Potential utilization of milk oligosaccharides of cows

and other domestic farm animals

Tadasu Urashima1, Takashi Terabayashi2, Minoru Morita3

1Obihiro University of Agriculture and Veterinary Medicine,
2Kitasato University, and 3Toko Pharmaceutical Industries Co.,Ltd.
What are lactose and milk oligosaccharides?
Human milk
1. Lactose 6%
2. Lipids 3.5%
3. Milk oligosaccharides 1.21.3%
4. Proteins 1.1%
5. Ash 0.2%

Human milk oligosaccharides

1213 g/L in mature milk
2224 g/L in colostrum
more than 200 varieties
more than 100 characterized structures
 Lactose and milk oligosaccharides

Lactose Sialyllactose
1,4 2,3 1,4

Fucosyllactose Lacto-N-neotetraose
1,2 1,4 1,4 1,3 1,4

Lacto-N-hexaose
1,4 1,6 : Galactose
: Glucose
1,4
: Fucose
: Sialic acid
1,4 1,3
: N-acethylglucosamine
Bio functions of human milk oligosaccharides

1. Anti infection as receptor analogues

Campylobacter jejuni, Entamoebahistolytica,
Streptococcus pneumoniae, enteropathogenic E. coli,
Uropathogenic E. coli, enterotoxigenic E. coli, etc

2. Prebiotics

3. Immuno modulating factor etc

The 13 core structures of human milk oligosaccharides
Structures Name
Gal(1-4)Glc
Lactose
Gal(1-3)GlcNAc(1-3)Gal(1-4)Glc
Lacto-N-tetraose
Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc
Lacto-N-neotetraose
Gal(1-4)GlcNAc(1-6)
Gal(1-3)GlcNAc(1-3)
Gal(1-4)Glc Lacto-N-hexaose
Gal(1-4)GlcNAc(1-6)
Gal(1-4)GlcNAc(1-3)
Gal(1-4)Glc Lacto-N-neohexaose
Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc para-Lacto-N-hexaose
Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc para-Lacto-N-neohexaose
Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-6)
Gal(1-3)GlcNAc(1-3)
Gal(1-4)Glc Lacto-N-octaose
Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-6)
Gal(1-4)GlcNAc(1-3)
Gal(1-4)Glc Lacto-N-neooctaose
Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-6)
Gal(1-3)GlcNAc(1-3)
Gal(1-4)Glc iso-Lacto-N-octaose
Gal(1-3)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc para-Lacto-N-octaose
Gal(1-4)GlcNAc(1-6)
Gal(1-4)GlcNAc(1-6)
Gal(1-3)GlcNAc(1-3)
Gal(1-3)GlcNAc(1-3)
Gal(1-4)Glc Lacto-N-decaose
Gal(1-4)GlcNAc(1-6)
Gal(1-4)GlcNAc(1-3)
Gal(1-4)GlcNAc(1-6)
Gal(1-3)GlcNAc(1-3)
Gal(1-4)Glc Lacto-N-neodecaose
 Bovine milk oligosaccharides (BMOs)
More than 1 g/L exists in colostrum immediately post partum

The concentration rapidly decreases 48 hr after partum to very low level

Around 40 varieties of structures have been characterized
Flow of this lecture

1. Historical aspects of BMO study

2. BMO chemical structures
3. Quantitative aspect of BMO
4. Our project of bovine colostrum (April, 2005March, 2010)
5. Potential utilization of bovine milk oligosaccharides
 Acidic BMO
Kuhn and Brossmer (1956)
Neu5Ac(2-3)Gal
Neu5Ac(2-6)Gal(1-4)Glc (6-SL)
Neu5Gc(2-3)Gal(1-4)Glc
Neu5Ac(2-6)Gal(1-4)GlcNAc (6-SLN)
Neu5Ac(2-8)Neu5Ac(2-3)Gal(1-4)Glc (DSL)
Cumar et al. (1965)
Gal(1-4)Glc-3-PO4
Schneier and Rafelson (1966)
Neu5Ac(2-3)Gal(1-4)Glc (3-SL)
Veh et al. (1981)
Neu5Gc(2-6)Gal(1-4)Glc
Neu5Gc(2-6)Gal(1-4)GlcNAc
Parkkinen and Finne (1985, 1987)
Neu5Ac(2-6)Gal(1-4)GlcNAc-1-PO4
Neu5Ac(2-6)Gal(1-4)GlcNAc-6-PO4
Neutral BMO

