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Electrophoresis. Author manuscript; available in PMC 2014 June 07.
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Electrophoresis. 2014 June ; 35(11): 17421750. doi:10.1002/elps.201300653.
Hydrolysis of milk gangliosides by infant-gut associated

bifidobacteria determined by microfluidic chips and highresolution mass spectrometry
Hyeyoung Lee1,2, Daniel Garrido2,3,4, David A. Mills2,3, and Daniela Barile1,2
1Department
of Food Science and Technology, University of California, Davis, CA 95616, United
States
2Foods
for Health Institute, University of California, Davis, CA 95616, United States
3Department
of Viticulture & Enology, University of California, Davis, CA 95616, United States
4Department
of Chemical and Bioprocesses Engineering, School of Engineering, Pontifical

Catholic University of Chile, Santiago, Chile
Abstract
Gangliosides are receiving considerable attention because they participate in diverse biological
processes. Milk gangliosides appear to block pathogen adhesion and modify the intestinal ecology
of newborns. However, the interaction of milk gangliosides with gut bifidobacteria has been little
investigated. The digestion products of a mixture of gangliosides isolated from milk following
incubation with six strains of bifidobacteria were studied using nanoHPLC ChipQ-TOF MS. To
understand ganglioside catabolism in vitro, the two major milk gangliosidesGM3 and GD3
remaining in the media after incubation with bifidobacteria were quantified. Individual
gangliosides were identified through post-processing precursor ion scans, and quantitated with the
find by molecular feature algorithm of MassHunter Qualitative Analysis software. B. infantis
and B. bifidum substantially degraded the GM3 and GD3, whereas B. longum subsp. longum and
B. animalis subsp. lactis only showed moderate degradation. MALDI FTICR MS analysis enabled
a deeper investigation of the degradation and identified ganglioside degradation specifically at the
outer portions of the glycan molecules. These results indicate that certain infant gut-associated
bifidobacteria have the ability to degrade milk gangliosides releasing sialic acid, and that these
glycolipids could play a prebiotic role in the infant gut.
Keywords
Bifidobacteria; Gangliosides; Mass spectrometry; Nano liquid chromatography
Correspondence: Daniela Barile, Department of Food Science and Technology, University of California, Davis, CA 95616, United
States, [email protected], Tel: +1-530-752-0976, Fax: +1-530-752-4759.
The authors have declared no conflict of interest.
Lee et al.
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1 Introduction
The oligosaccharides in milk and their glycoconjugates have gained considerable attention
because of their biological importance, including prebiotic activity [1, 2]. These
heterogeneous glycans consist of various monosaccharides, e.g., glucose, galactose, Nacetylglucosamine, and frequently, fucose and/or N-acetylneuraminic acid (Neu5Ac or sialic
acid) residues assembled via glycosidic linkages. Milk glycans are minimally affected by
gastric enzymes as they transit through the stomach and small intestine, but instead they can
deploy pathogen binding to the intestinal epithelium, and be utilized as a carbon source by
commensal bacteria in the gut [3, 4]. These glycans can be divided into two groups, neutral
and anionic, depending on the existence of anionic sialic acid residues in the molecule. Both
neutral and anionic milk oligosaccharides play an important role in the enrichment of gut
bifidobacteria in breast-fed infants [57].
Although the high concentrations of free oligosaccharides in milk can explain a substantial
part of their prebiotic effect, glyconjugates such as glycolipids and glycoproteins might
contribute to this function due to their carbohydrate moieties[8]. Acidic glycolipids
(gangliosides) are typically elongated from a lactose core (Gal1-4Glc) and contain at least
one sialic acid. Ganglioside synthesis occurs in a stepwise fashion, with an glucose and
galactose added to ceramide lipid and then subsequent sugars transferred by
glycosyltransferases from nucleotide sugar donors. The most abundant gangliosides in
human and bovine milk are monosialoganglioside GM3 (Neu5Ac2-3Gal1-4GlcCer) and
disialoganglioside GD3 (Neu5Ac2-8Neu5Ac2-3Gal1-4GlcCer) [9]. Milk gangliosides
exist in the milk fat globule membranes, which are degraded by diverse lipases in the
gastrointestinal tract, releasing lipids that are readily absorbed into the cells that line the
small intestine. However, only a small proportion of gangliosides seem to be absorbed after
oral ingestion [10]. In contrast, Park et al. [11] reported that a ganglioside-enriched diet
increased the content of total gangliosides in rat intestinal mucosa, plasma, and brains.
However, the degree of ganglioside bioavailability has not been well investigated. A recent
study showed that the bioaccessibility of gangliosides from human milk is rather low [12].
Therefore, their presence in human milk seems to correlate with a biological role in the
gastrointestinal tract [13]. Therefore, the interaction between gangliosides and gut bacteria
would seem to be biologically important.
A few studies showed the interaction between glycolipids and infant intestinal microbiota.
Larson et al. [14] first reported glycolipid excretion in the feces of newborn and young
children who were fed breast milk. They subsequently showed that extracellular
glycosidases from several gut bacteria degraded intestinal glycolipids [15, 16]. Rueda et al.
[17] showed in clinical studies that the addition of gangliosides, in concentrations similar to
those in human milk, to an adapted milk formula altered the microbial composition of feces
from preterm, newborn infants. These data suggest that fortification of infant formula with
gangliosides results in a growth-promoting effect on bifidobacteria.
However, to our knowledge, the specific ability of bifidobacteria to catabolize gangliosides
from milk has not been explored, and the fate of digested milk gangliosides is not well
understood. This is probably due to the lack of accurate analytical methods for quantitation
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of these molecules. Because of the amphipathic nature and structural complexity of

