Selective carbohydrate utilization by lactobacilli and bifidobacteria



Douwe van Sinderen, Room 4.05, Department of Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Western Road, Cork, Ireland. E-mail:



To evaluate the ability of specific carbohydrates, including commercially available products, to support the growth of representatives of two well-known groups of gut commensals, namely lactobacilli and bifidobacteria.

Methods and Results

Sixty-eight bacterial strains, representing 29 human-derived lactobacilli and 39 bifidobacteria (both human- and animal-derived), were tested for their ability to metabolize 10 different carbohydrates. Analysis of growth and metabolic activity was performed using a combination of diagnostic parameters, such as final OD600, final pH, fermentation end products and growth rate.


The data assembled in this study provide significant complementary and comparative information on the growth-promoting properties of a range of carbohydrates, while also investigating interspecies differences between lactobacilli and/or bifidobacteria with regard to their carbohydrate utilization abilities. Galacto-oligosaccharides (GOS) and lactulose were shown to support the most favourable growth characteristics, whereas relatively poor growth of lactobacilli and bifidobacteria was observed on inulin, maltodextrin and polydextrose. GOS/inulin (9 : 1) and fructo-oligosaccharides (FOS)/inulin mixtures supported mostly similar growth abilities to those obtained for GOS and FOS, respectively. Microbial consumption of GOS, as determined by high-performance anion-exchange chromatography with pulsed amperometric detection, was evident for both lactobacilli and bifidobacteria.

Significance and Impact of the Study

These results may allow for the rational prediction of lactobacilli and/or bifidobacteria to be used in conjunction with prebiotics, such as GOS, as synbiotics.


Since the pioneering work of Élie Metchnikoff advocated the consumption of fermented milk to eradicate putrefactive intestinal bacteria (Metchnikoff 1908), there has been an increased interest in the potential health benefits of probiotics. Currently, the most popular food-based strategy to (transiently) modulate the composition and/or metabolic activity of the intestinal microbiota, including bifidobacteria and lactobacilli, for the purpose of imparting beneficial effects, is via the dietary intake of ‘functional foods’ containing probiotics, prebiotics or their combination, synbiotics (Hoppu et al. 2001; Stanton et al. 2005; Macfarlane et al. 2008; Bosscher et al. 2009).

Prebiotic, a term first coined in 1995 by Gibson and Roberfroid, has been (re)defined to describe nondigestible dietary components that undergo selective colonic fermentation, ‘thus causing significant changes in the composition of the gut microflora with increased and reduced numbers of potentially health-promoting bacteria and potentially harmful species, respectively’ (Roberfroid 2007). Breast-fed infants harbour a colonic microbiota that is rich in bifidobacteria, a predominance, which is believed to be due to their ability to metabolize human milk oligosaccharides (HMOs) (Sela et al. 2008; Roger et al. 2010). This is believed to have beneficial effects on host health through the enhancement of defence mechanisms (i.e. antipathogenic properties) and the education and modulation of the immune system (Bode 2009; Bosscher et al. 2009; Fanaro et al. 2009; Fukuda et al. 2011; Kau et al. 2011; Fanning et al. 2012). In fact, a recently defined minimal human gut metagenome describes the bacterial functions involved in gut homoeostasis to include activities responsible for complex polysaccharide degradation, as well as the synthesis of short-chain fatty acids (SCFAs), vital amino acids and vitamins (Qin et al. 2010). Consequently, significant research efforts are currently focusing on the identification of oligosaccharides that can stimulate growth and/or metabolic activity of beneficial bacteria. Several prebiotic substrates, in particular fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin and lactulose, have already obtained scientific credibility due to their inclusion in human trials where they have demonstrated prebiotic effects (Menne et al. 2000; Kolida et al. 2002; Bosscher et al. 2006; Davis et al. 2010; Veereman-Wauters et al. 2011). For example, Fanaro et al. (2009) demonstrated that the supplementation of infant formula with 5 g l−1 GOS resulted in an increase in bifidobacterial numbers isolated per gram of stool. Ben et al. (2008) also demonstrated that the addition of low levels of GOS (0·24 g 100 ml−1) to the formula of infants significantly increased the numbers of both intestinal bifidobacteria and lactobacilli relative to infant formula without GOS, following 3 months of feeding, while no significant differences were observed between the GOS formula- and human milk-fed groups. A recent study by Maathuis et al. (2012) demonstrated the prebiotic attributes of a purified Vivinal GOS formulation in an in vitro colon model. The authors observed an increase in numbers of lactobacilli and bifidobacteria as well other beneficial bacteria, with a concomitant decrease in numbers of members of the genera Bacteroides, Prevotella and Lactococcus.

In the present study, we evaluated the ability of specific carbohydrates, including commercially available products, to support the growth of representatives of two well-known groups of gut commensals, namely lactobacilli and bifidobacteria, and analysed strain diversity in this regard. The data gathered in this study provide a significant amount of complementary and comparative information on the growth-promoting properties of a range of carbohydrates, while also investigating interspecies differences between lactobacilli and/or bifidobacteria with regard to their carbohydrate utilization abilities.

