Food Microbiology 33 (2013) 262e270
`
`Contents lists available at SciVerse ScienceDirect
`
`Food Microbiology
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f m
`
`Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis
`isolates
`Daniel Garrido a,d, Santiago Ruiz-Moyano a,d, Rogelio Jimenez-Espinoza c, Hyun-Ju Eom b,d,
`David E. Block b,c, David A. Mills a,b,d, *
`a Department of Food Science and Technology, University of California Davis, 1 Shields Ave., Davis, CA 95616, USA
`b Department of Viticulture and Enology, University of California Davis, 1 Shields Ave., Davis, CA 95616, USA
`c Department of Chemical Engineering, University of California Davis, 1 Shields Ave., Davis, CA 95616, USA
`d Foods for Health Institute, University of California Davis, 1 Shields Ave., Davis, CA 95616, USA
`
`a r t i c l e i n f o
`
`a b s t r a c t
`
`Article history:
`Received 19 September 2012
`Received in revised form
`5 October 2012
`Accepted 5 October 2012
`Available online 22 October 2012
`
`Keywords:
`Bifidobacterium longum subsp. infantis
`Prebiotics
`Galactooligosaccharides
`b-Galactosidase
`
`Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in
`the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are
`commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details
`on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these
`dietary interventions. In this study the consumption of galactooligosaccharides was studied for 22
`isolates of Bifidobacterium longum subsp. infantis, one of the most representative species in the infant gut
`microbiota. In general all isolates showed a vigorous growth on these oligosaccharides, but consumption
`of larger galactooligosaccharides was variable. Bifidobacterium infantis ATCC 15697 has five genes
`encoding b-galactosidases, and three of them were induced during bacterial growth on commercial
`galactooligosaccharides. Recombinant b-galactosidases from B. infantis ATCC 15697 displayed different
`preferences for b-galactosides such as 40 and 60-galactobiose, and four b-galactosidases in this strain
`released monosaccharides from galactooligosaccharides. Finally, we determined the amounts of short
`chain fatty acids produced by strain ATCC 15697 after growth on different prebiotics. We observed that
`biomass and product yields of substrate were higher for lactose and galactooligosaccharides, but the
`amount of acids produced per cell was larger after growth on human milk oligosaccharides. These results
`provide a molecular basis for galactooligosaccharide consumption in B. infantis, and also represent
`evidence for physiological differences in the metabolism of prebiotics that might have a differential
`impact on the host.
`
`Ó 2012 Elsevier Ltd. All rights reserved.
`
`1. Introduction
`
`The Bifidobacterium genus is composed of Gram-positive strictly
`anaerobic rods, which are common inhabitants of the intestinal
`tract of humans (Lee and O’Sullivan, 2010). They are dominant in
`the infant gut microbiota, especially in breast-fed infants
`(Yatsunenko et al., 2012), where they can represent up to the 90% of
`the total bacteria in this environment (Boesten et al., 2011).
`
`Abbreviations: GOS, galactooligosaccharides; HMO, human milk oligosaccha-
`rides; FOS, fructooligosaccharides; LNT, lacto-N-tetraose; SCFA, short chain fatty
`acids.
`* Corresponding author. Department of Viticulture and Enology, University of
`California Davis, 1 Shields Ave., Davis, CA 95616, USA. Tel.: þ1 530 754 7821;
`fax: þ1 530 752 0382.
`E-mail address: damills@ucdavis.edu (D.A. Mills).
`
`0740-0020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
`http://dx.doi.org/10.1016/j.fm.2012.10.003
`
`Bifidobacteria still represent a significant proportion of the adult
`gut microbiota (Eckburg et al., 2005), however different species can
`be found in both environments (Mangin et al., 2006; Roger et al.,
`2010).