gangliosides, classical analytical methods for their analysis use multiple chromatographic
steps along with extensive sample preparation, which generally requires large volumes of
samples. Moreover, the analytical methods often fail to detect both the ceramide and glycan
moieties simultaneously. Recently, mass spectrometry (MS) analyses of gangliosides were
conducted via soft ionization techniques, such as matrix-assisted laser desorption/ionization
(MALDI) and electrospray ionization (ESI). Often, upfront chromatographic separation of a
mixture of gangliosides makes it possible to detect more ions by minimizing ion suppression
by other polar lipids [9, 18].
In this study, we explored the ability of six representative bifidobacterial species to digest
milk gangliosides in vitro. Following incubation with the bacteria, the major milk
gangliosidesGM3 and GD3and their subspecies were extracted from growth media and
quantified using nanoHPLC Chip quadrupole-time of flight (Q-TOF) MS. Furthermore,
matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron
resonance (FTICR) MS was used to profile the degradation products. This is the first report
to show catabolism of milk gangliosides, other than free oligosaccharides, by various
Bifidobacterium species. The findings are suggestive that milk gangliosides may have a
prebiotic effect on these microorganisms in humans, especially in breast-fed infants.
2 Materials and methods

2.1 Chemicals and materials
HPLC grade methanol, isopropanol, and 2,5-dihydroxybenzoic acid were purchased from
Sigma (St. Louis, MA). Ammonium acetate and acetic acid were of analytical reagent grade
and from Merck (Darmstadt, Germany). The C8 and aminopropyl (NH2) silica gel cartridges
were obtained from Supelco (Bellefonte, PA).
2.2 Preparation of milk gangliosides
Bovine milk gangliosides were purified as previously described[9], but adapted for largescale purification. The distribution of the main bovine milk gangliosides and their
subspecies was previously reported [9].
2.3 Microorganisms and media

The Bifidobacterium strains used in this study were B. breve SC139, B. longum subsp.
infantis ATCC15697, B. bifidum SC555, B. longum subsp. longum SC596, B. adolescentis
UCD318, and B. animalis subsp. lactis JCM 10602. The strains were obtained from the
American Type Culture Collection (Manassas, VA), and the University of California, Davis
Viticulture and Enology Culture Collection (Davis, CA). Bacteria were routinely grown on
De Man, Rogosa, and Sharpe (MRS) broth supplemented with 0.05 % w/v L-cysteine
(Sigma-Aldrich, St. Louis, MO) under anaerobic conditions (Coy Laboratory Products,
Grass Lake, MI) at 37C in an atmosphere consisting of 5% carbon dioxide, 5% hydrogen,
and 90% nitrogen.
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2.4 Bifidobacterial incubation with milk gangliosides
To test if bifidobacteria could use or interact with milk glycolipids, 15 mg of milk