Materials and methods

Bacterial strains, media and culture conditions

Sixty-eight strains representing a variety of mostly human-derived lactobacilli and bifidobacteria (representing 21 and 12 different species, respectively) were included in this study (Table 1). For inoculum preparation and storage purposes, the Lactobacillus strains were cultivated anaerobically in Lactobacilli de Man-Rogosa and Sharpe (LMRS; Difco™, France) broth at their optimal growth temperature (30 or 37°C; specified in Table 1), from stock cultures. Cells from an overnight LMRS culture were then subcultured (1%, v/v) into fresh LMRS and incubated anaerobically for a further 16 h, after which they were collected by centrifugation at 5000 g for 5 min, washed twice with phosphate-buffered saline (PBS), before a growth experiment was undertaken (see below). Bifidobacterial strains were grown under anaerobic conditions in Reinforced Clostridium Medium (RCM) (Oxoid, Hampshire, England) at 37°C, from stock cultures. Cells from an overnight RCM culture were then subcultured (1%, v/v) into fresh RCM and incubated anaerobically for an additional 16 h before use in a growth experiment (see also below). The overnight cultures were inoculated into modified MRS (mMRS) medium made from first principles (de-Mann et al. 1960): trypticase peptone: 10 g l−1; granulated yeast extract: 2·5 g l−1; tryptose: 3 g l−1; K2HPO4: 3 g l−1; KH2PO4: 3 g l−1; triammonium citrate: 2 g l−1; pyruvic acid: 0·2 g l−1; cysteine HCl: 0·3 g l−1; Tween-80: 1 ml; MgSO4.7H2O: 0·575 g l−1; MnSO4.4H2O: 0·12 g l−1; FeSO4.7H2O: 0·034 g l−1. The pH of the medium was adjusted to 6·8 before autoclaving (121°C for 15 min). This mMRS medium is a complex growth medium, which does not support the growth of any of the strains in the absence of a supplemented carbohydrate. Solid LMRS medium was prepared by the addition of 1·5% (w/v) agar (Oxoid, Basingstoke, UK) to the LMRS broth. The purity of the lactobacilli and bifidobacterial cultures was routinely monitored by plating on LMRS agar or Reinforced Clostridium agar (RCA) (Oxoid), respectively. All strains were maintained at −80°C in the appropriate medium supplemented with 20% (v/v) glycerol as a cryoprotectant.

Table 1. Carbohydrate utilization by strains of Lactobacillus and Bifidobacterium
StrainGrowth Temp (°C)GlucoseLactoseLactuloseMalt-odextrinPolyde-xtroseFructo-oligosa-ccharidesFructo-oligosa-ccharides /inulinInulinGalacto-oligosac-charides /InulinGalacto-oligosac-charidesLactobacilli de Man-Rogosa and Sharpe /Reinforced Clostridium MediummMRS
  1. ATCC, The American Type Culture Collection; CCUG, Culture Collection of the University of Goteborg; CIP, Collection de l'Institut Pasteur; CRL, CERELA culture collection; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; JCM, Japan collection of micro-organisms; LMG, Belgian co-coordinated collection of micro-organisms; NCDO, National collection of dairy micro-organisms; NCFB, National collection of food bacteria; NCIMB, National collection of industrial and marine bacteria; NCTC, National collection of type cultures; UCC, University College Cork, Collection.

  2. a

    Strains isolated from commercial products. Lactobacillus acidophilus LA-5 and Bif. animalis subsp. lactis Bb-12 were obtained from a commercial product containing both strains. Bb-12 and LA-5 are registered trademarks of Chr. Hansen A/S. A minus sign (−) indicates that final OD600 <0·3; + indicates final OD600 = 0·3–0·5; ++ indicates final OD600 >0·5–0·8; and +++ final OD600 >0·8.

