Cross-Feeding among Probiotic Bacterial Strains on Prebiotic Inulin Involves the Extracellular exo-Inulinase of Lactobacillus paracasei Strain W20

An increasing number of β-(2-1)-fructans is commercially available. We first analyzed the structural composition of 9 commercial β-(2-1)-fructans (Table 1). Two types of compounds were found (6): fructan chains with a terminal sucrose unit on one end (GF type) and β-(2-1)-linked fructosyl residues only (FF type). GF-type compounds were present in all products analyzed, while FF-type compounds were found only in 5 of 9 (Table 1). When we compared the intensities of peaks obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis (data not shown), the peak responses for FF-type compounds were the highest in Frutalose OFP and FOS P1. These compounds were classified as oligofructoses. For Frutafit HD, Frutafit CLR, and Inulin P2, the signal responses for FF-type compounds were lower, while the numbers of GF-type compounds found in chromatograms increased. These fructans were thus classified as inulin-type β-(2-1)-fructans. The highest diversity in structural composition overall was found in the degree of polymerization (Table 1). Frutafit HD and Inulin P2 offered the broadest range of fructan compounds (DP 2 to DP 60), while Frutalose OFP, FOS P1, and scFOS P6 had a limited range (DP 2 to DP 7). Most preparations still contained some glucose and fructose monosaccharides (relative peak heights ranging between 0.5% and 8.2%) and sucrose (constituting 2% to 15% relative peak heights). The 1-kestose and nystose samples were pure and comprised only these specific DP-3 and DP-4 compounds, respectively. This comparative structural analysis of 9 commercial fructans provided a firm basis for the subsequent study of the utilization of specific compounds by probiotic bacteria.

Various probiotic bacteria were tested for their ability to grow on short-chain inulin (sc-inulin; Frutafit CLR). This prebiotic has been derived from inulin and comprises over 50 individual compounds with various DPs (see below), of both GF- and FF-type β-(2-1)-fructans (Table 1). Frutafit CLR thus shows all the structural variations that are unique to prebiotic β-(2-1)-fructans, enabling an analysis of their effects on the growth of individual probiotic bacteria. Various strains of lactobacilli and bifidobacteria, plus Enterococcus faecium and Pediococcus acidilactici, were tested. The growth responses of individual strains with sc-inulin varied greatly, e.g., in the lengths of the lag phase and in the final values for optical density at 600 nm (OD₆₀₀) reached (Fig. 1A to C). All strains grew well on glucose (positive control), reaching final (100%) OD₆₀₀ values at stationary phase of approximately 1.0 in microtiter plates and 2.0 in Hungate tubes, set as a relative OD₆₀₀ value of 1.00. When incubated with sc-inulin, most strains reached a stationary-phase relative OD₆₀₀ of around 0.4, indicating that sc-inulin compounds were supporting the growth of these strains only partly compared to that of the positive control (Fig. 1C).

Following bacterial growth, the culture supernatants were analyzed for the presence of any remaining components from the sc-inulin mixture. A carbohydrate analysis revealed four patterns. (i) L. salivarius W57, E. faecium W54, and P. acidilactici W143 specifically used only fructose, GF (sucrose), and GF2 (1-kestose) (Fig. 2A). (ii) Bifidobacteria utilized sc-inulin compounds with the preference F2 > F3 > F4 and GF > GF2 > GF3 > GF4, but only the F2 [β-d-Frup-(2→1)-d-Fru] and F3 [β-d-Frup-(2→1)-β-d-Frup-(2→1)-d-Fru] compounds were completely used, at 24 h for Bifidobacterium animalis subsp. lactis W51 and B. lactis W53 and at 36 h for B. lactis W52 (Fig. 2B). (iii) Lactobacillus acidophilus W37 utilized short-chain compounds of the FF and GF types. (iv) L. paracasei W20 utilized all GF- and FF-type compounds present in sc-inulin. These results clearly highlight that probiotic bacteria have diverse substrate specificities for the utilization of β-(2-1)-fructans.