Saito et al. (1985)

GalNAc(1-4)Glc
Gal(1-4)GlcNAc (N-acetyllactosamine)

Saito et al. (1987)

Gal(1-3)Gal(1-4)Glc (3-GL)
Gal(1-6)Gal(1-4)Glc (6-GL)
Gal(1-4)[Fuc(1-3)]GlcNAc

Urashima et al. (1991)

Gal(1-3)Gal(1-4)Glc (isoglobotriose)
GalNAc(1-3)Gal(1-4)Glc
Gal(1-3)[Gal(1-4)GlcNAc(1-6)]Gal(1-4)Glc (lacto-N-novopentaose 1)

These oligosaccharides have never been found in human milk.

Isoglobotriose is a major neutral BMO.
 GalNAc(1-3)Gal(1-4)Glc

13C-NMR of GalNAc(1-3)Gal(1-4)Glc, separated from bovine colostrum

Urashima et al. Biochim. Biophys. Acta 1073, 225 (1991)
 Recent study of BMO structures

Method for milk oligosaccharide profiling by 2-aminobenzamide

Labeling and hydrophilic interaction chromatography

K. Marino, J.A. Lane, J.L. Abraham, W.B. Struwe, D.J. Harvey,

M. Marotta, R.M. Hickey, P.M. Rudd

Glycobiology 21, 1317, 2011

Neutral Bovine Milk Oligosaccharides Characterized by Marino et al

1.GalNAc(1-4)GlcNAc

2. GalNAc(1-4)Glc

3. Gal (1-4)GlcNAc (N-acetyllactosamine)

4. Fuc(1-2)Gal(1-4)Glc (2-FL)

5. GalNAc(1-3)Gal(1-4)Glc

6. GlcNAc(1-3)Gal(1-4)Glc

7. Gal(1-6)Gal(1-4)Glc (6-GL)

8. Gal(1-3)Gal(1-4)Glc (isoglobotriose)

9. Gal(1-3)Gal(1-4)Glc (3-GL)

10. Gal(1-4)Gal(1-4)Glc (4-GL)

11. Fuc(1-2)Gal(1-4)Glc
GalNAc(1-3)

12. Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc (lacto-N-neotetraose)

13. Gal(1-4)GlcNAc(1-6)
Gal(1-4)Glc
GlcNAc(1-3)

14. Gal(1-4)GlcNAc(1-6)
Gal(1-3) Gal(1-4)Glc (lacto-N-novopentaose 1)
15. Gal(1-4)GlcNAc(1-6)
Gal(1-4)GlcNAc(1-3) Gal(1-4)Glc (lacto-N-neohexaose)
Acidic Bovine Milk Oligosaccharides Characterized by Marino et al.

16.Neu5Ac(2-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc

17. Neu5Ac(2-6)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc

18. Neu5Ac(2-3)Gal(1-4)GlcNAc(1-6)
Gal(1-4)Glc
GlcNAc(1-3)
19.
Gal(1-4)GlcNAc(1-6)
Neu5Ac(2-3) Gal(1-3)
Gal(1-4)Glc

20. Neu5Ac(2-6)Gal(1-4)GlcNAc(1-6)
Gal(1-4)Glc
GlcNAc(1-3)

21. Neu5Ac(2-6)Gal(1-4)GlcNAc(1-6)
Gal(1-3) Gal(1-4)Glc

22.

