Glycobiology of Human Milk Oligosaccharides - Glycom

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The First International Conference on the 

Glycobiology of Human Milk Oligosaccharides 

May 16 & 17, 2011 

Tivoli Hotel, Copenhagen, Denmark 

BOOK OF ABSTRACTS


The First International Conference on the 

Glycobiology of Human Milk Oligosaccharides 

May 16 & 17, 2011 

Tivoli Hotel, Copenhagen, Denmark 

Main topics 

Significance of Carbohydrates 

HMO Metabolism in Humans 

Microbial Colonization and HMOs metabolism by microbiota 

HMO Content & Composition: New Analytical Approaches 

Potential of HMOs & Principal Components in Health and Disease 

Organizing Committee 

Professor Clemens Kunz, University of Giessen, Germany 

Professor Sharon Donovan, University of Illinois, USA 

Christoph Röhrig, Judit Kovacs & Susanne Mau, Glycom A/S, Denmark 

Website: www.glycom.com

P 01 Contents 

P 03 Conference program 

P 05 Conference dinner 

CONTENTS 

Contents 

Abstracts of plenary lectures and oral communications 

P 07 CLEMENS KUNZ (Germany): Research on HMOs – A brief historical review 

P 08 HUDSON FREEZE (USA): Sweet solution: Sugars for health? 

P 09 PEDRO ANTONIO PRIETO et al. (Mexico): Human Milk Oligosaccharides: from synthesis to clinical tests and collateral 

observations 

P 10 TADASU URASHIMA et al. (Japan): Possible significance of the predominance of type 1 oligosaccharides, a human specific 

feature, in breast milk 

P 11 CLEMENS KUNZ et al. (Germany): In vivo 13C-labeling of milk oligosaccharides and their metabolism in infants 

P 12 SHARON M. DONOVAN et al. (USA): Transcriptome of the human infant intestinal ecosystem 

P 13 THIERRY HENNET et al. (Switzerland): The role of milk sialyllactose on intestinal bacterial colonization 

P 14 DAVID A. MILLS (USA): Nursing our microbiota: Interactions between bifidobacteria and milk oligosaccharides 

P 15 MOTOMITSU KITAOKA (Japan): Bifidobacterial enzymes involved in the metabolism of human milk oligosaccharides 

P 16 DAVID S. NEWBURG et al. (USA): Human milk oligosaccharides alter human faecal microbiota during in vitro 

fermentation 

P 17 DANIEL GARRIDO et al. (USA): Characterization of glycosyl hydrolases in bifidobacterium longum subsp. infantis active 

on human milk oligosaccharides 

P 18 DEVON KAVANAUGH et al. (Ireland): Increased adherence of bifidobacterium longum subsp. infantis to HT-29 cells 

following exposure to a predominant human milk oligosaccharide 

P 19 RUDOLF GEYER et al. (Germany): Strategies for screening and structural characterization of HMOs 

P 20 CARLITO B. LEBRILLA (USA): High throughput analysis and quantitation of free oligosaccharides 

P 21 JOHN S. KLASSEN et al. (Canada): Quantifying human milk oligosaccharide-protein interactions in vitro using 

electrospray ionization mass spectrometry 

P 22 DENNIS BLANK et al. (Germany): Lewis blood group detection from human milk oligosaccharide mass finger prints 

P 23 SHUAI WU et al. (USA): The development of an annotated structure library for human milk oligosaccharides 

P 24 UTE KRENGEL et al. (Norway): Blood group dependence of cholera infections 

P 25 NORBERT SPRENGER (Switzerland): Sialic acid utilization 

P 26 BING WANG (China): Nutritional significance of sialic acid in human milk: an essential nutrient for brain development 

and cognition 

P 27 LARS BODE et al. (USA): Human milk oligosaccharides in amebiasis and necrotizing enterocolitis 

P 28 SØRGE KELM et al. (Germany): Interactions of milk glycoconjugates with siglecs 

P 29 JASMINE GRINYER et al. (Australia): Protein-sugar interactions between secreted fluids and pathogens as a protective 

mechanism 

P 31 Short biographies of invited speakers 

1

Abstracts of posters 

CONTENTS 

Contents 

P 35 WAI YUEN CHEAH et al. (Australia): Role of sialic acid in innate immune protection provided by mammalian milk 

P 36 SHARON M. DONOVAN et al. (USA): Ascending colonic microbiota composition and SCFA patterns produced from in 

vitro fermentation of human milk oligosaccharides and prebiotics differ between formula-fed and sow-reared 

piglets 

P 37 VICTORIA DOTZ et al. (Germany): Oligosaccharide MALDI-MS profiles in milk and urine from mother-child pairs 

P 38 AMR EL-HAWEIT et al. (Canada): Human milk oligosaccharides as anti-adhesion candidates for clostridium difficile 

toxin 

P 39 SABRINA ETZOLD et al. (UK): Structure determination of bacterial mucus-binding proteins and their functional role 

in adhesion to host glycans 

P 40 LEONIDES FERNÁNDEZ et al. (Spain): Breast milk microbiota: is there a relationship with HMOs? 

P 41 SURI S. IYER (USA): Tailoring carbohydrates to capture toxins and pathogens 

P 42 TAKANE KATAYAMA et al. (Japan): α-L-Fucosynthase that specifically introduces Lewis a/x antigens into type-1/2 

chains 

P 43 JONATHAN A. LANE et al. (Ireland): A new methodology for screening of bacteria-carbohydrate interactions: anti- 

adhesive milk oligosaccharides as a case study 

P 44 MAGDALENA ORCZYK-PAWIŁOWICZ et al. (Poland): Sialylation and fucosylation of human milk α1-acid glycoprotein 

during the first two weeks of lactation 

P 45 MAGDALENA ORCZYK-PAWIŁOWICZ et al. (Poland): The relative amounts of fucose isoforms in oligosaccharides of 

human milk fibronectin 

P 46 KRISTINE A. SCHOLAND et al. (Germany): Effects of specific milk oligosaccharides on the expression of interleukin-8 

and marker enzymes of intestinal cell maturation 

P 47 KATELYN ZAK et al. (USA): In vivo production of fucose-α1, 2-lactose 

P 48 BETSY YANG et al. (USA & Taiwan): Sialylated galactosides of human milk as inhibitors of enterovirus 71 and A 

(H1N1) 2009 influenza infections 

P 49 ARDYTHE L. MORROW et al. (USA): The oligosaccharide (OS) phenotype of preterm infants predicts risk: A potential 

indication for HMOS administration? 

P 50 MAKSIM NAVAKOUSKI et al. (Russia): Natural antibodies against milk oligosaccharides 

P 51 GER T. RIJKERS et al. (The Netherlands): Primary prevention of allergic diseases by probiotics: impact of HMOs 

P 52 The Sponsor: Glycom 

P 53 List of participants 

2

08.00 Registration & Coffee 

CONFERENCE PROGRAM 

Conference Program 

Monday, May 16, 2011 

09.00 Welcome by John Theroux, Glycom & Clemens Kunz, University of Giessen, GERMANY 

09.15 

09.50 

10.25 


Chairs: Sharon Donovan, Bing Wang 

Research on HMOs – A brief historical review 

Clemens Kunz, Ph.D. – University of Giessen, GERMANY 

Sweet Solution: Sugars for Health? 

Hudson Freeze, Ph.D. – Sanford-Burnham Medical Research Institute, La Jolla, CA, USA 

Human Milk Oligosaccharides: From Synthesis to Clinical Tests and Collateral Observations 

Pedro Antonio Prieto, Ph.D. – Tecnológico de Monterrey, Health Sciences Division, Monterrey, MEXICO 

10.50 Break and Poster Viewing & Coffee 

11.15 

11.50 

12.25 

13.00 Lunch 

14.00 

14.35 


Chairs: David Mills, Norbert Sprenger 

Type 1 HMO Predominance: A Specific Feature in Human Milk 

Tadasu Urashima, Ph.D. – Obihiro University, Hokkaido, JAPAN 

In vivo 13 C-labeling of Milk Oligosaccharides and Metabolism in Infants 


Transcriptome of the Human Infant Intestinal Ecosystem 

Sharon Donovan Ph.D., R.D. – University of Illinois, Urbana, USA 

Microbial Colonization and HMOs Metabolism by Microbiota 

Chairs: Hudson Freeze, Carlito Lebrilla 

The Role of Milk Sialyllactose on Intestinal Bacterial Colonization 

Thierry Hennet, Ph.D. – University of Zurich, SWITZERLAND 

Nursing Our Microbiota: Interactions Between Bifidobacteria and Milk Oligosaccharides 

David Mills, Ph.D. – University of California, Davis, USA 


15.30 

16. 05 

16.30 

16.45 

Bifidobacterial Enzymes Involved in the Metabolism of Human Milk Oligosaccharides 

Motomitsu Kitaoka, Ph.D. – National Food Research Institute, Ibaraki, JAPAN 

Human Milk Oligosaccharides alter Human Fecal Microbiota During In Vitro Fermentation 

David S. Newburg, Ph.D. – Boston College, Chestnut Hill, MA, USA 

Characterization of Glycosyl Hydrolases in Bifidobacterium longum subsp. infantis active on Human 

Milk Oligosaccharides 

Daniel Garrido – University of California, Davis, USA 

Increased Adherence of Bifidobacterium longum subsp. infantis to HT-29 cells following Exposure to a 

Predominant Human Milk Oligosaccharide 

Devon Kavanaugh – Teagasc Food Research Centre, Cork, National University of Ireland, IRELAND 

17.00 END OF SESSION 

18.00 Meet in Lobby – Leave for Tivoli Gardens at 18.15 

19.00 Dinner at Tivoli Gardens (Reception opens at 18.30) 

3

08.30 

09.05 

09.40 

CONFERENCE PROGRAM 

Conference Program 

Tuesday, May 17, 2011 


Chairs: Tadasu Urashima, Lars Bode 

Strategies for Screening and Structural Characterization of HMOs 

Rudolf Geyer, Ph.D. – University of Giessen, GERMANY 

High Throughput Analysis and Quantitation of Free Oligosaccharides in Mammalian Milk 

Carlito Lebrilla, Ph.D. – University of California, Davis, USA 

Quantifying Human Milk Oligosaccharide-Protein Interactions In Vitro using Electrospray Ionization 

Mass Spectrometry 

John Klassen, Ph.D. – University of Alberta, CANADA 


10.25 

10.40 

10.55 

Lewis Blood Group Detection from Human Milk Oligosaccharide Mass Finger Prints 

Dennis Blank – University of Giessen, GERMANY 

The Development of an Annotated Structure Library for Human Milk Oligosaccharides 

Shuai Wu – University of California, Davis, USA 

Blood Group Dependence of Cholera Infections 

Ute Krengel, Ph.D. – University of Oslo, NORWAY 

11.10 Poster Session & Coffee 

13.00 Lunch 

14.00 

14.35 


Chairs: Rudolf Geyer, Thierry Hennet 

Sialic Acid Utilization 

Norbert Sprenger, Ph.D. – Nestlé Research Center, Lausanne, SWITZERLAND 

The Nutritional Significance of Sialic Acid on Neurodevelopment and Cognition 

Bing Wang, Ph.D. – University of Sydney, Australia; Xiamen University & Nestlé Research Center, Beijing, P. R. CHINA 

15.10 Coffee break 

15.20 

15.55 

16.20 

16.35 

Human Milk Oligosaccharides in Amebiasis and Necrotizing Enterocolitis 

Lars Bode, Ph.D. – University of California, San Diego, USA 

Interactions of Milk Glycoconjugates with Siglecs 

Sørge Kelm, PhD. – University Bremen, GERMANY 

Protein-Sugar Interactions between Secreted Fluids and Pathogens as a Protective Mechanism 

Jasmine Grinyer – Macquarie University, Sydney NSW, AUSTRALIA 

Summary of Conference 

Sharon Donovan Ph.D., R.D. – University of Illinois, Urbana, USA 


4

CONFERENCE DINNER 

Conference Dinner at Tivoli Gardens 

The Reception for the Conference Dinner starts on Monday evening at 18.30 in the H. C. Andersen 

Castle inside Tivoli Gardens (① on the map). The Dinner will start at 19.00. 

To enter Tivoli Gardens you will require the entrance & dinner ticket that you received together with 

your conference material if you signed up for the dinner in time. 

If you stay at the Tivoli Hotel we recommend that you come to the Lobby at 18.00. To Tivoli Gardens it 

is approximately a 10 minute walk from the Hotel. 

If you stay at another location, please use the map to find Tivoli Gardens and ask then at the entrance 

for the H. C. Andersen Castle (Tivoli Gardens is big and there are several restaurants). 

5

CONFERENCE DINNER 

Conference Dinner at Tivoli Gardens 

Menu 

Roasted scallop with lamb’s lettuce and pesto 

(Ristet kammusling med vårsalat og pesto) 

Fillet of beef with mild rose pepper, pancetta and beans 

(Oksefilet med mild rosenpeber, pancetta og bønner) 

Chocolate cake Marie José with preserved rhubarb and créme Anglaise 

(Chokoladekage Marie José med syltede rabarber og creme anglaise) 

Wines 

Casa Mayor Chile, Chardonnay and Merlot 

6

ABSTRACTS OF PLENARY LECTURES & ORAL COMMUNICATIONS 

Abstracts of Plenary Lectures & Oral Communications 

Research on HMOs – A brief historical review 

CLEMENS KUNZ 

Institute of Nutritional Science, University of Giessen, Germany 

7 


The early history of human milk oligosaccharides (HMOs) comprises research focusing on chemical milk 

composition in general, but also on physiological and nutritional aspects and chemical structure determinations. 

At the end of the 19th century, human milk and cow’s milk were thought to contain a mixture of lactoses 

differing strongly in their properties. Between the 1920s and 1950s, it was found that this lactose fraction was 

composed of a mixture of components called “gynolactose”. Their essential feature was the presence of nitrogen 

and hexosamines. Pediatricians observed that in feces of breast-fed infants compared to infants receiving 

formula, bifidobacteria were the predominant microorganisms; oligosaccharide fractions containing N- 

acetylglucosamine were growth promoting and thus named “bifidus factor”. At the same time, the structures of 

major HMOs such as lacto-N-tetraose, neo-lacto-N-tetraose and their fucosylated derivatives, as well as sialylated 

and fucosylated lactose have been identified. The last 30 years have been dominated by the development of 

powerful analytical tools to identify and characterize HMOs and by a vast number of functional studies in vitro. 

Today, due to the enormous biotechnological progress we are at the beginning of a new era focusing on specific 

effects of HMOs in animals and humans.

Sweet solution: Sugars for health? 

HUDSON FREEZE 


Sanford Children’s Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA 

There’s little surprise when sugars, energy and life appear in the same sentence, but sugars are much more than nutrition and 

energy. Complex carbohydrates, polysaccharides, glycans, call them what you will, are seldom well known and their 

physiological functions are usually underappreciated. Glycan synthesis begins with activation of selected monosaccharides 

and their localization next to biosynthetic enzymes for assembly, remodeling and recycling in the glycan biosphere. The flat 

metabolic chart inadequately reflects the complexity of the cellular organization, regulation and balance inherent in these 

competing and complimentary homeostatic processes. 

The shear complexity of grappling with human health drives medicine to define and conceptualize organ systems and 

enumerate their pathogenic symptoms. But the fundamental rules of basic science respect no artificial constructs. Combine 

that fact with an under appreciation of the diversity of glycan structure and function, and it leaves the current medical system 

ill-prepared to deal with glycan based pathologies arising from genetic or environmental insults. Here are a few examples of 

the consequences and complexities of faulty monosaccharide biosynthetic pathways. 

Congenital disorder of glycosylation (CDG) Type Ib (CDG-Ib) patients show failure to thrive, numerous gastrointestinal 

problems, hypoglycemia, and liver fibrosis due to a phosphomannose isomerase deficiency (MPI, Fruc-6-P→ Man-6-P). 

Providing oral mannose supplements reverse all symptoms except fibrosis because mannose bypasses the deficient step. 

Good. In mice, complete loss of MPI is embryonic lethal and additional mannose only hastens their demise due to Man-6-P 

accumulation and ATP depletion. Not good. Make a hypomorphic mouse line with patient-levels of residual MPI activity 

(15%) and embryos survive, but adult mice reach old age showing few if any pathologies; obviously not a model for CDG-Ib. 

But stress young mice on a high fat diet, and they gain far less weight than their wild-type brothers. That seems a good 

outcome. However, give a pregnant hypomorphic mouse mannose supplements in her drinking water and she aborts all the 

hypomorphic progeny. Reduce the amount of mannose and embryos survive, but half of the progeny are blind by 5 weeks of 

age, because of abnormal eye development during gestation. So mannose is good, bad, and ugly in different scenarios. What 

does this mean for MPI-deficient patients, heterozygous parents or individuals using mannose as a therapy or prevention of 

E. coli-based urinary tract infections? The answer is unknown, but cautious use of this simple sugar may be wise. 

Some patients who have a fucosylation deficiency due to the loss of a GDP-Fucose transporter in the Golgi normalize 

their elevated circulating neutrophils within a few days of being provided fucose. Other patients with mutations in the same 

gene are unresponsive. Experiments in mouse models of this disorder show that controlling leukocyte number depends on 

appropriate fucosylation of proteins in the critical Notch signaling pathway. 

