Physiology of Consumption of Human Milk Oligosaccharidesby Infant Gut-associated Bifidobacteria*□S

Received for publication, April 6, 2011, and in revised form, June 30, 2011 Published, JBC Papers in Press, August 9, 2011, DOI 10.1074/jbc.M111.248138

Sadaki Asakuma‡, Emi Hatakeyama§, Tadasu Urashima§, Erina Yoshida¶, Takane Katayama¶1, Kenji Yamamoto¶,Hidehiko Kumagai¶, Hisashi Ashida�, Junko Hirose**, and Motomitsu Kitaoka‡‡

From the ‡Intensive Grazing Research Team, National Agricultural Research Center for the Hokkaido Region, Sapporo,Hokkaido 062-8555, the §University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, the ¶Research Institutefor Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, the �Graduate School ofBiostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, the **Department of Life Style Studies, University of Shiga Prefecture,Hikone, Shiga 522-8533, and the ‡‡National Food Research Institute, National Agriculture and Food Research Organization,Tsukuba, Ibaraki 305-8642, Japan

The bifidogenic effect of human milk oligosaccharides(HMOs) has long been known, yet the precise mechanismunderlying it remains unresolved. Recent studies show thatsome species/subspecies of Bifidobacterium are equipped withgenetic and enzymatic sets dedicated to the utilization ofHMOs, and consequently they can growonHMOs; however, theability to metabolize HMOs has not been directly linked to theactualmetabolic behavior of the bacteria. In this report, we clar-ify the fate of each HMO during cultivation of infant gut-asso-ciated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifi-dobacterium longum subsp. infantis JCM1222,Bifidobacteriumlongum subsp. longum JCM1217, and Bifidobacterium breveJCM1192 were selected for this purpose and were grown onHMO media containing a main neutral oligosaccharide frac-tion. The mono- and oligosaccharides in the spent mediawere labeled with 2-anthranilic acid, and their concentra-tions were determined at various incubation times using nor-mal phase high performance liquid chromatography. Theresults reflect the metabolic abilities of the respective bifido-bacteria. B. bifidum used secretory glycosidases to degradeHMOs, whereas B. longum subsp. infantis assimilated allHMOs by incorporating them in their intact forms. B. longumsubsp. longum and B. breve consumed lacto-N-tetraose only.Interestingly,B. bifidum left degradedHMOmetabolites out-side of the cell even when the cells initiate vegetative growth,which indicates that the different species/subspecies canshare the produced sugars. The predominance of type 1chains in HMOs and the preferential use of type 1 HMO byinfant gut-associated bifidobacteria suggest the coevolutionof the bacteria with humans.

Human milk contains between 10 and 20 g/liter of oligosac-charides (the degree of polymerization (DP)2 �3) as the thirdmost abundant solid component after lactose and lipids (1).These oligosaccharides are collectively termed humanmilk oli-gosaccharides (HMOs). HMOs are characterized by their com-plex structures. About 200 molecular species have beendetected using a microfluidic chip separation coupled to massspectrometry (2), and among them, 115 structures have beendetermined so far (3). HMOs have been classified into 13 corestructures that consist of lactose, at the reducing end, elongatedby �1–3-linked lacto-N-biose I (Gal�1–3GlcNAc, LNB, type 1chain) and/or �1–3/6-linked N-acetyllactosamine (Gal�1-4GlcNAc, LacNAc, type 2 chain) (4, 5). These core structuresare frequently modified by fucose and sialic acid residues via�1–2/3/4 and �2–3/6 linkages, respectively. The unique fea-ture of HMOs is the predominance of type 1 chains, and such acomposition has not been observed in milk oligosaccharidesfrom other mammals, including anthropoids (Ref. 3 and refer-ences therein).HMOs are resistant to gastrointestinal digestion in host

infants, and thus the majority of HMOs reach the colon (6, 7),where they may serve as prebiotics to shape a healthy gut eco-system by stimulating the growth of beneficial microorganismsand by acting as receptor analogues to inhibit the binding ofvarious pathogens and toxins to epithelial cells (5, 8–10). Therapid and predominant colonization of a breast-fed infantintestine by bifidobacteria has long been known in pediatrics,and the selective growth of bifidobacteria has been attributed toHMOs (11, 12). Several groups have revealed through geneticor enzymatic approaches that particular infant gut-associatedbifidobacteria have dedicated pathways to assimilate HMOs(the details are described later) (13–16). However, our overallunderstanding of what structures of HMOs exert bifidogeniceffects, and how, remains incomplete.Ward et al. (17) and LoCascio et al. (18) examined the in vitro

fermentation abilities of several bifidobacteria on HMOs and

* This work was supported in part by a Grant-in-aid for Scientific Research byYoung Scientists (B) 22780072 from the Ministry of Education, Culture,Sports, Science and Technology, Japan, and grant-in-aid from The UrakamiFoundation.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1–S5.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) JF332149, JF332150, andJF332151.

1 To whom correspondence should be addressed: Research Institute forBioresources and Biotechnology, Ishikawa Prefectural University, Nonoi-chi, Ishikawa 921-8836, Japan. Tel.: 81-76-227-7513; Fax: 81-76-227-7557;E-mail: takane@ishikawa-pu.ac.jp.

2 The abbreviations used are: DP, degree of polymerization; HMO, humanmilk oligosaccharide; 2�-FL, 2�-fucosyllactose; 3-FL, 3-fucosyllactose; GLBP,galacto-N-biose/lacto-N-biose-I-binding protein; GNB, galacto-N-biose;LDFT, lactodifucotetraose; LNB, lacto-N-biose I; LNDFH, lacto-N-difuco-hexaose; LNFP, lacto-N-fucopentaose; LNT, lacto-N-tetraose; LNnT,lacto-N-neotetraose.

