recognizes sucrose as substrate (Schönert et al., 1999) and may contribute to sucrose hydrolysis in lactobacilli. Levansucrases synthesize FOS from sucrose but do not contribute to FOS metabolism (Tieking et al., 2005;van Hijum et al., 2006). With the exception of L. brevis, all genomes of Lactobacillus spp. analyzed harbored at least one functional sucrose metabolic pathway ( Table 3). The presence of two or more alternative pathways for metabolism of sucrose and higher FOS in most lactobacilli indicates that sucrose and higher FOS are highly preferred substrates. Many strains of L. sanfranciscensis do not metabolize sucrose; however, sucrose-negative strains L. sanfranciscensis in sourdough are generally associated with sucrose positive lactobacilli and thus take advantage of extracellular levansucrase activity of other strains (Tieking et al., 2003).
Glucansucrases and fructansucrases are the only extracellular enzymes capable of sucrose hydrolysis (Figure 2) but are the enzymes least frequently found in lactobacilli (Table 3). Glucansucrases and fructansucrases alternatively catalyze sucrose hydrolysis and oligo-or polysaccharide formation (van Hijum et al., 2006) and clearly serve ecological functions other than carbohydrate metabolism. Exopolysaccharide formation by glucansucrases and fructansucrases contributes to biofilm formation in intestinal ecosystems as well as the resistance of lactobacilli to chemical and physical stressors (Schwab and Gänzle, 2006;Walter et al., 2008). Correspondingly, their expression is induced by sucrose in some strains of L. reuteri but their expression in L. reuteri and L. sanfranciscensis was also reported to be constitutive or dependent on environmental stress (Tieking et al., 2005;Schwab and Gänzle, 2006;Teixeira et al., 2012). However, glucansucrases and levansucrases also contribute to sucrose metabolism. In L. sanfranciscensis, levansucrase is the only enzyme capable of sucrose conversion (Tieking et al., 2005; Table 3). Disruption of glucansucrases and levansucrase genes in L. reuteri impaired sucrose metabolism of L. reuteri TMW1.106 but not of L. reuteri LTH5448 (Schwab et al., 2007).
Sucrose phosphorylase exhibits high specificity for sucrose as substrate (Goedl et al., 2008). Sucrose phosphorolysis is energetically more favorable than sucrose hydrolysis because glucose is phosphorylated with inorganic phosphate and not at the expense of ATP (Figure 2). However, sucrose phosphorylase was less frequently identified in genomes of lactobacilli than sucrose hydrolases (Table 3). Sucrose phosphorylase is the only intracellular sucrose converting enzyme in L. reuteri and significantly contributes to sucrose metabolism in this species (Schwab et al., 2007).  Table 3. MsmEFGK (L. acidophilus), four-component ATP-binding cassette (ABC) transport system, imports FOS into the cytosol (Barrangou et al., 2003). ABC transport systems for FOS in lactobacilli transport glucose, fructose, and FOS with a degree of polymerization of 2-4 ( Kaplan and Hutkins, 2003). BfrA or ScrB (L. acidophilus) and SacA (L. plantarum), fructosidases, hydrolyze terminal β-d-fructofuranosides in (phospho-)β-d-fructofuranoside oligosaccharides (Barrangou et al., 2003;Ehrmann et al., 2003;Saulnier et al., 2007). ScrP (L. reuteri ), named GtfA in L. acidophilus, a sucrose phosphorylase, phosphorylyses sucrose to d-fructose, and α-d-glucose-1-phosphate (Barrangou et al., 2003;Schwab et al., 2007). Pts1BCA (L. plantarum), a sucrose phosphotransferase system, transports FOS into the cytosol while transferring a phosphoryl-moiety onto the glucose residue of the FOS. Pts1BCA transports FOS with a degree of polymerization of 3 and 4 (Saulnier et al., 2007). SacK1 (L. plantarum), a fructokinase, transfers a phosphate group to d-fructose, converting it to a d-fructose-6-phosphate (Saulnier et al., 2007). LevS (L. sanfranciscensis) named FtfA in L. reuteri, cell-wall bound levansucrase, hydrolyzes sucrose to glucose and fructose, also has a transferase activity which catalyzes the transfer of the fructose moiety of sucrose to a fructosyl-acceptor yielding FOS or levan (Tieking et al., 2005;van Hijum et al., 2006). GtfA (L. reuteri ), extracellular glucansucrase, hydrolyzes sucrose to glucose, and fructose, also has a transglucosylation activity which transfers the glucose moiety of sucrose to a glucan chain (van Hijum et al., 2006). Transport systems for sucrose in lactobacilli include the oligosaccharide transporter MsmEFGK and the sucrose phosphotransferase system Pts1BCA (Figure 2; Barrangou et al., 2003;Saulnier et al., 2007). MsmEFGK was identified only in L. acidophilus and L. crispatus (Table 3); its presence in L. acidophilus was attributed to acquisition