Gal(1-4)GlcNAc(1-6)
Neu5Ac(2-3) Gal(1-4)Glc
Gal(1-4)GlcNAc(1-3)

23.
Gal(1-4)GlcNAc(1-6)
Neu5Ac(2-6)
Gal(1-4)GlcNAc(1-3)
Gal(1-4)Glc

24. Neu5Ac(2-6)GalNAc(1-4) GlcNAc

25. Neu5Ac(2-3)Gal(1-4) Glc (3-SL)

Acidic Bovine Milk Oligosaccharides Characterized by Marino et al.
26. Neu5Gc(2-3)Gal(1-4)Glc

27. Neu5Ac(2-6)Gal(1-4)GlcNAc (6-SLN)

28. Neu5Gc(2-6)Gal(1-4)GlcNAc

29. Neu5Ac(2-6)Gal(1-4)Glc (6-SL)

30. Neu5Gc(2-6)Gal(1-4)Glc

31. Neu5Ac(2-6)Gal(1-4)Glc
GlcNAc(1-3)

32. Neu5Ac(2-3)Gal(1-4)Gal(1-4)Glc

33. Neu5Ac(2-6)Gal(1-4)Glc
Gal(1-3)

34. Neu5Ac(2-8)Neu5Ac(2-3)Gal(1-4)Glc (DSL)

35. Neu5Gc(2-8)Neu5Ac(2-3)Gal(1-4)Glc

36. Neu5Ac(2-8)Neu5Gc(2-3)Gal(1-4)Glc

37. Neu5Ac(2-8)Neu5Ac(2-3)Gal(1-4)GlcNAc
Differences between the milk oligosaccharides of human and cows
HMOs
2224 g/L in colostrum
1213 g/L in mature milk
more than 200 varieties
Gal(1-3)GlcNAc containing HMO (type) >Gal(1-4)GlcNAc containing HMO (type)
neutral HMO >acidic HMO

The ratio of fucosyl oligosaccharides to total HMOs is high

Gal(1-3)Gal(1-4)Glc GalNAc(1-3)Gal(1-4)Glc
Neu5Gc containing HMO Sialyl galactosyllactose

BMOs
more than 1 g/L in colostrum
small concentration in mature milk
around 40 varieties
typeBMOs, typeBMOs
acidic BMO > neutral BMO,70% ; Neu5Ac(2-3)Gal(1-4)Glc
The concentration of fucosyl oligosaccharides is very low
Gal(1-3)Gal(1-4)Glc, GalNAc(1-3)Gal(1-4)Glc
Neu5Gc containing BMO
Sialyl galactosyllactose
 Minor fucosyl BMOs characterizaed in recent study
Annotation and structural elucidation of bovine milk oligosaccharides and determination
of novel fucosylated structures
D.L.Aldredge, M.R.Geronimo, S.Hua, C.C.Nwosu, C.B.Lebrilla, D.Barile
Glycobiology 23,664,2013

Fuc

H2N1F1 HexNAc Gal Glc

Fuc

H4N1F1 Gal HexNAc Gal Gal Glc

Fuc

H3N2F1 HexNAc HexNAc

Gal Glc
Gal

R. Mehra, D. Barille, M. Marotta, C.B. Lebrilla, C. Chu, J.B. German

PLOS one, 9, e96049, 2014
Novel high-molecular weight fucosylated milk oligosaccharides identified in dairy streams
H3N4F1, H4N4F1, H3N5F1, H5N4F1, H4N5F1, H3N6F1
Quantitative aspect of BMOs
 0.2

6'SL concentration (g/L)

1.2
3SL concentration (g/L)
1.0

0.8

0.6 0.1

0.4

0.2

0 0
24 48 72 96 120 144 168 0 24 48 72 96 120 144 168
0
Time post partum (h) Time post partum (h)
0.2
6'SLN concentration (g/L)

0.1

0
0 24 48 72 96 120 144 168
Time post partum (h)

Changes in the concentrations of individual sialyloligosaccharides of bovine

colostrum during early lactation. 3SL, Neu5Ac(2-3)Gal(1-4)Glc;
6SL, Neu5Ac(2-6)Gal(1-4)Glc; 6SLN, Neu5Ac(2-6)Gal(1-4)GlcNAc. Values are
indicated as meansSD (n=4).