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analyzed the oligosaccharide compositions in spentmedia. Theresults showed that Bifidobacterium longum subsp. infantisATCC15697 (same as JCM1222 used in this study) can growvigorously in the presence of HMOs acting as carbon sources,whereas Bifidobacterium bifidum ATCC29521 (JCM1255),Bifidobacterium breve ATCC15700 (JCM1192), and B. longumsubsp. longumATCC15707 (JCM1217) did not grow.B. longumsubsp. infantis ATCC15697 preferentially consumed HMOswith DP �7 during the incubation period, whereas the otherspecies showed very limited utilization of HMOs (only a20–30% decrease of an oligosaccharide with an m/z 732.3,which corresponds to lacto-N-tetraose (Gal�1-3GlcNAc�1–3Gal�1–4Glc, LNT) or lacto-N-neotetraose(Gal�1–4GlcNAc�1–3Gal�1–4Glc, LNnT), was observed forB. breve and B. longum subsp. longum). More recently, Mar-cobal et al. (19) used a chemically synthesized medium to showthat B. longum subsp. infantis ATCC15697 can utilize HMOswith DP up to 12. Sela et al. (20) demonstrated that B. longumsubsp. infantisATCC15697 can use sialylatedHMOs as carbonsources. In all of these studies,matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance massspectrometry was employed for analyzing the consumption ofHMOs. This method is superior because of its high sensitivityand high throughput capacity; however,MALDI-Fourier trans-form ion cyclotron resonance MS is not suitable for the quan-tification of each HMO, and the technique cannot distinguishbetween several important isomers contained in HMOs, e.g.LNT versus LNnT and lacto-N-difucohexaose I (LNDFH I)(Fuc�1-2Gal�1-3(Fuc�1-4)GlcNAc�1-3Gal�1-4Glc) versuslacto-N-difucohexaose II (LNDFH II) (Gal�1–3(Fuc�1–4)-GlcNAc�1–3Gal�1–4(Fuc�1–3)Glc). In addition, the use ofthe matrix may interfere with the measurement of low molec-ular compounds, and therefore the approach may not be suita-ble for the detection ofHMOswithDP� 3 and the degradationproducts (mono- and disaccharides) of HMOs during cultiva-tion. This particular drawback is apparent in the recent reportswhere the presence of trisaccharides 2�-fucosyllactose (Fuc�1–2Gal�1–4Glc, 2�-FL) and 3-fucosyllactose (Gal�1–4(Fuc�1–3)Glc, 3-FL) are not described. 2�-FL is the most abundantHMOs unless the milk is derived from nonsecretor subjects (5,21).In this report, we have conducted refined HPLC profiling of

HMOconsumption by the infant gut-associated bifidobacteria,inwhich the concentrations of themain fractions ofHMOs (DPof 3–6) and the degraded intermediates (mono- and disaccha-rides) were determined. These results were then interpretedwith respect to themetabolic functionalities of the bifidobacte-ria. The physiology of HMOmetabolism in each bifidobacterialstrain is also discussed in detail. This is the first study thatunequivocally reveals the fate of each tested HMO during bac-terial fermentation and the results indicate that bifidobacterialspecies/subspecies share the degraded products.

EXPERIMENTAL PROCEDURES

Chemicals—The standard human milk oligosaccharides2�-FL, 3-FL, LNT, LNnT, lacto-N-fucopentaose I(Fuc�1–2Gal�1–3GlcNAc�1–3Gal�1–4Glc, LNFP I), LNFPII (Gal�1–3(Fuc�1–4)GlcNAc�1–3Gal�1–4Glc), LNFP III

(Gal�1–4(Fuc�1–3)GlcNAc�1–3Gal�1–4Glc), LNDFH I,and LNDFH II were purchased from Dextra Laboratory(Reading, UK). Lactodifucotetraose (Fuc�1–2Gal�1–4-(Fuc�1–3)Glc, LDFT) was synthesized by introducing an�1–3-fucosyl linkage into the Glc residue of 2�-FL using1,3–1,4-�-L-fucosynthase.3 LNB was synthesized as describedpreviously (22). Lacto-N-triose II (GlcNAc�1–3Gal�1–4Glc)was prepared by hydrolyzing LNnTby�-galactosidase (23), andthe triose was subsequently purified using Toyopearl HW-40F(Tosoh, Tokyo, Japan). Isomaltopentaose (IG5) was obtainedfrom Seikagaku Kogyo (Tokyo, Japan). Lactose monohydrate(Gal�1–4Glc, Lac) was from Kanto Chemical (Tokyo, Japan).L-Fucose (Fuc) and N-acetylglucosamine (GlcNAc) were fromNacalai Tesque (Kyoto, Japan), and glucose (Glc) and boric acidwere from Kishida Kagaku (Osaka, Japan). Galactose (Gal),2-anthranilic acid, and sodium acetate were purchased fromWako Pure Chemicals (Osaka, Japan). LacNAc, Lewis a/x tri-saccharides (Gal�1–3/4(Fuc�1–4/3)GlcNAc) and sodium cya-noborohydride (grade, 95%) were from Sigma. The purities ofthemono- and oligosaccharides other than LNB, lacto-N-trioseII, LDFT, and LNFP III were certified to be �95% by the man-ufacturers. The purities of LNB, lacto-N-triose II, LDFT, andLNFP III were checked (�95%) using anHPLC equippedwith acharged aerosol detector (Corona CAD, MA). All otherreagents were analytical grade.Preparation of Oligosaccharides from Human Milk—Fifty

seven healthy Japanese mothers who had not taken any antibi-otics for 1monthwere recruited from several midwife clinics inKyoto Prefecture between July and September 2008. They were32.7� 4.3 years old (mean� S.D.), with a range of 25–40 yearsof age, and had given birth to infants at term (gestational age of36–41 weeks). The mothers and their babies had no healthissues. Breastmilk samples were collected in sterile polypropyl-ene tubes at 74.9 � 27.2 days after delivery with a range of30–120 days. The samples (900ml in total) were combined andmixed with equal volumes of chloroform and methanol andstirred vigorously for 1 h at 25 °C to remove proteins and lipids.After standing for 16 h at 25 °C, the upper layer (�1.6 liters) wascollected and concentrated to �800 ml by evaporation. Thesolution was centrifuged, and the supernatant was lyophilizedto give 59.6 g of solid materials that contained mainly carbohy-drates. The crude carbohydrate preparation was dissolved in400 ml of distilled water, and aliquots (20 ml) were appliedevery 60 min for a total of 20 loadings to a Toyopearl HW-40Fgel filtration column (inner diameter, 5 � 80 cm) (Tosoh). Theelution was carried out using water at a flow rate of 10 ml/min.Fractions containing neutral oligosaccharides were collectedand combined as a pool of human milk oligosaccharides. Insome cases, fractions contained both lactose and trisaccharides.These fractions were separately collected, concentrated bylyophilization, and rechromatographed to remove the lactose.During the steps, almost all lactose (�99.5%)was removed. Thepurified fractionswere combinedwith the pool and then lyoph-ilized to give a final preparation of humanmilk oligosaccharides(4.74 g). Note that the sialyl oligosaccharides were eluted near