by lateral gene transfer (Barrangou et al., 2003). Both transport systems also internalize FOS with a degree of polymerization of 2 and 3 but have a very low affinity for FOS with a degree of polymerization of 4 or higher (Kaplan and Hutkins, 2003;Saulnier et al., 2007). Characterization of an ABC family transporter in L. paracasei demonstrated that its affinity strongly decreased in the order kestose > glucose or fructose > sucrose or nystose > fructofuranosylnystose (Kaplan and Hutkins, 2003). Irrespective of the presence of intracellular fructo-furanosidases with activity on high molecular weight fructans, transport limits metabolism of FOS in lactobacilli to di-, tri-, and tetrasaccharides. However, because many sucrose-metabolizing lactobacilli harbor neither MsmEFGK nor Pts1BCA, additional but uncharacterized sucrose transport systems exist. www.frontiersin.org
The β-galactosidases LacLM and LacZ, both classified in the GH2 family, hydrolyze a wide variety of β-(1 → 2, 3, 4, or 6) βGOS, including oligosaccharides with a degree of polymerization of 3-6 (Gänzle, 2012). LacLM or LacZ enzymes are active as multimeric enzymes (Schwab et al., 2010). The GH42 β-galactosidases LacA was cloned and characterized in L. acidophilus and was found to have only low activity on lactose or GOS (Schwab et al., 2010). In other GH42 family β-galactosidases from bifidobacteria and Carnobacterium piscicola, activity with lactose as substrate is low or absent. A contribution of LacA to βGOS metabolism in lactobacilli thus remains to be demonstrated.
βGOS are transported by the lactose permase LacS, which transports lactose in exchange with galactose, or in symport with protons (Poolman et al., 1992). Induction of lacS expression by βGOS with a DP of 2-6 in L. acidophilus was interpreted as indication that LacS transports higher βGOS as well as lactose, however, experimental evidence for tri-or tetrasaccharide transport by LacS is lacking. Lactose transport by LacS of S. thermophilus is inhibited by the disaccharide melibiose, indicating that αGOS are an alternative substrate for the transport enzyme (Poolman et al., 1992). L. rhamnosus and L. acidophilus were capable of acid production from galactosyllactose, but preferentially metabolized disaccharides over tri-and tetrasaccharides (Gopal et al., 2001). Because β-galactosidase exhibits no preference for disaccharides over tri-or tetrasaccharides, this preferential metabolism of βGOS with a lower degree of polymerization likely reflects transport limitations.
Metabolism of oligosaccharides with mixed αand βgalactosidic linkages requires combined activity of α-galactosidase and β-galactosidase (Figures 3 and 4). Likewise, hydrolysis of RFO by α-Gal releases sucrose and complete degradation of RFO is dependent on sucrose metabolic enzymes (Figure 4). Of the sucrose metabolic enzymes shown in Figure 2, levansucrase and the fructo-furanosidase BfrA also show activity with RFO as substrates van Hijum et al., 2006;Teixeira et al., 2012). Few strains of lactobacilli harbor levansucrase but not α-Gal, e.g., L. sanfranciscensis LTH2590. These strains convert RFO to αGOS by levansucrase activity without further metabolism of the galactosides ( Table 5; Teixeira et al., 2012). Glucansucrases and sucrose phosphorylase do not cleave RFO (Kim et al., 2003;van Hijum et al., 2006). In strains expressing melA, levansucrase, and sucrose phosphorylase or fructo-furanosidase, two alternative pathways for RFO degradation exist: (i) extracellular conversion of RFO to the corresponding αGOS and fructose by levansucrase, followed by αGOS uptake and hydrolysis; and (ii) RFO uptake, followed by hydrolysis to sucrose and galactose through MelA activity, and sucrose conversion by sucrose phosphorylase or fructo-furanosidase (Teixeira et al., 2012). Extracellular conversion by levansucrase is the preferred metabolic route in L. reuteri (Teixeira et al., 2012), presumably because of facilitated transport. However, raffinose induces the expression of sucrose phosphorylase in L. reuteri (Teixeira et al., 2012) and B. lactis (Trindade et al., 2003), demonstrating that both pathways exist in parallel.
Lactobacillus plantarum and L. gasseri harbor several systems for transport and metabolism of β-glucosides (Andersson et al., 2005;Francl et al., 2010). Bioinformatics analyses predicted the presence of two β-glucoside/cellobiose specific phosphotransferase systems with associated phospho-β-glucosidase in addition to a β-glucosidase in L. plantarum WCFS1 (Andersson et al., 2005). It remains to be established whether this multitude of transport and enzyme systems reflects adaptation to substrates differing in their linkage type or degree of polymerization.