`Bifidobacteria show remarkable adaptations to use and metab-
`olize complex oligosaccharides as a carbon and energy source (Lee
`and O’Sullivan, 2010). In breast-fed infants, the main carbon sour-
`ces available for the developing intestinal microbiota are human
`milk oligosaccharides (HMO; (Kunz et al., 2000)) and certain bifi-
`dobacteria can gain access to N- and O-glycans in milk proteins or
`mucins (Garrido et al., 2012b; Ruas-Madiedo et al., 2008). Only
`a few bacterial species have been shown to use these substrates
`(Marcobal et al., 2010), and the molecular mechanisms involved in
`HMO consumption in bifidobacteria are beginning to be under-
`stood (Garrido et al., 2012a). In adults, diet delivers the intestinal
`microbiota a great variety of oligo- and polysaccharides, which are
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`resistant to enzymatic degradation in the intestinal lumen and
`therefore reach distal portions of the intestine. Different Bifido-
`bacterium species are capable of metabolizing complex oligosac-
`charides usually from plant origin such as cellodextrins and
`amyloses (Pokusaeva et al., 2011), raffinose (Dinoto et al., 2006),
`arabinooligosaccharides (Lagaert et al., 2010; Van Laere et al., 1997),
`xylooligosaccharides (Gilad et al., 2010), fructooligosaccharides and
`inulin (Omori et al., 2010; Perrin et al., 2001; Rossi et al., 2005),
`galactans and galactooligosaccharides (GOS; (Barboza et al., 2009;
`Goulas et al., 2009a; Hinz et al., 2005; O’Connell Motherway et al.,
`2011)) among several others.
`Several infant formulas are supplemented with FOS and GOS
`with the aim of replicating some of the beneficial effects of human
`milk, in special its bifidogenic effect (Bakker-Zierikzee et al., 2005;
`Brunser et al., 2006). GOS are synthetically produced by microbial
`b-galactosidases (Gosling et al., 2010), which under specific
`conditions can perform transglycosylation reactions with lactose as
`the starting material. These enzymes are widespread in bifidobac-
`teria, and some of them have remarkable yields in GOS synthesis
`(Hinz et al., 2004; Hung and Lee, 2002; Rabiu et al., 2001). GOS can
`have a degree of polymerization (DP) between 3 and 15 (Barboza
`et al., 2009) and are composed of galactose oligomers in b1-3/4/6
`linkages with a terminal glucose residue (Coulier et al., 2009;
`Gosling et al., 2010). These substrates have been extensively studied
`for their prebiotic status, promoting the growth of beneficial
`microorganisms such as bifidobacteria and lactobacilli (Andersen
`et al., 2011; Davis et al., 2011), therefore providing putative health
`benefits (Gibson et al., 2004). GOS structures resemble galactan
`chains found in plant oligosaccharides abundant in adult diets,
`which might explain how these microorganisms consume GOS.
`The mechanisms by which infant bifidobacteria are enriched by
`these prebiotics probably include oligosaccharide transporters and
`b-galactosidases specific for these substrates. In Bifidobacterium
`longum subsp. infantis ATCC 15697, two genes encoding solute-
`binding proteins (SBPs) from ABC transporters were specifically
`induced during growth on GOS (Garrido et al., 2011). b-galactosi-
`dases are widespread enzymes in bifidobacteria, and they display
`diverse substrate specificities (Goulas et al., 2009b). Two b-galac-
`tosidases in Bifidobacterium infantis HL96 have been previously
`studied (Hung and Lee, 2002; Hung et al., 2001) regarding their
`transglycosylation properties, and recently two b-galactosidases in
`the strain ATCC 15697 were shown to be active on different linkages
`found in HMO (Yoshida et al., 2012), and their activity likely
`complements b-hexosaminidases in this bacterium (Garrido et al.,
`2012c). Of increasing interest are also the metabolites produced
`after bifidobacterial fermentation of sugars, especially short chain
`fatty acids (SCFA) such as acetate and lactate as they represent one
`of the main protective mechanisms of bifidobacteria for its host
`(Fukuda et al., 2011). In this study we have characterized the
`consumption of GOS in a panel of B. infantis isolates from infant
`feces, and we further investigated the mechanisms involved in GOS
`degradation and metabolism in the type strain ATCC 15697.
`
`2. Materials and methods
`
`2.1. Microorganisms and media
`
`Strains used in this study (Supplementary Table 1), were ob-
`tained from the American Type Culture Collection (Manassas, VA),
`and the University of California Davis Viticulture and Enology
`Culture Collection (Davis, CA). De Mann, Rogose and Sharp (MRS)
`broth supplemented with 0.05% w/v L-cysteine (SigmaeAldrich, St.
`Louis, MO) was used for routine growth of B.
`infantis under
`anaerobic conditions (Coy Laboratory Products, Grass Lake, MI) at
`37 C in an atmosphere consisting of 5% carbon dioxide, 5%
`
`hydrogen, and 90% nitrogen. Chemically competent Escherichia coli
`BL21 Star and Top10 cells were obtained from Invitrogen (Carlsbad,
`CA), and transformants were cultured at 37 C in Luria Broth with
`50 mg/ml carbenicillin (Teknova, Hollister CA) when necessary.