3 Haruko Sakurama, Yuji Honda, and Takane Katayama (Ishikawa PrefecturalUniversity), personal communication.

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the void fractions as polymeric compounds (24) and thereforewere not contained in this preparation, as confirmed by theperiodate-resorcinolmethod (25). The structures of the neutraloligosaccharides analyzed in this study are listed in Table 1.Bacterial Strains and Growth Conditions—Bacterial strains

used in this study were B. bifidum JCM1254, JCM1255T, andJCM7004, B. breve JCM1192T, B. longum subsp. infantisJCM1222T, andB. longum subsp. longum JCM1217T.All strainswere purchased from the Japan Collection of Microorganisms.Bididobacteria were anaerobically grown at 37 °C in GAMmedium (Nissui Pharmaceutical, Tokyo, Japan) or in basalmedium consisting of 0.2% yeast extract, 1.0% peptone, 0.5%sodium acetate, 0.2% diammonium citrate, 0.02% magnesiumsulfate, 0.2% dipotassium hydrogen phosphate, 0.1% cysteinehydrochloride, and 1% sugar (Fuc, Gal, Glc, GlcNAc, Lac, LNB,or HMOs). The basal medium was supplemented with 4%reducing reagent (2% cysteine hydrochloride and 11% sodiumcarbonate) prior to the inoculation of the bacteria. Themedium(10 ml) was inoculated with the bifidobacterial strain (equiva-lent to A600 � 0.1) and incubated under anaerobic conditions.For the analysis of HMO consumption, the samples (0.5 ml)were taken at the indicated times and centrifuged at 12,000 � gfor 1 min, and the resulting supernatants were immediatelyfrozen in liquid nitrogen. The samples were stored at �80 °Cuntil use. Growthwasmonitored bymeasuringA600 at the indi-cated times.Labeling of Oligosaccharides—The oligosaccharides were

fluorescence-labeled with 2-anthranilic acid as described byAnumula (26) with a slight modification, in which the labelingreagent was prepared by dissolving 2-anthranilic acid (30 mg)and solid sodium cyanoborohydride (20 mg) in 1 ml of metha-nol solution containing 4% sodium acetate (w/v) and 2% boricacid (w/v) (27, 28). Fifty �l of the bacterial culture supernatantwas mixed with 100 �l of the labeling reagent and 30 �l of theinternal standard solution (1 mg/ml of IG5). The mixture wasplaced at 80 °C for 45 min and cooled to room temperature.Labeled oligosaccharides were purified by solid extractionusing Discovery DPA-6S columns (50 mg) (Supelco, Poole,UK). The columns were pre-equilibrated twice with 1 ml ofacetonitrile, and the sampleswere loaded using gravity flow andallowed to drip through the column. The column was thenwashed four times with 1 ml of acetonitrile/water (99:1 by vol-ume), followed by 0.5ml of acetonitrile/water (97:3 by volume).The labeled oligosaccharides were eluted twice with 0.6 ml ofwater, dried by evaporation, and stored at either 4 or �30 °Cuntil use.Determination of the Concentration of Each Oligosaccharide—

The labeled oligosaccharides were dissolved in 1 ml of distilledwater and filtered through a polytetrafluoroethylene Millexmembrane (pore diameter 0.2 �m) (Millipore, MA). Ten �l ofsolution was injected into a Shimadzu LC-10AvpHPLC system(Kyoto, Japan). An amide-80 column (4.6 � 250 mm) (Tosoh)was used for the separation at 80 °C. Elution was performedwith a binary gradient of solvent A (85% acetonitrile) and sol-vent B (60% acetonitrile) in 100 mM ammonium formate (pH4.3) at a flow rate of 1.0 ml/min. The gradient was performedfrom 0 to 100% B for 90 min and then 0% B for 15 min toequilibrate the column before the next sample injection.

Labeled oligosaccharides were detected with excitation andemission wavelengths of 360 and 425 nm, respectively. Thestandard curve for each oligosaccharide was created in tripli-cate experiments using a solution containing both the internalstandard (1 mg/ml) and an oligosaccharide with varying con-centrations (0.125, 0.25, and 0.5 mg/ml for LNDFH II, 0.25, 0.5,and 1 mg/ml for the other saccharides). The peak areas of thesaccharides were standardized by comparing them with that ofthe internal standard, and the corrected values were used forthe calculations. The data represent means of two independentdeterminations.Molecular Cloning of the gltA (GLBP) Genes from B. bifidum