gangliosides extracted as above were first suspended in 3 mL of modified MRS (mMRS),
containing 0.05% L-cysteine. Each of the six Bifidobacterium species mentioned above was
incubated with four concentrations of the milk ganglioside preparation (0.5 mg/mL, 1.0
mg/mL, 1.5 mg/mL, and 2.0 mg/mL). Two l of each overnight culture grown on regular
MRS with 0.05% cysteine were used to inoculate 200 l of mMRS supplemented 0.05%
cysteine and with each sterile-filtered glycolipid concentration as the sole carbohydrate
source. Reactions were carried out in triplicate, and performed in 96-microwell plates.
Optical density was monitored in a PowerWavemicroplate spectrophotometer (BioTek
Instruments, Winoosky, VT) at 37 C for 60 h, reading culture absorbance at 600 nm.
Finally, cells were centrifuged at 10,000 g for 2 min, and supernatants were recovered and
stored at 80 C. Controls with no carbon source and no bacteria were included, as well as
tests for parallel growth of each strain on 2% lactose as the sole carbon source in the same
conditions. The OD reads values from the control with no carbon source were considered as
background and withdrawn from experimental OD reads.
2.5 Ganglioside purification

The gangliosides were recovered from the bacterial supernatant (2.0 mg/mL of gangliosides)
through two consecutive chloroform-methanol extractions. Briefly, a 50-L aliquot of
sample was mixed with 200 L methanol, 100 L chloroform, and 25 L water, and the
mixture shaken vigorously for 5 sec. After centrifugation at 3,000 g for 5 min, 30 L of
water were added. The supernatant was collected, the pellet re-extracted with 300 L of
3:4:8 water/chloroform/methanol (v:v:v), and the supernatants were pooled. The ganglioside
fractions were further purified through C8 solid phase extraction (SPE). Prior to use, the
cartridge was washed with 3 vol of methanol:isopropanol (1:1, v/v) and conditioned with
another 3 vol of methanol:water (1:1, v/v). The ganglioside samples were loaded into the
SPE cartridge and washed with 6 vol of methanol:water (1:1, v/v), eluted with 2 vol of
methanol:isopropanol (1:1, v/v), and dried in vacuo.
2.6 Neutral glycolipid extraction
Lipids were recovered from the incubation supernatant through Folch extraction. A 50-L
aliquot of a sample was mixed with 82.5 Lof methanol and 165 L of chloroform, and the
bottom layer was collected and dried. Neutral glycolipids were enriched through
aminopropyl (NH2) SPE [19]. The cartridge was washed with 3 vol of acetone:methanol
(9:1.35, v/v) and conditioned with another 3 vol of ethyl acetate:hexane (15:85, v/v). The
lipid fractions were loaded into the NH2 cartridge and washed with 6 vol of ethyl
acetate:hexane (15:85, v/v). The neutral glycolipids were eluted with 2 vol of
chloroform:methanol (23:1, v/v) and 2 vol of acetone:methanol (9:1.35, v/v). These two
fractions were combined and dried in vacuo.
2.7 nanoHPLC Chip Q-TOF analysis of gangliosides
Gangliosides in the supernatants were analyzed using a nanoHPLC Chip Q-TOF system
equipped with an Agilent 1200 series microwell-plate autosampler, capillary pump, nano
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pump, HPLC-Chip interface, and an Agilent 6520 Q-TOF MS (Agilent Technologies, Inc.,
Santa Clara, CA). The chip consisted of a 40-nL enrichment column and a 43 0.075-mm
ID analytical column. Both columns were packed with a ZORBAX C18 (5-m pore size)
stationary phase. The mobile phases were water (solvent A) and 15% isopropanol in
methanol (v/v) (solvent B), both containing 20 mM ammonium acetate and 0.1% acetic acid.
For the loading of the sample onto the enrichment column, the capillary pump was operated
at 3 L/min with 70% solvent B. A gradient-based chromatographic separation was
performed on the analytical column, driven by the nanoliter pump running at 300 nL/min.
The gradient used for this separation was as follows: 70% of B until 1 min after sample
injection (1 L), followed by a linear increase to 80% B after 3 min. The gradient further
increased in a linear manner over 40 min to 100% B and was kept at 100% B for 5 min. At
that point, the amount of solvent B was decreased to 70% and maintained at this level until
completion of the run.
The Agilent 6520 Q-TOF MS was operated in the negative-ion mode for MS scans and
MS/MS. The drying gas temperature and gas flow were 325C and 4 L/min, respectively.
Recorded mass ranges were m/z 5002500 for MS only and m/z 501500 for MS/MS.
Acquisition rates were 1 spectrum/s for MS and 3 spectra/s for MS/MS. All mass spectra
were internally calibrated with reference masses at m/z 680.036 and 1279.995. Datadependent MS/MS analysis was performed, with collision energies set at 40V. The precursor
ions yielding 1000 counts/s ion intensity or more were automatically selected for MS/MS.
Data analyses were performed with MassHunter Qualitative Analysis software ver. B.04.01
(Agilent Technologies, Inc., Santa Clara, CA). Molecular Feature Extraction was performed
through the Find by Molecular Feature function for the non-targeted approach. The
software was capable of generating a peak list (m/z, retention time and peak area), taking
into account all ions exceeding 1000 counts. The m/z correlated to the gangliosides; the
intensity of a particular mass indicated the relative amount of specific gangliosides
remaining after incubation with bacteria. A focused post-processing precursor ion-scan
analysis was performed through the Find by Auto MS/MS to detect gangliosides
specifically. In the negative-ion mode, Neu5Ac ion (m/z 290.095) was the fragment ion
chosen to determine the Q1 masses representing gangliosides. Due to the high mass
accuracy and resolution of the Q-TOF mass analyzer, it was possible to extract an exact
compound chromatogram with a narrow mass window for gangliosides, thus excluding the
false positives.
Relative GM3 and GD3 abundances were calculated with respect to the uninoculated control
by normalizing the summed abundance of each GM3 and GD3 spectra in ion counts in the
supernatant from the gangliosides incubated with bacteria (bacteria sample) to that of the
control using the following equation:
Eqn.
1
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Eqn.
where API is absolute peak intensity and n is the number of identified gangliosides.
2.8 MALDI-FTICR MS analysis
Neutral glycolipids were profiled via a HiRes MALDI FTICR MS instrument with an
external MALDI source, a 355-nm pulsed Nd:YAG laser, a quadrupole ion guide, and a 7
Tesla superconducting magnet (IonSpec, Irvine, CA). A 1-L aliquot of each sample (in
50% chloroform in methanol) was spotted on a stainless steel probe, followed by the
addition of 0.5 L of 0.01M NaCl and 1L of the 2,5-dihydroxybenzoic acid matrix. The
samples were mixed on the probe surface and dried under vacuum prior to analysis. Ions
were desorbed from the sample target plate by laser shots. In the ion cyclotron resonance
cell, the ions were excited and detected. Acquisition scans were performed on each sample
in the positive mode. Transients were acquired with IonSpec OMEGA software.
3 Results and discussion