Lactobacillus acidophilus ATCC435637++++++++++++++++
Lact. acidophilus LA5®a37++++++++++++++++++++
Lact. acidophilus NCFMa37++++++++++++++++++++
Lactobacillus amylovorus DSM2055237+++++++++++++++++++++
Lactobacillus antri DSM1604137+++++++++++++++++++
Lactobacillus brevis DSM2005430++++++++
Lactobacillus bulgaricus 37+++++++++
Lactobacillus casei DN-144-001a37++++++++++++++++++++++
Lact. casei Shirotaa37+++++++++++++++++++++++++
Lactobacillus curvatus NCDO273930++++++
Lactobacillus delbrueckii subsp. lactis DSM2007337++++++++++++++++++
Lactobacillus fermentum CECT5716a30++++++++++++++++++
Lact. fermentum DSM2005530+++++++
Lactobacillus gasseri CR15937+++++++++++++++++++
Lact. gasseri DW00137++++++++++++++++++++++
Lactobacillus gastricus DSM1604537++++++++++++++++++++++
Lactobacillus johnsonii LA-1a37+++++++++++++++++
Lactobacillus kalixensis DSM1604337++++++++++++++++++++++
Lactobacillus oris DSM486437+++++++++++++++++++++
Lactobacillus parabuchneri DSM570730++++++++++++++++++
Lactobacillus paracasei CRL43137++++++++++++++++++++++++
Lactobacillus plantarumNCDO32630+++++++
Lactobacillus reuteri ATCC 5573037+++++++++++++++++++++
Lact. reuteri DSM 1793837+++++++++++++++++++++
Lact. reuteri DSM2001637+++++++++++++++++++++++
Lact. reuteri DSM2005337++++++++++++++++++++
Lactobacillus rhamnosus GG®a37+++++++++++++++++++++++
Lactobacillus sakei DSM2010030++++++++++++++++
Lactobacillus ultunensis DSM1604737++++++++++++++++++
Bifidobacterium adolescentis CIP64·6137+++++++++++++++++++++++++++++
Bif. adolescentis DSM2008337+++++++++++++++++++++++
Bif. adolescentis LMG1050237++++++++++++++++++++++++++++++
Bif. adolescentis NCFB220437+++++++++++++++++++++++++
Bif. adolescentis NCFB222937+++++++++++++++++++++++++++
Bifidobacterium animalis DSM2010537+++++++++++++++++++++++
Bif. animalis JCM2009737++++++++++++++++++++++++
Bif. animalis subsp. lactis Bb12®a37++++++++++++++++++++++++++++++
Bif. animalis subsp. lactis Bl818a37+++++++++++++++++++++++++++++++
Bifidobacterium bifidum LMG1104137+++++++++++++++++++++++++++
Bif. bifidum NCIMB881037++++++++++++++++++++++++++++
Bifidobacterium breve CCUG4387837+++++++++++++++++
Bif. breve JCM701737+++++++++++++++++++++
Bif. breve JCM701937+++++++++++++++++++++++++++++++
Bif. breve NCFB1181537+++++++++++++++++++++++
Bif. breve NCFB225737++++++++++++++++++++++++++++++
Bif. breve NCFB225837+++++++++++++++++++++++++++
Bif. breve NCIMB881537+++++++++++++++++++++++++++
Bif. breve UCC200337+++++++++++++++++++++++++++
Bif. breve Yakulta37++++++++++++++++++++++++++
Bifidobacterium dentium NCFB284337+++++++++++++++++++++++++++++
Bifidobacterium globosum JCM582037++++++++++++++++++++
Bif. globosum JCM709237++++++++++++++++++++
Bifidobacterium infantis NCDO220537++++++++++++++++++++++++++++
Bifidobacterium longum CCUG1513737++++++++++++++++++++++++++++
Bif. longum CCUG3069837+++++++++++++++++++++++++
Bif. longum CIP64·6337++++++++++++++++++++++++++++++
Bif. longum JCM705037++++++++++++++++++++++++++
Bif. longum JCM705237++++++++++++++++++++++++++++
Bif. longum JCM705337+++++++++++++++++++
Bif. longum JCM705637++++++++++++++++++++++
Bif. longum NCIMB880937++++++++++++++++++++++++
Bif. longum Onkena37++++++++++++++++++++++++++++
Bif. longum/infantis CCUG1815737++++++++++++++++++++++++++++
Bifidobacterium pseudocatenulatum LMG1050537+++++++++++++++++++++++++
Bif. pseudocatenulatum NCIMB 881137++++++++++++++++++++++++++++
Bifidobacterium pseudolongum DSM2009537++++++++++++++++++++++++++
Bif. pseudolongum NCIMB224437+++++++++++++++++++++
Bifidobacterium thermophilum JCM702737++++++++++++++++++++++++++++

Sources, reported purity and preparation of carbohydrate solutions

The commercial sources of the carbohydrates tested were as follows: glucose (served as a positive control as it supports good growth of all strains used in this study), lactose and lactulose were obtained from Sigma-Aldrich (Steinheim, Germany); maltodextrin (potato-derived) from Cargill-Cerestar; polydextrose (Litesse®; 90% pure, 10% glucose and sorbitol) obtained from Danisco, Wellingborough, UK; FOS (Raftilose P95®; 5% glucose, fructose and sucrose), inulin (Raftiline HP, 100% pure) and FOS/inulin mixture (Synergy 1; ratio 50 : 50 FOS : inulin; contains 8% glucose, fructose and sucrose) sourced from Orafti, Tienen, Belgium; GOS (which represents purified Vivinal® GOS; contains 97% GOS, 0·7% galactose, 1·6% glucose and 0·7% lactose) and GOS/inulin [ratio 9 : 1, GOS : inulin (Raftiline HP)] supplied by FrieslandCampina Domo, Amersfoort, the Netherlands. For the preparation of GOS, Vivinal® GOS (typical composition: 59% GOS, 21% lactose, 19% glucose, 1% galactose) was enzymatically treated with a lactase to hydrolyse the lactose into glucose and galactose, after which the monosaccharides were removed by nanofiltration.