JCM1254, JCM1255, and JCM7004—The gltA genes encodinggalacto-N-biose (GNB)/lacto-N-biose I (LNB)-binding pro-teins (GLBPs) were amplified by high fidelity PCR involvingPrimeStar MAX DNA polymerase (Takara Bio, Shiga, Japan)using the genomic DNAs of B. bifidum JCM1254, JCM1255,and JCM7004 as the templates. The primers used were5�-CACGACGAGGAGGCAACATCCAT-3� and 5�-GTG-GTCTTGGGTGATGGACGGAA-3�. The amplified frag-ments were inserted into pMW118 (Nippon Gene, Tokyo,Japan) for sequencing. PCR was done in two separate tubes foreach gltA gene, and the sequences of the two independentlyamplified fragments were determined. The nucleotidesequences of the gltA genes from JCM1254, JCM1255, andJCM7004 were deposited in GenBankTM under the accessionnumbers of JF332149, JF332150, and JF332151, respectively.Immunoblotting—Anti-GLBP antibodies were prepared by

immunization of rabbits with the purified recombinant GLBP(29). The immunoglobulin G fraction was purified by proteinA-Sepharose CL-6B column chromatography (GEHealthcare).Immunoblotting was performed as described previously (30)with slight modifications. In brief, the bifidobacterial cells weregrown in the basal media containing lactose as a carbon source,harvested by centrifugation, suspended in 100 mM sodiumphosphate buffer (pH 7), and disrupted by sonication. The cell-free extracts (10 �g) were subjected to SDS-PAGE, and subse-quently electroblotted to polyvinylidene fluoride membrane(Millipore). Anti-GLBP antibodies and anti-rabbit immuno-globulin, horseradish peroxidase-linked whole antibodies(from donkey) (GE Healthcare) were used in 3,000- and 2,000-fold dilutions, respectively. The ECL chemiluminescent detec-tion agent (GE Healthcare) was used to visualize thecross-reaction.Enzyme Assay—HMO degrading activities were examined

using 2�-FL, Lewis a/x, Lac, LNT, LNnT, and lacto-N-triose II asthe substrates to detect the presence of 1,2-�-L-fucosidase, 1,3–1,4-�-L-fucosidase, �-galactosidase (lactase), lacto-N-biosi-dase, �-galactosidase (specific for type 2 chains), and �-N-acetylhexosaminidase, respectively. The bifidobacterial cellswere grown in basal medium containing lactose as a carbonsource, harvested by centrifugation, suspended in 50 mM

sodiumphosphate buffer (pH 6.5), and disrupted by sonication.The cell-free extracts were ultracentrifuged at 100,000 � g for60 min to separate the cytoplasmic factions from the cell wallfractions. Each fraction (250 �g) was incubated with 2 mM sub-strates in a total volume of 10 �l at 37 °C for 30 min (B. bifidumand B. longum subsp. infantis) or 3 h (B. longum subsp. longum

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and B. breve) to determine the localization of the enzymes.Lacto-N-biosidase activity of B. longum subsp. longum waslost when the cells were disrupted by sonication, and there-fore, the intact cell suspension was regarded as the cell wallfraction. The reaction products were analyzed by thin layerchromatography (TLC) using a silica gel 60 aluminum sheet(Merck). The sugars were visualized as described previously(23).Ethical Consideration—This study was reviewed and ap-

proved by the Ethics Committee of the University of Shiga Pre-fecture, and it followed the Declaration of Helsinki. Informedconsent was obtained from all donors.

RESULTS

Preparation and Analysis of HMOs

Asmentioned above, HMOs are classified into 13 core struc-tures, and the core structures are frequently modified by fucoseand sialic acid via �1–2/3/4 and �2–3/6 linkages, respectively.The final HMOs prepared in this study contained neutral oli-gosaccharides with DP between 3 and 8 and appeared not tocontain larger oligosaccharides and acidic (sialylated) oligosac-charides that are known to represent minor components(�10% of the total HMOs) (31). The purified HMOs wereadded to the medium at a concentration of 1%, and their con-sumptions by bifidobacteria were monitored using normalphase HPLC. Fig. 1 shows the HPLC profiles of mono- andoligosaccharides in the spent media of B. bifidum JCM 1254 atdifferent times. The main HMOs were successfully separatedunder the conditions employed, with the exception of LNFP IIand III. At 0 h, monosaccharides (Fuc, Gal, and Glc), Lac (smallcontaminants derived from the HMO preparation step) andoligosaccharides with DP of 7 and 8 (not identified) were alsodetected as minor peaks. Table 1 presents the list of oligosac-charides analyzed in this study and also shows their initialconcentrations in the culture. The total oligosaccharide con-centration was estimated to be 10.66 g/liter, which is con-sistent with the medium composition (1% carbon source).The concentrations of the respective oligosaccharides andthe ratios between them were not significantly different fromthose reported previously (31) and may reflect the lactationperiods of the donors (74.9 � 27.2 days) (24, 32, 33).

Strain Description

In the genus Bifidobacterium, B. bifidum, B. longum subsp.infantis, B. longum subsp. longum, and B. breve are frequentlyisolated from the feces of breast-fed infants (11, 34).We usedB.bifidum JCM1254, B. longum subsp. infantis JCM1222, B.longum subsp. longum JCM1217, and B. breve JCM1192 strainsto evaluate how these infant gut-associated bifidobacteria con-sume HMOs. They are the type strains of their respective spe-cies/subspecies except for B. bifidum. The type strain of B. bifi-dum (JCM1255) was found not to possess the functional GNB/LNB transporter (described later), whereas all of the otherbifidobacterial strains examined had the transporters, as shownby Western blot analysis using anti-GLBP antibodies (Table 2

FIGURE 1. HPLC profiles of the HMOs consumption by B. bifidum JCM1254cells at different times. The sugars were derivatized by 2-anthranilic acid,solid phase-purified, and analyzed as described under the “Experimental Pro-cedures.” The peaks (1–14) were identified by comparing their retentiontimes with those of the standard sugars.

TABLE 1Structures of oligosaccharides and their initial concentrations in the culture mediumNote that the oligosaccharides were added at concentrations of 1% (w/v) of the culture.

