In Vitro Fermentation of Breast Milk Oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2006, p. 4497–4499
Vol. 72, No. 6
0099-2240/06/$08.00?0 doi:10.1128/AEM.02515-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
In Vitro Fermentation of Breast Milk Oligosaccharides by
Bi?dobacterium infantis and Lactobacillus gasseri
Robert E. Ward,1 Milady Nin
˜onuevo,2 David A. Mills,3 Carlito B. Lebrilla,2 and J. Bruce German1,4
Departments of Food Science and Technology,1 Chemistry,2 and Viticulture and Enology,3 University of California,
Davis, California 95616, and Nestle´ Research Centre, Lausanne, Switzerland4
Received 25 October 2005/Accepted 26 March 2006
It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bi?dobacteria, yet
this activity has not been adequately investigated. In this study, Bi?dobacterium infantis was shown to ferment
puri?ed HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment
HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant
intestine.
Human milk is unique because of the high concentration
(Kluyveromyces fragilis) was added, and the solution was incu-
and diversity of human milk oligosaccharides (HMO). HMO
bated for 1 h at 37°C. Then the solution was extracted with 4
are the third most abundant component of human milk (9),
volumes of chloroform-methanol (2:1, vol/vol), and the aque-
and at least 130 different masses have been identi?ed (14). In
ous layer was collected.
vitro, HMO are resistant to catabolism by host hydrolases (3,
As described by Redmond and Packer (12), monosaccha-
4), and based on the mass balance between consumption and
rides and disaccharides were removed by selective adsorption
excretion, Chaturvedi et al. (1) calculated that 97% of HMO
of HMO, using solid-phase extraction with graphitized nonpo-
pass through infants undigested, while Coppa et al. (2) esti-
rous graphitized carbon cartridges (Supelco Inc., Bellefonte,
mated that 40 to 50% of HMO pass through infants undi-
PA). The oligosaccharides that were retained were eluted with
gested.
water-acetonitrile (60:40) containing 0.01% tri?uouroacetic acid.
A prebiotic function was attributed to HMO based on stud-
The residual lactose and glucose contents in the eluant were
ies done in the 1950s with Bi?dobacterium bi?dus subsp. penn-
determined enzymatically (R-Biopharm, South Marshall, MI).
sylvannicus (6, 7), yet the measurements were based on growth
The bacteria used were B. infantis ATCC 15697 and L.
enhancement, not fermentability, as the media contained lac-
gasseri ATCC 33323. The medium used was L-cysteine-supple-
tose. Direct fermentation of HMO by Bi?dobacterium spp.
mented MRS with either 2% (wt/vol) glucose, 2% (wt/vol)
and/or Lactobacillus spp. has not been demonstrated yet, and
inulin, or 2% (wt/vol) HMO carbohydrate. Bacteria were
there are many questions about the metabolic fate of these
grown in triplicate cultures in 7.5 ml of broth in screw-cap
molecules. Do subpopulations mediate speci?c functions, such
culture tubes (13 by 100 mm) at 37°C. Sterile ?ltered carbo-
as pathogen binding or prebiotic activity? Are speci?c bacterial
hydrate was added to autoclaved media, and tubes were inoc-
species required for catabolism in the gut? The goals of this
ulated with a sterile loop from frozen stock preparations. The
investigation were to determine the fermentability of HMO by
negative controls included inoculated medium lacking carbo-
two representative species of breast-fed infant microbiota, Bi-
hydrate and uninoculated medium. Optical density was mea-
?dobacterium infantis and Lactobacillus gasseri (8, 10) and to
sured with a Klett-Summerson colorimeter (Klett Manufactur-
characterize the changes in the HMO after bacterial growth.
ing Co., Inc., New York, NY) using a no. 45 (green) ?lter.
Determination of the biological basis underlying the HMO
After growth, tubes were centrifuged at 2,000 ? g for 30 min.
abundance in human milk should be of general interest in
The supernatant was collected and used for analysis.
human nutrition (15).
Mass spectrometry (MS) was performed with a HiRes ma-
Pooled milk was provided by the Mother’s Milk Bank of San
trix-assisted laser desorption ionization—Fourier transform
Jose, CA, and the Mother’s Milk Bank of Austin, TX. Oligo-
MS (MALDI-FTMS) instrument (IonSpec Corp., Irvine, CA).
saccharides were extracted as described by Gnoth et al. (5),
Media (100 ?l) containing HMO were concentrated using non-
with modi?cations. One liter of milk was centrifuged at 5,000 ? g
porous graphitized carbon cartridge solid-phase extraction col-
for 30 min at 4°C, and the fat was removed. Ethanol (2 liters)
umns and were eluted with 20% acetonitrile in water. The
was added, and the solution was incubated overnight at 4°C.
eluants were dried and reconstituted in 40 ?l deionized water.