A 5% stock solution of each of the carbohydrates to be tested was prepared by dissolving the particular sugar in distilled water. The obtained stock solution was then sterilized by membrane filtration, using ministart filters (pore size, 0·45 μm; Sartorius AG, Göttingen, Germany), and stored at 4°C. Inulin proved difficult to dissolve and required heating to 50°C prior to filter sterilization.

Evaluation of bacterial growth on oligosaccharides

Carbohydrate-dependent growth by a given culture was evaluated in 96-well microtitre plates. Overnight cultures (10 ml) of Lactobacillus strains were prepared as described above. An aliquot (representing 1 × 107 CFU ml−1) was inoculated into 5 ml of mMRS medium supplemented with 0·5% final concentration (v/v) of an individual carbohydrate solution, representing the sole carbon source in the medium. The mMRS without any supplemented carbohydrate source and commercially obtained LMRS (Difco) were used as negative and positive controls, respectively. A similar procedure was employed to test growth potential and characteristics of the bifidobacterial strains, albeit that they were not subjected to a wash step. In this case, the freshly prepared mMRS medium was supplemented with 0·5% final concentration (v/v) of an individual carbohydrate solution plus 0·05% (v/v) l-cysteine HCl (a redox potential-lowering compound). For the bifidobacteria, mMRS without any added carbohydrate and commercially obtained RCM (Reinforced Clostridial Medium; Oxoid) were used as negative and positive controls, respectively.

All cultures were incubated under anaerobic conditions in a modular atmosphere-controlled system (Davidson and Hardy Ltd, Dublin, Ireland), at the desired temperature (30 or 37°C; as indicated in Table 1) for 24 h. The optical density at 600 nm (final OD600) was monitored using a Powerwave™ Microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) in conjunction with Gen5™ Microplate software for data collection and analysis. The plate reader was run in discontinuous mode, with absorbance readings captured at 30-min intervals, and each read was preceded by 10-s shaking step at medium speed to mix the culture. Cultures were grown in biologically independent duplicates, and the resulting growth data were expressed as the mean of these replicates.

Maximum growth rate determinations and pH determinations

Growth rates (μ) were calculated for strain–carbohydrate combinations that demonstrated good growth (final OD600 >0·8) according to a previously published formula (Rada et al. 2008): μ = (Inx2 − Inx1)/(t2 − t1), where x2 and x1 were absorbance values measured within the exponential phase of growth at times t2 and t1, respectively. The pH measurements were performed on the culture medium following 24 h of growth using a standard pH meter.

Analysis of GOS or FOS utilization by HPAEC-PAD

In order to demonstrate possible selective metabolic utilization of particular GOS or FOS components by lactobacilli and bifidobacteria, the carbohydrate composition profile of postfermentation cell-free supernatants was determined using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), employing a Dionex ICS-3000 system (Dionex, Sunnyvale, CA, USA). Oligosaccharide fractions (25 μl aliquots) were separated on a CarboPac PA1 (Dionex) analytical-exchange column with dimensions 250 mm × 4 mm with a CarboPac PA1 guard column (Dionex) with dimensions 50 mm × 4 mm and a Dionex ED40 detector in the pulsed amperometric detection (PAD) mode. Elution was performed at a constant flow rate of 1·0 ml min−1 at 30°C using the following eluents for the analysis: (A) 200 mmol l−1 NaOH, (B) 100 mmol l−1 NaOH, 550 mmol l−1 sodium acetate and (C) Milli-Q water. The following linear gradient of sodium acetate was used: 100 mmol l−1 NaOH: 0–50 min, 0 mmol l−1; 50–51 min, 16 mmol l−1; 51–56 min, 100 mmol l−1; 56·1–61 min, 0 mmol l−1. The obtained chromatographic profile of nonfermented GOS or FOS was used for comparison to evaluate GOS or FOS utilization, respectively, from samples, and the Chromeleon software Ver. 6·70 (Dionex Corp.) enabled the integration and evaluation of the chromatograms.

Bioinformatics used for heat maps and clustering of data

Clustering of the data relating to the final OD600 of strains grown on various carbon sources was performed in order to search for potential correlations between strains or species and the acquired information on growth/metabolic characteristics. Hierarchical clustering was performed using the complete linkage clustering method implemented in Genesis software and employing default settings (Sturn et al. 2002).


Comparative growth on different carbon sources

All carbohydrates included in this study (except glucose and lactose, which were included as positive controls) were selected for this in vitro study as they are deemed to possess prebiotic properties and are thus expected to support growth/metabolism of beneficial gut commensals.