Oligosaccharidea Structure Concentrationb

g/liter mM

Lac Gal�1–4Glc 0.45 � 0.04 1.25 � 0.112�-FL Fuc�1–2Gal�1–4Glc 1.48 � 0.15 3.03 � 0.313-FL Gal�1–4(Fuc�1–3)Glc 1.40 � 0.15 2.86 � 0.30LDFT Fuc�1–2Gal�1–4(Fuc�1–3)Glc 0.80 � 0.11 1.27 � 0.17LNT Gal�1–3GlcNAc�1–3Gal�1–4Glc 1.11 � 0.13 1.57 � 0.18LNnT Gal�1–4GlcNAc�1–3Gal�1–4Glc 0.32 � 0.06 0.45 � 0.08LNFP I Fuc�1–2Gal�1–3GlcNAc�1–3Gal�1–4Glc 0.67 � 0.07 0.78 � 0.08LNFP II � III Gal�1–3(Fuc�1–4)GlcNAc�1–3Gal�1–4Glc (LNFP II) 1.78 � 0.19 2.09 � 0.22

Gal�1–4(Fuc�1–3)GlcNAc�1–3Gal�1–4Glc (LNFP III)LNDFH I Fuc�1–2Gal�1–3(Fuc�1–4)GlcNAc�1–3Gal�1–4Glc 2.53 � 0.25 2.53 � 0.25LNDFH II Gal�1–3(Fuc�1–4)GlcNAc�1–3Gal�1–4(Fuc�1–3)Glc 0.12 � 0.01 0.12 � 0.01Total 10.66 � 0.81

a The abbreviations used is as follows: Lac, lactose.b Values represent mean � S.D. of the data obtained from duplicate measurements of the four different bacterial cultures at 0 h.

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and supplemental Fig. S1A). The sequence analysis revealedthat the dysfunction of GLBP inB. bifidum JCM1255 is becauseof a frameshift mutation within the C-terminal region, whichwas caused by the duplication of a 17-bp sequence in its gene(supplemental Fig. S1B). Given the results, strain JCM1254 wasemployed in this study as the representative of B. bifidum. Theintact GLBP genes (gltA) are also found inB. bifidum JCM7004,S17, and PRL2010 (supplemental Fig. S1) (16, 35).The occurrence and localization of HMO-degrading en-

zymes in each bifidobacterial strain are summarized in Table 2and supplemental Fig. S2. B. bifidum JCM1254 secretes 1,2-�-L-fucosidase and 1,3–1,4-�-L-fucosidase to unmask the corestructures of HMOs. The core structure with the type 1 chain,e.g. LNT, is hydrolyzed by an extracellular lacto-N-biosidase toproduce LNB and Lac (supplemental Fig. S2). The liberatedLNB is subsequently incorporated into cells by the GNB/LNBtransporter and enters the GNB/LNB pathway (describedbelow) (22, 36). Lacmay be hydrolyzed by an extracellular�-ga-lactosidase intoGal andGlc (23) or directly transported into thecells by a yet unidentified Lac transporter. The type 2 corestructure is considered to be sequentially hydrolyzed by extra-cellular �-galactosidase and �-N-acetylhexosaminidase. Forexample, LNnT is first degraded into Gal and lacto-N-triose II,and the latter is subsequently cleaved to GlcNAc and Lac (sup-plemental Fig. S2) (23). In contrast to B. bifidum, which has arepertoire of extracellular enzymes, B. longum subsp. infantisJCM1222 is equipped with intracellular enzymes that degradeHMOs (14). The organism possesses a large gene cluster (43kb), the so-called HMO cluster-1, in its genome, and thecluster contains the homologous genes of 1,2-�-L-fucosidase(Blon_2335), 1,3–1,4-�-L-fucosidase (Blon_2336), �-galactosi-dase (Blon_2334), and�-N-acetylhexosaminidase (Blon_2355).These genes encode enzymes that do not have signal peptides atthe N termini and anchoring motifs at the C termini, and toreflect these sequential properties, all enzymatic activities weredetected in the cytoplasmic fraction (supplemental Fig. S2).The cluster also contains various ABC transporter genes thatare thought to be involved in importing HMOs (14). In addi-tion,B. longum subsp. infantis JCM1222 has aGNB/LNB trans-porter (supplemental Fig. S1A) at a locus different from theHMO cluster-1 (14). The strain does not possess a homolog oflacto-N-biosidase, and accordingly, the activity was notdetected. B. longum subsp. longum JCM1217 and B. breveJCM1192 did not exhibit �-L-fucosidase activities; however,the activities of �-galactosidase, which can liberate Gal from

both LNT and LNnT, and �-N-acetylhexosaminidase, whichliberates GlcNAc from lacto-N-triose II, were detected intheir cytoplasmic fractions. Both of the strains could expressthe functional GNB/LNB transporter, as their cell-freeextracts specifically cross-reacted with the anti-GLBP anti-bodies (supplemental Fig. S1A). B. longum subsp. longumJCM1217 expressed a secretory lacto-N-biosidase, whereasB. breve JCM1192 did not show this activity (supplementalFig. S2) (15).

Growth of Bifidobacteria on HMOs

Fig. 2 shows the growth curves of the bifidobacteria in thepresence of HMOs as the carbon sources. B. longum subsp.infantis JCM1222 grew fast, and the cell density reached amax-imum (A600 �2) within 10 h of incubation.B. bifidum JCM1254also exhibited significant growth and entered a stationary phaseafter 15 h. In contrast, B. longum subsp. longum JCM1217 andB. breve JCM1192 did not show considerable growth, and theA600 of their cultures did not exceed 0.3; however a slightincrease of the A600 was observed for both strains. Note that allof the strains grew well when Glc was added as a carbon source(supplemental Table S1), although no growth was observed inthe absence of sugars.

Utilization of HMOs by Each Bifidobacterial Strain

Samples of each bifidobacterial strain grown on HMOs werewithdrawn at different times, and the mono- and oligosaccha-ride concentrations in the supernatants were determined. The

FIGURE 2. Growth of the bifidobacteria in the basal media containingHMOs as carbon sources. Open squares, B. longum subsp. infantis JCM1222;closed diamonds, B. bifidum JCM1254; open triangles, B. longum subsp. longumJCM1217; closed circles, B. breve JCM1192.