`
`2.2. Consumption of GOS by B. infantis isolates
`
`Bifidobacterium isolates in Supplementary Table 1 were grown
`overnight in MRS and inoculated at 5% in modified MRS (mMRS),
`containing no carbon source, and supplemented with 0.05%
`cysteine (Sigma) and 0.5% commercial GOS (Purimune, GTC Nutri-
`tion, Golden, CO) or 0.5% lactose as a growth control. Growth was
`monitored using a PowerWave microplate spectrophotometer
`(BioTek Instruments, Winoosky, VT) at 37 C for 48 h, reading
`absorbance at 600 nm. Each experiment was done in triplicate, and
`controls with no carbon source and no bacteria were subtracted
`from growth values. Aliquots of the reactions (1 ml) were spotted in
`TLC Silica gel plates (Sigma). A mixture of n-propanol, acetic acid
`and water in a 2:1:1 ratio was used as solvent. Plates were dryed
`and sprayed with 0.5% a-naphthol and 5% H2SO4 in ethanol, and
`developed at 150 C for 10 min.
`
`2.3. Gene expression analysis
`
`For RNA extraction, B. infantis ATCC 15697 was grown on the
`chemically defined media Zhang-Block-Mills 1 (ZMB-1; (Zhang
`et al., 2009)) to which 2% w/v of glucose (Sigma), lactose (Sigma),
`GOS (GTC nutrition) or purified HMO (Ward et al., 2006) were
`added. Growth was monitored in a plate reader as described above.
`Cells at exponential phase were pelleted at 12,000  g for 2 min,
`resuspended in 1 ml of RNA later (Ambion, Austin, TX), stored at
`4 C overnight and then at 80 C until use. RNA extraction was
`performed using the RNAqueous Ambion kit (Ambion) and cDNA
`was obtained from 10 mg of RNA using the High Capacity cDNA
`Reverse Transcription kit (Applied Biosystems). For the relative
`quantification of b-galactosidase and related genes, the Fast Sybr
`Green Master Mix (Applied Biosystems, Foster City, CA) was used,
`using the gene Blon_0393, cysteinyl-tRNA synthetase, as the
`endogenous control (Parche et al., 2006). Reaction conditions were
`as recommended by manufacturer. Primer efficiency was normal-
`ized in each plate using standard curves. The Primer3 software was
`used for primer design (Supplementary Table 2), and the Q-gene
`software was used for relative quantification analysis.
`
`2.4. b-galactosidase gene cloning
`
`B. infantis ATCC 15697 genomic DNA was obtained from over-
`night cultures on MRS, using the MasterPure Gram Positive DNA
`Purification Kit (Epicentre Biotechnologies, Madison, WI), following
`the manufacturer instructions. Primers used for PCR cloning are
`shown in Supplementary Table 2. PCR reactions contained 0.2 mM
`dNTPs (Fermentas, Glen Burnie, MD), 1 ng DNA, 0.5 mM of each
`primer, and 2 U of Phusion DNA Polymerase (Finnzymes, Vantaa,
`Finland) in a 150 ml final volume. PCR was performed in a Verity 96
`well thermal cycler (Applied Biosystems), using the following
`program: initial denaturation at 98 C for 2 min; 35 cycles of
`denaturation at 98 C 30 s, annealing at 58 C for 30 s, and exten-
`sion at 72 C 3 min; and a final extension at 72 C for 7 min. PCR
`products were gel purified (Qiaquick Gel Extraction Kit, Qiagen,
`Valencia, CA) and cloned into pET101 using the Champion pET101
`Directional TOPO Expression Kit (Invitrogen), following manufac-
`turer instructions. Plasmids were transformed into BL21 star E. coli
`cells as well as Top10 cells for plasmid storage, and transformants
`were confirmed for the correct insert sequence by plasmid
`sequencing using primers T7prom and T7term (Invitrogen).