Growth results obtained for strains of Lactobacillus

All 29 tested Lactobacillus strains demonstrated appreciable growth (i.e. reached a final OD600 >0·7) on mMRS supplemented with glucose, as compared to 24 strains that were capable of equivalent growth on lactose (Tables 1 and S1). Distinctive growth profiles were produced for the lactobacilli when the remaining eight carbohydrates were used as the sole carbon/energy source in mMRS. To exemplify the strain dependency of the obtained profiles, the four tested Lactobacillus acidophilus species (Lactobacillus johnsonii LA-1; Lact. acidophilus ATCC4356; Lact. acidophilus LA5; and Lact. acidophilus NCFM) all grew well in the presence of glucose and lactose. They are unable to efficiently utilize maltodextrin, polydextrose or inulin, but do possess variable utilization capabilities for lactulose, FOS, FOS/inulin, GOS/inulin (9 : 1) and GOS. Growth of Lactobacillus casei Shirota was supported (final OD600 >0·3) by all the tested carbohydrates (as did the other commercial probiotic lactobacilli strains that are used as active ingredients in various functional foods, e.g. Lact. casei DN-144-001 and Lactobacillus rhamnosus GG), and for this reason was chosen as a representative strain to highlight the observed growth characteristics across the 10 carbohydrates (Fig. 1a). Lactulose, a semisynthetic disaccharide produced by the isomerization of lactose (Panesar and Kumari 2011), supported intermediate to good growth for 15 of the lactobacilli strains tested. From the in vitro growth data obtained, it is evident that maltodextrin, polydextrose, FOS, FOS/inulin and inulin are rather poor in vitro growth substrates for the majority of the tested lactobacilli. Just four (Lact. acidophilus LA5; Lact. acidophilus NCFM; Lact. casei DN-144-001; and Lactobacillus paracasei CRL431) of the 29 lactobacilli tested were capable of growth (final OD600 >0·5) in the presence of FOS, while Lact. paracasei CRL431 was the only tested Lactobacillus strain capable of reaching a higher final OD600 on FOS compared to that obtained for GOS (1·380 ± 0·093 vs 0·865 ± 0·078, respectively). Another interesting observation is that FOS/inulin performs marginally better as a growth substrate as compared to its individual components, FOS and inulin (e.g. Lactobacillus gasseri DW001; Fig. 1b). Lactobacillus casei Shirota reached a final OD600 on GOS of 1·454 ± 0·499, GOS/inulin (9 : 1) of 1·094 ± 0·117 and inulin of 0·397 ± 0·023. These final OD600 values suggest that this strain is utilizing the GOS component of GOS/inulin as it displays poor growth on inulin alone. Lactobacillus casei Shirota was shown to reach a final OD600 of 0·474 ± 0·033 when grown on FOS, and together, these findings indicate that GOS imparts a superior growth-promoting ability to 23 of the tested lactobacilli strains as compared to FOS or inulin (additional examples of this growth are observed for Lact. gasseri DW001, Lactobacillus bulgaricus and Lactobacillus antri DSM16041, and displayed in Fig. 1b–d).

Figure 1.

Final OD600 reached by Lacobacillus casei Shirota (a) grown on modified MRS supplemented with 0·5% of the relevant carbohydrates as the sole carbon source. Lacobacillus gasseri DW001 (b) reaches a higher final optical density on 0·5% fructo-oligosaccharides (FOS)/inulin (50 : 50) compared to 0·5% FOS and 0·5% inulin when used as separate supplements. Carbohydrate utilization fingerprint of Lacobacillus bulgaricus (c) and Lacobacillus antri DSM16041 (d), grown separately on mMRS supplemented with either 0·5% of glucose, FOS, inulin or galacto-oligosaccharides (GOS) as the sole carbon source, highlighting excellent growth-promoting properties of GOS as compared to FOS or inulin.