The precipitate was removed by centrifugation at 5,000 ? g for
For analysis, 2 ?l of a sample was spotted with 2 ?l of 0.4 M
30 min at 4°C, and the solvent was removed by rotary evapo-
2,5-dihydroxybenzoic acid and 1 ?l of 0.01 M NaCl.
ration. The concentration of the solution was adjusted to 0.05
Separation of HMO from lactose and monosaccharides was
M with phosphate buffer (pH 6.8), 3,000 U ?-galactosidase
con?rmed by thin-layer chromatography (data not shown).
From 1 liter of milk, the yield was 2.5 g of solids. Glucose and
lactose accounted for ?2% of the total carbohydrate. The
* Corresponding author. Mailing address: Department of Food Sci-
protein and fat contents were not measured.
ence and Technology, University of California, Davis, CA 95616. Phone:
(530) 752-1486. Fax: (530) 752-4759. E-mail: jbgerman@ucdavis.edu.
The growth assay was suitable for fermentation studies as
4497

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4498
WARD ET AL.
APPL. ENVIRON. MICROBIOL.
density resulting from fermentation. B. infantis (Fig. 1, top
panel) grew best on the HMO and began exponential growth
on glucose only after 40 h. L. gasseri (Fig. 1, bottom panel)
grew only on glucose. It was surprising that neither bacterium
fermented inulin, as this compound is a well-documented sub-
strate for bi?dobacteria (13). However, the degree of polymer-
ization of the inulin used in this study was about 30, and Perrin
et al. (11) found that B. infantis metabolized ?5% of the inulin
when the degree of polymerization was ?27.
Figure 2 shows the mass spectrometry data for both bacteria
and the control. These data indicate that the reduction in
HMO with B. infantis was more pronounced than the reduction
in HMO with L. gasseri. The change in the pH of the HMO-
containing media after L. gasseri growth was slight, and the low
cell density observed indicated that this strain did not ferment
much of the HMO. However, the analytes with m/z 1462 and
m/z 1827 were clearly different in the control and L. gasseri
media. At least two explanations could account for this obser-
vation. First, MALDI-FTMS detects intact masses, and it is
possible that L. gasseri partially catabolized these analytes
without fermentation of the resulting monosaccharides. Alter-
natively, it is also possible that these analytes were selectively
FIG. 1. Growth of B. infantis ATCC 15697 and L. gasseri ATCC
adsorbed to the L. gasseri cells that were present in the media,
33323 on glucose (Œ), HMO (?), and inulin (}). The initial pHs of the
which were then removed prior to analysis.
MRS broth media were 6.13 (HMO), 6.44 (inulin), and 6.47 (glucose).
HMO polymers are composed of ?ve monosaccharides
The ?nal pHs for B. infantis were 4.63 (HMO), 5.05 (glucose), and 6.46
(inulin). The ?nal pHs for L. gasseri were 5.83 (HMO), 3.94 (glucose),
linked by at least 12 different glycosidic bonds, and the degree
and 6.13 (inulin).
of polymerization ranges from 3 to 32. Consequently, complete
catabolism of these molecules should require an extensive set
of glycosidases and membrane transporters. The increase in
there was very little growth without added carbohydrate. Fig-
the cell density of B. infantis, coupled with the reduction in the
ure 1 shows growth curves for both bacterial species on all
pH and the disappearance of HMO species in the spent media,
three substrates. The ?nal pHs are indicated in the ?gure
indicates some of the HMO were fermented. Based on the
legend and in general are consistent with the change in optical
results of this work, it appears that B. infantis has at least some
FIG. 2. Results of MALDI-FTMS analysis of HMO in control and spent media. The inset shows the constituent monosaccharides for the HMO
species detected. Hex, hexose; HexNAc, hexosamine; Fuc, fucose.

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IN VITRO FERMENTATION OF HMO
4499
of the enzymes necessary for catabolism of HMO. While our
using a novel high performance liquid chromatography-mass spectrometry
results support the hypothesis that HMO may function as a
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Arch. Biochem. Biophys. 48:202–208.
this is the case, the differential detection of HMO in the feces
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This research was supported in part by an NIH-funded fellowship
ti?cation and detection methods. J. Pediatr. Gastroenterol. Nutr. 30:61–67.
under the Training Program in Biomolecular Technology (grant T32-
9. Kunz, C., S. Rudloff, W. Baier, N. Klein, and S. Strobel. 2000. Oligosaccha-
GM08799) at the University of California, Davis, by the California
rides in human milk: structural, functional, and metabolic aspects. Annu.
Dairy Foods Foundation, UC Discovery, NIEHS Superfund grant P42
Rev. Nutr. 20:699–722.
ES04699, and by the CHARGE study (grant P01 ES11269).
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