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`2.5. Recombinant protein expression and purification
`
`2.8. Evaluation of relative affinities of b-galactosidases
`
`E. coli BL21 transformants were grown in 200 ml LB broth with
`50 mg/ml carbenicillin in a shaker at 250 rpm (Innova-4000, New
`Brunswick Scientific, Edison, NJ) at 37 C until cultures reached an
`O.D. of 0.6. Recombinant proteins were induced for 6 h with the
`following optimized conditions: 0.5 mM IPTG (USB) at 24 C for
`Blon_0268 and Blon_2416; 0.5 mM IPTG at 28 C for Blon_2016;
`1 mM IPTG at 24 C (Blon_2123) and 0.5 mM IPTG at 28 C for
`Blon_2334. Cultures were centrifuged in 50 ml falcon tubes at
`4000 rpm in an Eppendorf 5804 centrifuge (Eppendorf,
`Hauppauge, NY) for 20 min at 4 C, and pellets were kept at 80 C
`until use. Cells were resuspended in Bugbuster Protein Extraction
`Reagent (EMD Chemicals), using 5 ml of the buffer for every 100 ml
`of culture. Lysozyme (Sigma Aldrich, 50 ml of 50 mg/ml stock), and
`DNAse I (Roche Applied Sciences; 20 ml of 10,000 U stock) were
`added to help in bacterial lysis. The suspensions were vortexed and
`incubated for 10 min at room temperature, and centrifuged for
`20 min at 13,200 rpm at 4 C. Supernatants were recovered and
`applied to 1 ml Bio-Scale Mini Profinity IMAC cartridges, connected
`to an EP-1 Econo-pump (Bio-Rad, Hercules CA). Protein purification
`was performed as recommended by the manufacturer, but proteins
`were eluted using an imidazole gradient between 20 and 250 mM
`in the washing buffer. Recombinant b-galactosidases were checked
`for purity and correct molecular weight using 10% SDS-PAGE gels
`(Bio-Rad). Elution buffer was exchanged for PBS using Amicon
`Ultra-15 Centrifugal Filter Units, with a cut-off of 50 kDa (Milli-
`pore). Protein concentrations were determined using the Bio-Rad
`protein assay, with a standard curve using Bovine Serum Albumin
`(Sigma).
`
`2.6. Determination of enzymatic kinetic parameters
`
`Enzymatic assays were carried out using ortho-nitrophenyl-b-
`galactoside (ONPG; Sigma) at a concentration of 2 mg/ml and 1e
`10 mg of each recombinant enzyme. Optimum pH for each
`enzyme was determined using McIlvaine buffers, with values from
`4.0 to 8.0. Reactions were performed in triplicate in 96 microwell
`plates, and contained 80 ml of each buffer, 15 ml of substrate, and 5 ml
`of enzyme. Reactions were incubated for 10 min at 37 C, and
`stopped adding equal volumes of 1 M Na2CO3. Absorbance at
`420 nm was determined using a Synergy 2 microplate reader
`(Biotek). For determination of optimum reaction temperatures,
`enzymatic assays were performed at optimum pH and at 4 C,
`30 C, 37 C, 45 C, 55 C and 65 C. Relative activity was deter-
`mined from OD420 values. Kinetic constants were obtained using
`substrate concentrations in the range of 0.1e4 mM of ONPG and 1e
`100 mg of each enzyme. Reactions were performed at optimum pH
`and temperature, and times were preestablished to fall within the
`initial rate of reaction. Amounts of o-nitrophenol produced in each
`reaction were calculated from a standard curve and OD420 values.
`Non-linear regression was used to determine Km and Vmax, fitting
`the experimental values to the Michaelis Menten equation, using
`the tool Solver on Microsoft Excel.
`
`2.7. b-galactosidase substrate specificity determination
`
`Recombinant enzymes were coincubated in phosphate buffer
`and 2 mg of the following substrates at their optimum pH and
`temperatures: D-lactose (Sigma), 30 galactosyl lactose (Carbosynth,
`Berkshire UK), Galb1-4Gal (40 galactobiose; V-labs, Covington, LA),
`Galb1-6Gal (60 galactobiose; V-labs), and 10 mg of commercial GOS
`(GTC Nutrition). Reactions were carried out in 10 ml for the specified
`times at 37 C, and inactivated at 95 C for 5 min. 1 ml of each
`reaction was analyzed in TLC plates as described above.