TABLE 2Occurrence and localization of HMO-degrading enzymes in the bifidobacterial strains used in this study

Activitiesa B. bifidum JCM1254B. longum subsp. infantis

JCM1222B. longum subsp. longum

JCM1217 B. breve JCM1192

1,2-�-L-Fucosidase � (extrab) � (intrac) NDd ND1,3–1,4-�-L-Fucosidase � (extra) � (intra) ND ND�-Galactosidase � (intra, extra) � (intra) � (intra) � (intra)�-N-Acetylhexosaminidase � (intra, extra) � (intra) � (intra) � (intra)Lacto-N-biosidase � (extra) ND � (extra) NDGLBPe (GNB/LNB transporter) � � � �

a The HMO degrading activities of these strains were examined as described under “Experimental Procedures” (see also supplemental Fig. S2).b extra means extracellular.c intra means intracellular.d NDmeans not detected.e The presence of GLBP was examined by Western blot analysis as described under “Experimental Procedures” (see supplemental Fig. S1).

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saccharide concentrations were plotted as a function of theincubation period for B. bifidum JCM1254 (Figs. 1 and 3), B.longum subsp. infantis JCM1222 (Fig. 4 and supplemental Fig.S3), B. longum subsp. longum JCM1217 (Fig. 5 and supplemen-tal Fig. S4), and B. breve JCM1192 (Fig. 6 and supplemental Fig.S5).B. bifidum JCM1254—The concentrations of 2�-FL as well as

LNT rapidly decreased during the early logarithmic phase (5 h)with a concomitant increase of LNB and Lac. 2�-FL, LNB, andLNT had disappeared by the mid-exponential phase (10 h),although trace amounts of Lac remained. The concentrationsof LDFT, LNnT, LNFP I, LNFP II � III, LNDFH I, and LNDFHII gradually decreased during the early to mid-exponentialphase periods. The concentration of 3-FL slightly increased bythemid-logarithmic phase and thendecreased.All oligosaccha-rides were completely consumed after 25 h of incubation. Atthis time, the cells had ceased growing. The concentrations ofFuc and Gal increased and remained unchanged during thegrowth, whereas Glc was found to increase by the mid-expo-nential phase and then decreased by the end of the growthperiod. GlcNAc was not detected throughout the culturingperiod.B. longum subsp. infantis JCM1222—The concentrations of

2�-FL, 3-FL, LDFT, LNT, LNnT, LNFP I, LNFP II� III, LNDFHI, and LNDFH II rapidly decreased when the cells entered thelogarithmic phase (3 h), and negligible levels were detected bythemid-exponential phase (6.5–7.5 h). A transient increasewas

observed for Lac, Fuc, Gal, and Glc at the mid-exponentialphase with peak values at 5.5 h. The majority of the mono- andoligosaccharides were consumed by the end of the growthperiod (12.5 h). GlcNAc and LNB were not detected at anyincubation times.

FIGURE 3. Changes in the concentrations of mono- and oligosaccharidesin the culture of B. bifidum JCM1254 grown in HMO medium. The sampleswere taken at the indicated times and analyzed. A, monosaccharide concen-trations: open squares, Fuc; closed circles, Gal; open triangles, Glc; closed dia-monds, GlcNAc. B, di- and trisaccharide concentrations: open triangles, Lac;closed circles, LNB; open squares, 2�-FL; closed diamonds, 3-FL. C, tetrasaccha-ride concentrations: closed circles, LDFT; open triangles, LNT; open squares,LNnT. D, penta- and hexasaccharide concentrations: closed circles, LNFP I;open triangles, LNFP II � III; closed diamonds, LNDFH I; open squares, LNDFH II.

FIGURE 4. Changes in the concentrations of mono- and oligosaccharidesin the culture of B. longum subsp. infantis JCM1222 grown in HMOmedium. The samples were taken at the indicated times and analyzed. Thesymbols used are the same as described in the legend of Fig. 3.

FIGURE 5. Changes in the concentrations of mono- and oligosaccharidesin the culture of B. longum subsp. longum JCM1217 grown in HMOmedium. The samples were taken at the indicated times and analyzed. Thesymbols used are the same as described in the legend of Fig. 3.

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B. longum subsp. longum JCM1217 and B. breve JCM1192—In the spent media of B. longum subsp. longum JCM1217, arapid decrease in the concentration of LNT was observed from0 to 11 h, with a concomitant increase in the concentration ofLac. The other oligosaccharides remained rather constant dur-ing the cultivation period, although a slight and slow decreaseof LNFP I occurred. GlcNAc and LNB were not detected, andthe Fuc, Gal, and Glc concentrations were at the lowest levelsthroughout the cultivation period. In the culture of B. breveJCM1192, the LNT concentration also immediately decreasedbetween 0 and 9 h of incubation. The concentrations of theother oligosaccharides did not change during the cultivationexcept for a decrease of the Lac concentration. Fuc, Gal, Glc,GlcNAc, and LNB were at negligible levels throughout thegrowth period.

DISCUSSION

Intestinal microflora of breast-fed infants is formed as aresult of a tripartite relationship between mother’s milk, theinfant, and bacteria, and it is commonly accepted that bifido-bacteria dominate the infant gut ecosystem, although the ratioof occupancy varies in different studies (11, 34). The populationof bifidobacteria in a gut ecosystem has been found to drasti-cally decrease after weaning, and the gut microflora becomesadult-like, indicating the presence of bifidogenic compounds inhuman milk. HMOs have apparently no nutritional value forinfants even thought they are included as the third most abun-dant component in human milk (8–10). Recent genetic andenzymatic studies indicate that particular bifidobacteria pos-sess the metabolic capabilities to degrade HMOs (38). Conse-quently, this observation indicates that HMOs support thegrowth of select bifidobacteria in the gut of infants (3, 14).How-

ever, the structural diversity of HMOs and the lack of a simpleand reliable method for quantifying HMOs and their metabo-lites (mono- and disaccharides) hamper a comprehensiveunderstanding of the consumption of HMOs by bifidobacteria.In this study, using a normal phase HPLC approach with a pre-column labeling method, we have elucidated how each of themain neutral HMOcomponents is consumed by the infant gut-associated bifidobacteria.Procedure for Glycoprofiling—Amethod for the quantitation

