International Dairy Journal 22 (2012) 141e146

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International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj

Review

The role of oligosaccharides from human milk and other sources in prevention
of pathogen adhesion
Rita M. Hickey*
Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland

a r t i c l e i n f o a b s t r a c t

Article history: Adhesion of pathogens is required for colonization and subsequent development of disease, and when
Received 30 November 2010 adhered, they are more likely to survive, as their resistance to cleansing mechanisms, bacteriolytic
Received in revised form enzymes and antibiotics is higher than in the free state. Therefore, prevention of adhesion at an early
11 August 2011
stage after exposure of the host should prevent disease. Many oligosaccharides from human milk are
Accepted 18 September 2011
considered to be soluble receptor analogues of epithelial cell surface carbohydrates; they display
structural homology to host cell receptors and thus function as receptor decoys to which pathogens bind
instead of to the host. Most research in this area has focused on human milk oligosaccharides; however,
the techniques used can readily be applied to the investigation of oligosaccharides produced in other
milk types, e.g., milk of domestic animals. These milk types may therefore become an attractive source of
anti-adhesive oligosaccharides with potential for future incorporation into functional foods.
2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2. Bacterial anti-adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .142
3. Fungal anti-adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143
4. Viral anti-adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143
5. Potential of milk oligosaccharides for use as food ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143
6. Perspectives and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .144
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

1. Introduction other uncharacterised effects (for reviews see: Bode, 2006; Hickey,
2009; Kunz & Rudloff, 2006; Newburg, Ruiz-Palacios, & Morrow,
Oligosaccharides are the third largest solid component of 2005). However, there are very few commercial products on the
human milk following lactose and lipids, with concentrations market that capitalise on these functions. This is mainly due to the
reaching up to 50 g L
1 or more in colostrum to an average of fact that the large quantities of human milk oligosaccharides
10e15 g L
1 in mature milk (Kunz & Rudloff, 2008; Kunz, Rudloff, required for clinical trials are unavailable. In contrast, commercial
Schad, & Braun, 1999). Given their abundance, it became clear oligosaccharides such as galacto-oligosaccharides (GOS) and fruc-
that human milk oligosaccharides have specic biological func- tooligosaccharides (FOS) are present in certain products such as
tions. Such functions may include prebiotic activity, anti-adhesive infant formula, which are currently marketed based on prebiotic
activity, anti-inammatory properties, modication of the entire health claims (Fanaro et al., 2005). However, the structure and
complement of cell surface sugars, a role in brain development, composition of commercial oligosaccharides is very different from
inuencing growth-related characteristics of intestinal cells and the structure and composition of human milk oligosaccharides.
For instance, certain biological properties, such as prevention of
pathogen adhesion, seem ascribed mainly to human milk oligosac-
* Tel.: 353 (0) 25 42227; fax: 353 (0) 25 42340. charides given that a single group of oligosaccharides (galacto-
E-mail address: rita.hickey@teagasc.ie. oligosaccharides or other) invariably cannot match the anti-adhesive

0958-6946/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2011.09.012
142 R.M. Hickey / International Dairy Journal 22 (2012) 141e146