L. paracasei W20 was clearly exceptional by consuming all GF- and FF-type compounds present in sc-inulin. To analyze its fructan metabolism in more detail, we grew L. paracasei W20 on sc-inulin and GF3 (both GF-type fructans) and Frutalose oligofructose (FF-type fructan). An analysis of the changes in fructan compositions over time revealed a preferential degradation of fructose and all sc-inulin compounds larger than a DP of 3 in culture supernatants harvested from L. paracasei W20 at the mid-exponential phase (Fig. 1A) (L. paracasei, t = 12 h), while sucrose (GF) and 1-kestose (GF2) were present in relatively large amounts (Fig. 3A). Also, the degradation of the pure GF3 compound nystose by L. paracasei W20 first resulted in the release of 1-kestose (not shown) in the supernatant, followed by sucrose GF (Fig. 3C). In time, oligofructose degradation resulted in the formation of GF and GF2 and of the disaccharide F2 (Fig. 3B). All compounds had been consumed completely in stationary-phase samples (not shown). These results show that L. paracasei W20 degrades β-(2-1)-fructans in a sequential manner, leading to temporary increases of GF and GF2 in the case of GF-type fructans and F2 in the case of FF-type fructans.

Following growth on several β-(2-1)-fructans, we detected β-fructosidase activity in L. paracasei W20 culture supernatants. This activity degraded sc-inulin into sucrose, fructose, and glucose (see Fig. S1 in the supplemental material). The supernatants obtained from the growth of L. paracasei W20 on glucose or fructose clearly had much lower activities. Via a search of the Carbohydrate-Active enZYmes Database (www.cazy.org) (dbCAN [21]), the genome of L. paracasei W20 was found to encode one putative β-fructosidase protein of 1,031 amino acids (family GH32) including a cell wall anchor motif for Gram-positive bacteria (LPQAG) (GenBank accession no. MH047828). The subcellular localization of this protein is predicted to be >99% extracellular. The protein sequence showed 97% identity to β-fructosidases and sucrose-6-phosphorylases present in various strains of L. paracasei and L. casei and around 30% identity to exo-inulinases from Bacteroides species and Bacillus subtilis (22, 23). To characterize the activity of this putative GH32 enzyme, we cloned the predicted catalytic domain (residues 188 to 739) into the pET15bLIC vector which added an N-terminal 6-His tag (called LpGH32). The overexpression of LpGH32 in Escherichia coli BL21 yielded a dominant band by SDS-PAGE at the expected molecular mass of 61 kDa; this protein was purified using His-trap column affinity chromatography. The incubation of 0.9 μg/ml (excess amount) of the purified LpGH32 with β-(2-1)-fructans revealed activity on inulin and oligofructose (only at pH 5.0), which was observed before with culture supernatants. The final products of enzymatic conversion after 40 h were identified as monosaccharides (Fig. 4A, inulin and oligofructose) and subsequently separated and identified by HPAEC-PAD as glucose and fructose. This activity was similar to what was observed before within the culture supernatants of L. paracasei W20 (Fig. S1). We further tested the activity of the enzyme with sucrose and the β-(2-6)-fructan levan (Fig. 4A, levan and sucrose). While all sucrose was converted into fructose and glucose after 40 h, levan was not a substrate at pH 4.0 and only partly converted into fructose at pH 5.0 (indicated by a remaining spot at the bottom of the thin-layer chromatography [TLC] plate) (Fig. 4A, levan, lanes 3 and 4). Product formation at 40 h was also analyzed by adding a minimal amount (0.15 μg/ml) of LpGH32 to incubations with inulin. Besides the monosaccharides glucose and fructose, GF sucrose and GF2 1-kestose accumulated from inulin conversion (Fig. 4B). With oligofructose, we observed an increase of DP-2 F2 plus DP-2 sucrose and DP-3 1-kestose (derived from GF impurities present in the oligofructose) (Fig. 4C). These results show that larger β-(2-1)-fructan chains are preferably converted by the LpGH32 enzyme, leading to an intermediate accumulation of shorter DP-2 to DP-3 β-(2-1)-fructan chains.

Our results show that L. paracasei W20 employs an extracellular exo-inulinase (EC 3.2.1.80, family GH32) to degrade prebiotic β-(2-1)-fructans (LpGH32). Among the bacteria tested in this study, this enzyme accounts for the ability of L. paracasei W20 to degrade β-(2-1)-fructans independent of their structural parameters (GF and FF types; DP 2 to DP 60). Due to the preference of LpGH32 to degrade fructan chains larger than a DP of 3 first, seen both with the purified enzyme and in supernatants of L. paracasei W20, the intermediate breakdown products GF and GF2 for GF-type fructans and GF, GF2, and F2 for FF-type fructans accumulated (Fig. 3 and 4).