T. Nakamura et al., J. Dairy Sci., 86, 1315, 2003

Bovine milk oligosaccharide analysis by HPLC-HRSRM: typical total ion current trace of oligosaccharide
Standards (a) followed by their extracted ion chromatograms (c-f) and total ion current trace of bovine
Milk oligosaccharide (b) followed by the extracted ion chromatograms (g-j).

B. Fong, K. Ma & P. McJarrow: J. Agric. Food. Chem., 59, 9788, 2011

Concentrations of Oligosaccharides in Samples of Bovine Milk, Bovine Colistrum,
and Infant Formula Number of samples and coefficient of variation in parentheses

3SL ( mg/L) 6SL (mg/L) 6SLN ( mg/L) DSL ( mg/L) GNL ( mg/L)

a a
Skim milk 1 51 4 (30,9%) 6.3 0.4 (8,6%) 0.13 0.02 (2,14%) 1.5 0.1 (2,6%) 2.6 0.3 (2,10%)

Skim milk 2 a a
55 4 (2, 6%) 9 0.2 (2, 2%) 0.10 0.02 (2, 17%) 2.1 0.3 (2, 14%) 3.4 0.4 (2, 13%)

Homogenized milk a a
48 4 (6, 8%) 9.6 0.8 (3, 8%) 0.1 0.03 (3, 14%) 3.1 0.2 (3, 6%) 2.4 0.1 (3, 2%)

Unpasteurized milk a
47 4 (6, 9%) 3.6 0.3 (2, 9%) <LOD (2) 0.54 0.01 (2,14%) <LOD (2%)

Mature milk
94-119 67-88 145-176 41-77
(Martin-Sosa et al.)
Mature milk
35-50 14-25 9-12 2-7 3-4
(?g/mL, McJarrow and van
Amelsfort-Schoonbeek)
Mature milk
30 25 12 ND
(7 d post partum, Nakamura et al.)
Colostrum (second milking) a
1245 82 (7, 85 6 (7, 7%) 119 7 (7, 6%) 126 8 (2, 8%) 1 0.1 (2, 12%)
7%)

Colostrum (fourth milking) a

739 53 (5, 7%) 73 2 (5, 2%) 117 10 (5, 8%) 80 7 (2, 9%) 1 0.1 (2, 10%)

Colostrum
354 147 210 135
(Martin-Sosa et al.)
 Development of the innovative utilizations
of bovine colostrum components

Financially supported by the Research and Development Program for New

Bio-IndustryInitiatives, the Bio-oriented Technology Research Advancement
Institution (BRAIN), National Agriculture and Food Research Organization (NARO),
Japan

April, 2005March, 2010

1. Obihiro University of Agriculture & Veterinary Medicine, Graduate course of Food Hygiene

2. Obihiro University of Agriculture & Veterinary Medicine, Protozoa Disease Institution

3. Kitasato University

4. Toko Pharmaceutical Industries Co.Ltd

5. National Animal Hygiene Institution

1. Anti influenza activity by the condensation products of sialyl
oligosaccharides from bovine colostrum with fatty acids

Dr. Takashi Terabayashi (Kitasato University)

Dr. Minoru Morita (Toko Pharmaceutical Industries Co. LtD)

2. Biofunctional proteins from bovine colostrum

Bovine colostrum odrant binding protein

Dr. Kenji Fukuda (Obihiro University of Agriculture & Veterinary Medicine)
Poster :
Binding of influenza A virus to the host cell via sialyl-galactosylglyco-chain on the
receptor
Neuraminidase(NA)

Hemagglunitin
(HA) Neu5Ac-Gal-

Avian influenza A virus Human influenza A virus

Neu5Aca2-3Gal- Neu5Aca2-6Gal-
Competitive inhibition against binding of the viral HA to the receptor
sugar chain