Growth results obtained for strains of Bifidobacterium

One major difference between lactobacilli and bifidobacteria is that the majority of the bifidobacterial strains were capable of reasonable to good growth (final OD600 ≥0·5) on eight of the 10 tested substrates, representing a higher number as compared to the lactobacilli, where the latter were able to support reasonable to good growth (final OD600 ≥0·5) on just four carbohydrates (Table 1, S1 and S2). Lactulose, maltodextrin, FOS, GOS and the GOS/inulin (9 : 1) mixture supported the most prolific growth abilities, while inulin and polydextrose appeared to be rather poor substrates for bifidobacterial growth (Table 1). All bifidobacterial strains tested were capable of growth reaching a final OD600 ≥0·7 in mMRS supplemented with glucose, lactose or lactulose. This is exemplified by Bifidobacterium longum JCM7050 that reaches a final OD600 of 1·320 ± 0·108 on glucose, 1·414 ± 0·132 on lactose and 1·203 ± 0·001 on lactulose (Fig 2a and Table S2). Merely 11 of the bifidobacterial strains tested demonstrated appreciable levels of growth (final OD600 >0·5) on polydextrose, while inulin appears to be a rather poor growth substrate for most bifidobacterial strains tested, supporting the growth of just nine strains to a final OD600 >0·5. Just two strains, Bifidobacterium breve JCM7019 and Bif. longum CIP64·63, exhibited good growth (final OD600 values of 1·009 ± 0·031 and 0·840 ± 0·159, respectively) on inulin. Growth of the bifidobacterial strains on maltodextrin, FOS, FOS/inulin, GOS/inulin (9 : 1) and GOS all produced easily interpretable and differentiating growth profiles. These profiles were, again, as expected and rather strain dependent as illustrated by the nine Bif. breve strains included in this study. All strains demonstrated variable growth on these carbohydrates ranging from no/very poor growth (-) to good growth (+++) depending on the substrate and strain involved (Table 1). Thirty-two strains exhibited reasonable to good growth (final OD600 >0·5) in maltodextrin (an oligosaccharide consisting of d-glucose linked in chains of varying lengths), while 34 of the bifidobacterial strains showed good growth in the presence of FOS. This is mirrored in the growth-sustaining properties observed for the FOS : inulin mixture (Synergy 1), where 30 of the bifidobacterial strains exhibited similar growth abilities. Finally, 36 strains grew to high final optical densities in GOS, and 31 strains in GOS/inulin (9 : 1). As an example, using the same representative strain as above, Bif. longum JCM7050 reached a final OD600 on GOS of 1·000 ± 0·136, GOS/inulin (9 : 1) of 0·965 ± 0·175 and inulin of 0·407 ± 0·069 (Fig. 2a). These final OD600 values again highlight this strain's preference for the GOS components of GOS/inulin as it does not grow on inulin alone, but does reach a similar final OD600 when grown in the presence of the GOS or GOS/inulin (9 : 1). Another strain that illustrates that GOS is on par with FOS, with regard to its growth-sustaining abilities, is Bif. longum CCUG18157, which reached a final optical density of 0·950 ± 0·052 on FOS and an equally high final OD600 of 0·916 ± 0·201 on GOS (Fig. 2b).

Figure 2.

(a) Final OD600 reached by Bifidobacterium longum JCM7050 grown on mMRS supplemented with 0·5% of the relevant carbohydrates as the sole carbon source. (b) Final OD600 reached by Bif. longum/infantis CCUG18157 grown on mMRS supplemented with 0·5% of glucose, fructo-oligosaccharides (FOS), inulin or galacto-oligosaccharides (GOS) as the sole carbon source, again highlighting excellent growth-promoting properties of GOS which is on par with FOS and better than inulin for the majority of strains.

Final pH values and growth rates

The pH value of the growth medium following 24 h of growth was determined, which demonstrated the expected inverse correlation between the final OD600 value reached for a particular strain and the final pH of the corresponding medium in which the strain was grown: the higher the final OD600 value obtained, the lower the final pH of the medium (Tables S3 and S4). Lactobacillus johnsonii LA-1 exemplifies the fact that in the presence of maltodextrin this strain reaches a final OD600 of 0·115 ± 0·020 and the pH of the medium is 6·48; however, in the presence of a suitable growth substrate such as glucose, the strain reaches a final OD600 of 1·374 ± 0·030 while the pH decreases to 4·14. This was indeed found for the majority of the strains, with just a few exceptions. Such an exception is illustrated by Lact. antri DSM16041, which in the presence of GOS/inulin (9 : 1) and GOS reaches a final OD600 of 1·228 ± 0·142 and 1·240 ± 0·045 and a pH of 6·41 and 5·20, respectively.

Growth rates (μ) (Tables S5 and S6) are critical for the evaluation of suitable prebiotic carbohydrates as they establish the substrate requirements and specificities of an individual strain. These findings provide valuable insights into how a particular prebiotic may influence a micro-organism's ability to compete with other bacteria in the colon. A wide variety of growth rates were observed across the different strain and carbohydrate combinations with values ranging from 0 (when no growth on a particular carbohydrate was observed) to a maximum of 0·886 h−1 (Lactobacillus reuteri ATCC55730 grown on 0·5% glucose) for the lactobacilli, and a maximum of 1·150 h−1 (Bif. longum CCUG30698 grown on 0·5% glucose) for the tested bifidobacteria. Growth rates were compared within the results of a given test strain, rather than comparatively between strains, due to the large variability in growth rates with any carbon source between strains. Notably, 14 Lactobacillus strains grew more rapidly on GOS (when ignoring growth on glucose and lactose) than on any other tested carbohydrate substrate. The same trend was observed for 17 bifidobacterial strains which also achieved a higher growth rate on GOS (disregarding growth rates on glucose and lactose) as compared to other sugars, while only two bifidobacterial strains exhibited faster growth on FOS when compared to other tested carbohydrates (again with the exception of glucose and lactose).