We searched bacterial genomes for genes involved in known pathways of fructose utilization, sucrose utilization, and fructooligosaccharide/raffinose utilization (22, 24–31) to explain the strain differences in utilization of GF-type and FF-type components. Specifically, we identified key glycoside hydrolases and transporters involved in fructose metabolism. We found that these strains differed highly when comparing genes from all three pathways mentioned above (Fig. 5 and Table S1). None of these strains combined all three pathways; rather, one or two pathways were predicted by the genomic annotation. The genomes of L. paracasei W20 and L. salivarius W57 both encode a fructose utilization pathway with a PTS transport system, which may account for the uptake of the free fructose released by LpGH32. The L. salivarius W57 genome contains a predicted sucrose utilization system with a sucrose PTS transport system, which was not found in the L. paracasei W20 genome. The Bifidobacterium strains studied lacked a fructose utilization pathway (also observed experimentally, bifidobacterial strains tested could not grow on pure fructose) (Fig. 1B), but sucrose utilization was predicted in the bifidobacterial representative strain B. longum NCC 2705, with a sucrose permease and a FOS/raffinose utilization pathway with an ABC transporter and an intracellular β-fructosidase (Fig. 5 and Table S1). On the basis of the results from the genome analyses of these probiotic strains, we hypothesize that the diverse distribution of these pathways may enable interspecies interactions resulting in cross-feeding during the degradation of inulin. If interspecies interactions occur, these may generate interesting biotechnological opportunities in the application of combinations of probiotic strains with the prebiotic inulin as novel synbiotic mixtures on the basis of interspecies cross-feeding properties.

To test the interspecies cross-feeding hypothesis and to identify possible involvement of particular enzymes and transporters harbored by probiotic bacteria in the degradation of prebiotic fructan components, we studied the interspecies interactions of the two probiotic strains L. paracasei W20 and L. salivarius W57. We chose to use these strains on the basis of the results of their genome analysis and their different carbohydrate-utilization profiles: while L. salivarius incubated with sc-inulin showed very specific utilization of fructan compounds with a DP of <4 (Fig. 2A), L. paracasei was able to utilize all components of β-(2-1)-fructans, employing an extracellular exo-inulinase. Both strains differ at the transporter level, with a single sucrose transporter present in L. salivarius W57 (PTS_ScrA) (Fig. 5) but lacking in L. paracasei W20.

To identify any preference of L. salivarius W57 for GF-type fructans with distinct sizes, the strain was grown on pure 1-kestose (GF2, DP-3 FOS) and nystose (GF3, DP-4 FOS). The results show that L. salivarius only grew on 1-kestose, while L. paracasei grew on both 1-kestose and nystose compounds (Fig. 6A). Nystose thus is the smallest DP fructooligosaccharide that demonstrates selectivity between the two Lactobacillus strains and therefore requires the activity of LpGH32 in order to be metabolized. We then looked for the possible beneficial cross-feeding effects of LpGH32 by adding the purified enzyme to L. salivarius W57 cultures incubated with nystose. L. salivarius W57 was unable to grow on nystose (Fig. 6A), while the addition of LpGH32 enabled the strain to completely metabolize this DP-4 fructan (Fig. 6B). To identify the products of LpGH32 produced by L. paracasei W20 that are potentially available for cross-feeding by L. salivarius W57, we grew L. paracasei W20 and L. salivarius W57 on several LpGH32 inulin products. While L. salivarius W57 grew equally well on fructose, sucrose, and 1-kestose, reaching stationary phase after 5 h, L. paracasei W20 used fructose rapidly (stationary after 9 h) but grew quite poorly on 1-kestose (after a long lag phase of 12 h) and on sucrose (Fig. 6C). These results suggest that L. paracasei W20 overall has a high affinity for fructose, while the GF-type compounds sucrose and GF2 1-kestose are used at a lower preference. Therefore, sucrose and 1-kestose liberated by LpGH32 activity may be available as cross-feeding products from sc-inulin for metabolism by other bacteria that are in close proximity to L. paracasei.

To test for in vivo cross-feeding, we cocultured L. salivarius W57 and L. paracasei W20 on two types of β-(2-1)-fructans, sc-inulin containing mainly GF-type fructan chains and oligofructose (Frutalose OFP) containing mostly pure fructose-composed β-(2-1)-fructan chains. In the case of sc-inulin, coculturing resulted in a clear increase in CFU/ml for L. salivarius W57 in the cocultures compared to that in the single cultures (Fig. 7A). Cross-feeding thus occurred between the two strains, stimulating the growth of L. salivarius W57, which benefited from the presence of L. paracasei W20, employing the extracellular GH32 enzyme for the degradation of the GF-type fructan sc-inulin (resulting in an accumulation of short-chain FOS DP-2 to DP-3 compounds). The degradation of oligofructose yields free fructose, which does not result in cross-feeding of L. salivarius W57 (Fig. 7B).