Supplements of N-acetylglucosamine were reported to reverse serious intestinal pathology some among children with 

Crohn’s disease. These findings led to testing oral N-acetylglucosamine as a treatment for T-cell mediated autoimmunity in a 

mouse model with good results. 

Recently, a deficiency in the ER-localized glucose-6-P’ase3 (G6PC3) was found to cause ER stress and compromise 

glycosylation of gp91 component of the NADPH oxidase, resulting in neutropenia. Moreover, patients with glycogen storage 

disorder (GSD) 1b due to a glucose-6-P translocase deficiency show similar neutrophil dysfunction. These results suggest that 

glucose homeostasis will be important in not only nutrition, but also in the biosynthesis of both N- and O-glycans. 

These examples show that manipulating the metabolic flux of monosaccharides can have marked influences on protein 

glycosylation and health in different organ systems. Basic glycobiology underlies and interconnects multiple medical 

disciplines for the benefit of the most important stakeholders, the patients and their families. 

Supported by The Rocket Fund and a Sanford Professorship in Glycobiology 

8


Human Milk Oligosaccharides From Synthesis to Clinical Tests and Collateral Observations 

PEDRO ANTONIO PRIETO 1 AND JAMES LEACH 2 

1 Tecnológico de Monterrey, Health Sciences Division, Monterrey, Mexico 66250 

2 Abbott Nutrition, Columbus, Ohio, United States 43219 

During the decade of the 90s and the first years of the present century, a group of scientists and engineers 

sponsored by Abbott Laboratories embarked in the endeavor of synthesizing and testing human milk 

oligosaccharides. By the year 2000 it was possible to synthesize and test kilogram amounts of the core 

oligosaccharide Lacto-N-neotetraose which, in turn, allowed testing of this structure in clinical settings. In 

parallel fashion, we explored the production of oligosaccharides in the milk of transgenic animals, in vitro 

prebiotic activity of carbohydrates and an extensive analysis of human milk samples to ascertain their contents 

of neutral oligosaccharide structures. Some of our findings emerged from fortuitous observations such as the 

ability of dried yeast to drive oligosaccharide synthesis in the presence of suitable glycosyltransferases; others 

were predicted from previously published accounts such as the variability of oligosaccharide content in human 

milk samples. Yet other observations were unexpected and contradicted previously published data such as the 

existence of oligosaccharide profiles devoid of fucose-containing structures and the ability of fucosylated 

glycoconjugates to shot down milk production in transgenic rabbits. Taken together, the results that emerged 

from this project demonstrate that there are no longer obstacles for the clinical testing of certain oligosaccharide 

structures and that transgenic expression of secondary gene products in milk is possible in some species but not 

in others. The present account summarizes the evolution of a project from its inception to the evaluation of 

scientifically relevant facts that emerged as byproducts in the quest of attaining access to free soluble 

carbohydrate structures from human milk. 

9 

Funded by: Abbott Nutrition, Tecnológico de Monterrey


10 


Possible significance of the predominance of type 1 oligosaccharides, a human specific feature, 

in breast milk 

TADASU URASHIMA 1, SADAKI ASAKUMA 2, MICHAEL MESSER 3, OLAV T. OFTEDAL 4 

1 Obihiro University of Agriculture & Veterinary Medicine, Obihiro, Hokkaido, Japan. 2 National Agriculture Research 

Center for Hokkaido Region, Sapporo, Japan. 3 School of Molecular and Microbial Biosciences, The University of 

Sydney, Australia. 4 Smithsonian Environmental Research Center, Edgewater, USA. 

Human milk and colostrum contain 12 ~ 13 g/L and 22 ~ 24 g/L of oligosaccharides, respectively. The chemical 

structures of 115 human milk oligosaccharides (HMOs) have been characterized to date. We determined the 

concentrations of 10 neutral and 9 acidic colostrum HMOs collected during the first 3 days of lactation, using 

reverse phase HPLC after derivatization with 2-aminopyridine or 1-methyl-3-phenyl-5-pyrazolon. 1,2 The 

predominant oligosaccharides were found to be Fuc(α1-2)Gal(β1-4)Glc (2'-FL), Fuc(α1-2)Gal(β1-3)GlcNAc(β1- 

3)Gal(β1-4)Glc (LNFP1), Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc (LNDFH1) and Gal(β1- 

3)GlcNAc(β1-3)Gal(β1-4)Glc (LNT), the concentration of each of which was 1 ~ 3 g/L. As these HMOs, other than 

2'-FL, all contain Gal(β1-3)GlcNAc (LNB, type 1), we conclude that HMOs containing the type 1 structure 

predominate over other those containing Gal(β1-4)GlcNAc (LacNAc, type 2). This appears to be a feature that is 

specific to humans since the milk/colostrum of other species, including apes and monkeys, either contain only 

type 2 oligosaccharides or the type 2 predominate over the type 1. 3,4,5 

It was shown in another study that the relative concentration of LNT, when compared with other HMOs, is 

significantly lower in the feces of breast fed infants than in breast milk, suggesting that LNT is metabolized in 

preference to other HMOs within the infant colon. 6 In addition, specific oligosaccharides such as Gal(β1- 

4)GlcNAc(β1-6)Gal(β1-4)Glc or Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)Gal(β1-4)Glc were found in these feces. These 

type 2 oligosaccharides were presumably formed from LNH (Gal(β1-3)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1- 

6)]Gal(β1-4)Glc) or monofucosyl LNH (Gal(β1-3)GlcNAc(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1- 

4)Glc) by the release of LNB during the passage of HMOs through the infant colon, and were relatively resistant 

to further degradation. 

We hypothesize that LNB released from type 1 in preference to type 2 HMOs by Bifidobacterium bifidum 

lacto-N-biosidase can be utilized by other strains of bifidobacteria within the colon of fed infants. The 

predominance of type 1 HMOs may therefore promote the growth of beneficial colonic bifidobacteria, an effect 

which may be stronger than that for type 2 HMOs. Thus acquisition of the predominance of these prebiotic type 1 

HMOs may have had a selective advantage, in terms of survival of newborn human infants, during human 

evolution. 

1S. Asakuma et al., Biosci. Biotechnol. Biochem. 71, 1447-1451, 2007. 

2S. Asakuma et al., Eur. J. Clin. Nutr. 62, 488-494, 2008. 

3T. Urashima et al., In: Comprehensive Glycoscience (J. P. Y. Kamerling ed.), pp. 695-724, Elservier, Amserdam, 2007. 

4T. Urashima et al., Glycobiology 19, 499-508, 2009. 

5K. Goto et al., Glycoconj. J. 27, 703-715, 2010. 

6S. Albrecht et al., Electrophoresis 31, 1264-1273, 2010.


In vivo 13 C-labeling of milk oligosaccharides and their metabolism in infants 

CLEMENS KUNZ 1 AND SILVIA RUDLOFF 2 

1 Institute of Nutritional Science, University of Giessen, Wilhelmstrasse 20, 35392 Giessen, Germany 

2 Department of Pediatrics, University of Giessen, Feulgenstrasse 12, 35392 Giessen, Germany 

There is increasing evidence that HMOs may be of particular importance for the infant. Functions which are 

discussed include anti-adhesive and anti-inflammatory effects, an influence on brain development or preventive 

effects with regard to certain diseases. 

With regard to animal and human studies more knowledge on the metabolic fate of HMOs is needed. The use of 

stable isotope ( 13 C)-labelled HMOs facilitates such investigations as it allows the sensitive determination of the 

13 C-enrichment in biological samples such as feces, blood or urine. The combination of isotope ratio mass 

spectrometry and other mass spectrometric methods is well suited to answer metabolic questions in humans 

and animals. In a previous study, we orally applied 13 C-galactose to lactating mothers investigating whether or 

not this monosaccharide was directly used for the biosynthesis of the large amounts of lactose (50-70 g/L) as 

well as of oligosaccharides (5-15 g/L) in milk without being metabolized first-pass by the liver. We hypothesized 

that it could be of advantage to offer galactose instead of glucose to the lactating mammary cell because (a) milk 

biosynthesis can be turned on to a maximum within few minutes, (b) the amount of milk produced by a woman 

can reach up to several liters a day and (c) galactose but not glucose is a main component of HMOs. As the 

resulting in vivo- labelling of HMOs was very effective, we subsequently addressed metabolic questions in the 

infant by analyzing the 13 C-enrichment in faeces or in urine. The data obtained so far will be compared with the 

current knowledge on metabolic aspects. 

Interest in metabolic questions with regard to HMOs in infants started about 40 years ago when Lundblad 

and co-workers in Lund (Sweden) and Strecker´s group in Lille (France) observed that renal excretion of 

oligosaccharides in an infant was partly influenced by the oligosaccharide pattern of the mother’s milk. Today, 

questions regarding HMOs which are of particular importance in the infant comprise (i) their potential 

utilization by the gut microbiota, (ii) physiological processes within the whole digestive tract (from mouth to 

colon), (iii) mechanisms of intestinal absorption of HMOs and (iv) their renal excretion. 

Supported by the German Research Foundation (DFG Ru 529/7-3 and Ku 781/8-3). 

11


Transcriptome of the Human Infant Intestinal Ecosystem 

SHARON M. DONOVAN 1, MEI WANG 1, SHUAI WU 2, CARLITO B. LEBRILLA 2, SCOTT L. SCHWARTZ 3,4, IVAN V. IVANOV 3,4, 

LAURIE A. DAVIDSON 3, JENNIFER S. GOLDSBY 3, DAVID B. DAHL 5, EDWARD R. DOUGHERTY 6, IDDO FRIEDBERG 7, 

DAMIR HERMAN 8 AND ROBERT S. CHAPKIN 2 

1 Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801 USA. 2 Department of 

Chemistry, University of California, Davis, CA 95616 USA. 3 Program in Integrative Nutrition, Center for 

Environmental & Rural Health, 4 Departments of Statistics, 5 Veterinary Physiology & Pharmacology, and 6 Electrical 

Engineering, Texas A&M University, College Station, TX, 77843 USA. 7 Departments of Microbiology and Computer 

Science & Software Engineering, Miami University, Oxford, OH 45056 USA. 8 Winthrop Rockefeller Cancer Institute, 

University of Arkansas, Little Rock, AR 72204 USA 

Our long-term goal is to use non-invasive approaches to define how early nutrition influences intestinal 

development and shapes host-microbe interactions in the intestine of breast- (BF) and formula (FF) -fed infants. 

Human milk contains a rich diversity of oligosaccharides (HMO) that serve as substrates for fermentation, act as 

prebiotics for beneficial bacteria, block the attachment of pathogens and modulate immune development of the 

infant. We have developed a novel molecular methodology that utilizes stool samples containing intact sloughed 

epithelial cells to quantify intestinal gene expression profiles in the developing human neonate (AJP 

2010;298:G582-9). Stool samples were collected from 3-month-old FF (n=10) and BF (n=12) infants and gene 

expression was assessed by microarray analysis. Linear Discriminant Analysis (LDA) identified single genes and 

the two- to three-gene combinations that distinguished the feeding groups. In addition, putative "master" 

regulatory genes were identified using Coefficient of Determination analysis. Recently, this database was 

extended to include breastmilk HMO composition determined by GC/MS, infant stool short chain fatty acids 

(SCFA) by GC and infant microbiota composition and gene expression (in a subset of samples, n=6/group) by 

Roche 454 metagenomic pyrosequencing. 2-Fucosyllactose (2’FL) was predominant in the milk of 3 mothers, 

whereas lacto-N-tetraose (LNT) predominated in 3 mothers. Total SCFA and propionate concentrations were 

greater in stool from FF vs. BF infants. Similar to host mRNA expression, bacterial DNA phylogenetic profiles 

provided strong feature sets that clearly classified FF vs. BF babies. Several quantitative approaches for 

integrating host cell gene expression and the bacterial phyla profiles were applied. First, LDA was used to predict 

single gene expression for each of the 16,853 host genes using the percentage of Firmicutes and Actinobacteria 

present in stool as covariates. For 394 genes, all model coefficients were significant (R 2 >0.7; q-values 

0.7, q-values


Microbial Colonization and HMOs Metabolism by Microbiota 

The role of milk sialyllactose on intestinal bacterial colonization 

ANDREA FUHRER 1, NORBERT SPRENGER 2, EKATERINA KURAKEVICH 1, LUBOR BORSIG 1, YEN-LIN HUANG 1, 

CHRISTOPHE CHASSARD 3 AND THIERRY HENNET 1 

1 Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Switzerland 

2 Nestlé Research Center, Vers-chez-les-Blanc, Lausanne, Switzerland 

3 Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Switzerland 

Milk oligosaccharides influence the composition of intestinal microbiota and thereby mucosal inflammation. 

Recently, we have shown that mice fed on milk deficient for sialyl(2,3)lactose were more resistant to dextran 

sulfate sodium (DSS)-induced colitis. By contrast, the exposure to milk containing or deficient for 

sialyl(2,3)lactose had no impact on the development of mucosal leukocyte populations. The resistance to DSS- 

induced colitis was specifically related to sialyl(2,3)lactose since mice exposed to sialyl(2,6)lactose-deficient 

milk did not react differently than mice exposed to normal milk. Considering the long term protection of 

sialyl(2,3)lactose-deficient feeding, we have analysed and compared the composition of intestinal microbiota in 

mice exposed to normal or sialyl(2,3)lactose-deficient milk during lactation. Using temperature-gradient gel 

electrophoresis and real-time PCR, we did find that milk sialyl(2,3)lactose mainly affected the colonization of 

the intestine by clostridial cluster IV bacteria. The resulting intestinal microbiota presented different metabolic 

properties as shown by changes at the level of short-chain fatty acid production in the caecum of mice exposed to 

either normal milk or sialyl(2,3)lactose-deficient milk. The relationship between intestinal microbiota and the 

severity of DSS-induced colitis was demonstrated by reconstituting germ-free mice with intestinal microbiota 

isolated from mice fed on normal milk or sialyl(2,3)lactose-deficient milk. Germ-free mice harbouring 

microbiota from sialyl(2,3)lactose-deficient fed mice were more resistant to DSS-induced colitis than germ-free 

mice reconstituted with standard intestinal microbiota. Our study shows that the contact to milk 

sialyl(2,3)lactose during infancy affects the bacterial colonization of the intestine, thereby influencing the 

susceptibility to DSS-induced colitis in adult mice. 

Funded by: Zurich Center for Integrative Human Physiology, Nestlé 

13


Nursing our Microbiota: Interactions between Bifidobacteria and Milk Oligosaccharides 

DAVID A. MILLS 

Department of Viticulture and Enology, University of California Davis, Davis, California, USA 

Bifidobacteria are commonly used as probiotics in dairy foods. Select bifidobacterial species are also early 

colonizers of the breast-fed infant colon, however the mechanism for this enrichment is unclear. We have 

previously shown that Bifidobacterium longum ssp. infantis is a prototypical bifidobacterial species that can 

readily utilize human milk oligosaccharides as a sole carbon source. Mass spectrometry-based glycoprofiling has 

revealed that numerous B. infantis strains preferentially consume small mass oligosaccharides, abundant in 

human milks. Genome sequencing revealed that B. infantis possesses a bias towards genes required to utilize 

mammalian-derived carbohydrates. Many of these genomic features encode enzymes that are active on milk 

oligosaccharides including a novel 40-kb region dedicated to oligosaccharide utilization. Biochemical and 

molecular characterization of the encoded glycosidases and transport proteins have further resolved the 

mechanism by which B. infantis selectively imports and catabolizes milk oligosaccharides. Expression studies 

indicate that many of these key functions are only induced during growth on milk oligosaccharides and not 

expressed during growth on other prebiotics. In addition, key cell surface oligosaccharide binding proteins in B. 

infantis bind both milk oligosaccharides and epithelial cell surface glycans. Moreover, growth on milk 

oligosaccharides results in significant increases in binding of B. infantis to intestinal cells in vitro. Additional 

sequencing of numerous B. infantis isolates has confirmed that these genomic features are common among the 

infantis subspecies and likely constitute a competitive colonization strategy employed by these unique 

bifidobacteria. By detailed characterization of the molecular mechanisms responsible, these studies provide a 

conceptual framework for bifidobacterial persistence and host-interaction in the infant gastrointestinal tract 

mediated, in part, through consumption of human milk oligosaccharides. 

14


Bifidobacterial enzymes involved in the metabolism of human milk oligosaccharides 

MOTOMITSU KITAOKA 

National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305- 

8642, Japan 

Galactosyl-β1,3-N-acetylhexosamine phosphorylase (GLNBP, EC 2.4.1.211), which was first found in a cell free 

extract of Bifidobacterium bifidum, catalyzes the reversible phosphorolysis of galacto-N-biose (Galβ1,3GalNAc, 

GNB) and lacto-N-biose I (Galβ1,3GlcNAc, LNB). During the cloning of lnpA gene encoding GLNBP from 

Bifidobacterium longum subsp. longum, we found a gene cluster encoding the intracellular pathway specific to 

GNB and LNB, the GNB/LNB pathway, involving N-acetylhexosamine 1-kinase, UDP-glucose-hexose/HexNAc 1- 

phosphate uridilyl transferase, and UDP-glucose/GlcNAc 4-epimerase as well as GLNBP. Both the galactose and 

N-acetylhexosamine parts of GNB and LNB are converted into the compounds to be entered the glycolytic 

pathway. Genes encoding the components of GNB/LNB specific transporter are placed upstream of the genes 

encoding these enzymes. 