`
`Equimolar concentrations (0.2 mM) of ONPG (Sigma), lactose
`(Sigma), 40 galactobiose (V-labs), 60 galactobiose (V-labs), 30 galac-
`tosyl lactose (Carbosynth, UK) and lacto-N-tetraose (V-labs) were
`coincubated with the same amount (1e20 mg) of each of the five
`recombinant b-galactosidases for 10 min at
`their optimum
`temperatures and pH in McIlvaine buffer in a 10 ml volume. Reac-
`tions were inactivated by incubation at 95 C for 5 min. The
`Galactose Assay Kit (Biovision, Mountain View CA) was used to
`quantify galactose concentrations present
`in each sample,
`following the manufacturer instructions. Fluorescence was quan-
`tified using a standard curve in a Synergy 2 microplate reader.
`Values were normalized considering the amount of galactose
`released from ONPG as 100%.
`
`2.9. Production of SCFA by B. infantis ATCC 15697
`
`The production of acetate, lactate and formate by B. infantis was
`tested on seven different substrates: glucose (Sigma), lactose, HMO,
`LNT (V-labs), GOS, FOS (raftilose Synergy 1, Orafti, Malvern, PA) and
`inulin (raftiline HP, Orafti, Malvern, PA). B. infantis ATCC 15697 was
`grown overnight in MRS and inoculated at 5% in modified MRS,
`replacing sodium acetate by sodium chloride (10 g/l peptone, 5 g/l
`yeast extract, 5 g/l sodium chloride, 2 g/l ammonium citrate, 0.2 g/l
`magnesium sulfate, 0.05 g/l manganese sulfate, 2 g/l dipotassium
`phosphate, 1 g/l tween 80, and 0.05% cysteine). Media was sup-
`plemented with equal amounts (2% w/v) of each carbon source
`mentioned above. The incubations were carried out at 37 C in
`anaerobic chamber in triplicate (Coy Laboratory Products, Grass
`Lake, MI). After 48 h of incubation the final optical density was
`measured in a spectrophotometer at 600 nm ((Shimadzu Scientific
`Instruments, Columbia MD), and the media was recovered by
`centrifuging at 12,000  g for 10 min). Supernatants were analyzed
`using the L-lactate assay kit (Bioassay Systems, Hayward, CA),
`acetate assay kit (Bioassay Systems), and formate assay kit (Bio-
`vision, Milpitas, CA) according to the manufacturer instructions. For
`calculation of fermentation kinetic parameters, OD values were
`converted to dry weight in a ratio of 0.39 (Rossi et al., 2005), and
`these values were used with the amounts of SCFA in grams and
`initial concentration of each substrate (2 g/100 ml) to estimate the
`product yield of substrate (YP/S), the biomass yield of substrate (Yx/
`S) and the product yield of biomass (YP/x). Substrate consumption
`was assumed to be 100%.
`
`3. Results
`
`3.1. Consumption of GOS by B. infantis isolates
`
`Twenty-two strains of B. infantis isolated from infant feces were
`studied in vitro for their ability to grow using 0.5% commercial GOS
`as the sole carbohydrate source. All isolates excepting SC97 showed
`a moderate to vigorous growth on GOS (Fig. 1). We also analyzed
`the supernatants of the fermentations in TLC plates (Fig. 2). All
`isolates were able to deplete the small amount of mono- and di-
`saccharides found in commercial GOS, and DP3 was also consumed
`to a great extent. In general, disappearance of individual GOS with
`DP >3 was correlated with a higher final OD600, and conversely
`lower growth on GOS was related to a lack of consumption of larger
`GOS. For example, strains SC30, SC145 and UCD302 displayed the
`higher OD values on GOS and a noticeable reduction in all DP
`compared to the control with no bacteria added (Fig. 2). These
`results indicate that consumption of larger DP GOS is strain-
`dependent.
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`Fig. 1. In vitro growth of strains of B. infantis using 0.5% commercial GOS as the sole media carbon source.
`
`3.2. Distribution of b-galactosidases in B. infantis
`
`To provide molecular details on the consumption of GOS in
`B. infantis, we focused on the genome of B. infantis ATCC 15697,
`which contains five genes predicted to encode b-galactosidases, EC
`3.2.1.23 (Table 1). Some of these genes are in the proximity of
`carbohydrate transporters (Supplementary Fig. 1), suggesting a co-
`regulated transcription. For example Blon_0268 and Blon_2334
`are located next to sugar permeases, and Blon_2416 is in a gene
`cluster containing an ABC transporter with affinity for oligosaccha-
`rides and a Glycosyl Hydrolase (GH) family 43. Blon_2334 is part of
`the HMO cluster I (Sela et al., 2008), which contains several genes
`predicted to be important in metabolism of HMO in this bacterium.