The commercial probiotic bacterial strains Lactobacillus paracasei subsp. paracasei W20 (L. paracasei W20), Lactobacillus acidophilus W37, Lactobacillus salivarius W57, Lactobacillus casei W56, Enterococcus faecium W54, Pediococcus acidilactici W143, Bifidobacterium animalis subsp. lactis W51 (B. lactis W51), Bifidobacterium animalis subsp. lactis W52 (B. lactis W52), and Bifidobacterium animalis subsp. lactis W53 (B. lactis W53) were provided by Winclove Probiotics B.V. (Amsterdam, The Netherlands). All strains belonging to lactic acid bacteria (LAB) were maintained in de Man-Rogosa-Sharpe (MRS) broth (Oxoid, Basingstoke, UK) containing 2% glucose as the carbon source. Liquid cultures (5 ml) were inoculated in anaerobic culture glass tubes (Boom, Meppel, The Netherlands) under anaerobic conditions provided by the Hungate system with nitrogen (36) and incubated at 37°C. For permanent storage, fresh 200-μl aliquots of the cultures were diluted with sterile glycerol to 15% (vol/vol) in 1.5-ml tubes, and the tubes were maintained at −80°C. The purity of the cultures was frequently checked by spreading aliquots on agar plates prepared with MRS and Luria-Bertani (LB) medium and by light microscopy. For growth experiments with β-(2-1)-fructans, modified MRS medium (mMRS) was used with LAB strains prepared according to reference 32. In brief, 1 liter mMRS contained 10 g peptone, 2.5 g granulated yeast extract, 3 g tryptose, 1 ml Tween 80, 3 g K₂HPO₄, 3 g KH₂PO₄, 2 g ammonium citrate, 0.2 g pyruvic acid sodium salt, 0.575 g MgSO₄·7H₂O, 0.12 g MnSO₄·H₂O, and 0.034 g FeSO₄·7H₂O. The components were dissolved in double-distilled H₂O by bringing the medium to a boil and then adding 0.5 g/liter cysteine-HCl and adjusting the pH to 6.8. The medium was sterilized by autoclaving (15 min at 121°C).

Bifidobacterial strains were maintained in Bifidobacterium medium (BM) that was prepared according to reference 37. One liter BM contained 10 g Trypticase peptone, 2.5 g yeast extract, 3 g tryptose, 3 g K₂HPO₄, 3 g KH₂PO₄, 2 g triammonium citrate, 0.3 g pyruvic acid, 1 ml Tween 80, 0.574 g MgSO₄·H₂O, 0.12 g MnSO₄·H₂O, and 5 g NaCl in 1 liter of distilled water. After autoclaving, the BM was supplemented with 0.05% (wt/vol) filter-sterilized cysteine-HCl, and strains were grown at 37°C under anaerobic conditions maintained by 100% CO₂.

Native inulin (Frutafit HD; inulin of DP of 2 to 60, ≥90% [wt/wt]; average chain length, 11), long-chain inulin (lc-inulin, Frutafit TEX; inulin ≥99.5% [wt/wt]; average chain length, 22), short-chain inulin (sc-inulin, Frutafit CLR; inulin/oligofructose, ≥85% [wt/wt]; average chain length, 7), and oligofructose (Frutalose OFP; oligofructose, ≥92% ± 2% [wt/wt]; average chain length, 7) were provided by SENSUS (Roosendaal, The Netherlands). 1-Kestose (≥98%) and nystose (≥98%) were purchased from CarboSynth (Compton, UK). FOS P1 (oligofructose of DP of 2 to 8, ≥93% [wt/wt]), Inulin P2 (inulin of DP of 2 to 60, ≥90% [wt/wt]; average chain length, 10), and scFOS P6 (oligofructose of DP of 3 to 5, ≥95% [wt/wt]) were obtained from Winclove Probiotics B.V. Levan was produced by sucrose conversion with B. subtilis as described previously (38). Carbohydrates were dissolved to 1% (wt/vol) in Milli-Q water and filter sterilized using 0.2-μm cellulose acetate membrane filters (VWR International, Radnor, PA); lc-inulin was dissolved and sterilized by autoclaving.