Since oligosaccharides containing LNB in their structure (type I) are predominant in HMOs, the possession of 

extracellular enzymes that liberate LNB from HMOs by bifidobacteria explains how HMOs promote the 

bifidobacterial growth. Such extracellular enzymatic system of B. bifidum has been identified including α1,2- 

fucosidase, α1,3/4-fucosidase, sialidase, and lacto-N-biosidase. 

Bifidobacterial enzymes related to their metabolism of HMOs are useful tool for preparing compounds related to 

HMOs. For instance LNB and GNB were produced from sucrose and GlcNAc/GalNAc in one-pot using four 

bifidobacterial enzyme including GLNBP. From 10 L of the reaction mixture, 1.4 kg of LNB was isolated as the 

crystals. The procedure does not contain any chromatographic techniques and is ready to be scaling-up. 

LNB promote growth of particular strains of bifidobacteria that possess GLNBP gene, but not effective to other 

bifidobacteria and most lactic acid bacteria. Most bifidobacterial strains that are usually isolated from feces of 

infants possess the gene, suggesting the function of the LNB structure in HMOs for the growth of bifidobacteria in 

intestines of breast-fed infants. On the other hand, Bifidobacterium adolescentis, the major habitant in adult 

intestine, does not have GLNBP and does not utilize LNB. 

Funded by: The Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry 

15


Human milk oligosaccharides alter human faecal microbiota during in vitro fermentation 

ZHUO-TENG YU AND DAVID S. NEWBURG 

Higgins hall - Boston College, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA 

Aims: 

The effect of the human milk oligosaccharide fraction (HMOS) on infant fecal microbiota cultured in vitro was 

investigated to determine the prebiotic activity of HMOS. 

Methods: 

Slurries of infant feces were cultured in the presence or absence of HMOS. Differences in the resulting microbiota 

culture were measured. 

Results: 

HMOS supplemented cultures developed significantly lower pH than their unsupplemented negative controls, 

and even lower than fructooligosaccharide (FOS) supplemented positive controls. After fermentation, HMOS 

supplemented cultures contained higher lactic acid concentrations than the negative control cultures. HMOS 

supplemented microbiota contained significantly more Bifidobacteria and Lactobacillus sp. HMOS 

supplementation significantly decreased the number of E. coli, Clostridium perfringens, and Clostridium difficile. In 

vitro adhesion assays in the presence and absence of HMOS in the medium demonstrate that HMOS significantly 

reduces binding of C. perfringens to Caco-2 cells relative to untreated cells, and relative to binding by symbionts 

(e.g., bifidobacteria) in the presence of HMOS. 

Conclusions: 

HMOS supplementation of infant fecal bacteria in culture stimulate growth of Bifidobacteria and Lactobacilli, and 

inhibit the growth of clostridial pathogens. HMOS also inhibit binding by C. perfringens to intestinal epithelial 

cells. These changes in microbiota community composition are highly reminiscent of differences in microbiota of 

breastfed infants relative to microbiota of prematurely weaned infants. 

Significance: 

These data suggest that the characteristic breastfed microbiome may be attributed largely to the HMOS fraction 

of human milk, and that the HMOS fraction of human milk is highly prebotic. 

Keywords:human microbiota; HMOS; prebiotics; in vitro fermentation; cell adhesion 

16


Characterization of Glycosyl Hydrolases in Bifidobacterium longum subsp. infantis active on 

Human Milk Oligosaccharides 

DANIEL GARRIDO 1,5,6,7, DAVID A. SELA 2,5,6,7, SHUAI WU 3,5,6, ROGELIO JIMENEZ-ESPINOZA 4,7, HYUN-JU EOM 2,5,6,7, J. 

BRUCE GERMAN 1,5,6,7, DAVID E. BLOCK 2,4,7, CARLITO B. LEBRILLA 3,5,6,7 AND DAVID A. MILLS 2,5,6,7 

Departments of 1 Food Science and Technology, 2 Viticulture and Enology, 3 Chemistry and 4 Chemical Engineering, 

University of California Davis, CA USA; 5 Foods for Health Institute, University of California Davis, 6 Functional 

Glycobiology Program, University of California Davis, 7 Robert Mondavi Institute for Wine and Food Sciences, 

University of California Davis, USA 

Human milk provides the infant with several protective mechanisms that encourage its proper development and 

growth. Human milk contains a high concentration of complex oligosaccharides, which putatively serve as 

prebiotics, favoring the growth of specific members of the infant gut microbiota. As expected, bacteria residing in 

this environment have evolved molecular mechanisms for the utilization of these complex substrates. This 

includes transport mechanisms and glycosyl hydrolases active on complex oligosaccharides. Bifidobacterium 

longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota previously 

characterized by its ability to consume several oligosaccharides found in human milk. The genome of this 

microorganism revealed various enzymatic systems potentially active on complex host-derived oligosaccharides, 

including -fucosidases, -hexosaminidases and -galactosidases. Five genes encode for -fucosidases in B. 

infantis. Among these, Blon_0248, Blon_0426 and Blon_2336 were specific for 1-3-fucose linkages, while 

Blon_2335 was active on substrates containing 1-2 fucose. The latter two genes were induced by growth on 

HMO or lacto-N-tetraose, the most abundant isomer in HMO. Among five genes encoding -galactosidases, 

Blon_2016 was induced by LNT and was specific for Galb1-3GlcNAc linkages, abundant in type 1 HMO. On the 

other hand Blon_2334 was active on Galb1-4GlcNAc, motif characteristic of type 2 HMO. Both enzymes showed 

high Kcat values using ONPG. On the other hand, three -glucosaminidases showed activity on HMO with a 

general -hexosaminidase activity, but with different kinetic efficiencies. One enzyme, Blon_2355, was induced 

by HMO isomers such as LNT. Finally, these results provide a more complete picture of how B. infantis is able to 

consume host milk oligosaccharides, as well as it identifies a number of genes likely associated with 

consumption of host glycans. 

17


Increased adherence of Bifidobacterium longum subsp. infantis to HT-29 cells following exposure 

to a predominant human milk oligosaccharide 

DEVON KAVANAUGH 1,2,3, LOKESH JOSHI 2,3, MARIAN KANE 2,3 AND RITA M. HICKEY 1,3 

1 Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland 

2 National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland 

3 Alimentary Glycoscience Research Cluster, Ireland 

The intestinal microbiota is vital to human health and nutrition as evident by its contributions to nutrient supply 

through degradation of non-digestible food components as well as through the provision of colonisation 

resistance to enteropathogens and modulation of mucosal immunity. Bifidobacteria are a member of the 

dominant microbiota and can represent up to 80% of cultivable bacteria in infants and 25% in adults. 

Bifidobacteria have demonstrated a capacity to digest human milk oligosaccharides (HMOs) via specific 

adaptations, possess the ability to influence pro-inflammatory immune responses, and have been assessed for 

their efficacy in the treatment and prevention of many human and animal gastrointestinal disorders. 

Furthermore, a recent study employing human milk, in which the fat content was removed; demonstrated that 

growth of a specific strain of Bifidobacterium in the treated human milk resulted in an increased genetic 

expression of putative type II binding fimbriae which are implicated in bacterial colonisation. As the majority of 

oligosaccharides in breast milk are able to traverse the GI tract and reach the colon undigested, perhaps HMOs 

may contribute not only to selective growth of commensal bacteria, but to their specific adhesive and colonising 

ability, as well. In the current study, selected human-milk derived oligosaccharides and commercially available 

prebiotics were assayed for their ability to promote the adhesion of Bifidobacterium to the human intestinal cells. 

Using these human cell monolayers, we found that pre-incubation of Bifidobacterium with some HMOs 

significantly enhanced bacterial adhesion compared to other treatments. Putative mechanisms of how HMOs 

influence host-commensal interactions were investigated and will be presented in the poster. 

18



Strategies for Screening and Structural Characterization of HMOs 

DENNIS BLANK 1, SABINE GEBHARDT 2, KAI MAASS 1, CLEMENS KUNZ 2 AND RUDOLF GEYER 1 

1 Institute of Biochemistry, Faculty of Medicine, Justus Liebig University of Giessen, Giessen, Germany 

2 Institute of Nutritional Science, Justus Liebig University of Giessen, Giessen, Germany 

Human milk oligosaccharides (HMOs) represent a highly heterogeneous class of carbohydrates which are widely 

accepted to be beneficial for human milk fed infants because of their assumed anti-inflammatory, anti-infective 

and immune stimulating properties. The structural heterogeneity of HMOs strongly depends on the expression of 

specific glycosyltransferases in correlation to the mother’s Lewis blood group and secretor status. Hence, 

detailed information on the structural features of these carbohydrates is a prerequisite for future studies on 

potential biological functions of these glycans. 

In order to facilitate structural and/or compositional assignments we have established an experimental protocol 

for mass spectrometric profiling of HMOs starting from fifty microliters of human milk only which allows a rapid 

screening of milk samples from different individual donors. Moreover, diagnostically relevant fragment ions of 

selected key compositional species could be identified by tandem mass spectrometry enabling a correlation 

between the respective HMO pattern and the Lewis blood group of the mother. Based on this information 

conclusions on the expression of specific carbohydrate epitopes for each individual milk sample can be easily 

drawn without the need of a blood sample. 

Characterization of novel HMO structures, however, still requires a different strategy and additional analytical 

tools. To this end glycan fractions were fluorescently tagged with 2-aminobenzamide, separated by 2- 

dimensional HPLC and analyzed in native as well as permethylated state by different mass spectrometric 

techniques. In addition, linkage positions of individual monosaccharides were assigned by digestion with specific 

exoglycosidases or chemical defucosylation in conjunction with GC/MS linkage analysis. Application of this 

approach allowed the complete structural characterization of two novel HMO isomers, i.e., a difucosylated 

octaose and a trifucosylated decaose. 

19


High throughput analysis and quantitation of free oligosaccharides 

CARLITO B. LEBRILLA 

University of California, Davis, CA, USA 

Free oligosaccharides in human milk are involved in several functions necessary for the infant’s health and 

development. A hallmark of these compounds is the large structural diversity, which is compounded by the 

complicated structures typical of oligosaccharides. For this reason research in this area has been hampered by 

the lack of rapid methods for structural analysis. Research in our laboratory is focused at developing methods 

for the rapid analysis and quantitation of milk oligosaccharides. While estimates of the number of 

oligosaccharides range into the tens of thousands, nanoflow liquid chromatography analysis of milk 

oligosaccharides suggests there are only a few hundred structures. A library of structure is being developed by 

systematically determining the structures of a number of oligosaccharides. Furthermore, nanoLC with tandem 

MS provides a rapid method for structural identification of known structures. In this way, hundreds of 

structures can be monitored with complete structures of nearly 100 known. By having a library of known 

structures, biological questions are examined including the role of oligosaccharides as prebiotics and as 

pathogen blocks. 

20


Quantifying human milk oligosaccharide-protein interactions in vitro using electrospray 

ionization mass spectrometry 

AMR EL-HAWIET, GLEN SHOEMAKER, ELENA N. KITOVA, RAMBOD DANESHFAR AND JOHN S. KLASSEN 

Department of Chemistry, University of Alberta, Edmonton, AB, Canada 

The direct electrospray ionization mass spectrometry (ESI-MS) assay has emerged as a powerful tool for 

quantifying the association constants for protein-carbohydrate interactions in vitro. The assay is based on the 

direct detection and quantification of free and ligand-bound protein ions by ESI-MS for solutions of known initial 

concentrations of protein and ligand. The technique boasts a number of strengths, including its simplicity (no 

labeling or immobilization required), speed (measurements can usually be completed within 1-2 min), and the 

unique ability to provide direct insight into stoichiometry and to study multiple binding equilibria 

simultaneously. Additionally, when performed using nanoflow ESI, which operates at solution flow rates in the 

nL/min range, the ESI-MS assay affords high sensitivity, normally consuming picomoles or less of analyte per 

analysis. A brief overview of the ESI-MS assay will be presented, along with recent methodological advances that 

overcome the major sources of error in the binding measurements. Several examples illustrating the application 

of the assay for quantifying binding between bacterial proteins (e.g., toxins and adhesins) and human milk 

oligosaccharides will be given. A new high-throughput ESI-MS approach for screening carbohydrate libraries 

against target proteins will also be described. The “catch and release” ESI-MS assay involves incubating the 

target protein with the library of carbohydrates, detecting the protein-carbohydrate complexes by ESI-MS, 

activating the complexes to release the carbohydrate ligand, followed by fragmentation of the ligand. The 

identification of specific ligands is based on the measured molecular weight and the fragmentation spectrum. 

Collision cross section measurements also aid in ligand identification. Several examples highlighting the 

application of the “catch and release” ESI-MS assay for the discovery of carbohydrate ligands for a variety of 

bacterial proteins, including Clostridium difficile toxins A and B, will be presented. 

21


Lewis Blood Group Detection from Human Milk Oligosaccharide Mass Finger Prints 

DENNIS BLANK 1, KAI MAASS 1, VIKTORIA DOTZ 2, SABINE GEBHARDT 2, CLEMENS KUNZ 2 AND RUDOLF GEYER 1 

1 Institute of Biochemistry, Faculty of Medicine, Justus Liebig University of Giessen, Friedrichstrasse 24, 35392 

Giessen, Germany 

2 Institute of Nutritional Science, Justus Liebig University of Giessen, Wilhelmstrasse 20, 35392 Giessen, Germany 

The structural diversity of human milk oligosaccharides (HMOs) strongly corresponds with the Lewis (Le) blood 

group status of the individual donor. The three different Lewis blood groups, i.e., Le(a−b+), Le(a+b−) and 

Le(a−b−) exhibit different expression levels of fucosyltransferases which result in a great structural variety, in 

particular, in the case of neutral fucosylated HMO species. These differences in the oligosaccharide patterns are 

the basis for our mass spectrometric approach which allows us to assign a milk sample to one of the three 

groups. Starting from fifty microliters of human milk the established method provides a time and material saving 

way for a Lewis blood group classification of milk samples. The relative abundance of diagnostically relevant 

compositional species, such as, Hex2Fuc2, Hex3HexNAc1Fuc2 and Hex4HexNAc2Fuc3 in MS profile spectra is used 

as a first step for the identification. In a second step MS/MS analyses of characteristic precursor ions are 

performed. For each Lewis blood group, specific mass profiles and fragment ion patterns could be identified 

allowing a rapid classification without the need of a blood sample. Furthermore, the outlined protocol can be 

used for rapid screening in clinical studies to gain detailed information about neutral as well as acidic 

oligosaccharide patterns of specific samples, allowing also a relative quantification of individual compositional 

glycan species. Moreover, the described analytical approach enables an easy quality control of milk samples 

acquired from milk banks. 

22


The development of an annotated structure library for human milk oligosaccharides 

SHUAI WU 1, NANNAN TAO 1, RUDI GRIMM 2, J. BRUCE GERMAN 3 AND CARLITO B. LEBRILLA 1 

1 Department of Chemistry, University of California, Davis, CA, USA . 2 Agilent Technologies, Palo Alto, CA, USA 

3 Department of Food Science and Technology, University of California, Davis, CA, USA 

Key Words: 

Human Milk Oligosaccharides; Structure Library; Mass Spectrometry; Exoglycosidase Digestion. 

Background and Significance: 

Human milk oligosaccharides (HMOs) are known as prebiotics stimulating the growth of beneficial intestinal bacteria, and as 

receptor analogs inhibiting the binding of pathogens with cell surface glycans. In addition, the development of a balanced 

intestinal microflora system may play an important role in modulating the postnatal immune system. Furthermore, 

HMOs involved in intestinal absorption and renal excretion may also enhance mineral absorption and promote 

postnatal brain development. Since the absorption, metabolism, and function of oligosaccharides have a strong correlation 

with their structures, a better understanding of HMO structures will provide important insights into their biological 

functions. Almost all HMOs have a lactose core at the reducing end, and they show heterogeneity in terms of composition, 

linkages, and branching resulting in a large variety of structures. Elucidating the structures of HMOs has remained a 

formidable challenge. 

Mass spectrometry provides the most sensitive and rapid method for characterizing HMOs. The combination of nano- 

flow liquid chromatography (nano-LC) and mass spectrometry (MS) provides orthogonal dimensions involving retention 

times, accurate masses, and tandem MS. A HMO structure library has been constructed and annotated using nano- 

LC/MS/MS system. Together with Agilent database software, the HMO library serves as a novel tool for milk research as 

it enables users to identify oligosaccharides very easily and quickly by retention time and mass spectral data. Moreover, it 

provides an important reference to study the secretor and Lewis status of the mother and also the absorption and 

metabolism of HMOs in their offspring. 