`These enzymes belong to either GH2 or GH42, as defined by the
`Carbohydrate-Active Enzymes database (www.cazy.org; (Cantarel
`et al., 2009)). The presence of these genes in some of the B. infantis
`isolates used in this study has been previously determined by
`comparative genome hybridization ((LoCascio et al., 2010);
`Supplementary Table 3). While Blon_2016, Blon_2123 and
`
`Blon_2334 were present in all the strains, Blon_0268 was only found
`in strain UCD301 and Blon_2416 was lacking in strains UCD298,
`UCD299 and UCD300, altogether with Blon_2414, an upstream gene
`encoding a SBP induced by GOS in strain ATCC 15697.
`
`3.3. Gene expression of b-galactosidases in B. infantis on GOS
`
`A relative quantification of the expression levels for each b-
`galactosidase gene was performed on B. infantis grown to expo-
`nential phase using lactose, glucose, GOS or HMO as the sole carbon
`source. Results were normalized to gene expression levels of cells
`growing on lactose (Fig. 3). We considered significant a level of
`induction more than two fold, as previously determined to be
`comparable to proteomic data (Garrido et al., 2011). Two genes,
`Blon_2123 and Blon_2416, were repressed at least four fold in cells
`grown on glucose. In contrast, Blon_0268 was induced eight fold
`during growth on glucose relative to lactose. When a pool of HMO
`was used as the sole carbon source, none of the enzymes was
`induced relative to lactose. However, Blon_2016 and Blon_2334 are
`
`Fig. 2. Analysis of the consumption of GOS by B. infantis isolates by TLC. Supernatants represent strains in Fig. 1, and a control prepared under the same conditions but with no
`bacteria added was included (GOS lane). Plates were run in n-butanol-acetic acid-water 2:1:1 and developed with a-naphthol. Numbers correspond to strains in Supplementary
`Table 1.
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`Table 1
`Enzyme kinetic parameters and optimums for B. infantis b-galactosidases.
`1)
`kcat (s
`
`Optimum
`pH
`5.0
`5.0
`5.0e6.0
`7.5
`5.0e6.5
`
`Optimum
`temperature
`45 Ce55 C
`45 C
`45 Ce55 C
`37 C
`45 C
`
`Blon_0268
`Blon_2016
`Blon_2123
`Blon_2334
`Blon_2416
`
`Km (mM)
`
`0.60
`0.70
`1.06
`0.29
`1.09
`
`5.11
`1365.58
`269.92
`420.04
`7.21
`
`kcat/Km
`1 M(s
`1)
`
`8.50  103
`1.94  106
`2.5  105
`1.47  106
`6.58  103
`
`3.5. Relative affinities and substrate specificity of B. infantis b-
`galactosidases
`
`We also studied the activity of these enzymes on common b-
`galactosides. 4- and 6-galactobiose (Galb1-4Gal and Galb1-6Gal)
`and galactosyl-lactose (Galb1-3Galb1-4Glc) are common products
`in transglycosylation reactions (Gosling et al., 2010). Moreover,
`plant polysaccharides also contain these linkages as building
`blocks. As observed on TLC plates and considering the relative
`affinities for these substrates (Table 2), Blon_0268 displayed an
`overall preference for 60 galactobiose (Fig. 4A, lanes 2e4; 9e11 and
`Fig. 4C, lane 3). Conversely, Blon_2416 showed a preference for 40
`galactobiose over galactosyl lactose (Fig. 4B lanes 6e7 and 13e14;
`Fig. 4C,
`lane 7). Blon_2123 was only partially active on
`60galactobiose.
`Blon_2334 showed higher lactase activity compared to the other
`B. infantis b-galactosidases (Fig. 4A, lanes 20e22). Blon_0268 and
`Blon_2016 showed only a minor activity on this substrate (Fig. 4A,
`lanes 17e19 and Table 2). Blon_2016 releases galactose very effi-
`ciently from type 1 HMO such as lacto-N-tetraose (LNT; Galb1-
`3GlcNAcb1-3Galb1-4Glc), and it had the highest enzymatic effi-
`ciency given by the kcat/Km ratio on ONPG (Table 1). The results
`presented in this study indicate that this enzyme can cleave other
`galactosyl linkages, such as those found in 40 and 60 galactobiose
`(Fig. 4C, lanes 2e3 and 8e9), 30 galactosyl lactose (Fig. 4C, lane 4),
`and GOS (Fig. 4D, lanes 4e5), displaying however a preference for
`LNT (Table 2).