Mothers milk and humans intestines contain high contents of gangliosides, and they may
have a key role in neonatal health as they are involved in modification of intestinal
microbiota and the promotion of intestinal immunity development in the neonate [13, 20].
Disialoganglioside GD3 and monosialolganglioside GM3 are the major gangliosides in
human milk. GD3 is the predominant form in colostrum and it decreases during lactation,
whereas GM3 is the main ganglioside in milk during late lactation [21, 22]. In normal
physiological processes, changes occur in the molecular compositions of the gangliosides.
The GD3 increases in cells during development and cell differentiation [23, 24]. Changes of
the gangliosides depend on the balance between the activities of the enzymes in the
biosynthetic and degradative pathways. To ensure proper functioning, cells regulate the
amounts of gangliosides in the body by intracellular compartmentalization, rapid turnover,
and chemical modification [25]. Because their biological functions depend on their
structures, GM3 and GD3 have different roles in physiological processes. Ganglioside GM3
is an enterocyte receptor analogue for pathogens such as enterotoxigenic and
enteropathogenic Escherichia coli, and it seems to have a protective role relative to
infections, whereas GD3 is involved in modulating immunity [2629]. Therefore, we
assume that gut bacteria may contribute to the chemical modulation of ingested gangliosides
and ultimately change their biological functions.
We evaluated the ability of six species of intestinal bifidobacteria to grow in the presence of
purified bovine milk gangliosides in vitro. Bifidobacterium is the most abundant genus in
the infant gut microbiota in breast-fed infants [3033]. B. breve, B. longum subsp. infantis,
B. bifidum, and B. longum subsp. longum are the dominant bifidobacteria isolated from
breast-fed infants, whereas bifidobacteria species in feces from bottle-fed infants are more
diverse and include B. adolescentis, which is often found in feces of adults [30, 3335]. The
representative bifidobacteria species were incubated in ganglioside-containing media, and
growth curves showed that gangliosides stimulated the growth of some bifidobacteria. Some
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of the strains, especially B. bifidum, B. breve, and B. longum subsp. infantis, grew to varying
extents with gangliosides as their sole carbon source, as shown by low-to-medium optical
density (OD) values during growth over a 60-h time course (Figure 1). The other
bifidobacterial strains did not seem to grow on any of the concentrations tested (data not
shown). The results indicated that the three bifidobacterial species may cleave the glycan
moiety of these lipids and hypothetically use the glycan portion as a carbon source. Human
milk oligosaccharides have been reported to be utilized by B. longum subsp. infantis, B.
bifidum, and to a lesser extent, strains of B. breve [6, 36]. The utilization of milk
oligosaccharides was explained to result from enzymatic processing of these molecules by
glycosylhydrolases, including sialidases [1, 7, 37]. Therefore, it is probable that species of
bifidobacteria utilize milk gangliosides as carbon sources by cleaving the glycan parts of the
molecule with glycosylhydrolases.
Mass spectrometry analysis of the milk gangliosides remaining after incubation with
Bifidobacterium species provided a detailed representation of the consumption of milk GM3
and GD3 gangliosides. Recently, glycoprofiling of free milk oligosaccharides after
bifidobacteria digestion was conducted by nanoHPLC Chip Q-TOF MS [38]. Here we
developed a nanoHPLC method for milk ganglioside profiling. The nanoLC separations of
gangliosides using microchip-based columns were successfully achieved. Mixtures of
gangliosides extracted from media after incubation with different bifidobacteria species
were separated on a C18 stationary phase. The nanoLC-MS with high mass accuracy and
high retention time reproducibility, along with MS/MS capabilities, provided accurate
assignment and quantitative results.
Figure 2 shows the extracted compound chromatograms (ECCs) of the recovered