Methods and Results: 

The HMOs were extracted from pooled milk sample, then reduced with NaBH4 solution, and followed by desalting with solid 

phase extraction. Sample profiling was performed on the HPLC-Chip/TOF MS instrument, which separates HMOs on a nano- 

LC column integrated in a micro-chip. The results generated retention times, accurate masses, monosaccharide 

compositions, and relative abundances for 224 HMOs. Thirty-two standard milk oligosaccharides were obtained from 

commercial sources and introduced into the Chip/TOF under the identical condition. The corresponding structures in the 

milk pool were determined by matching the retention time and accurate mass against the standards. For de novo structural 

analysis, the sample was separated into fractions using standard HPLC. Each fraction was analyzed by MALDI FT-ICR and 

Chip/TOF to identify the major oligosaccharides in the fraction. IRMPD in the FT-ICR were introduced to study the 

branching and connectivity of the structures. Chip/QTOF provides nano-LC separation with online MS/MS. The 

collision energy applied was scaled to the size of the oligosaccharides with higher collision energy for larger HMOs. The 

fragmentation of isomers under identical collision energy generated different tandem mass spectra. The spectra were distinct 

and could be used to identify the oligosaccharides in a different sample. The exact linkages were determined by employing 

sequential exoglycosidase digestion. So far, 75 known HMO structures yielding a total of over 200 entries are included in the 

library and input into Agilent database software. Accurate mass, retention time, and MS/MS spectra are used to identify all 

the oligosaccharide entries. 

Funded by: NIH R0 HD061923, NIH R01 HD059127, NIH R01 GM049077 

23


Blood Group Dependence of Cholera Infections 

ÅSA HOLMNER 1,2, ALASDAIR MACKENZIE 1 AND UTE KRENGEL 1 

1 Department of Chemistry, University of Oslo, Norway 

2 Current address: Department of Biomedical Engineering & Informatics, Västerbotten County Council, SE-901 85 

Umeå, Sweden 

The cholera toxin from Vibrio cholerae and the heat-labile enterotoxin from Escherichia coli are major virulence 

factors causing cholera and the milder traveler’s diarrhea, respectively. The two toxins share extensive structural 

and functional similarities, but they also display distinct characteristics, which find an expression in their 

biological function. My group is investigating these two toxins by protein crystallography and complementary 

methods. We are interested in elucidating the molecular determinants of these toxins, which determine their 

specific characteristics. A question that we have recently become especially interested in is the origin of the 

blood group dependence of many infectious diseases, and in particular the molecular basis of cholera blood 

group dependence (Holmer et al., 2010). Since human blood group antigens are very similar in structure to 

human milk oligosaccharides, breast feeding might have a more direct beneficiary effect by binding inhibition, in 

addition to stimulating immunological protection. 

In response to increases in temperature, sea level and precipitation variability in already exposed regions of the 

world, water-borne diseases are likely to become an increasing threat. The identification of individuals at 

particular risk for severe disease will hence become increasingly important for crisis minimization. Furthermore, 

understanding of the molecular basis of blood group dependence may pave the way to the development of novel 

intervention tools tailor-made for those groups of the population that are at highest risk. 

Å. Holmner, A. Mackenzie & U. Krengel (2010). Molecular basis of cholera blood-group dependence and implications for a world 

characterized by climate change. FEBS. Lett. 584, 2548-2555. 

24 

Funded by: University of Oslo

Sialic acid utilization 

NORBERT SPRENGER 

Nestlé Research Center, Lausanne, Switzerland 



Postnatal mammalian development encounters milk as a key environmental variable and yet the sole nutrient 

source. Milk is generally considered to have co-evolved with the developmental needs of the suckling newborn. 

One evolutionary conserved constituent of milk is sialic acid generally displayed on glyco-conjugates and free 

glycans. During postnatal development high sialic acid need was proposed to be unmet by the endogenous sialic 

acid synthetic capacity. Hence, milk sialic acid was proposed to serve as conditional nutrient for the newborn. 

Using a neonatal rat model, we revisited the relationship between the sialic acid content of milk and sialic acid 

metabolism in gut, liver and brain, the major sites of sialic acid synthesis and display. At the other end of 

ontogeny, reduced sialylation in brain, saliva and immune system is observed in the elderly. Thus, in analogy to 

the neonates the hypothesis that endogenous synthetic capacity cannot keep up with the need in this age group. 

Using an aged rat model, we explored sialic acid display and synthesis in gut, brain and liver, and further 

investigated the enteric nervous system and stimulated salivation function to assess neuronal function. The data 

propose a functional dietary role of sialic acid as a building block for sialylation and beyond. 

25


Nutritional Significance of Sialic Acid in Human Milk: an Essential Nutrient for Brain 

Development and Cognition 

BING WANG 1,2,3 

1 Human Nutrition Unit, University of Sydney, NSW 2006 Australia 

2 School of Medicine, Xiamen University, Xiamen 361005, P. R. China 

3 Nestlé Research Centre Beijing, P. R. China 

Sialic acid, a family of 9-carbon acidic sugar molecules, are key monosaccharide units in brain gangliosides and 

glycoproteins, including the polysialic acid (polySia) glycotope on neural cell adhesion molecules (NCAM). 

Human milk is one of nature’s richest sources of sialic acid (~1 g/L) and cow’s milk based infant formulas 

contain very little amounts (0~0.25g/L) (1). Gangliosides and polysialylated NCAM in the brain have an 

important role in cell-to-cell interactions, neuronal outgrowth, modifying synaptic connectivity, and memory 

formation. The alpha 2,8 sialyltransferase IV (ST8SiaIV) is one of two key enzymes for synthesizing polySia on 

NCAM. In rodents, the level of NCAM polysialylation increases with learning behavior. The liver can synthesise 

sialic acid from glucose, but the activity of the limiting enzyme, UDP-N-acetylglucosamine-2-epimerase (GNE) is 

low during the neonatal period. An exogenous source of sialic acid may be critical under conditions of extremely 

rapid brain growth, particularly during the first months after birth. We have now demonstrated that dietary 

sialic acid supplementation increased the levels of sialic acid in neural tissues, leading to enhanced learning and 

memory in piglets, and to up-regulate expression of two learning related genes, Gne and ST8SiaIV (1,2). Global 

gene transcription profiling also supports dietary sialic acid supplementation has effects on brain development 

and cognition in piglets. We have also found that sialic acid concentration in the frontal cortex of breastfed 

infants is higher than the levels of formula-fed infants (1). Taken together, milk sialic acid may be a limiting 

nutrient in the neonatal period, facilitating optimal cognitive development in young animals. An exogenous 

source of sialic acid enhances brain development, providing a mechanism to explain the link between breast- 

feeding and higher intelligence. 

References: 

1) Wang B. Annu. Rev. Nutr 2009;29:177. 

2) Wang B et al. Am J Clin Nutr. 2007;85:561-9. 

26


Human Milk Oligosaccharides in Amebiasis and Necrotizing Enterocolitis 

EVELYN JANTSCHER-KRENN 1,2, MONICA ZHEREBTSOV 2, TINEKE LAUWAET 3, CAROLINE NISSAN 1,2, KERSTIN GOTH 4, 

YIGIT GUNAR 4, ANATOLY GRISHIN 4, HENRI FORD 4, SHARON REED 3, FRANCES GILLIN 3 AND LARS BODE 1,2 

1 Division of Neonatology, 2 Division of Gastroenterology and Nutrition, Department of Pediatrics, 3 Department of 

Pathology, University of California, San Diego, 4 Saban Research Institute, University of Southern California, USA 

Human Milk Oligosaccharides (HMO) are highly abundant in human milk but not in infant formula, which 

triggers the questions whether and how HMO benefit the breast-fed infant. Breast-fed infants are at lower risk to 

develop such devastating disorders as amebiasis or necrotizing enterocolitis (NEC), and we hypothesized that 

HMO contribute to the beneficial effects of breast-feeding in the context of these disorders. 

Amebiasis, caused by the protozoan Entamoeba (E.) histolytica, is the third leading cause of death by parasitic 

diseases, surpassed only by malaria and schistosomiasis. Worldwide, approximately 50 million people are 

infected with E. histolytica, resulting in nearly 100,000 deaths annually. E. histolytica resides in the colon where 

it uses a specific Gal/GalNAc lectin to attach to and destroy the host’s epithelial cells. Here, we show that specific 

HMO but also Galactooligosaccharides (GOS) prevent E. histolytica attachment and cytotoxicity. These results 

suggest that HMO contribute to the lower incidence of amebiasis in breast-fed infants compared to formula-fed 

infants. The results also suggest the use of safe, inexpensive and readily available GOS as a novel agent to treat or 

even prevent amebiasis. 

Necrotizing Enterocolitis (NEC) is one of the most common and often fatal intestinal disorders in preterm 

infants. Almost 10% of very-low-birth-weight infants (


Interactions of milk glycoconjugates with siglecs 

HENDRIK KOLIWER-BRANDL 1, NADJA SIEGERT 2, ALEXANDER TOLKACH 2, ULRICH KULOZIK 2 AND SØRGE KELM 1 

1 Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University Bremen, 28334 

Bremen, Germany. 2 Institute for Food Process Engineering and Dairy Science, Technische Universität München, 

Weihenstephaner Berg 1, 85354 Freising-Weihenstephan, Germany 

Siglecs are a family of sialic aid-binding immunoglobulin-like lectins occurring mainly on cells of the immune 

system. Their main functions are probably in the regulation of processes leading to the activation of these cells [1] . 

Consistent with this hypothesis is the observation that most siglecs have tyrosine-based inhibitory motifs 

(ITIMs) within their cytoplasmic domains. Probably their sialic acid-binding activities play important roles in 

these processes. Of particular interest in this context is the interplay of cis-interactions with glycoconjugates on 

their own cell surfaces with trans-interactions with glycoconjugates on opposing cells and in the extracellular 

space. Therefore, sialylated compounds like oligosaccharides, glycoproteins and glycolipids like those occurring 

in human and animal milk have the potential to modulate siglec-regulated activation processes in the immune 

system. 

We have used ELISA-like hapten-inhibition assays [2] to investigate the interactions of milk glycoconjugates 

with Siglec-2, preferentially binding to 2,6-linked sialic acids, and with Siglec-4, which binds preferentially to 2,3- 

linked sialic acids. As possible sources for bioactive sialylated structures we looked at complex and crude 

mixtures from milk fractionation processes. The complementary linkage specificities of these lectins allow the 

determination of both 2,3- and 2,6-linked sialic acids even in the presence of high lactose concentrations, as they 

are common in milk products [3] . Using these assays, the presence of bioactive sialoglycoconjugates has been 

determined in fractions from bovine milk. 

Whereas whole milk from local dairy showed no inhibition, samples from milk fractionation processes like 

buttermilk, skim milk, lactose-reduced serum, whey proteins as well as caseins and GMP (glycomacropeptides) 

were significantly inhibitory, indicating a higher Sia content in these samples. A loss of inhibitory, active 

sialoglycoconjugates during fractionation processes was observed for buttermilk samples treated at different 

temperatures. Particularly, low pH-values after heat treatment appear to be possible reasons for loss of activity. 

In summary, the interaction with Siglec-4 was less sensitive to temperature- and pH-treatment of the samples, 

whereas Siglec-2 has lost its inhibition potency completely. The loss of Siglec-2 binding activity could be caused 

either by a selective hydrolysis of the α2,6-linkage of sialic acids or by conformational changes in the 

glycoconjugate carrier molecules leading to sterically less favored presentations of sialylated glycans serving as 

recognition determinants. 

[1]Crocker PR, Paulson JC, Varki A. Siglecs and their roles in the immune system. Nat Rev Immunol. 2007 Apr;7(4):255-66. 

[2]Bock, N., & Kelm, S. (2006). Binding and inhibition assays for Siglecs. Methods in Molecular Biology, 347, 359–375. 

[3]Koliwer-Brandl, H., Siegert, N., Umnus, K., Kelm, A., Tolkach, A., Kulozik, U., Kuballa, J., Cartellieri, S., & Kelm, S. (2011). Lectin 

inhibition assays for the analysis of bioactive milk sialoglycoconjugates. International Dairy Journal, in press. 

Funding by: German Federal Ministry for Education and Research (BMBF, project BioChangePLUS 031632A), the Tönjes-Vagt 

28 

Foundation (project XXI)


Protein-sugar interactions between secreted fluids and pathogens as a protective mechanism 

JASMINE GRINYER 1,2, WAI YUEN CHEAH 1,2, DANIEL KOLARICH 1,3 AND NICOLLE PACKER 1,2 

1 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney NSW, Australia 2109 

2 Biomolecular Frontiers Research Centre, Macquarie University, Sydney NSW, Australia 2109 

3 Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Glycoproteomics Group, 

Arnimallee 22, 14195 Berlin, Germany 

The oral environment, comprising the mouth, teeth, tongue and buccal cells, is home to many different bacterial 

species. Streptococcus gordonii is an early coloniser of tooth enamel, whereas S. mutans colonises later to cause 

dental caries. Bacterial lectins play a role in the initial attachment and biofilm formation by binding to 

glycoproteins on the saliva-coated enamel of teeth. The human innate immune system has several mechanisms 

for preventing bacterial colonisation on teeth. One such mechanism is thought to be provided by the 

glycoproteins in saliva and human milk by protecting the oral cavity from streptococcal species by binding to and 

removing these bacteria when swallowed. The removal of bacteria from the oral environment is thus in constant 

competition with bacterial binding and infection of the oral cavity. 

We have developed two techniques to monitor bacterial binding, one using fluorescent microscopy to visualise 

bacterial attachment to saliva-coated hydroxyapatite, and a fluorescent assay on PVDF membrane in a 96-well 

plate format to quantitate streptococcal binding to proteins from saliva and human milk. We found that S. 

gordonii binds better to saliva and milk proteins/glycoproteins than S. mutans, and S. gordonii binding is 

reduced after treatment with neuraminidase to remove the sialic acid residues from the glycoproteins. Structural 

determination of saliva and milk protein glycosylation and confirmation of the removal of sialic acids by 

neuraminidase was conducted using LC-ESI-MS. Free sialic acid was also shown to modify streptococcal 

adherence to saliva and milk. We have shown that sialic acid residues are involved in S. gordonii binding (but not 

S.mutans) to saliva and milk glycoproteins, indicating that these secretions can act as a decoy to protect the oral 

environment against infection. 

29 

Funded by: Macquarie University

NOTES 

30

SHORT BIOGRAPHIES OF INVITED SPEAKERS 

Short Biographies of Invited Speakers 

CLEMENS KUNZ – Institute of Nutritional Science, University of Giessen, Germany 

Clemens Kunz received his graduate degrees in nutrition from the University of Bonn where he did his 

PhD on vitamin D and metabolites in milk at the Department of Pediatrics in 1984. He then joined the 

group of Prof. Heinz Egge at the Institute of Physiological Chemistry getting his first scientific contact 

with HMOs and has been fascinated by these unique components since then. Subsequently, he held a 

postdoctoral fellowship at the University of California, Davis, USA from 1986 to 1989 in Prof. Bo 

Lönnerdal’s group focusing on milk proteins and glycoproteins. He then became leader of the working 

group Clinical Chemistry at the Research Institute for Child Nutrition in Dortmund and a Professor of Physiological Chemistry at 

the University of Bonn, continuing his research in the field of HMOs with a focus on metabolic aspects. In 1999 he was appointed 

Professor of Human Nutrition at the Justus-Liebig-University of Giessen. His current research focuses on structural, functional 

and metabolic aspects of HMOs including the use of stable isotopes and on the bioavailability and metabolism of polyphenols 

from berries in healthy humans. His work yielded several research awards and has continuously been funded by the German 

Research Foundation (DFG), the Federal Ministry of Science and Education (BMBF) and the Hessian Ministry of Science and Arts 

(HMWK). 

HUDSON FREEZE – Sanford-Burnham Medical Research Institute La Jolla, CA, USA 

Hudson Freeze earned his Ph.D. from the University of California at San Diego in 1976. Subsequently 

he held fellowships in Biology, Medicine and Neurosciences and later joined the faculty at the same 

institution. In 1988, Dr. Freeze was recruited to Sanford-Burnham Medical Research Institute 

and served as the Director of the Glycobiology and Carbohydrate Chemistry Program from 2000- 

2008. His work focuses on pathology resulting from faulty glycosylation, the process of adding sugar 

chains to proteins and lipids. Carbohydrates are required for proper secretion and targeting of thousands of proteins, an often 

overlooked fact of biology. He is driven by the search for novel therapeutics to treat patients with mutations leading to 

glycosylation defects called Congenital Disorders of Glycosylation or CDGs. 