of bacterial consumption of HMOs was first developed byNinonuevo et al. (2) and LoCascio et al. (18). In that method,HMOs in the control media and in the spent media werereduced with NaBH4 and NaBD4, respectively, mixed in equalvolumes, and then subjected to MALDI-Fourier transform ioncyclotron resonance MS analysis. Quantitation of each HMOwas carried out by comparing the deuterium/hydrogen ratio ofeach peak in the mass spectrum, and the values were expressedas the percentage consumed. This method is very simple androbust, but it is not a suitable quantification approach and can-not differentiate between the glycosyl linkage isomers. In addi-tion, mono- to trisaccharides are difficult to analyze, perhapsbecause of matrix interference. Conversely, we have recentlydeveloped the quantification method of HMOs using a normalphase HPLC method with pre-column labeling with 2-amino-pyridine or 1-methyl-3-phenyl-5-pyrazolone (39, 40). Reversephase HPLC using 2-anthranilic acid as the labeling reagent isalso found to be useful in the analysis ofHMOs (28). The advan-tages of these methods are the high sensitivity toward sugars(regardless of their sizes) and the good separation of isomers. Inthis study, we selected normal phase HPLC and pre-columnlabeling with 2-anthranilic acid because the fluorescence inten-sities of the labeled standard mono- to hexasaccharides werefound to linearly increase as a function of their concentrations(data not shown). The effectiveness of this method is validatedin the results presented in Table 1 and Fig. 1.Physiology of HMOs Degradation in Bifidobacteria—The

presence of the metabolic pathway dedicated to the HMOassimilation was first described on B. longum subsp. longumJCM1217 and B. bifidum JCM1254 (13). The pathway (theGNB/LNB pathway) involves the five proteins/enzymesrequired for the uptake and subsequent degradation of LNB (abuilding block of type 1 HMOs), i.e. the GNB/LNB transporter,GNB/LNB phosphorylase, N-acetylhexosamine 1-kinase,UDP-glucose-hexose-1-phosphate uridylyltransferase, andUDP-galactose epimerase (37). In addition to this pathway, B.bifidum JCM1254 possesses extracellular lacto-N-biosidase,1,2-�-L-fucosidase, 1,3–1,4-�-L-fucosidase, sialidase, �-galac-tosidase, and �-N-acetylhexosaminidase (Table 2 and supple-mental Fig. S2) (15, 23, 41–43). Using these enzymes, the orga-nism can degrade a range of HMOs. Recent reports on thegenomic sequences of the two B. bifidum strains PRL2010 andS17 revealed that they are also equipped with the same reper-toire of enzymes asB. bifidum JCM1254 (16, 35), suggesting theconservation of these enzymes in this species. The type strain ofB. bifidum JCM1255, however, lacks the functional GLBP (sup-plemental Fig. S1). The importance of the GNB/LNB trans-porter for the assimilation of HMOs can be inferred from thepaper of Turroni et al. (16). They showed by comparative

FIGURE 6. Changes in the concentrations of mono- and oligosaccharidesin the culture of B. breve JCM1192 grown in HMO medium. The sampleswere taken at the indicated times and analyzed. The symbols used are thesame as described in the legend of Fig. 3.

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genomic analysis that the absence of BBPR_1056 (an integralmembrane protein of the GNB/LNB transporter) correlateswith a reduced growth on mucin (a good source of GNB).Indeed, B. bifidum JCM1255 did not show significant growthon HMOs (17), whereas robust growth was observed forJCM1254 (Fig. 2) and PRL2010 (16). B. longum subsp. longumJCM1217 possesses extracellular lacto-N-biosidase and intra-cellular �-galactosidase and �-N-acetylhexosaminidase inaddition to the GNB/LNB pathway, but it does not appear tohave �-L-fucosidase (Table 2 and supplemental Fig. S2). Lacto-N-biosidase activity of B. longum subsp. longum is strain-de-pendent as described previously (15), but intracellular �-galac-tosidase and �-N-acetylhexosaminidase could be commonlypresent in this subspecies as B. longum subsp. longumNCC2705 and DJO10A have the corresponding genes (44, 45).Sela et al. (14) found that the genome ofB. longum subsp. infan-tis ATCC15697 (JCM1222) contains, in addition to the genesfor the GNB/LNB pathway, the genetic suite (HMO cluster-1)that apparently enables this organism to assimilate all kinds ofHMOs in different ways to that of B. bifidum. The cluster isconserved amongmany strains ofB. longum subsp. infantis, andthe presence of this cluster is highly correlated with the growthability on HMOs (46). Considering the absence of the lacto-N-biosidase inB. longum subsp. infantisATCC15697 (Table 2 andsupplemental Fig. S2), this subspecies probably uses the HMOcluster-1, not the GNB/LNB pathway, to assimilate HMOs.Compared with the above-mentioned species/subspecies, thephysiology of B. breve has not been considered in associationwith the metabolism of HMOs. The draft genome sequences ofB. breve JCM1192 and DSM20213 contain the genes encodingthe putative intracellular 1,2-�-L-fucosidase, sialidase, �-galac-tosidase, and �-N-acetylhexosaminidase, and all of the genesfor the GNB/LNB pathway, but do not include the genes forlacto-N-biosidase and 1,3–1,4-�-L-fucosidase. In the cell-freeextracts of B. breve JCM1192, the expression of GLBP wasdetected, and the activities of �-galactosidase and �-N-acetyl-hexosaminidase were detected in its cytoplasmic fraction; how-ever, 1,2-�-L-fucosidase activity was not detected (Table 2 andsupplemental Figs. S1 and S2).Thesemetabolic capabilities explain the growth properties of