`infantis b-galactosidases with
`Finally, after incubation of B.
`commercial GOS for 1 h, we observed that four of them displayed
`significant hydrolytic activity on these prebiotics, as observed by an
`increase in time in the amount of galactose and glucose released
`from commercial GOS (Fig. 4A, lanes 23e30 and Fig. 4D).
`
`3.6. Production of SCFA by B. infantis
`
`Sugar metabolism in bifidobacteria differs from other bacterial
`metabolic pathways (Fushinobu, 2010) and is characterized by the
`presence of fructose-6-P phosphoketolase, an enzyme that gener-
`ates acetyl-P and erythrose-4-P from fructose-6-P. This pathway,
`termed the “bifid shunt”, produces 2.5 mol of ATP per mole of
`glucose, as well as 3 mol of acetate and 2 mol of lactate that are
`
`constitutively expressed at high levels when B. infantis grows on
`HMO and lactose (Sela et al., 2008; Yoshida et al., 2012). Finally,
`growth on commercial GOS had the greatest impact on the tran-
`scription of B. infantis b-galactosidases, with Blon_2334 induced
`over tenfold and Blon_0268 and Blon_2416 also induced over two
`fold by these oligosaccharides.
`We also analyzed the expression levels for genes adjacent to
`Blon_0268 and Blon_2334 (Supplementary Fig. 2), encoding for
`transporters of the major facilitator superfamily. Even though the
`carbohydrate affinity of these transporters (Blon_0267, Blon_2331
`and Blon_2332) is unknown, their induction by GOS, as well as
`glucose (Blon_0268), is suggestive that their affinities are related to
`these substrates.
`
`3.4. Kinetic parameters of b-galactosidases in B. infantis
`
`In order to study some of the properties of these glycosyl
`hydrolases, they were cloned and expressed in E. coli, and purified
`with an N-terminal his-tag. ONPG was used for evaluating different
`kinetic parameters. Optimum pH values were relatively acidic for
`Blon_0268 and Blon_2016, and more neutral for the other three
`enzymes (Table 1). As observed with other b-galactosidases, the
`optimum temperature varied between 45 C and 55 C for all
`enzymes, except Blon_2334 which showed the highest activity at
`37 C. Using these conditions, kinetic parameters were determined
`using ONPG. Turnover rates were the highest for Blon_2016, and
`together with Blon_2334, these glycosidases showed the greatest
`1 M
`1. These
`kinetic efficiency given by a kcat/Km ratio over 1 106 s
`values are in agreement with those obtained by Yoshida et al.
`(2012), and they were 1000 times higher than the kinetic effi-
`ciencies observed for Blon_0268 and Blon_2416 on ONPG.
`
`Fig. 3. Relative quantification of the gene expression of B. infantis b-galactosidases after logarithmic growth on the substrates indicated in the x-axis. Dashed lines indicate a two-
`fold change in gene expression.
`
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`267
`
`Table 2
`Relative affinities of B. infantis b-galactosidases for individual substrates. Results are
`expressed as a percentage normalized to the amount of galactose released from
`ONPG.
`
`produced, growth on HMO, under these conditions we observed
`the highest yield of product over biomass, which indicates that
`B. infantis produces more acetate and lactate per cell. Formate is
`another end product of bifidobacterial fermentation of carbohy-
`drates, and growth on inulin, FOS and glucose led to a significant
`production of this organic acid. Finally, the acetate:lactate ratio was
`determined for each substrate. Considering that the theoretical
`value in bifidobacteria is 1.5, we observed that growth on FOS and
`inulin, as well as HMO and LNT to a lesser extent, led to a consid-
`erable increase in this value.
`
`Blon_0268 Blon_2016 Blon_2123 Blon_2334 Blon_2416
`100
`100
`100
`100
`100
`ONPG
`78.9
`19.9
`128.1
`Lactose
`e
`e
`40 galactobiose
`9.6
`e267.6
`e
`e
`60 galactobiose 153.2
`60.3
`137.5
`e
`30Gal-lac
`18.7
`11.6
`e
`e
`LNT
`123.4
`14.5
`e
`e
`
`e
`
`e
`
`6.1
`
`either released to the media or used in de novo fatty acid synthesis
`(Sela et al., 2010). These yields are variable among different strains
`and growth conditions. Here, we determined the amounts and
`yields of SCFA produced by B. infantis ATCC 15697 after growth on
`different prebiotics including GOS as the sole carbon source.