Human milk contains about 1% oligosaccharides. Human milk oligosaccharides consist of d-glucose, d-galactose, Nacetylglucosamine, L-fucose, and sialic acid and have a degree of polymerization of 3-32. The combination of different monomers, linkage types, and different degrees of branching or polymerization allows for a vast number of different structures. More than 100 different structures were identified and the composition of milk oligosaccharides is dependent on the mother (Kunz et al., 2000;Kobata, 2010). Human milk oligosaccharides generally consist of lactose at the reducing end and are elongated with galactose, Nacetylglucosamine, fucose, and sialic acid. Core structures include galactosyllactose, fucosyllactose, lacto-N -fucopentaose, and sialyllactose (Kunz et al., 2000;Kobata, 2010). These oligosaccharides are not degraded by β-galactosidases of lactobacilli (Schwab and Gänzle, 2011). Studies with purified human milk oligosaccharides demonstrate that lactobacilli utilize fucose and Nacetylglucosamine but not human milk oligosaccharides as carbon source (Ward et al., 2006;Schwab and Gänzle, 2011). Weak growth of L. acidophilus on human milk oligosaccharides was observed for L. acidophilus (Marcobal et al., 2010), which may reflect metabolism of α-galactosyllactose or β-galactosyllactose, or the ability to release few of the fucosyl-or N -acetylglucosaminyl-residues after cell lysis and release of intracellular glycosyl hydrolases. The www.frontiersin.org inability of lactobacilli to grow on human milk oligosaccharides contrasts the metabolic toolset of bifidobacteria, which are highly adapted to growth on human milk oligosaccharides as carbon source (González et al., 2008;Sela et al., 2008).

CONCLUSION
Lactobacilli are well-equipped to metabolize oligosaccharides that occur in their habitats, including plants, milk, and the (upper) intestinal tract of humans and animals. This metabolic diversity is remarkable for a group of organisms that have evolved by reduction of genome size (Makarova et al., 2006). Metabolic pathways for disaccharide metabolism often also enable the metabolism of tri-and tetrasaccharides. However, with the exception of amylase and levansucrase, metabolic enzymes for oligosaccharide conversion are intracellular and oligosaccharide metabolism is limited by transport. Starch and related α-glucans are the only group of compounds for which a metabolic pathway dedicated to oligo-and polysaccharide metabolism was retained. This general restriction to intracellular glycosyl hydrolases clearly differentiates lactobacilli from other bacteria that adapted to intestinal habitats, particularly Bifidobacterium spp., which maintain a more extensive toolset for extracellular hydrolysis and transport of complex carbohydrates (Sela et al., 2008;van den Broek et al., 2008). The divergent approach of bifidobacteria and lactobacilli to carbohydrate fermentation may reflect their respective dominance in the human colon, characterized by a limited availability of monoand disaccharides, and the upper intestinal tract of animals, which offers a rich supply of oligosaccharides (Sela et al., 2008;Walter, 2008).
The capacity of individual strains and species of lactobacilli for oligosaccharide metabolism differs substantially. This metabolic diversity conforms to the phylogenetic diversity in the genus Lactobacillus. Several species metabolize a large diversity of different carbon sources, including all major categories of oligosaccharides. Well-characterized representatives include L. acidophilus, L. casei, and L. plantarum. Oligosaccharides are preferentially metabolized by phosphotransferase/phospho-glycosyl hydrolase systems and oligosaccharide metabolism is repressed by glucose (Andersson et al., 2005;Barrangou et al., 2006;Monedero et al., 2008;Francl et al., 2010). Other species in this continuum of metabolic diversity, however, exhibit more restricted carbohydrate fermentation patterns. An extreme is the "nothing but maltose or sucrose" diet of several strains of L. sanfranciscensis, which is partially reflected in the genome-sequenced strain L. sanfranciscensis TMW1.304 (Table 1). In this group of strains, oligosaccharides are preferentially metabolized by permease/phosphorylase systems and oligosaccharide metabolic enzymes are not repressed by glucose (Tieking et al., 2005;Schwab et al., 2007;Teixeira et al., 2012). Remarkably, both groups -broad versus narrow spectrum of oligosaccharide fermentation -are represented in intestinal habitats (e.g., L. acidophilus and L. reuteri) as well as food fermentations (e.g., L. plantarum and L. sanfranciscensis). Further insight into oligosaccharide metabolism in lactobacilli is dependent on the biochemical characterization of metabolic enzymes and their substrate specificity -particularly transport enzymes -and the sound quantification of oligosaccharide consumption during metabolism of lactobacilli in model substrates, and in food or intestinal ecosystems.
Glycosyl hydrolases and glycosyl phosphorylases of lactic acid bacteria have evolved as an important tool in the (chemo-)enzymatic synthesis of functional oligosaccharides or sugar derivatives (e.g., van Hijum et al., 2006;Goedl et al., 2008;Black et al., 2012). Further insight into the diversity and catalytic properties of carbohydrate-active enzymes of lactobacilli will further improve this toolset for food-related and other applications.