TADASU URASHIMA – Obihiro University, Hokkaido, Japan 

Tadasu Urashima was born in Hiroshima at 12/3/1957. Degrees: (1) undergraduate course: Tokyo 

University of Agriculture and Technology, 1980. (2) master course: Tohoku University, 1982. (3) Ph.D. 

course: Tohoku University, 1986. Past Positions: (1) Assistant Professor at Obihiro University of 

Agriculture and Veterinary Medicine, April 1986. (2) Associate Professor at Obihiro University of 

Agriculture and Veterinary Medicine, April 1994. (3) Professor at Obihiro University of Agriculture and 

Veterinary Medicine, August 2003. Present position: Professor at Obihiro University of Agriculture and Veterinary Medicine, 

Graduate School of Animal and Food Hygiene. Studies: (1) 1986-present: Characterization of milk oligosaccharides of domestic 

farm animals. (2) 1991: Beta N-acetylglycosaminyltransferase activity in lactating mammary glands of tammar wallaby, Sydney 

University, under Dr. Michael Messer. (3) 1995-present: Characterization of milk oligosaccharides of captured wild animals 

including primate species, Canoidea species, elephant, Felidae species, Cetacea species etc, with Dr. Olav Oftedal. (4) 2000- 

present: Determination of each human milk oligosaccharide concentration. (5) 2009-present: Determination of each milk 

oligosaccharide level in the broth of Bifidobacteria, with Dr. Motomitsu Kitaoka, Dr. Takane Katayama, Dr. Kenji Yamamoto and 

Dr. Sadaki Asakuma. (6) 1994-present: Extracellular polysaccharides produced by dairy lactic acid bacteria, with Dr. Bill Bubb 

and Dr. Kenji Fukuda. (7) 2006-2010: Bioactive components in bovine colostrum, with Dr. Takashi Terabayashi, Dr. Minoru 

Morita and Dr. Kenji Fukuda. 

31



SHARON M. DONOVAN – Department of Food Science and Human Nutrition, University of 

Illinois, Urbana, IL, USA 

Sharon Donovan received her B.S. and Ph.D. in Nutrition from the University of California, Davis. She is 

also a Registered Dietitian. After completing a post-doctoral fellowship in Pediatric Endocrinology at 

Stanford University School of Medicine, she accepted a faculty position at the University of Illinois, 

Urbana in 1991. She was promoted to Professor in 2001 and, in 2003, was named the first recipient of 

the Melissa M. Noel Endowed Chair in Nutrition and Health at the University of Illinois. Her research 

focuses on pediatric nutrition, with an emphasis on optimization of neonatal intestinal development. She compares the 

biological effects of human milk and infant formulas on intestinal function in human infants, neonatal piglets and in various 

models of intestinal disease. She has published over 100 peer-reviewed publications, review articles and conference proceedings. 

Her research is funded by NIH, USDA and private industry and foundations. 

THIERRY HENNET – Institute of Physiology and Center for Integrative Human Physiology, 

University of Zurich, Switzerland 

Thierry Hennet is a Professor in the Institute of Physiology and Center for Integrative Human 

Physiology at the University of Zurich. His research interest is on Defects of glycosylation that lead to 

diseases with variable clinical manifestations, encompassing neurological disorders, congenital 

muscular dystrophies, connective tissue disorders, immune deficiencies, and coagulopathie. 

His group investigates the roles of specific glycoconjugates and glycosyltransferase enzymes in health and disease. To this end, 

they combine the study of genetic animal models to biochemical assays performed in cell culture systems. In addition, he studies 

the molecular basis of novel types of Congenital Disorders of Glycosylation (CDG), a family of diseases characterized by abnormal 

biosynthesis of several classes of glycoconjugates. 

DAVID A. MILLS – Department of Viticulture and Enology, University of California Davis, Davis, 

California, USA 

David Mills is a Professor in the Department of Viticulture and Enology in the Robert Mondavi Institute 

for Wine and Food Sciences at the University of California at Davis. For over 20 years Dr. Mills has 

studied the molecular biology of lactic acid bacteria involved in food and beverage fermentations or 

active as probiotics in intestinal health. In the last decade, Mills led the Lactic Acid Bacteria Genomics 

Consortium which resulted in a seminal comparative analysis and release of key genome sequences of 

food-grade lactic acid bacteria and bifidobacteria. More recently with Bruce German and Carlito Lebrilla, Dr. Mills formed the UC 

Davis Milk Bioactives Program, a multidisciplinary effort to characterize the influence of milk glycans on intestinal health. Dr. 

Mills has served as a Waksman Foundation Lecturer and Chair of the Food Microbiology Division of the American Society for 

Microbiology. He currently serves as an associate editor for the journal Microbiology. In 2010 Dr. Mills was awarded the Cargill 

Flavor Systems Specialties Award from the American Dairy Science Association. 

Dr. Mills obtained a Bachelors of Science in Biochemistry from the University of Wisconsin-Madison. In 1991, he obtained a 

Masters degree in Biochemistry with Dr. Michael Flickinger at University of Minnesota, followed by a PhD in Microbiology in 

1995 working with Dr. Gary Dunny and Dr. Larry McKay. After postdoctoral studies at North Carolina State University with Dr. 

Todd Klaenhammer, Dr. Mills took a faculty position at UC Davis. 

32



MOTOMITSU KITAOKA – National Food Research Institute, National Agriculture and Food 

Research Organization, Tsukuba, Ibaraki 305-8642, Japan 

Motomitsu Kitaoka is a research leader of the enzyme laboratory at the National Food Research 

Institute of the National Agriculture and Food Research Organization, Japan. He obtained a Ph. D. from 

the University of Tokyo in 1993. After completing a post-doctoral fellowship at the Iowa State 

University, he moved to the National Food Research Institute in 1998. He has been working on 

carbohydrate degrading enzymes for more than 20 years, especially on sugar phosphorylases. He has 

been pursuing practical procedures to produce oligosaccharides, including a large-scale preparation of lacto-N-biose I. He is also 

interested in rational mutation to convert glycoside hydrolytic enzymes into catalysts for the syntheses of glycosides. 

RUDOLF GEYER – Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University 

of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany 

Rudolf Geyer studied Chemistry at the University of Darmstadt and the University of Freiburg in 

Germany. After graduation he worked for his Ph.D. at the Max-Planck-Institute of Immunology in 

Freiburg and received his degree in Biochemistry in 1977 at the University of Freiburg. He then 

moved to Giessen, Germany, were he worked as a research assistant and later as a university 

assistant at the Institute of Biochemistry of the Giessen University Medical School. In 1990 he was appointed as a professor of 

biochemistry and leader of the Glycobiology Unit at the same institute. His present research interests are mainly focused on 

structures and putative functions of glycoconjugates from parasitic helminths as well as on the carbohydrate structures of 

mammalian and invertebrate glycoproteins and human milk oligosaccharides. 

CARLITO B. LEBRILLA – University of California, Davis, CA, USA 

Carlito Lebrilla is a Professor at the University of California, Davis in the Department of Chemistry and 

Biochemistry and Molecular Medicine at the School of Medicine. He is currently the Chair of the 

Chemistry Department. He was born in the Philippines. He received his BS degree from the University 

of California, Irvine and Ph.D. from the University of California, Berkeley. He was an Alexander von 

Humboldt Fellow and a NSF-NATO Fellow at the Technical University in Berlin. He returned to the UC 

Irvine as a President’s Fellow and has been at UC Davis since 1989. His research is in Analytical 

Chemistry, primarily mass spectrometry with applications to clinical glycomics and biofunctional food. He has over 225 peer- 

reviewed publications. He is also co-editor of Mass Spectrometry Reviews and has been on the editorial board of Mass 

Spectrometry Reviews, Journal of American Society for Mass Spectrometry, European Mass Spectrometry, and International 

Journal of Mass Spectrometry. 

33



NORBERT SPRENGER – Nestlé Research Center, Lausanne, Switzerland 

Norbert Sprenger studied biology at the University of Basel, Switzerland, and received 

his B.S. in Developmental Biology and his Ph.D. in Molecular Plant Physiology with Profs. 

A. Wiemken and T. Boller for his studies on plant storage, transport and stress 

carbohydrates. He then took a first post-doctoral position in plant molecular physiology 

in the laboratory of Prof. F. Keller at the University of Zurich, Switzerland. For a second 

post-doctoral fellowship, financed by grants from the Swiss National Science Foundation, Novartis and the Human Frontier 

Science Program Organisation, he joined the laboratory of Prof. C. Somerville at Stanford University at the Carnegie Institute for 

Science department of plant biology, where he studied plant cell wall synthesis and physiology using molecular genetics 

approaches. He then switched to mammalian physiology accepting a position as research scientist in Glycobiology at the Nestlé 

Research Center in Lausanne, Switzerland. His research focuses on functional glycans for pediatric and medical nutrition, with an 

emphasis on intestinal development and barrier function. 

BING WANG – Human Nutrition Unit, University of Sydney, Australia; School of Medicine, 

Xiamen University, P. R. China; Nestlé Research Centre Beijing, P. R. China 

Bing Wang received her M.D. degree from Tianjin Medical University, China, and a Ph.D. in Science 

(Biochemistry) from the University of Sydney, Australia. She is a currently an Honorary Associate in the 

School of Molecular & Biosciences at the University of Sydney, a Min-Jiang Scholar and Adjunct 

Professor of Molecular Medicine, School of Medicine, Xiamen University, China and a Senior Research 

Scientist at the Nestlé Research Centre-Beijing. She is an internationally recognized expert in 

nutritional glycobiology. She pioneered the development of the piglet as a model system to elucidate the molecular mechanisms 

underlying the role of milk sialic acid in brain development and cognition. She is also a registered Nutritionist of the Nutrition 

Society Australia. Her studies have been carried out at both the gene and biochemical levels on the functional effect of new food 

ingredients. 

LARS BODE – Division of Neonatology and Division of Gastroenterology and Nutrition, 

Department of Pediatrics, University of California, San Diego, USA 

Lars Bode received his M.S. and Ph.D. in Nutrition from the Justus-Liebig University, Giessen, Germany. 

After determining structural differences in the lipid composition of human and bovine milk gangliosides 

as a Master student, Lars joined Dr. Clemens Kunz’s lab as a PhD student and worked with Dr. Silvia 

Rudloff to study the effects of human milk oligosaccharides on selectin-mediated cell-cell interactions in 

the immune system. After a predoctoral fellowship at the Institute of Child Health, University College 

London, Lars joint Dr. Hudson Freeze’s lab as a postdoctoral fellow in the Glycobiology and Carbohydrate Chemistry Program at 

the Burnham Institute for Medical Research. In 2009 the University of California, San Diego, School of Medicine recruited Lars as 

an Assistant Professor of Pediatrics in the Division of Neonatology and the Division of Gastroenterology and Nutrition, where his 

lab develops a new research program to elucidate functions and biosynthesis of human milk oligosaccharides. Lars is an affiliate 

of the Glycobiology Research and Training Center and the Digestive Disease Research Development Center at the University of 

California, San Diego. Lars is the Chair of the Lactation Research Interest Group of the American Society for Nutrition and on the 

Executive Board of the International Society for Research on Human Milk and Lactation. Lars’ research is supported by an NIH 

Pathway to Independence Award, and funded by NIH and private industry. 

34

ABSTRACTS OF POSTERS 

Abstracts of Posters 

Role of Sialic acid in Innate Immune Protection provided by Mammalian Milk 

WAI YUEN CHEAH 1, KATHERINE WONGTRA-KUL KISH 1, DANIEL KOLARICH 2, JASMINE GRINYER 1, NICOLLE PACKER 1 

1 Department of Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, Sydney, NSW, 

Australia 

2 Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Berlin, Germany 

Sialic acid contained in mammalian milk has been reported to have an immune protective role for the infant. 

Human milk contains a large amount of sialic acid compared with bovine milk, with sialic acids found on 

glycoproteins, glycolipids and free oligosaccharides. However, the mechanism by which this monosaccharide 

inhibits infection remains unknown. We have investigated how milk confers innate immune protection to the 

infant gut through binding of gastrointestinal-associated bacteria and compare the difference in binding of 

specific bacteria to human and bovine milk glycoconjugates. 

Different methods to fractionate human and bovine milk were explored to determine which fraction contains 

bacterial binding glycoconjugates. Milk fractions were adhered to PVDF membranes in a 96 well format. 

Fluorescently-labelled bacteria were added to the wells and the binding of the bacteria to the fractions was 

quantified by fluorescence. To determine whether the glycans were involved in this interaction, in particular 

sialylated glycans, the glycoconjugate structures were altered using exoglycosidases and the effect on bacterial 

binding was measured. Exoglycosidase activities were confirmed by glycan profiling of the N- and O-glycans 

released by PNGase F and β-elimination, respectively, of milk glycoproteins using graphitised carbon LC-ESI- 

MS/MS. The inhibition of binding of the bacteria to the milk glycoconjugates was tested by pre-incubation of the 

bacteria with free oligosaccharides. 

We found that specific bacteria bind differently to human and bovine milk glycoconjugates. Most bacteria had 

higher binding affinity to the milk protein fraction than the lipid fraction. Bacterial binding was inhibited both by 

removal of sialic acid with sialidase and by pre-incubation of the bacteria with sialic acid. This confirms that 

sialic acid can competitively inhibit bacterial adhesion to both human and bovine milk glycoconjugates. These 

data indicate the importance of sialylated milk glycoconjugates in binding to gastrointestinal-associated bacteria 

and providing innate immune protection to the infant’s gut. 

35 

Funded by: Macquarie University


Ascending colonic microbiota composition and SCFA patterns produced from in vitro 

fermentation of human milk oligosaccharides and prebiotics differ between formula-fed and 

sow-reared piglets 

MIN LI 1, LAURA L. BAUER 2, XIN CHEN 1, MEI WANG 1, THERESA KUHLENSCHMIDT 3, MARK S. KUHLENSCHMIDT 3, 

GEORGE C. FAHEY JR. 2 AND SHARON M. DONOVAN 1 

1 Department of Food Science and Human Nutrition, 2 Department of Animal Sciences, 3 Department of Pathobiology, 

University of Illinois, Urbana, IL 61801, USA 

The objective was to compare the effect of piglet age and sow-rearing versus formula feeding on in vitro 

fermentation characteristics and gut microbial modulation properties of human milk oligosaccharides (HMO), 

lacto-N-neotetraose (LNnT), a predominant HMO, and three commonly used prebiotics: a 1:2 mixture of 

galactooligosaccharide (GOS) and polydextrose (PDX), and short-chain fructooligosaccharides (scFOS). Ascending 

colon contents were obtained from four donor groups: 9 and 17 day-old formula-fed (FF9, FF17) and sow-reared 

(SR9, SR17) piglets. The changes in pH and gas, short chain fatty acid (SCFA) and lactate production were 

determined following 0, 4, 8, and 12 h of incubation. The pH change and total SCFA, acetate and propionate 

production were greater in the FF than SR, and in the 9- compared to the 17-day-old piglets, regardless of diet. 

However, SR produced higher amounts of gas, butyrate and lactate than FF piglets. For most donors, the pH 

change was greatest for scFOS, and least for GOS/PDX. The fermentation of LNnT produced larger amounts of gas, 

total SCFA, acetate, and butyrate than did the other substrates, whereas higher amounts of propionate and lactate 

were observed from HMO and scFOS fermentation respectively. Gut microbial populations were assessed by 16S 

rRNA V3 gene denaturing gradient gel electrophoresis (DGGE) analysis and group-specific real-time PCR. Global 

gut microbial structures differed among four piglet groups prior to fermentation. SR had higher concentration of 

Bifidobacterium and Lactobacillus, whereas FF had greater amounts of Clostridium cluster IV and XIVa. The 

concentrations of these four bacteria groups were higher in 9- than 17-day-old piglets. Changes in microbial 

patterns during fermentation were assessed by DGGE. Bands that increased in density on most substrates were 

identified by sequencing as related to Bacteroides vulgatus, Collinsella aerofaciens, Anaerovibrio sp., and species 

belong to Clostridium cluster IV, XIVa and XVIII. The concentrations of Bifidobacterium, Clostridium cluster IV and 

XIVa and B. vulgatus detected by qPCR increased after 8 and 12h fermentation on most substrates. In summary, 

piglet diet and age affect gut microbial populations, which leads to different bacterial fermentation patterns. HMO 

and LNnT have potential prebiotic effects due to their ability to be fermented to SCFA and modulate microbial 

populations. 

36 

Funded by: NIH R01 HD061929


Oligosaccharide MALDI-MS profiles in milk and urine from mother-child pairs 

VIKTORIA DOTZ 1, SILVIA RUDLOFF 1,2, DENNIS BLANK 3, SABINE GEBHARDT 1, KAI MAASS 3, RUDOLF GEYER 3 AND 

CLEMENS KUNZ 1 

1 Institute of Nutritional Science, University of Giessen, Wilhelmstrasse 20, 35392 Giessen, Germany 

²Department of Pediatrics, University of Giessen, Feulgenstrasse 12, 35392 Giessen, Germany 

³Institute of Biochemistry, Faculty of Medicine, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany 

Human milk contains a large variety of complex lactose-based oligosaccharides (HMO) in concentrations ranging 

from 10 to 20 g/L. The presence of different neutral HMO depends on the activity of specific glycosyltransferases 

in the mammary gland. Therefore, the presence of 1-2-, 1-3- and/or 1-4-fucosylated core oligosaccharide 

structures is determined by the secretor status and the Lewis (Le) phenotype of the mother (1,2). So far, little 

information exists about the metabolic fate of HMO in newborns. In previous studies, we found a renal excretion 

of about 1-2 % of the ingested LNT and LNFPII in exclusively breast-fed preterm infants (3). Here, we report on 

the excretion profile of HMO in term infants. 