Evaluation of GOS degradation using HPAEC-PAD

Microbial consumption of GOS was evident for most lactobacilli and bifidobacteria with no apparent selectivity for the different GOS fractions as quantified by HPAEC-PAD. The HPAEC profile of the reference GOS mixture indicated a high abundance of different oligosaccharides with different retention times in the GOS mixture (Fig. 3a). The carbohydrate identity of three peaks, that is, those corresponding to glucose, lactose and galactose, was established on the basis of reference compounds – glucose and/or galactose (both of these carbohydrates have a retention time of approximately 5·4 min under the experimental conditions used, while lactose has a retention time of ~11 min) (Fig. 3 I). Microbial consumption of GOS was evident for 23 of the lactobacilli, as indicated by the obvious reduction in GOS peak size and number. Due to the nonmolar response of the HPAEC detector, no quantitative conclusions on the rate of degradation of GOS and the consumption of its constituents can be drawn from these results; however, we can demonstrate the differential utilization of GOS components in the case of three different strains (Fig. 3). A representative Lactobacillus strain, Lact. casei DN-144-001, is able to utilize individual GOS components to achieve a final OD600 of 1·193 ± 0·018 (Fig. 3 II). Interestingly, eight of the 29 tested lactobacilli (Lactobacillus amylovorus DSM20552; Lactobacillus delbrueckii subsp. lactis DSM20552; Lactobacillus kalixensis DSM16043; Lactobacillus fermentum CETC5716; Lact. reuteri DSM20016; Lact. reuteri DSM17938; Lact. acidophilus NCFM; and Lact. johnsonii LA-1) each exhibited galactose accumulation in the culture broth when GOS was the sole carbohydrate source (as observed in the HPAEC chromatograms displayed for Lact. amylovorus DSM20552 in Fig. 3 III). This would suggest that the rate of galactose release in the medium was higher than the rate of galactose uptake and/or intracellular galactose metabolism, leading to a net accumulation in the medium. Three bifidobacterial strains (Bif. breve CCUG43878; Bifidobacterium globosum JCM7092; and Bifidobacterium pseudolongum NCIMB2244) did not exhibit good growth in the presence of GOS. However, the remaining 36 strains were all shown to grow well on GOS as the sole carbon source, as illustrated by Bif. breve NCFB11815 (Fig. 3 IV).

Figure 3.

(I) The high-performance anion-exchange chromatography (HPAEC) profile of the reference galacto-oligosaccharides (GOS) mixture indicated a high abundance of different oligosaccharides with different retention times (II) Qualitative GOS degradation pattern for Lacobacillus casei DN-144-001 which grows well on GOS. This HPAEC chromatogram highlights the ability of the strain to utilize individual GOS components to achieve a final OD600 of 1·193. (III) Lactobacillus amylovorus DSM20552 demonstrates galactose accumulation in the cultural broth. (IV) Qualitative GOS degradation pattern for Bifidobacterium breve NCFB11815 which also grows well on GOS. This HPAEC chromatogram highlights the ability of the strain to utilize individual GOS components to achieve a final OD600 of 0·752.

Correlations and clustering

In the case of lactobacilli, even though no absolute correlations were observed, some general tendencies could be discerned. Carbohydrate metabolism has been recognized as being encoded by hypervariable regions in lactobacilli and these areas are commonly related to niche adaptation or survival (Berger et al. 2007). Based on hierarchical clustering, three major divisions (Fig. 4a, clusters Lba, Lbb and Lbc) can be distinguished among the Lactobacillus strains. The strains were assigned to clusters that occurred at the first major branching point of the dendrogram. The first of these (Fig. 4a, cluster Lba) contains five of the six strains that exhibited optimal growth at 30°C; four strains (Lactobacillus curvatus NCDO2739; Lact. fermentum DSM20055; Lactobacillus brevis DSM20054; and Lactobacillus plantarum NCDO326) were clustered together, linked by their sole ability to utilize glucose. Members of clusters Lbb and Lbc are grouped based on their ability to utilize particular carbohydrates, namely lactose and GOS, and may support the notion that adaptation to growth in nutrient-rich environments is the principal driving force behind the duplication or acquisition of genes during the evolutionary process (Ochman 2005; Makarova et al. 2006; Makarova and Koonin 2007).

Figure 4.

(a) Heat map and cluster analysis of final optical density (final OD600) data received following 24 h of growth of 29 different Lactobacillus strains (a), and 39 different bifidobacterial strains (b), grown on a variety of carbohydrate substrates as the sole carbon source. Colour range is correlated with growth rate of a given sample (ranging from OD600 0 to 2·0 or 0 to 1·5 for the Lactobacilli and Bifidobacteria respectively, which correlates in colour from black to white). The result of the cluster analysis is indicated on the left of the figure as a dendrogram. Each horizontal row corresponds to one strain, and each column represents a carbohydrate. The colour gradient, which goes from black to white, depicts the level of growth achieved by the strain.

Based on hierarchical clustering, two major divisions are evident for the tested bifidobacteria (Fig. 4b, clusters Bifa and Bifb), and strains were again assigned to clusters that occurred at the first major branching point of the dendrogram. This variation suggests environmental selection or adaptation in carbohydrate metabolism, even the GIT itself is a succession of varying habitats corresponding to different compartments (e.g. oral cavity, the stomach and the small/large intestine). Based on this, we observed different preferences of desirable substrates among strains isolated from the infant gut (Fig. 4b). Cluster Bifa contains 14 of the strains, including six strains that were originally isolated from animal faecal samples.