the respective bifidobacterial strains and reflect the fates ofHMOs and their metabolites during cultivations. In B. bifidum,secretory 1,2-�-L-fucosidase and 1,3–1,4-�-L-fucosidase act onfucosylated HMOs (2�-FL, 3-FL, LDFT, LNFP I/II/III, andLNDFH I/II) to unmask the core structures (Lac, LNT, andLNnT). The rapid decrease of 2�-FL compared with LDFTand the slight increase of 3-FL during the incubation period canbe explained by the facts that the specific activity of 1,2-�-L-fucosidase for 2�-FL is considerably higher than for LDFT andthat the catalytic efficiency (kcat/Km) of 1,2-�-L-fucosidase issignificantly higher than that of 1,3–1,4-�-L-fucosidase (41, 42),although the expression levels of two �-L-fucosidases may dif-fer. 1,2-�-L-Fucosidase of B. bifidum is also known to prefer2�-FL over LNFP I (41), which agrees with the faster decrease of2�-FL than LNFP I. The combined actions of the two �-L-fuco-sidases resulted in an increase in the concentration of Fuc in themedium. However, B. bifidum JCM1254 could not use Fuc as acarbon source (supplemental Table 1), although the genomic

sequencing of the strain PRL2010 indicated the presence of aFuc permease (16). The rapid hydrolysis of 2�-FL and LNTcaused an increase in the levels of Lac and LNB.The presence ofLNB indicates that LNT was actually hydrolyzed by extracellu-lar lacto-N-biosidase, a critical enzyme for the subsequentfunctioning of theGNB/LNBpathway. Interestingly, the resultsshow that the organism leaves these intermediates outside ofthe cell at a time when the cells have begun to grow (Fig. 2).Considering the initial concentrations of 2�-FL, LNT, andLNFP I (3.0, 1.6, and 0.8 mM) (Table 1), the observed increasesin the concentrations of Lac (2.3 g/liter, 6.7 mM) and LNB (0.50g/liter, 1.3 mM) at 5 h are significant. The results imply thatthese two metabolites are shared with different bacterial spe-cies that are not capable of fully metabolizing HMOs but canutilize Lac and LNB as carbon sources (supplemental Table S1)(47). The increase in themonosaccharide concentrations, espe-cially Gal (0.7 g/liter, 3.9 mM at 31 h), is also interesting. Theextracellular �-galactosidase from B. bifidum can liberate Galfrom Lac and type 2 chains of HMOs (supplemental Fig. S2)(23), and B. bifidum is able to utilize Gal as the sole carbonsource (supplemental Table S1); however, a significant amountof Gal was left unconsumed. The result also suggests that themonosaccharide is shared between bacterial species.Conversely, the HMO consumption behavior of B. longum

subsp. infantis JCM1222 was simple. The concentrations of allHMOs were reduced to an equal extent when the cells began togrow. The results strongly suggest that the organism incorpo-rates HMOs in their intact forms and then successivelydegrades these compounds in the cytoplasm from the nonre-ducing ends, as deduced by Sela et al. (14). Considering theintracellular localization of the HMOs degrading glycosidasesin this subspecies (Table 2 and supplemental Fig. S2), the tran-sient increase in the levels of monosaccharides and Lac isintriguing. We do not have a clear answer to this observation,but it is likely that the cells excrete these saccharides to counterthe changes in the osmotic pressure caused by the rapid incor-poration of HMOs. B. longum subsp. infantis JCM1222 doesnot possess the fucose utilization genes and accordingly couldnot grow on Fuc (supplemental Table 1); however, its genomeencodes a putative Fuc permease (Blon_2307) (14).B. longum subsp. longum JCM1217 and B. breve JCM 1192

showed slight growth and consumed LNT only. The action ofextracellular lacto-N-biosidase to degrade LNT in B. longumsubsp. longum can be inferred by the transient increase in theconcentration of Lac as observed for B. bifidum. Here, theincrease in the concentration of Lac (0.5 g/liter, 1.5 mM) wasconsistent with a decrease in the concentration of LNT (1.6mM) (Table 1). LNB was not detected, indicating the preferen-tial utilization of LNBover Lac by this organismusing theGNB/LNB pathway (13, 15). In contrast, B. breve appears to incorpo-rate LNT in the intact form, because no intermediates (Gal, Lac,LNB, and lacto-N-triose II) were formed. A previous study onGLBP from B. longum subsp. longum demonstrated that theprotein can bind LNT, although its affinity was significantlylower than those for GNB and LNB (37). The low affinity ofGLBP from B. longum subsp. longum for LNT could be due tosteric hindrance caused by lysine 83, which is located at theentrance of the cavity. Interestingly, in the GLBP gene (gltA)

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encoded in the draft genome of B. breve JCM 1192, the corre-sponding residue is replaced with an asparagine (data notshown), which may enable the GLBP of this strain to bind LNTmore efficiently. Examination of the complete and draftgenomes of bifidobacteria indicates that this position variesamong species and strains. We cannot rule out the possibilitythat B. breve JCM 1192 has the other degradation pathway forLNT; however, the absence of secretory lacto-N-biosidase,�-galactosidase, and �-N-acetylhexosaminidase (supplementalFig. S2) suggests the direct intake of LNT by the strain.Concluding Remarks—The above description indicates the

preferential use of type 1 HMOs by bifidobacteria except for B.longum subsp. infantis, which equally incorporates type 1 andtype 2 HMOs. The conservation of the GNB/LNB pathway inthe four infant gut-associated bifidobacteria and the predomi-nance of type 1 structures found inHMOs imply that bifidobac-teria have coevolved with humans; the presence of these bacte-ria appears to be beneficial to the well being of the infants(8–10). During evolution, B. bifidum and B. longum subsp.infantis appear to have acquired an advanced ability to assimi-lateHMOs.However, significant amounts ofHMOmetabolites(mono- and disaccharides) remained unconsumed in the spentmedia of B. bifidum grown on HMOs, even when the cells didnot attain their maximum growth. The results strongly suggestthe symbiotic sharing of these metabolites between the bacte-ria. In this sense, it is interesting to note that LNB is able toselectively stimulate the growth of bifidobacteria, but not theother genera such as Clostridia, Enterococci, and Lactobacillus(47, 48).

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Motomitsu KitaokaKatayama, Kenji Yamamoto, Hidehiko Kumagai, Hisashi Ashida, Junko Hirose and

Sadaki Asakuma, Emi Hatakeyama, Tadasu Urashima, Erina Yoshida, TakaneGut-associated Bifidobacteria

Physiology of Consumption of Human Milk Oligosaccharides by Infant

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