The aims of the study are (i) to compare the HMO pattern in milk of exclusively breastfeeding women with the 

urinary oligosaccharide profile of their infants and (ii) to investigate whether the infants´ urinary HMO profile 

resembles the pattern of Lewis-specific HMO in their mothers´ milk. 

After application of an oral 13 C-galactose bolus to 8 lactating mothers, milk at each nursing and urine of the 

infants was collected for 36 hours (4). For analytical purposes, 50 µL of a single milk sample and 500 µL of the 

infant urine sample were used for solid phase extraction prior to MALDI-MS of the non-derivatised HMO fraction. 

Lewis blood group determination was performed on all milk samples. 

Resulting from their characteristic MS profile as well as MS/MS spectra, the following Lewis phenotypes were 

found: Milk from 6 women was Le(a−b+), one sample was Le(a+b−) and one presumably Le(a−b−) or Le(a−b+). 

In the corresponding urinary samples of the infants, the same characteristic MS profiles were observed with 

regard to relative HMO peak intensities. However, HMO in the lower mass range were more predominant in 

urine, particularly for the infant whose mother was Le(a+b−). Moreover, MS/MS data revealed that none of the 

Le(a−b+)-specific HMO were detected either in the milk of the Le(a+b−) mother or in the urine of her infant. 

MS/MS of urine from infants fed with Le(a−b+)-milk confirmed the presence of Le(a−b+)-specific HMO. 

Our data indicate that the HMO patterns in urine from breast-fed infants reflect those from their mothers’ milk 

suggesting a strong association to the mothers´ Lewis phenotype determined in milk. 

Literature: 

1) Kunz et al., Annu Rev Nutr 2000; 2) Thurl et al., Br J Nutr 2010; 3) Rudloff et al. Acta Paediatr 1996; 4) Rudloff 

et al., Glycobiol 2006 

Funded by: German Research Foundation Ru 529/7-3 and Ku 781/8-3 

37


Human Milk Oligosaccharides as Anti-adhesion Candidates for Clostridium difficile Toxin 

AMR EL-HAWEIT 1, ELENA N. KITOVA 1, PAVEL KITOV 1, LUIZ EUGENIO 2, KENNETH K.S. NG 2, GEORGE L. MULVEY 3, 

TANIS C. DINGLE 3, ADAM SZPACENKO 1, GLEN D. ARMSTRONG 3 AND JOHN S. KLASSEN 1 

1 Department of Chemistry, University of Alberta, Edmonton, AB, Canada. 

2 Department of Biological Sciences, University of Calgary, Calgary, AB, Canada. 

3 Department of Microbiology & Infectious Diseases, University of Calgary, Calgary, AB, Canada. †Alberta Ingenuity 

Centre for Carbohydrate Science 

Human milk oligosaccharides (HMOs) play an important role in protecting neonates from pathogenic 

microorganism especially the enteric bacteria. This is believed to be due to their ability to act as soluble 

receptors that inhibit the pathogen binding to their host cell target ligands. Clostridium difficile is the leading 

cause of hospital acquired diarrhea and pseudomembranous colitis in Europe and North America. The key 

virulence determinants of C. difficile are the two exotoxins, toxin A (TcdA) and toxin B (TcdB). Those exotoxins 

are believed to bind to carbohydrate receptors on the intestinal epithelium. Here, we describe the first 

quantitative study of the binding of HMOs to the C. difficile toxins. The direct electrospray ionization mass 

spectrometry (ESI-MS) assay has been applied to determine the binding affinities of recombinant subfragments 

TcdA and TcdB of C. difficile toxins against a library of twenty one oligosaccharides representing, the most 

common neutral and acidic HMOs. The results of the ESI-MS measurements indicate that both toxins fragments 

bind specifically to several HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested 

bind to both toxins: Fuc(1-2)Gal(1-4)Glc, Gal(1-3)GlcNAc(1-3)Gal(1-4)Glc, Fuc(1-2)Gal(1-3)GlcNAc(1- 

3)Gal(1-4)Glc, Gal(1-3)[Fuc(1-4)]GlcNAc(1-3)Gal(1-4)Glc and Gal(1-4)[Fuc(1-3)]GlcNAc(1-3)Gal(1- 

4)Glc. Although, the binding of the HMOs is uniformly weak, with apparent affinities ≤3000 M -1 , a number of 

HMOs bind to the toxins with a much higher affinity than the only known natural receptor, 

Gal(1,3)Gal(1,4)Glc, for which binding constants for both A2 and B1 were found to be ~500 M -1 . The results 

of the molecular docking study suggest that the general mode of carbohydrate recognition may be conserved 

between TcdA and TcdB, with a lactose disaccharide occupying the central portion of the carbohydrate binding 

site for both toxins. Analysis of the docked structures also helps to explain how the differences in distributions of 

negatively and positively charged side chains in the binding pocket of TcdA and TcdB influence ligand binding 

specificity. The Verocytotoxicity neutralization assay reveals that HMO fractions extracted from human milk do 

not significantly inhibit the cytotoxic effects of TcdA nor TcdB. The absence of protection is attributed to the 

weak intrinsic affinities that the toxins exhibit towards the HMOs. 

Funded by: 1. Alberta Ingenuity Centre for Carbohydrate Science (AICCS) 

38 

2. Natural Sciences and Engineering Research Council (NSERC) 

3. University of Alberta


Structure determination of bacterial mucus-binding proteins and their functional role in 

adhesion to host glycans 

SABRINA ETZOLD 1, DONALD MACKENZIE 1, LOUISE E. TAILFORD 1, ROB FIELD 2, ANDREW HEMMINGS 3 AND 

NATHALIE JUGE 1 

1 Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK 

2 John Innes Centre, Norwich Research Park, NR4 7UH, UK 

3 University of East Anglia, Norwich NR4 7TJ, UK 

The mucus layer covers the epithelial cells of the gastrointestinal (GI) tract and protects the underlying mucosa 

from the lumen content. The main structural components of mucus are highly glycosylated mucin proteins 

carrying mainly O-linked glycan chains characterised by a high level of structural complexity and diversity. 

Protein-carbohydrate interactions are believed to play an important role in the adhesion of resident gut bacteria 

to the mucus layer. However, the nature of the ligands and the specificity of the interaction remain to be 

elucidated. Our research focuses on lactobacilli mucus-binding proteins (MUB) whose presence on the bacterial 

cell-surface contributes to bacterial attachment to the protective mucus layer. MUBs are composed of tandemly- 

arranged mucus-binding amino acid sequence repeats (Mubs). The 353 kDa MUB from the L. reuteri strain ATCC 

53608 consists of two types of repeats, Mub1 and Mub2, present in six and eight copies, respectively. We 

determined the first crystal structure of a type 2 Mub repeat (184 amino acids) at 1.8-Å, displaying high 

structural similarity to the repeat-unit of the Peptostreptococcus magnus Protein L (PpL), an immunoglobuling 

(Ig)-binding surface protein, and we showed that Mub repeats were able to interact with a number of 

mammalian Igs in vitro. Our current work is to obtain structural information on type 1 Mub repeats and multi- 

repeat modules to gain further insight into the structural organisation of MUB. In addition, we focus on the 

characterisation of the Mub repeat interaction with mucins and mucin glycans, whose structure can be similar to 

oligosaccharides found in milk, using a range of biophysical methods including in vitro binding assays, 

carbohydrate arrays and Isothermal Titration Calorimetry. Elucidating the molecular basis of host-bacteria 

interaction is crucial for the understanding of host colonisation and the beneficial role of lactobacilli in the GI 

tract. 

39 

Funded by: BBSRC, NRP


Breast milk microbiota: is there a relationship with HMOs? 

ANA SOTO, NIVIA CÁRDENAS, SUSANA DELGADO, JUAN MIGUEL RODRÍGUEZ AND LEONIDES FERNÁNDEZ 

Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid, 

Avda. Puerta de Hierro, Madrid, Spain 

Breast milk is a complex biological fluid that provides all the nutritional requirements for the first months of life 

but, additionally, educates the infant immune system and confers protection against pathogens. These effects 

result from the synergistic action of many bioactive molecules, such as cytokines, cellular components, 

oligosaccharides or lipids. While it is well known that breast milk is rich in oligosaccharides, it has only recently 

been accepted that human milk also constitutes a source of commensal and probiotic bacteria which seems to 

play an important role in gut colonization and modulation of the infant gut. 

In recent years, analyses of the bacterial diversity of human milk have revealed that this biological fluid is an 

important source of live staphylococci, streptococci, bifidobacteria and lactic acid bacteria to the infant gut. In 

contrast to other bacteria, these seem to be uniquely adapted to reside in the human digestive tract and to 

interact with us in symbiosis from the time we are born. Traditionally it was considered that bacteria present in 

breast milk were acquired by mere skin contamination. However, it has been found that lactobacilli and 

enterococcal isolates present in human milk are genotypically different from those isolated in the skin within the 

same host. Furthermore, several studies suggest the existence of a mammary microbiota during late pregnancy 

and lactation. Live bacteria from the maternal gut could colonize the mammary gland through an endogenous 

route (the so-called entero-mammary pathway), involving maternal dendritic cells and macrophages. 

In this context, the global objective of this work was to ascertain if there is a relationship between human milk 

oligosaccharides and microbiota. For that purpose, carbohydrate utilization was studied in a variety of lactic acid 

bacteria and bifidobacteria isolated from human milk of healthy donors, and compared with their enzymatic 

activity on chromogenic substrates. Genome sequences of selected bacteria were analyzed to identify possible 

genes involved in the metabolism of carbohydrates, especially those related to HMOs. 

Funded by: AGL2010-15420 (2010-2013), RM2009-00009-00-00 

40


Tailoring carbohydrates to capture toxins and pathogens 

SURI S. IYER 

Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA 

Over the past few years, our group has been harnessing their recognition properties to develop high affinity 

ligands for toxins and pathogens. Carbohydrates are excellent recognition molecules and modulate essential 

communication processes as cell adhesion, proliferation, migration, differentiation and metastasis. Synthetic 

carbohydrates can be used for capturing pathogens as they are highly robust under a variety of conditions, 

refrigeration is not required and can be stored for extended period (over months), inexpensive, not subject to 

lot-to-lot variation, can be manipulated to suit any sensor platform as these are small molecules and not subject 

to antigenic drift because toxicity is intimately associated with the cell binding sites. However, synthetic 

carbohydrates have “thought” to suffer from selectivity and sensitivity issues for practical applications. We have 

recently demonstrated that it is possible to tailor glycoconjugates to achieve high selectivity and sensitivity 

towards specific analytes. We have synthesized a library of tailored carbohydrates using a novel modular 

strategy for the rapid production of these molecules (Bioorg. Med. Chem. Lett., 2007, 17, 2459-64). We have 

demonstrated that synthetic carbohydrates differentiate between closely related strains of Shiga toxin, (Angew. 

Chem. Int. Ed. Engl. 2008, 47, 1265-68; Highlighted in Chemical and Engineering News, 2008, 86, 42) influenza (J. 

Am. Chem. Soc. 2008, 130, 8169–71) and Escherichia coli. (Chembiochem, 2008, 9, 2433-42). Our other relevant 

publications on the molecular nature of carbohydrate-protein interactions include ChemBioChem, 2009, 10, 

1486-89; Biochemistry, 2010, 49, 1649-57; Medicinal Research Reviews, 2010, 30, 327-93; Bioconj. Chem., 2010, 

21, 1486-93 and Biochemistry, 2010, 49, 5954-67. Recently, we have developed a simple, rapid, and sensitive 

glycan-based magnetic relaxation switch assay for the detection of glycan binding proteins. We demonstrated 

that magnetic relaxation switch assays can be used to detect toxins in a complex medium such as stool and 

environmental samples. (Anal. Chem., 2010, 82, 7430-7435). Our efforts in this area towards the development of 

potential diagnostics and therapeutics using natural and modified oligosaccharides will be the focus of this 

presentation. 

Funded by: NSF CAREER GRANT, NSF CHE 0845005; NIAID U01 AI075498 

41


α-L-Fucosynthase that specifically introduces Lewis a/x antigens into type-1/2 chains 

TAKANE KATAYAMA 1, HARUKO SAKURAMA 1, YUJI HONDA 1, MOTOMITSU KITAOKA 2 AND KENJI YAMAMOTO 1 

1 Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921- 

8836, Japan 

2 National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305- 

8642, Japan 

Lewis a (Le a ) and Lewis x (Le x ) blood group antigens attached at the non-reducing ends of glycoconjugates 

control various pivotal biological events; therefore, the efficient synthesis of the antigens is important to 

understand their physiological roles. Here, we show that the nucleophile-mutant D703S of 1,3-1,4-α-L-fucosidase 

from Bifidobacterium bifidum exclusively synthesizes Le a and Le x trisaccharides when incubated with β-L- 

fucopyranosyl fluoride (FucF) as a donor and lacto-N-biose I (Galβ1-3GlcNAc) and N-acetyllactosamine (Galβ1- 

4GlcNAc) as acceptors, respectively, with yields of 56% against the added FucF. The synthase also recognized 

lactose, 2’-fucosyllactose and lacto-N-tetraose as acceptors, and specifically produced 3-fucosyllactose, 

difucosyllactose and lacto-N-fucopentaose II, respectively. In contrast, the enzyme did not accept 

monosaccharides, cellobiose, Glc1-4GlcNAc, N,N’-diacetylchitobiose or galacto-N-biose as substrates. The 

results revealed that the enzyme specifically recognizes the disaccharide structures [Galβ1-3/4GlcNAc(Glc)] at 

the non-reducing ends and attaches a Fuc residue via an α-(1→3/4)-linkage to the GlcNAc/Glc residue. The strict 

regio- and acceptor specificity of this 1,3-1,4-α-L-fucosynthase is unique and should serve as a potentially 

powerful tool to specifically introduce Le a and Le x epitopes in type-1 and type-2 chains of glycans, respectively. 

Funded by: Grant-in-Aid for Scientific Research by Young Scientists (B) 22780072 from Ministry of Education, Culture, 

42 

Sports, Science and Technology, Japan


A new methodology for screening of bacteria-carbohydrate interactions: anti-adhesive milk 

oligosaccharides as a case study 

JONATHAN A. LANE 1, 2, KARINA MARIÑO 3, PAULINE M. RUDD 3, STEPHEN D. CARRINGTON 2 AND RITA M. HICKEY 1 

1 Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland. 2 Veterinary Science Centre, University 

College Dublin, Belfield, Dublin 4, Ireland. 3 Dublin Oxford Glycobiology Group, National Institute for Bioprocessing 

Research & Training (NIBRT), Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland 

Carbohydrates have been shown to inhibit the initial recognition events leading to adhesion and colonization of host 

tissues by pathogens and therefore have potential to prevent infection. Some of the most efficient anti-adhesion agents 

identified to date are present in foodstuffs - as best exemplified by human milk carbohydrates which protect newborns 

against infections. Such carbohydrates can display structural homology to host cell receptors or pathogenic lectins, 

thus functioning as receptor decoys. However, the large amounts of human milk carbohydrates which are required for 

intervention or clinical studies are unavailable; therefore researchers are beginning to focus their attention on sources 

of carbohydrates other than those from human milk. For instance, the milk from domestic animals, honey, fermented 

products, commensal and food-grade bacteria all contain carbohydrates which are structurally similar to human milk 

carbohydrates and have been in some cases shown to prevent pathogen binding to host cells. However, current 

screening methods for the identification of anti-adhesive oligosaccharides have limitations: they are time-consuming 

and require large amounts of oligosaccharides. Therefore, there is a need to develop analytical techniques that can 

quickly screen for, and structurally define, anti-adhesive oligosaccharides prior to using cell line models of infection. 

Considering this, we have developed a rapid method for screening complex oligosaccharide mixtures for potential anti- 

adhesive activity against bacteria. This label-free approach involves the brief and iterative exposure of a mixture of 

free oligosaccharide to aliquots of a defined bacterial population. These organisms act as an “affinity matrix” to 

progressively deplete glycans with the specific capacity to bind to the surface of these microbes, permitting their 

identification through comparison with untreated glycan profiles. As a case study, the free oligosaccharides from the 

colostrum of Holstein Friesian cows were screened for interactions with E. coli O157:H7 cells. Reductions in 

oligosaccharide concentrations were determined by High pH Anion Exchange Chromatography. The use of the 

depletion assay showed selective bacterial interaction with lactose, 3’-sialyllactose, disialyllactose, and 

sialyllactosamine in a population dependent manner, in line with previous results obtained by different methodologies. 

The depletion assay methodology was validated by inhibition studies performed using HT-29 cells. The structures of 

all potential anti-adhesives in the bovine mixture were confirmed in a fast and sensitive manner by HILIC-HPLC and 

offline mass spectrometry (ESI-MS/MS). The methodology proposed here represents a novel approach for the 

discovery of anti-adhesive oligosaccharides in diverse feedstocks. Initial screening can be undertaken in hours, 

whereas conventional approaches may take days if not weeks. The depletion assay, combined with the HILIC 

methodology, facilitates the structural and functional characterization of animal milk oligosaccharides and could be 

used to detect any bacteria-oligosaccharide interactions. Indeed, our approach could be exploited for the discovery of 

free oligosaccharides with the capacity to bind whole bacterial cells from food sources other than milk. 