gangliosides from the uninoculated ganglioside control (Fig. 2A), the mMRS media
containing B. breve (Fig. 2B), B. longum subsp. infantis (Fig. 2C), and B. bifidum (Fig. 2D).
The separation of milk gangliosides was performed effectively with the C18 stationary
phase. The stationary phase separated the native gangliosides on the basis of the chain length
of the ceramides and the degree of saturation, as shown previously [18]. Thirteen GM3
(peak shaded in blue) and 15 GD3 (peak shaded in pink) peaks were found by the Find by
Auto MS/MS algorithm. No gangliosides other than GM3 and GD3 were found. Peak
assignments with retention times are presented in Supporting Information Table 1S. The
ECCs indicated that B. longum subsp. infantis and B. bifidum significantly depleted the
initial amounts of milk gangliosides.
The data obtained from quantitative analysis of three Bifidobacterium species are shown in
Figure 3 and in Supporting Information Table 1S. Some bifidobacteria species depleted
ganglioside GD3 and its subspecies from the culture medium. Specifically, B. longum subsp.
infantis and B. bifidum degraded most of the GD3. There was less GM3 in gangliosides
incubated with B. longum subsp. infantis and B. bifidum than in the control without bacteria.
Interestingly, B. bifidum depleted most of the GD3, and only two GM3 subspecies were
detected. Regarding the structures of the ceramides, GD3 with d34:1 and d41:1 were the
most abundant subspecies in the non-inoculated milk ganglioside control. It is worth noting
that there was not much difference in degree of catabolism among the gangliosides with
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different ceramide chains, implying that the bacteria accessed the ganglioside molecules
according to their sugar groups rather than their ceramide chains.