Recent studies indicate that diet alterations can dramatically change the overall composition and structure of endogenous microbial communities in the gut (Muegge et al. 2011). The work described here represents a comparative analysis of an extensive array of in vitro growth and metabolic characteristics for a diverse collection of lactobacilli and bifidobacterial strains. These strains have been obtained from various culture collections and commercial sources. In terms of supporting growth of the tested bacteria, it was shown that GOS [including GOS/inulin (9 : 1)] is on par with, or better than, the prebiotic FOS, as well as other commercially available prebiotics, such as lactulose (in the case of the lactobacilli) and a mixture of FOS/inulin for both genera. This notion is re-enforced when one considers that the preparations of FOS and FOS/inulin still contain considerable amounts of mono- and disaccharides (glucose and fructose), which may have caused some exaggeration of the observed growth abilities. Previous in vivo studies have demonstrated that both lactobacilli and bifidobacteria can metabolize GOS (Ben et al. 2008; Konstantinov et al. 2008; Fanaro et al. 2009; Walton et al. 2012). These studies adopted a commercially available galacto-oligosaccharide mixture (Vivinal® GOS) containing digestible monosaccharides (i.e. galactose and glucose) and the disaccharide lactose, corresponding to 41% of dry matter content in addition to GOS. These digestible mono- and disaccharides are expected to be taken up in the human small intestine and therefore do not reach the large intestine for microbial fermentation.

From this study, microbial consumption of GOS was evident for lactobacilli and bifidobacteria with no apparent selectivity for the different GOS components. GOS is typically synthesized by reverse hydrolysis of lactose by the enzyme β-galactosidase (lactase), which is generally derived from a member of the Bacillus genus or from Kluyveromyces lactis or Streptococcus thermophilus (reviewed by Park and Oh 2010). A recent study demonstrated the use of recombinant β-galactosidase from Lact. plantarum WCFS1 to synthesize the prebiotic galacto-oligosaccharide (GOS) that is predicted to enhance the growth of lactobacilli in vivo. This prediction was attributed to the formation of a GOS mixture that contains an increased (relative to other analysed GOS) level of β-(1→6)-linked oligosaccharides, which supports the growth of bifidobacteria in mixed culture populations (Iqbal et al. 2010). The GOS used in this study is composed mainly of β-(1→4)-linked oligosaccharides (Coulier et al. 2009), and specific GOS mixtures may therefore possess more targeted specificities with regard to growth-promoting activity (e.g. by adjusting the amounts and ration of β-(1→4)- and β-(1→6)-linked oligosaccharides).

It is worth noting that although not all the strains have the ability to utilize (the majority of the) GOS components, this would not rule out the ability of intestinal communities to metabolize GOS, especially in the case of lactobacilli where they may cross-feed in the GIT environment. This notion is supported by the work of Xu et al. (2007), who found that fermentation of complex carbohydrates in the gut is due to the combined action of several bacteria. Our study shows that particular prebiotics promote the growth of certain strains/species, but not others. This is an important finding when deducing the prebiotic capabilities of various carbohydrates in order to enhance the gut microbiota diversity.

The data presented here illustrate that the success of prebiotics relies on the exploitation of differences in substrate preferences and competitive abilities among different members of the gut microbiota. Indeed, one has to keep in mind that pure culture models do not reflect the environmental experiences that confront bacteria in the mammalian host. Metabolic studies, such as the one described here, focus on the physiological implications of different oligosaccharide utilization in order to understand the bacteria. Our conclusions are at the moment based on a reasonably large number of investigated strains. Despite this, the number of strains is actually too small to decipher reliable ecological predictions and would require further, more extensive, investigations. The variations observed indicate niche-specific clustering of both bifidobacteria and lactobacilli and could have in vivo implications. Recently, elegant studies such as those employing in vitro colonic models (Maathuis et al. 2012) and in vivo clinical trials (Walton et al. 2012) have demonstrated that Vivinal GOS is bifidogenic while also increasing the numbers of Lactobacillus sp. These in vitro and in vivo studies highlight the potential for prebiotics to selectively enrich for probiotic strains and in doing so have the capacity to prevent pathogen colonization and promote gastrointestinal health. This research can pave the way towards a better understanding of the probiotic/prebiotic relationship through the facilitation of the rational design of prebiotics with a high degree of selectivity. In the current metagenomic and metatranscriptomic era, clinical trials involving prebiotic or symbiotic food formulations will undoubtedly unveil the beneficial changes in microbiota composition and activity.


This work was financially supported by FrieslandCampina. DvS and MO'CM (also a HRB postdoctoral fellow–Grant no. PDTM/20011/9) are members of the Alimentary Pharmabiotic Centre, which is a research centre funded by Science Foundation Ireland (SFI), through the Irish Government's National Development Plan (grant nos. 02/CE/B124 and 07/CE/B1368).