Growth experiments with LAB strains were carried out using 96-well microtiter plates (Greiner Bio-one, Frickenhausen, Germany). Prior to the inoculation of the plates, cultures of bacterial strains grown overnight were harvested by centrifugation (2,300 × g for 2 min) and diluted 25-fold in 2× mMRS. Separately, carbohydrate solutions were added in triplicates to wells of plates, while glucose and double-distilled water served as the positive and negative controls, respectively. Subsequently, diluted bacterial suspensions and plates containing carbohydrate solutions were transferred to an anaerobic glove box (Bohlender, Grünsfeld, Germany) with constant nitrogen flow, and cultures were inoculated with 2% (vol/vol) of bacteria (total volume of cultures per well, 160 to 200 μl) to provide anaerobic conditions in microtiter plates. Then, the plate was sealed with an airtight transparent seal (Simport, Beloeil, Canada) and placed in a plate reader (at 37°C) for the monitoring of the OD₆₀₀ at 5-min intervals with continuous shaking at medium speed. The plate reader was installed inside an AtmosBag (Sigma-Aldrich, Schnelldorf, Germany) and constantly flushed with nitrogen throughout the OD₆₀₀ measurements. For growing L. salivarius W57 in the presence of the GH32 enzyme from L. paracasei W20 (LpGH32), 10 μl of the recombinantly produced and purified enzyme was added per well, yielding a final concentration of 0.5 μg/ml. For coculture experiments, cultures were inoculated with 1% (vol/vol) of overnight cultures of L. salivarius W57 and L. paracasei W20. To determine the CFU/ml, bacterial cultures were harvested at 18 h (L. paracasei W20 single cultures and L. paracasei plus L. salivarius cocultures) and 8 h (L. salivarius W57 single cultures). Harvested cultures were diluted to appropriate levels and spread on MRS agar (MRS medium supplemented with 1.5% [wt/vol] agar). Subsequently, the plates were incubated anaerobically in jars using the GasPak EZ container system (BD, Sparks, MD). The plates incubated with both strains were kept at 18°C (growth of L. paracasei W20 only) and 37°C (growth of both strains).

Bifidobacterial strains were grown in 3-ml cultures in BM supplemented with 0.5% (wt/vol) carbohydrates using anaerobic glass tubes and maintained under anaerobic conditions with 100% CO₂ at 37°C. BM with and without 5 mg/ml glucose was used as the positive and negative controls, respectively. Growth was followed over time by measuring the OD₆₀₀ manually directly in anaerobic glass tubes using the cell density meter WPA CO 8000 (Biochrom, Cambridge, UK).

FOS composition was analyzed using thin-layer chromatography (TLC) as reported previously (39). In brief, samples of 1 μl were spotted up to 3 times on TLC sheets (silica gel 60 F254; Merck, Darmstadt, Germany) that were developed with n-butanol-ethanol-water (5:5:3 [vol/vol]). The bands were visualized by urea staining. FOS composition was determined by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) by diluting the samples to an appropriate concentration in 80% (vol/vol) dimethyl sulfoxide. The samples were analyzed on a CarboPac PA1 column (4 mm by 250 mm; Dionex, Sunnyvale, CA) using a linear gradient (buffer A, 0.1 M NaOH; buffer B, 0.6 M NaOAc in 0.1 M NaOH): 0 min, 0% B; 10 min, 22% B; 25 min, 40% B; 25.1 min, 100% B. Glucose, fructose, 1-kestose, and nystose were used as the standards to determine monosaccharides and short oligosaccharides (40). Inulin synthesized by the InuJ enzyme (DP range, 2 to 16; inulin polymer) was used to annotate any larger oligosaccharides (39).

After 24 h of growth on FOS, cultures of probiotic bacteria were centrifuged (10,000 × g for 15 min). Subsequently, the supernatants were filtered using 0.45-μm cellulose acetate filters (VWR International) and mixed 1:1 with 10 mg/ml sc-inulin in 1.5-ml reaction tubes. After incubating for 72 h at 37°C, the (changes in the) sc-inulin compositions were analyzed by TLC and HPAEC-PAD. The culture supernatants from growth on glucose served as the negative controls.

Genomic DNA isolation from L. paracasei W20 was performed using a GenElute bacterial genomic DNA kit (Sigma-Aldrich). Genomic DNA (10 ng) was used as the template in PCRs to amplify the gene encoding the GH32 enzyme from L. paracasei W20. The primers used in PCRs were LPGH32-Fwd (5′-CAGGGACCCGGTCCATACCGAAACCAGTATCACTACTCAAGTAGC-3′) and LPGH32-Rev (5′-CGAGGAGAAGCCCGGTTAGTTCCAAATTGAAGTAATTGGATTGATAGTTAAGTCGC-3′).