OH COOH
HO OH OH
OH
HO O O HO OH
O O O
AcHN OH OH O
HN
OH n
O
Neu5Aca2-3Gal1-4Glc1-Cer
Ganglioside GM3

Anti-influenza virus activity

Ganglioside analogue
( single tailed amphiphile )
OH COOH
HO OH OH
OH
HO O O HO OH
O O
AcHN OH OH O
OH New type !
Neu5Aca2-3Gal1-4Glc -NH-C12 Anti-influenza virus reagent
3-sialyllactose Anti-influenza virus activity
(3-SL)

[Terabayashi T., Carbohydrate Res., 341 (2006) 2246-2253]

 Synthesis of N-sialyllactosyl-acylamide
OH COOH
HO OH OH
OH
HO O O HO OH
O O
AcHN OH O
OH
OH 3- and/or 6- sialyllactoses were substituted
3 -sialyllactose (3- SL)
by an amino group in saturated NH4HCO3 / H2O,
amination with starring for four days at room temperature.

OH COOH
HO OH OH
OH
HO O O HO NH2
O O removal of NH4HCO3 by rotary evaporation
AcHN OH OH O
OH
3 -sialyllactosylamine (3 SL-NH2)

acyl amidation with long chain fatty acids

acyl amidation
( CH3(CH2)nCOOH; n = 12 ~ 18),
using a reagent for amidation, DMT-MM*
OH COOH
HO OH OH
OH O
HO O O HO NH C
O O
AcHN OH OH O
OH
N-3 sialyllactosyl-mylistinamide (3-SL-N-C14)

* DMT-MM; 4-(4,6-dimathoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride

 IC50 of N-sialyllactosyl-acylamide

Inhibitor IC50 (mg/ml)

3-SL-N-C12 286.5
3-SL-N-C14 56.5 21.9
3-SL-N-C16 22.5 4.7
3-SL-N-C18 4.0
6-SL-N-C14 99.4 51.6
6-SLN-N-C14 85.3 11.1
Oseltamivir10.9 0.13
Zanamivir20.5
Amantadine 3 30.9

1Tamiful(Roche); oseltamivir phosphate

2Relenza (GlaaxoSmithKline); zanamivir hydrate
31-Aminoadamantane (Aldorich)

Anti-influenza virus activity was evaluated by plaque reduction assays,

using MDCK (Madin-Darby canine kidney) cells and Influenza virus A/PR/8/34 (H1N1).
 The anti-influenzavirus A/PR/8/34 effect of each
sialyllactosyl-acylamide substance (Survival ratio)
Oseltamivir
100
3'-SL-N-C14
Survival (%)

6'-SL-N-C14
3'-SL-N-C18
50

6'-SLN-N-C14

0 1 2 3 4 5 6 7 8 9
Days after infection

The 1 mg/kg of each substance was

administered to mice twice per day.
The action of sialyllactosyl-acylamide substance to the viral replication
in the lung tissue of influenza virusA/PR/8/34 infection mice
(PFU/ml)
15000

10000

5000

0
Potential utilization of bovine milk oligosaccharides

1. Separation of oligosaccharides from bovine colostrum or lactose removed

cheese whey using membrane technology
R. Mehra et al., PLOS one, 9, e96049, 2014

2. Anti infection agents

Against Campylobacter jejuni
J.A. Lane et al., Int. J. Food Microbiol. 157, 182, 2012
Against Escherichia coli P1422
J.A. Lane et al., J. Microbiol. Methods 90, 53, 2012
Against rotavirus
S.N. Hester et al., Br. J. Nutr. 110, 1233, 2013
3. Immuno modulating agents
Transcritional response of HT-29 intestinal epithelial cells
J.A. Lane et al., Br. J. Nutr. 110, 2127, 2013

Activation for intestinal VD11Ccells

E. Kurakevich et al., PNAS, 110, 17444, 2013

4. Stimulating agents for maturation of intestinal epithelial cells

H.D. Holscher et al., J. Nutr. 144, 586, 2014
Thank you