Figure 4 shows the sum of the ion intensities of GM3 and GD3 subspecies. B. longum subsp.
infantis and B. bifidum consumed 63% (from 100% to 37%) and 100% (from 100% to 0%)
of GD3, respectively, whereas B. longum subsp. longum, B. adolescentis, and B. animalis
subsp. lactis degraded moderate amounts of GD3, i.e., 30% (from 100 to 70%), 28% (from
100 to 72%), and 48% (from 100 to 52), respectively. The catabolic capacity of the bacteria
towards sialylated oligosaccharides in human milk was previously determined by
monitoring the sialidase activities. B. longum subsp. infantis and B. bifidum exhibited high
sialidase activities, which may explain the high degradation of gangliosides by these two
strains [6, 7, 37]. B. breve is known to have sialidases, which preferentially consume select
acidic oligosaccharides in human milk [38]; however, this strain was not able to utilize the
sialic acids in gangliosides. The ion intensity of GM3 was increased 123% for B. breve and
108% for B. longum subsp. longum, but it was decreased by 42% (from 100 to 58%) for B.
longum subsp. infantis and by 99% (from 100 to 1%) for B. bifidum. In the case of
gangliosides incubated with B. breve and B. longum subsp. longum, the increased GM3
could be the result of sialidase hydrolysis products from GD3. B. longum subsp. infantis and
B. bifidum might exhibit strong 2-3 sialidase activity, which would even degrade GM3 [7].
B. longum subsp. infantis and B. bifidum had strong 2-8 sialidase activity, which degraded
most of the GD3. It was previously shown that B. longum subsp. infantis and B. bifidum
utilized the oligosaccharides in human milk, and the genome sequence of the species
provided an explanation for this phenotype [3941]. These two strains possess a number of
genes that are involved in the consumption of complex carbohydrates, including 2-3 and
2-6 sialidase genes [7, 42]. However, the two species are known to have different strategies
[43, 44]. B. longum subsp. infantis transfers oligosaccharides via binding protein, and they
are degraded by intracellular glycosidases; whereas B. bifidum utilizes and degrades human
milk oligosaccharides at their extracellular surface. Therefore, it is likely that the two
species utilize gangliosides in different ways. For example, B. bifidum may cleave sialic
acids in the gangliosides terminal positions and transport them into the cell. Regarding the
behavior of B. longum subsp. infantis towards gangliosides, we have observed that this
strain is able to degrade GM3 and GD3. Interestingly, most of the glycosylhydrolases in this
bacterium are intracellular, which suggest that the gangliosides are processed inside the
bacterium. Although B. breve and B. longum subsp. longum have weak glycosidase
activities, a recent study showed that B. breve seems to use -sialidase in the preferential
consumption of selected acidic oligosaccharides in human milk [38, 43]. In the present
study, B. longum subsp. longum consumed GD3 to a lesser extent, and no gangliosides were
utilized by B. breve.
To elucidate the glycolipid remaining after their incubation with bifidobacteria, we profiled
the neutral glycolipid and ceramide fractions using MALDI FTICR MS (Figure 5). Whereas
no neutral glycolipids were found in the uninoculated ganglioside control, distinct
lactosylceramides were observed in most of the media incubated with bifidobacteria. For
example, spectra of the media containing B. longum subsp. infantis, displayed several peaks
at m/z of 884.607, 954.688, 968.705, 982.717, and 996.734, which corresponded to LacCer
Lee et al.
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(d34:1), LacCer (d39:1), LacCer (d40:1), LacCer (d41:1), and LacCer (d42:1), respectively
(Supporting Information Table 2S). The structures of their oligosaccharides were confirmed
by tandem MS (data not shown). This high-resolution MS analysis showed the degradation
of specific gangliosides at the outer points of the glycan, leaving lactosylceramide. It is
known that gangliosides and neutral glycolipids are excreted in the feces of infants during
the first months of life[14]. GM3 is the dominant fecal ganglioside in the feces of breast-fed
infants, and the glycolipids are not fully degraded in infants until 23 months after birth, at
which time the microbiota are dominated by bifidobacteria. Therefore, probably the growth
of gut bifidobacteria and the digestion of gangliosides result in the appearance of
lactosylceramide in feces at the time of weaning [14].
3.1 Concluding Remarks
In view of the many biological activities (e.g. as inhibitors of bacterial toxins and enteric
pathogens) of gangliosides, their degradation by subsets of the microbiota in infant feces
would seem to be biologically important. Investigation of the degradation of gangliosides in
vitro by six Bifidobacterium species showed that specific species actively hydrolyzed sialic
acid moieties in gangliosides leaving behind lactosylceramides. The growth of certain
Bifidobacterium species may be explained, at least in part, by their utilization of sialic acid.
Ganglioside catabolism results in the release of free sialic acids in the intestinal tract that
may change the glycolipid profile in the guts intestinal epithelial cells and feces. The results
lead the authors to speculate that the gangliosides in milk, or their degradation products such
as sialic acids, may exert a selective, prebiotic activity on specific bifidobacteria species in
infants.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The authors thank Shelby Punu and Dr. Santiago Ruiz-Moyano for lab assistance, Dr. Carlito B. Lebrilla for
support during this study, and Cora J. Dillard for critical reading of the manuscript. This research was supported in
part by funding from the Bill and Melinda Gates Foundation and National Institutes of Health awards
R01HD061923 and R01AT007079. DG was supported in part by a Fulbright-Conicyt Chile scholarship and a
National Milk Producers Federation scholarship. DAM acknowledges support as the Peter J. Shields Endowed
Chair in Dairy Food Science.
Abbreviations
MS
mass spectrometry
FTICR
Fourier transform ion cyclotron resonance
MRS
De Man, Rogosa, and Sharpe medium
Neu5Ac
N-acetylneuraminic acid
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Lee et al.
Page 12