Jonathan Lane is in receipt of a Teagasc Walsh Fellowship. This work was funded by the Department of Agriculture and Food, 

Ireland, under the Food Institutional Research Measure, project reference number 05/R&D/TD/368 and done in 

collaboration with the Alimentary Glycoscience Research Cluster (AGRC) Science Foundation Ireland under Grant No. 

43 

08/SRC/B1393


Sialylation and fucosylation of human milk α1-acid glycoprotein during the first two weeks of 

lactation 

MAGDALENA ORCZYK-PAWIŁOWICZ 1 AND LIDIA HIRNLE 2 

1 Department of Chemistry and Immunochemistry, Wrocław Medical University, Bujwida 44A, 50-345 Wrocław, 

Poland, 2 Department of Obstetrics and Gynaecology, Clinic of Reproduction and Obstetrics, Wrocław Medical 

University, Dyrekcyjna 5/7, 50-328 Wrocław, Poland 

The biological function of α1-acid glycoprotein (AGP), acute phase glycoprotein (40% of sugars), is not clear, 

although a number of activities in vitro and in vivo indicate that it may immunomodulate an inflammatory 

response and, at the same time, act as a plasma transport protein. Oligosaccharides of AGP are reported to 

influence its biological activities, and modulate cell-to-cell interactions and cellular signaling. Under pathological 

conditions, AGP may serve as a general protective agent in infections and against toxins by binding to toxic lectin 

endotoxins and bacterial lipopolysaccharide. AGP might also play anti-inflammatory and immunomodulatory 

roles in inflammation and cancer through its N-glycans, especially its highly branched and α1,3-fucosylated N- 

glycans. Van Dijk and coworkers [1998] have suggested that AGP molecules expressing sialo-Lewis x 

glycodeterminant may interfere with leukocyte-endothelial interactions by binding to E- or P-selectin, 

manifesting its anti-inflammatory properties. 

The aim of the present work was to study the alterations in relative amounts of sialic acids and fucoses linked by 

different anomeric linkages to subterminal N-glycans of human milk AGP in comparison to plasma AGP, and in 

relation to stages of human lactation during the first two weeks. The relative amounts of fucosyl- and sialyl- 

glycotopes on AGP were analyzed in human milk samples by lectin-ELISA using fucose- (LCA, Lens culinaris, LTA, 

Tetragonolobus purpureus and UEA-1,Ulex europaeus) and sialic acid (MAA, Maackia amurensis and SNA, 

Sambucus nigra)-specific biotinylated lectins. 

We have found that the sialylation and fucosylation patterns of human milk AGP were different than those 

reported for human plasma AGP. The observed changes were qualitative and quantitative. The normal plasma 

AGP showed very low expression of fucosylated forms. In contrast, the human milk AGP was highly decorated 

with fucoses. The fucosylation pattern of normal plasma AGP is limited to the innermost α1,6-fucose, whereas 

milk AGP contained higher relative amounts of the innermost α1,6-linked fucose and α1,2-and α1,3-linked 

fucoses on outer arms. However, among milk AGPs, no significant differences between samples of milk taken 

during the first and the second week of lactation were observed. The sialylation of human milk AGP was higher 

than normal plasma AGP for both types, α2,3- and α2,6-, of sialic acid linkages with a predominance of α2,6- 

sialylated form. 

We can hypothesize that terminal sugars of milk AGP are biologically active, with anti-inflammatory properties 

in modulation of inflammatory processes: the highly sialylated and fucosylated oligosaccharides of milk AGP 

could ensure homeostasis during the first weeks of lactation. However, the obtained results are preliminary, they 

provide the starting point for further structural and functional studies. In future, it seems to be interesting to 

analyze oligosaccharides of human milk AGP in inflammatory diseases, e.g. mastitis. 

44 

Funded by: partly by UDA-POKL.04.01.01-00-010/08-01


The relative amounts of fucose isoforms in oligosaccharides of human milk fibronectin 

MAGDALENA ORCZYK-PAWIŁOWICZ 1, LIDIA HIRNLE 2 AND IWONA KĄTNIK-PRASTOWSKA 1 

1 Department of Chemistry and Immunochemistry, Wrocław Medical University, Bujwida 44A, 50-345 Wrocław, 

Poland, 2 Department of Obstetrics and Gynaecology, Clinic of Reproduction and Obstetrics, Wrocław Medical 

University, Dyrekcyjna 5/7, 50-328 Wrocław, Poland 

Fibronectin (FN), a multidomain and multifunctional large glycoprotein which is engaged in some cellular 

biological functions, e.g. adhesion, migration, proliferation, tissue repair, extracellular matrix remodelling. It is 

possible thanks FN multiple domain interactions with its natural ligands, such as fibrin, heparin, collagen, C- 

reactive-protein, and complement components. Moreover, FN serves binding sites to many pathogens through 

peptide and/or oligosaccharide sequences, thus being important for bacterial adhesion and colonization to host 

tissue. 

In human organism FN is an abundant component. FN produced by various cells (e.g. fibroblasts, chondrocytes, 

lymphocytes, endothelial cells) is known to be trapped into insoluble multimeric fibrills, while that present in 

plasma originate from hepatocyte synthesizes, and is released to blood as a compact globular dimer. 

Human FN contains 5-9% of oligosaccharides mainly N- type, and to a lesser degree as O-type. The extent and 

type of FN glycosylation varies depending on the tissue source and cell type, and human condition. The plasma 

and cellular FNs differ regarding the number of antennae, and the degree of sialylation and α1,6-fucosylation on 

the innermost GlcNAc of the chitobiose core. Plasma-derived oligosaccharides can be bi- and tri-antennary, 

heavily sialylated and weakly fucosylated. In contrast, cellular FN has bi-antennary glycans, largely fucosylated, 

but weakly sialylated. The expression of the α1,2-linked fucosylated glycotope of FN seems to be dependent on 

the site of FN synthesis. FN produced by hepatocytes and released into the blood lacks α1,2-linked fucose, 

whereas the amniotic and seminal FNs are heavily decorated by α1,2-fucoses. 

The N-glycosylation confers FN stability and protects against proteolytic degradation, and also may act as 

modulators of FN affinity to some substrates: a lack of oligosaccharides on FN markedly enhanced its ability to 

promote adhesion and spreading of fibroblasts, concomitantly increasing affinity to gelatin. The mucin-type O- 

glycans might play a significant role in segregating the neighboring domains and maintaining the topology and 

function of FN domains. 

This communication focuses on the analysis of the relative degree of α1,6, α1,3, and α1,2-linked fucoses on FN 

present in human milk samples during the first two weeks of lactation of healthy mothers who delivered healthy 

newborns. The relative amounts of exposed fucosylated glycotopes were analyzed by lectin-ELISA using LCA 

(Lens culinaris), LTA (Tetragonolobus purpureus), and UEA-1 (Ulex europaeus) lectins with well-defined sugar 

specificity. The analyses indicate the significant differences in the expositions of fucosylated glycotopes on FN 

present in blood plasma and human milk. It was found that the milk FN was heavily decorated with α1,6-, α1,3-, 

and α1,2-linked fucoses, whereas the plasma FN negligible. Since the α1,2-fucosylated glycans are known to be 

implicated in interaction between host and some pathogens, it can be postulated that α1,2-fucosylated FN in 

human milk can act as a natural inhibitor against pathogenic bacteria colonization. The α1,2-fucosylated 

glycotopes of soluble milk glycoproteins could constitute one of the element of an innate immune system for 

breastfed infants. 

45


Effects of specific milk oligosaccharides on the expression of interleukin-8 and marker enzymes 

of intestinal cell maturation 

KRISTINE ANNA SCHOLAND 1, SABINE KUNTZ 2, CLEMENS KUNZ 2, KLAUS-PETER ZIMMER 1 AND SILVIA RUDLOFF 1,2 

1 Department of Pediatrics, University of Giessen, Feulgenstr. 12, D-35392 Giessen, Germany 

2 Institute of Nutritional Science, University of Giessen, Wilhelmstr. 20, D-35392 Giessen, Germany 

The microbial colonization of the neonatal gut has a major impact on the development of intestinal functions, 

nutrient bioavailability and the immune system. Human milk oligosaccharides (HMO) may not only be substrates 

for specific bacterial species, but also resemble their adhesion molecules or ligands which may enable them to 

directly affect gut maturation or influence inflammatory processes. 

Therefore, we investigated the expression of trehalase (Treh), a disaccharidase regarded as a general marker for 

gut maturation, and of fucosyltransferase (Fut) 1 and 2 being involved in the postnatal shift of intestinal surface 

glycosylation from sialylated towards more fucosylated structures. An inflammatory response was determined 

as interleukin (IL)-8 expression. Human intestinal cells (Caco-2, HT-29) were incubated with 2 mmol of 2’- or 3- 

fucosyllactose (FL), lacto-N-tetraose (LNT), neo-LNT, lacto-N-fucopentaose (LNFP) I or III, 3’- and 6’-sialyllactose 

(SL) or disialyl-LNT for 24 h. mRNA expression of Treh, Fut1, Fut2 and IL-8 was determined using real-time RT- 

PCR. 

In HT-29 cells, HMO did not affect the expression of Fut1 and Fut2; Treh was merely expressed in these cells 

confirming HT-29 as a non-differentiating cell line. In Caco-2 cells, however, Treh expression increased along 

with their differentiation over 14 days post confluence. Whereas HMO except for FL showed the tendency to 

reduce Treh expression, Fut1 expression in Caco-2 cells seemed slightly upregulated by most HMO tested; Fut2 

expression remained unchanged. Stimulation of Caco-2 cells with the proinflammatory cytokine tumor necrosis 

factor alpha (TNF-α) itself had no influence on marker enzyme expression. HMO, in particular LNT, LNFP I and III 

as well as 3’-SL decreased Treh expression indicating an inhibitory effect of these oligosaccharides on cell 

differentiation. Fut1 expression, on the other hand, was slightly enhanced by sialylated HMO in stimulated Caco- 

2 cells; again, there was no effect on Fut2 expression. 

With regard to IL-8 expression, disialyl-LNT as well as LNFP III may have the potential to modulate cytokine 

production. Whereas IL-8 expression seemed to be decreased in the presence of disialyl-LNT, LNFP III slightly 

enhanced IL-8 expression. 

These results confirm previous observations [1] indicating that HMO directly modulate intestinal cell 

differentiation. In addition, HMO may also have an impact on cytokine production. 

[1] Kuntz S, Rudloff, S, Kunz C. (2008) Br J Nutr 99: 462-471 

46

In vivo production of fucose-α1, 2-lactose 

KATELYN ZAK AND PENG GEORGE WANG 

The Ohio State University, Columbus, Ohio, USA 


The third largest component of human milk is complex, minimally metabolized oligosaccharides that serve to 

protect the infant’s health both by promoting infant intestinal microbiota and warding away harmful pathogens. 

The mother’s blood type cause structural variations and change HMO concentrations change lactation. While 

HMOs can promote growth of infant intestinal microbiota, their best characterized function is their prebiotic 

effects. HMOs have anti-adhesive effects prohibiting pathogens from binding to intestinal epithelial cells as well 

as modifying surface glycans that pathogens use to facilitate their entry. Fucosylated oligosaccharides comprise 

eighty percent of all HMOs of which 2-linked fucosyloligosaccharides appear the most biologically significant. 

The current hurdle in HMO research is the lack of large scale synthesis of HMOs to further test their biological 

significance. From clinical trials, fucose-α1, 2-lactose protects against diarrhea significantly better than non-2- 

linked fucosyloligosaccharides. Fucose-α1, 2-lactose may be produced in large scale quantities by recombinant 

E. coli which utilize enzymes FKP (fucokinase pyrophosphorylase) and WbsJ (α1, 2-fucoslytransferase) to 

enzymatically synthesize fucose-α1, 2-lactose. FKP is a bifunctional enzyme which catalyzes the reaction 

between fucose, ATP, and GTP to produce the expensive and hard to obtain GDP-fucose, meanwhile WbsJ is a 

known α1, 2-FucT which demonstrates promiscuous acceptor substrate specificity. This method is cost effective 

by regenerating the sugar nucleotides required for the synthesis and require only inexpensive, commercially 

available, fucose and lactose. 

47 

Funded by: NIH R01 HD061935


Sialylated galactosides of human milk as inhibitors of enterovirus 71 and A (H1N1) 2009 

influenza infections 

BETSY YANG 1,2, HAU CHUANG 2 AND RON-FU CHEN 2 

1 University of North Carolina, Class of 2014, Chapel Hill, NC 27509, USA 

2 Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 833, Taiwan 

Background and specific aims: 

Many viruses recognize specific sugar residues, such as sialylated glycosides, as the infection receptors. Human 

milk contains many sialylated oligoglycosides which may block viral infections to gastrointestinal tract. We 

postulated in the illustration below that sialic acid (SA)-linked alpha 2,3 galactosides (SA-2,3Gal) and SA 

2,6Gal from human milk ( ) could inhibit infection of 

enterovirus 71 (EV71) or swine A(H1N1) influenza. We 

have previously shown that SA-2,6Gal and/or SA-2,3Gal 

from human milk specifically inhibited EV71 infection to 

48 

Flu 

EV71 

Sialylated glycans 

Intestinal cells 

DLD-1 intestinal cells (Virol J. 2009 Sep 15; 6:141). This study has been extended to investigate whether SA- 

2,6Gal and/or SA-2,3Gal from human milk inhibited swine A(H1N1) influenza infection to DLD-1 intestinal 

cells. 

Results: 

EV71 specifically infected DLD-1 intestinal cells but not K562 myeloid cells. Pretreatment of DLD-1 cells with 

sialidase (2mU, 2 hours) significantly reduced 20-fold EV71 replication (p


The oligosaccharide (OS) phenotype of preterm infants predicts risk: A potential indication for 

HMOS administration? 

ARDYTHE L. MORROW, KURT R. SCHIBLER, JAREEN MEINZEN-DERR, DIANA TAFT AND BARBARA DAVIDSON 

Perinatal Institute, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Ohio, 

USA 

The human milk oligosaccharides (HMOS) are analogs of oligosaccharides (OS) expressed on infant mucosal 

surfaces and in saliva. Understanding the role of HMOs to prevent morbidity and mortality in breastfed infants 

also requires understanding the innately expressed OS in the infant. Using banked samples from an extant 

cohort, we previously reported that absent or low expression of salivary H antigen (Fuc1,2 oligosaccharide 

produced by transferases of the FUT2 gene) – an indirect measure of intestinal expression – significantly predicts 

risk of necrotizing enterocolitis or death in preterm infants (J Pediatrics, 2011). 

To confirm and extend that finding, we launched a prospective study of infants under 33 weeks’ gestational age 

enrolled in three level III neonatal intensive care units (NICUs) in Cincinnati, Ohio (study ongoing). In the first 

175 enrolled infants, we analyzed saliva samples at day of life 8 by enzyme-linked immunoassay to determine H 

antigen and other oligosaccharide epitopes as predictors of necrotizing enterocolitis or death. FUT2 genotype 

was determined independently, and was consistent with salivary phenotype. Using the same cut-point, we again 

found that low or absent H antigen significantly predicted subsequent necrotizing enterocolitis or death (odds 

ratio =7.7; p=0.004), controlling for low expression of Lewis a (Fuc1,4 oligosaccharide produced by 

transferases of the FUT3 gene), infant gestational age and birthweight. 

We hypothesize that low or absent H antigen is a biomarker for aberrant intestinal colonization of the preterm 

infant. The innate risk that we have identified in relation to low or absent H antigen expression in preterm 

infants suggests a prophylactic role for HMOS, which are rich in fucosylated oligosaccharide, and stimulate the 

growth of beneficial bacteria. Analysis of the intestinal microbiome and the role of maternal HMOS is underway 

in a subset of infants with longitudinal samples. 

Sponsored by U.S. National Institute of Child Health and Human Development, HD 13021 and HD 059140 

49


Natural antibodies against milk oligosaccharides 

MAKSIM NAVAKOUSKI, NADEZHDA SHILOVA, POLINA OBUKHOVA, NAILYA KHASBIULLINA AND NICOLAI BOVIN 

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia 

Recently anti-glycan antibodies of 106 adult healthy donors’ sera were profiled with the help of printed 

glycan array (PGA) which contained about 200 glycans. It was shown that natural human anti-glycan antibodies 

bind at least 160 glycans [1], including milk oligosaccharides: LNT (95% of examined donors), LNnT (90%), 2`- 

fucosylLac (90%), Le b -Lac (80%), Lac (75%), 3`SL (60%), 6`SL (35%). The glycans were immobilized on the slide 

surface as N-glycine derivatives. In addition, the strong interaction between sera and some glycans closely 

related to milk OS was found, e.g. with SiaLe C (99%), Le C (95%), P1 (75%), Le D (50%), asialo-GM1 (100%), GA2 

(95%), Le Y -Lac (90%). 