`Supernatants reached exponential phase at similar times and were
`recovered at 48 h, where all cultures reached stationary phase. In
`order to compare production of SCFA across the different
`substrates, three kinetic yield coefficients were calculated (Table 3),
`estimating a 100% substrate consumption, which is supported by
`the efficient oligosaccharide consumption of GOS (Barboza et al.,
`2009), FOS and inulin (Perrin et al., 2001), and lactose, glucose,
`HMO and LNT (Asakuma et al., 2011; LoCascio et al., 2007; Sela
`et al., 2012) by B. infantis.
`We observed that the product and biomass yields of substrate
`were higher during growth on lactose, GOS and glucose (Table 3),
`indicating that under these conditions B. infantis produces biomass
`and acids more efficiently. Although less biomass and SCFA were
`
`4. Discussion
`
`GOS are important food supplements that, among other health
`benefits, stimulate the growth of beneficial microorganisms in the
`gastrointestinal tract. In this work we studied several aspects of the
`utilization of commercial prebiotic GOS by B. infantis, a represen-
`tative species of the developing infant gut microbiota. As a dietary
`substrate, GOS is not degraded by host intestinal enzymes.
`However, its relatively simple composition, consisting of repeats of
`b1-3/4/6 galactosyl
`linkages, allows several bacteria,
`including
`Bacteroides sp. and Clostridium sp. to degrade and consume GOS
`in vitro (Gibson et al., 2004). Surprisingly, GOS consumption in
`human volunteers resulted in significant and consistent enrich-
`ment of mainly one genus, Bifidobacterium, as shown recently
`(Davis et al., 2011). Several species of this genus have been shown to
`grow on these prebiotics as the sole media carbon source (Goulas
`et al., 2007; Hopkins et al., 1998; Van Laere et al., 2000), and for
`example it has been shown by MALDI-FT-ICR mass spectrometry
`
`Fig. 4. Determination of the substrate specificities of B. infantis b-galactosidases for different galactosyl linkages by TLC indicated in the bottom of the figure. (A) Coincubations of
`Blon_0268 and Blon_2334 for 5, 20 and 60 min with different b-galactosides. Lanes 1 and 8: standards; lanes 2e4: Blon_0268 with 4-galactobiose; lanes 5e7: Blon_2334 with 4-
`galactobiose, 5-20e60 min; lanes 9e11: Blon_0268 with 6-galactobiose; lanes 12e14: Blon_2334 with 6-galactobiose. Lane 15: Lactose and galactose; lane 16: glucose; lanes 17e19:
`Blon_0268 with lactose; lanes 20e22: Blon_2334 with lactose. Lane 23: Commercial GOS; lane 24: lactose and galactose; lanes 25e27: Blon_0268 with GOS; lanes 28e30:
`Blon_2334 with GOS. (B) Coincubations of Blon_2016, Blon_2123 and Blon_2416 with 40 or 60 galactobiose for 200 and 600. Lanes 1 and 8: galactose and 40 or 60 galactobiose
`standards; lanes 2e3: Blon_2016 on 4-galactobiose; lanes 4e5: Blon_2123 on 4-galactobiose; lanes 6e7: Blon_2416 on 4-galactobiose; lanes 9e10: Blon_2016 on 6-galactobiose;
`lanes 11e12: Blon_2123 on 6-galactobiose; lanes 13e14: Blon_2416 on 6-galactobiose. (C) Glycolytic activity on 30 galactosyl lactose. Enzymes were incubated with this substrate for
`600. Lane 1: 30 galactosyl lactose; lane 2: galactose and lactose; lane 3: Blon_0268; lane 4: Blon_2016; lane 5: Blon_2123; lane 6: Blon_2334; lane 7: Blon_2416. (D) Coincubations of
`Blon_2016, Blon_2123 and Blon_2416 with commercial GOS for 200 and 600. Lane 1: GOS; lane 2: lactose and galactose; lane 3: glucose; lanes 4e5: Blon_2016; lanes 6e7: Blon_2123;
`lanes 8e9: Blon_2416. Gal: galactose, Glc: glucose, Lac: lactose.
`
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`
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`Kinetic parameters of B. infant