The underlined complementary overhangs were used for joining inserts with vector pET15b, which was modified to enable ligation independent cloning (LIC) of the amplified DNA (41). Phire Hot Start II DNA polymerase (Thermo Scientific, Waltham, MA) was used for amplification in an MJ Mini personal thermal cycler (Bio-Rad, Hercules, CA). The reaction mixtures contained 0.05% (vol/vol) dimethyl sulfoxide. PCR mixtures yielding sufficient amplification of the desired gene were cleaned up using an Illustra GFX PCR DNA and Gel Band purification kit (GE Healthcare, Buckinghamshire, UK). Purified PCR products were then cloned into pET15b according to LIC procedures. In brief, the insert and vector were treated with T4 DNA polymerase for 60 min at room temperature (RT), followed by inactivation of the enzyme (20 min at 75°C). T4 DNA polymerase-treated vector and insert were ligated for 15 min at RT, EDTA was added to a 10 mM final concentration, and the ligation mixture was incubated for another 5 min at RT. Subsequently, the ligation mixture was transformed into E. coli TOP10 competent cells, and positive clones were checked by isolating plasmid DNA followed by digestions with NcoI/XhoI. For the overexpression of the recombinant protein, plasmid DNA containing the desired gene was transformed into E. coli BL21 Star (DE3), and precultures of transformed E. coli BL21 Star (DE3) were grown overnight in 5 ml LB medium containing 50 μg/ml ampicillin. Subsequently, expression cultures of 250 ml LB containing 100 μg/ml ampicillin were inoculated with 1% (vol/vol) of the precultures. The flasks were put into incubator shakers with agitation at 210 rpm at 37°C, and growth was allowed until the OD₆₀₀ reached 0.5. Then, the cultures were put on ice and expression was started by adding 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside). Expression was continued in incubator shakers at 18°C and 160 rpm for 16 h. Bacterial pellets were collected by centrifugation (10 min at 3,500 × g), resuspended in 20 ml 20 mM Tris HCl (pH 8.0) supplemented with 1 mM CaCl₂, 5 mM β-mercaptoethanol, and 4 mM imidazole, and sonicated 10 rounds for 15 s using a Soniprep 150 Plus (MSE, Lower Sydenham, UK). Between every sonication step, the suspension was put on ice for 30 s. Afterwards, the cell extract was centrifuged (15,000 × g for 20 min) and added to a HisTrap HP 1-ml column (GE Healthcare) connected to a peristaltic pump for protein purification. The column was equilibrated with 1.5 ml buffer A (20 mM Tris HCl [pH 8.0] supplemented with 500 mM NaCl, 3 mM CaCl₂, and 3 mM MgCl₂), eluted with 1.5 ml of elution buffer (containing 500 mM imidazole), and again equilibrated with 1.5 ml buffer A. Then, the protein sample was applied to the column, washed with 5 column volumes of buffer A, and eluted with 1.5 ml each of imidazole of 10 to 500 mM. Protein content and purity of collected fractions were analyzed by running samples by SDS-PAGE using 12% (vol/vol) polyacrylamide gels. Subsequently, the elution fractions with the highest content of purified protein were combined and dialyzed overnight against 100 mM sodium acetate buffer (pH 5.4) as described previously (39).

Substrates (10 mg/ml) were incubated with excess and minimal amounts of LpGH32 using 100 mM citric acid buffer at pH 4.0 and pH 5.0. Reaction volumes (100 μl) were supplemented with 0.01% (wt/vol) NaN₃ to prevent microbial growth, and incubations were carried out at 37°C for 40 h. Product formation through GH32 enzyme activity was checked by TLC and HPAEC-PAD.

All growth experiments are biological triplicates; the OD₆₀₀ values are expressed as averages. Medium without bacterial inoculation was used to obtain blank values during OD₆₀₀ measurements. Differences in the growth results between L. salivarius W57 and L. paracasei W20 in single and coculture were assessed by running nonparametric unpaired t tests. Tests yielding P values of <0.05 were considered significantly different.

The putative fructose and fructan utilization gene clusters of L. paracasei W20 were assigned the following accession numbers: fructose utilization, MH035726; fructan utilization, MH047828. The putative fructose and sucrose utilization gene clusters of L. salivarius W57 were assigned the following accession numbers: fructose utilization, MH047826; sucrose utilization, MH047827.