Figure 1.
Growth curves over a 60-h time course for three Bifidobacterium species grown in MRS
medium supplemented with 0.5mg/mL, 1.0mg/mL, 1.5mg/mL and 2.0mg/mL milk
gangliosides. (A) B. breve, (B) B. longum subsp. infantis, and (C) B. bifidum. Growth was
measured as optical density (OD) of the media at 600nm. Experiments were carried out in
triplicate. Controls consisted of uninoculated medium with milk gangliosides.
Lee et al.
Page 13

Figure 2.
Extracted compound chromatograms (ECCs) of GD3 and GM3 gangliosides remaining in

media after bacterial fermentation.(A) Uninoculated control, (B) B. breve, (C) B. longum
subsp. infantis, and (D) B. bifidum. ECCs show the elution profile of gangliosides via nanoLC/MS.
Lee et al.
Page 14

Figure 3.
nanoHPLCQ-TOF profiles of GM3 and GD3 gangliosides extracted from media after
incubation with bifidobacteria. (A) Ganglioside GM3 and (B) ganglioside GD3.
Lee et al.
Page 15

Figure 4.
Total GM3 and GD3 extracted from media following incubation with Bifidobacterium
species. The control was medium containing the gangliosides incubated without
bifidobacteria. Calculations were based on Equation 1 and Equation 2 for GM3 and GD3,
respectively.

Lee et al.
Page 16

Figure 5.
Positive mode MALDI FTICR MS spectra of the neutral glycolipid fraction of (A) control
and (B) B. longum subsp. infantis-containing medium following incubation. Asterisks (*)
indicate lactosylceramides. The ions are [M+Na]+ forms.

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Published in final edited form as:

Hydrolysis of milk gangliosides by infant-gut associated

of Food Science and Technology, University of California, Davis, CA 95616, United

for Health Institute, University of California, Davis, CA 95616, United States

of Viticulture & Enology, University of California, Davis, CA 95616, United States

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of Chemical and Bioprocesses Engineering, School of Engineering, Pontifical

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Electrophoresis. Author manuscript; available in PMC 2014 June 07.

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of these molecules. Because of the amphipathic nature and structural complexity of

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2 Materials and methods

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2.3 Microorganisms and media

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

2.4 Bifidobacterial incubation with milk gangliosides

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To test if bifidobacteria could use or interact with milk glycolipids, 15 mg of milk

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2.5 Ganglioside purification

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Electrophoresis. Author manuscript; available in PMC 2014 June 07.

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Electrophoresis. Author manuscript; available in PMC 2014 June 07.

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3 Results and discussion

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

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Figure 2 shows the extracted compound chromatograms (ECCs) of the recovered

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

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Fourier transform ion cyclotron resonance

De Man, Rogosa, and Sharpe medium

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

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Electrophoresis. Author manuscript; available in PMC 2014 June 07.

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Electrophoresis. Author manuscript; available in PMC 2014 June 07.

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Extracted compound chromatograms (ECCs) of GD3 and GM3 gangliosides remaining in

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

NIH-PA Author Manuscript

NIH-PA Author Manuscript

Electrophoresis. Author manuscript; available in PMC 2014 June 07.