Anti-milk OS antibodies (anti-milkOS), namely against LNT, LNnT, Le B Lac and Lac were detected in 

blood of 3-day infants receiving mixed feeding. These anti-milkOS belong to IgG class only and thus seem to be 

maternal. 

Currently accumulated data don’t allow us to figure out the origin of natural anti-milkOS. On one hand a 

reason of their appearance might be natural glycation conjugates of milk OS with proteins due to elevated 

concentrations of both components in colostrum; the conjugates could work as immunogens. On the other hand, 

the antibodies actually could be directed to different epitopes, in other words, milk OS play a role of mimetics for 

unknown yet antigens. 

1. Huflejt, M.E., et al., Anti-carbohydrate antibodies of normal sera: findings, surprises and challenges. 

Mol Immunol, 2009. 46(15): p. 3037-49. 

Funded by: European Commission’s Marie Curie program (the EuroGlycoArrays ITN) 

50


Primary prevention of allergic diseases by probiotics: impact of HMOs 

GER T. RIJKERS 1,2, TITIA NIERS 1, NICOLE RUTTEN 2, SASKIA VAN HEMERT 3, MAARTEN HOEKSTRA 1, ARINE VLIEGER 2 

1 University Medical Center Utrecht, 2 St. Antonius Hospital, Nieuwegein, 3 Winclove Bioindustries, Amsterdam, The 

Netherlands 

Modification of the intestinal microbiota by administration of probiotic bacteria may be a potential approach to 

prevent allergic disease. We aimed to study primary prevention of allergic disease in high-risk children by 

perinatal supplementation of selected probiotic bacteria. In a double-blind, randomized, placebo-controlled trial, 

a mixture of probiotic bacteria selected by in vitro screening (Bifidobacterium bifidum, Bifidobacterium lactis, 

and Lactococcus lactis, as Ecologic®Panda) was prenatally administered to mothers of high-risk children 

(positive family history of allergic disease) and to their offspring for the first 12 months of life. Probiotics were 

administered daily in a dose of 1 x 109 cfu per strain. The placebo-group received an equivalent amount of 

carrier material (rice starch plus maltodextran) daily. Up to 80% of mothers breast fed their babies for an 

average of 7.1 months. Parental-reported eczema during the first 3 months of life was significantly lower in the 

intervention group compared with placebo, 6/50 vs. 15/52 (P=0.035), a relative risk reduction of 58%. The 

number needed to treat was 5.9 at age 3 and 12 months and 6.7 at age 2 years. Similarly, physician-confirmed 

eczema during the first 3 months was 3/50 and 11/52 in the intervention and control groups, respectively 

(P=0.021). The intervention group was significantly more frequently colonized with higher numbers of Lc. lactis. 

In 30% of babies in the placebo group Lc. Lactis was found, which may a consequence of breast feeding. At age 3 

months, in vitro production of IL-5 in anti-CD2/CD28 mAb-stimulated whole blood was decreased in the 

probiotic group compared with placebo (P=0.04). In conclusion, Ecologic®Panda reduced the incidence of 

eczema in high-risk children at 3 months of life, an effect which seems to be sustained throughout the first 2 

years. Additional studies at 5-6 years assessing lung function and asthma development are ongoing. 

We are planning to study the potential synergistic effect of HMOs on immunomodulation by probiotics. To that 

end we will first determine in in vitro studies which HMOs show the highest potential to modulate the neonatal 

immune system for induction of Th1 and Treg cells. 

Funded by: The Wilhelmina Children's Hospital and the Ministery of Economic Affairs 

51

THE SPONSOR 

Glycom A/S 


Glycom is a privately held company founded in 2005 in Copenhagen, Denmark whose focus is to synthesize and 

commercialize human milk oligosaccharides (HMOs) along with their precursors and intermediates. 

HMOs have been the object of scientific curiosity for over 40 years. Developed over millions of years of human 

evolution, HMOs are the 3rd largest component of mother’s milk and are attributed with many of its wonderful 

health effects. Until now, study of these natural biopharmaceuticals has been limited and commercialization has 

been blocked by lack of available material and high costs – with virtually none available from extractive sources 

and only a few synthesized in extremely low quantities and extremely high costs. 

Glycom’s breakthrough synthetic chemistry has changed this equation permanently. We have developed efficient 

chemical and enzymatic technologies for synthesis of HMOs representing over 50% of total HMO concentration in 

typical samples of mother’s milk. The leading HMOs in the Glycom pipeline have established robust scale up 

technology in industrial scales and GMP conditions, with glycosylations in multi cubic meter reactors and 

production in ton scale and in pure form suitable for human consumption. The opportunity for large scale, 

widespread study and commercialization of HMOs has arrived as a result of our efforts and the support of our 

partners and investors. 

In order to efficiently develop HMOs, Glycom has also developed breakthrough synthesis of the four mono and 

disaccharide precursors/building blocks of HMOs. Two of these precursors – sialic acid and L-fucose – are among the 

handful of basic natural human sugars. None of them are currently available in sufficient quantities and costs for 

high volume commercial applications – including HMO synthesis. As with the HMOs themselves, the Glycom 

technology for the first wave precursors has been scaled up to industrial conditions. As a result, Glycom is already 

the world’s largest producer of L-fucose and has already achieved cost levels that are a small fraction of its current 

market price. Other precursors are in line for similar breakthroughs. 

52

ALBERMANN, CHRISTOPH 

Institute of Microbiology, University of Stuttgart, Germany 

christoph.albermann@imb.uni-stuttgart.de 

BEAUPREZ, JOERI 

Ghent University, Belgium 

joeri.beauprez@ugent.be 

BERTELSEN, HANS 

Arla Foods, Videbæk, Denmark 

hans.bertelsen@arlafoods.com 

BODE, LARS 

University of California, San Diego, USA 

lbode@ucsd.edu 

BØTTKJÆR, KIM 

Glycom A/S, Kongens Lyngby, Denmark 

kib@fihpartners.com 

BØJSTRUP, MARIE 

Carlsberg Laboratory, Copenhagen, Denmark 

mab@crc.dk 

CILIEBORG, MALENE 

University of Copenhagen, Denmark 

macilie@life.ku.dk 

CONTRACTOR, NIKHAT 

Pfizer Nutrition, Collegeville, PA, USA 

nikhat.contractor@pfizer.com 

DONOVAN, SHARON M. 

University of Illinois, Urbana, USA 

sdonovan@illinois.edu 

DEKANY, GYULA 


gyula.dekany@glycom.com 

DROUILLON, MARGRIET 

University of Ghent, Ghent, Belgium 

Margriet.Drouillon@UGent.be 

EL-HAWIET, AMR 

University of Alberta, Canada 

elhawiet@ualberta.ca 

FERNANDEZ, LEONIDES 

Dept. Food Science & Tech, Universidad Complutense de Madrid, Spain 

leonides@vet.ucm.es 

FISCHER, LUTZ 

University of Hohenheim, Germany 

lutz.fischer@uni-hohenheim.de 

GARRIDO, DANIEL 

University of California, Davis, USA 

dagarrido@ucdavis.edu 

GRINYER, JASMINE 

Faculty of Science, Macquarie University, Australia 

jasmine.grinyer@mq.edu.au 

HENNET, THIERRY 

Universität Zürich, Switzerland 

thennet@access.uzh.ch 

LIST OF PARTICIPANTS 

List of Participants 

53 

AUSTIN, SEAN 


sean.austin@rdls.nestle.com 

BERING, STINE BRANDT 

Department of Human Nutrition, Copenhagen University, Denmark 

sbs@life.ku.dk 

BLANK, DENNIS 

Justus-Liebig University, Giessen, Germany 

dennis.blank@chemie.uni-giessen.de 

BOYLE, ROBERT 

Department of Paediatrics Imperial College, London, UK 

r.boyle@nhs.net 

BRASSART, DOMINIQUE 

Nestec Ltd., Vevey, Switzerland 

dominique.brassart@nestle.com 

CHEAH, WAI YUEN 

Faculty of Science, Macquarie University, Australia 

wai.cheah@mq.edu.au 

COPPA, GIOVANNI V. 

Polytechnic University of Marche, Ancona, Italy 

g.v.coppa@univpm.it 

CRISÀ, ALESSANDRA 

C.R.A., Monterotondo, Italy 

alessandra.crisa@entecra.it 

DAVIS, STEVEN 

Abbott Nutrition, Columbus, OH, USA 

steven_davis@abbott.com 

DOTZ, VIKTORIA 

Justus-Liebig University of Giessen, Germany 

viktoria.dotz@uni-giessen.de 

DUNCAN, PETER 


monique.unternaehrer@rdls.nestle.com 

ETZOLD, SABRINA 

Norwich Research Park, Institute of Food Research, Norwich, UK 

sabrina.etzold@bbsrc.ac.uk 

FICHOT, MARIE-CLAIRE 

Nestec Ltd., Vevey, Switzerland 

marie-claire.fichot@nestle.com 

FREEZE, HUDSON 

Sanford-Burnham Medical Research Institute, La Jolla, USA 

Hudson@sandfordburnham.org 

GEYER, RUDOLF 

Justus Liebig Universität Giessen, Germany 

rudolf.geyer@biochemie.med.uni.giessen.de 

HALTRICH, DIETMAR 

University of Natural Resources and Life Sciences, Vienna, Austria 

dietmar.haltrich@boku.ac.at 

HESTER, SHELLY 


sndavis2@illinois.edu

HINDSGAUL, OLE 


hindsgaul@crc.dk 

HORLACHER, PETER 

Cognis, Illertissen, Germany 

Peter.Horlacher@cognis.com 

JØRGENSEN, JULIE BØCK 


julie_boeck@hotmail.com 

KATAYAMA, TAKANE 

Ishikawa Prefectural University, Nonoichi, Japan 

takane@ishikawa-pu.ac.jp 

KELM, SØRGE 

University Bremen, Germany 

skelm@uni-bremen.de 

KLARENBEEK, BERT 

Friesland Campina, Beilen, The Netherlands 

bert.klarenbeek@frieslandcampina.com 

Kovacs, JUDIT 


judit.kovacs@glycom.com 

KRISTENSEN, METTE BACH 

Arla Foods, Viby J., Denmark 

mekre@arlafoods.com 

KVISTGAARD, ANNE STAUDT 


askv@arlafoods.com 

LEBRILLA, CARLITO 


cblebrilla@ucdavis.edu 

MANURUNG, SARMAULI 

Danish Technical University, Veterinary Institute, Denmark 

saem@vet.dtu.dk 

MAU, SUSANNE 


susanne.mau@glycom.com 

MILLS, DAVID A. 


damills@ucdavis.edu 

MUNBLIT, DANIEL 

Imperial College, London Clinical Medicine, London, UK 

daniel.munblit08@imperial.ac.uk 

MØRKBAK, ANNE LOUISE 


anmor@arlafoods.com 

NEWBURG, DAVID S. 

Boston College, MA, USA 

david.newburg@bc.edu 

ORCZYK-PAWILOXICZ, MAGDALENA 

Dept. Chemistry & Immunology, Wroclaw Medical University, Poland 

morczyk@immchem.am.wroc.pl 

PETERBAUER, CLEMENS 

University of Natural Resources and Life Sciences, Vienna, Austria 

clemens.peterbauer@boku.ac.at 


54 

HOLSCHER, HANNAH D. 


hholsche@illinois.edu 

IYER, SURI S. 

University of Cincinnati, Ohio, USA 

suri.iyer@uc.edu 

KAMERLING, JOHANNIS P. 

University of Utrecht, Utrecht, The Netherlands 

j.p.kamerling@uu.nl 

KAVANAUGH, DEVON 

Teagasc Food Research Centre, Cork, Ireland 

devon.kavanaugh@teagasc.ie 

KITAOKA, MOTOMITSU 

National Food Research Institute, Tsukuba, Japan 

mkitaoka@affrc.go.jp 

KLASSEN, JOHN S. 

University of Alberta, Canada 

john.klassen@ualberta.ca 

KRENGEL, UTE 

Department of Chemistry, University of Oslo, Norway 

ute.krengel@kjemi.uio.no 

KUNZ, CLEMENS 

Justus Liebig Universität Giessen, Germany 

clemens.kunz@ernaehrung.uni-giessen.de 

LANE, JONATHAN 

Moorepark Food Research Centre, Cork, Ireland 

Jonathan.Lane@teagasc.ie 

LI, YANQI 

Department of Human Nutrition, University of Copenhagen, Denmark 

yli@life.ku.dk 

MARIOTTA, MARIAROSARIA 

Teagasc Food Research Centre, Cork, Ireland 

mariarosaria.marotta@teagasc.ie 

MIKLUS, MICHAEL 

PBM Nutritional, Georgia, USA 

mmiklus@pbmnutritionals.com 

MORROW, ARDYTHE L. 

Cincinnati Children's Hospital, Ohio, USA 

ardythe.morrow@cchmc.org 

MUTUNGI, GISELLA 

Pfizer Nutrition, Collegeville, PA, USA 

gisella.mutungi@pfizer.com 

NAVAKOUSKI, MAKSIM 

Institute of Bioorganic Chemistry, Moscow, Russia 

maxushob@gmail.com 

NIELSEN, DENNIS S. 

Department of Food Science, University of Copenhagen, Denmark 

dn@life.ku.dk 

PERONI, DIEGO 

Chorus S.p.A., Clinica Pediatrica, Verona, Italy 

r.cosenza@lachorus.com 

PRIETO, PEDRO ANTONIO 

Technologico de Monterrey, Mexico 

paprieto@itesm.mx

PUTZE, JOHANNES 

Children's University Hospital Mannheim, Germany 

johannes.putze@medma.uni-heidelberg.de 

RÖHRIG, CHRISTOPH H. 


christoph.roehrig@glycom.com 

SANGILD, PER TORP 


psa@life.ku.dk 

SCHOLAND, KRISTINE ANNA 

University Giessen, Germany 

kristine.a.scholand@paediat.med.uni-giessen.de 

SKALKAM, MARIA 

Oxie, Sweden 

maria.lena.skalkam@post.au.dk 

SPRENGER, NORBERT 


norbert.sprenger@rdls.nestle.com 

STAUDACHER, ERIKA 

University Vienna, Austria 

erika.staudacher@boku.ac.at 

TAPPENDEN, KELLY 


tappende@illinois.edu 

THIEM, JOACHIM 

University of Hamburg, Germany 

thiem@chemie.uni-hamburg.de 

URASHIMA, TADASU 

Obihiro University of Agriculture & Veterinary Medicin, Japan 

urashima@obihiro.ac.jp 

VAN LEEUWEN, SANDER 

University of Groningen, Groningen, The Netherlands 

s.s.van.leeuwen@rug.nl 

WANG, BING 

University of Sydney, Australia. Xiamen University & Nestlé Research 

Center, Beijing, P. R. China 

bing.wang@sydney.edu.au 

WELMAN, ALAN 

Fonterra Coop. Group, Palmerston, New Zealand 

alan.welman@fonterra.com 

YANG, KUENDER D. 

Department of Medical Research, Taiwan 

yangkd.yeh@hotmail.com 

YEH, SHI-HUI 

Chang Gung Institute of Technology, Taiwan 

yangkd.yeh@msa.hinet.net 

ZAK, KATELYN 

Ohio State University, Columbus, USA 

zak.27@osu.edu 


55 

RIJKERS, GER T. 

St. Antonius Hospital, Dept. Medical Microbiology, The Netherlands 

g.rijkers@antoniusziekenhuis.nl 

SALCEDO DOMÍNGUEZ, JAIME 

Facultad de Farmacia, Universidad de Valencia, Spain 

jaime.salcedo@uv.es 

SCHOEMAKER, RUUD 

Friesland Campina, Deventer, The Netherlands 

ruud.schoemaker@frieslandcampina.com 

SCHWAB, CLARISSA 

University of Vienna, Austria 

clarissa.schwab@univie.ac.at 

SOETAERT, WIM 

Ghent University, Belgium 

wim.soetaert@ugent.be 

STAHL, BERND 

Danone Research, Friedrichsdorf, Germany 

bernd.stahl@danone.com 

TANAKA, HIDENORI 


tanaka@crc.dk 

THEROUX, JOHN 


jt@glycom.com 

THURL, STEPHAN 

Fulda University of Applied Sciences, Fulda, Germany 

stephan.thurl@lt.hs-fulda.de 

VAN LEUSEN, ELLEN 

Friesland Campina, Beilen, The Netherlands 

ellen.vanleusen@frieslandcampina.com 

WALKER, CAREY D. 

Mead Johnson Nutrition, Evansville, IN, USA 

carey.walker@mjn.com 

WEICHERT, STEFAN 

Children's University Hospital, Mannheim, Germany 

stefan.weichert@medma.uni-heidelberg.de 

WU, SHUAI 

University of Davis, CA, USA 

shuwu@ucdavis.edu 

YANG, BETSY YAH-HAN 

University of North Carolina, Durham, USA 

yaha727@gmail.com 

YUE, KE 

Justus-Liebig University of Giessen, Germany 

yueke1983@yahoo.com.cn 

ØSTERGAARD, METTE VIBERG 

University of Copenhagen, Department of Human Nutrition, Denmark 

mevo@life.ku.dk

NOTES

NOTES

Glycobiology of Human Milk Oligosaccharides - Glycom

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