properties of the extremely diverse human milk oligosaccharides Table 1

structures. In fact, human milk oligosaccharides structurally mimic Free oligosaccharides and glycoconjugates from human milk that inhibit pathogen
adherence.
epithelial cell surface carbohydrates and thus function as decoys to
which pathogens can bind instead of the host, thereby preventing Species Decoy References
infection (Kunz, Rudloff, Baier, Klein, & Strobel, 2000). Indeed, Bacteria
oligosaccharides have been identied as ideal candidates for pre- Escherichia coli Fucosyloligosaccharide Newburg et al., 2004
Camplyobacter jejuni Fucosyl a1,2 oligosaccharide Cervantes, Newburg, &
venting pathogen adhesion as they are unlikely to be toxic. From
Ruiz-Palacios, 1995
a medical point of view, using such molecules in disease prevention Listeria monocytogenes Neutral oligosaccharides Coppa, Bruni, Zampini,
may be safer when compared with current approaches such as the Galeazzi, & Gabrielli,
use of preventative antibiotics, as the pathogens are not killed but 2003
just made non-infective, thus less selection pressure is produced Streptococcus Lacto-N-neotetraose (LNnT) Andersson et al., 1983;
pneumoniae Andersson, Porras,
which in turn decreases the emergence of resistance to oligosac- Hanson, Lagergard,
charides (Newburg et al., 2005; Pieters, 2007). These molecules can & Svanborg-Eden, 1986
withstand the low pH of the gut and resist degradation through Haemophilus inuenzae Sialylated oligosaccharides Reddy et al., 1996;
enzymes from the pancreas and brush border membrane (Bode, St Geme 1994
Helicobacter pylori 30 -sialyllactose Mysore et al., 1999
2006). Moreover, with each suckling, the infants orogas-
Streptococcus mutans Glycomacropeptide Kawakami, 1997
trointestinal tract is rinsed with 100e500 mg of oligosaccharides S. suis Sialylated oligosaccharides Liukkonen, Haataja,
that can effectively reach the sites where pathogen adhesion is Tikkanen, Kelm, &
known to occur (Kunz et al., 2000). Finne, 1992
Oligosaccharide expression in human milk is determined S. sobrinus Casein-derived components Neeser et al., 1994
S. sanguis Glycomacropeptide (GMP) Neeser et al., 1994
genetically and synthesis is linked to the presence of enzymes in Mycoplasma pneumoniae 30 -sialyllactose Roberts et al., 1989
the mammary acinar cells, whose expression varies among 0 0
Pseudomonas aeruginosa 3 - and 6 -sialyllactose Thomas & Brooks, 2004
different populations (Newburg, 2000). These enzymes or related Neisseria meningitidis Neutral human Hakkarainen et al.,
enzymes also function in the production of glycoproteins that are oligosaccharides 2005
Salmonella fyris Neutral oligosaccharides Coppa et al., 2006
present on the red blood cells and mucosal epithelial cells or which
Salmonella typhimurium Glycomacropeptide (GMP) Brck et al., 2006
occur as free antigens in other biological uids such as blood, saliva Vibrio cholerae Neutral oligosaccharides Coppa et al., 2006
and intestinal contents (Huang et al., 2005). Polymorphisms of the Shigella Globotriaosylceramide (Gb3) Newburg, Ashkenazi,
secretor and Lewis genes are known to determine expression of the & Cleary, 1992
Lewis blood group type and the biosynthetic pathways involved Fungi
(Morrow et al., 2004a). Interestingly, a number of studies have Candida albicans Fuc-a1-2Galb Brassart et al., 1991
shown that the heterogeneous expression of oligosaccharides and Viruses
related glycoconjugate structures by women from different histo- HIV Lewis X Naarding et al., 2005
blood groups is associated with varying risk of infectious diseases Noroviruses Lacto-N-difucohexose Morrow et al., 2004b
among individuals (Newburg et al., 2004). For instance, the risk of (LDFH-I)
Rotavirus Lactadherin Newburg et al., 1998
hemolytic uremic syndrome in individuals of the P blood group is
Inuenza virus 30 - and 60 -sialyllactose Kunz & Rudloff, 1993
higher (Newburg et al., 1993), while individuals of blood group O
are more predisposed to cholera and to Norwalk virus (Glass et al.,
1985; Hutson, Atmar, Graham, & Estes, 2002). The risk of infection
with Helicobacter pylori and inuenza virus has been linked to on interactions between bacteria and milk oligosaccharides (Hueso,
Lewis and secretor histo-blood group genotypes (Boat, Davis, Stern, Martn-Sosa, & Martn, 2005; Lane, Mehra, Carrington, & Hickey,
& Cheng, 1978; Ikehara et al., 2001). This occurrence may account 2010; Morrow Ruiz-Palacios, Jiang & Newburg, 2005; Newburg,
for the incidence of specic blood group types in areas where 2009; Newburg et al., 2005). The most important information on
certain pathogens are common. For example, in the mestizo pop- milk oligosaccharides and their effects on bacterialehost interac-
ulation a low occurrence of non-secretors is known to occur that tions found in these studies is summarised below.
may which may be due to an increased risk for infants receiving It is now widely accepted that breastfed infants have a lower
milk containing less defensive a1,2-linked oligosaccharides incidence of acute gastroenteritis when compared with their
(Newburg et al., 2004). Indeed, many human milk oligosaccharides bottle-fed counterparts. Coppa et al. (2006) investigated the ability
are known to possess anti-adhesive properties, as shown in Table 1. of oligosaccharides to inhibit the adhesion of three intestinal
Oligosaccharides from domestic animal milk could also be an pathogens namely Salmonella enterica serovar fyris, enteropatho-
acceptable source for molecules with biological functions close to genic Escherichia coli (EPEC) serotype O119 and Vibrio cholerae to
those of human milk oligosaccharides. In addition, non-milk Caco-2 cells. The study demonstrated that the neutral low molec-
oligosaccharides such as GOS (derived from enzymatic synthesis ular weight fractions had an anti-adhesive effect on Salmonella and
based on lactose) and FOS (derived from vegetable plants) should E. coli while the neutral high-molecular-weight fraction inhibited
not be excluded. The structure of GOS is based on lactose and has the adhesion of E. coli O119 and V. cholerae. The acidic oligosac-
some similarities to the core molecules of human milk oligosac- charide fraction was shown to have anti-adhesive activity against
charides (Boehm & Stahl, 2007). Several types of pathogens are all three pathogens. The authors concluded that human milk
known to be targeted by human milk oligosaccharides (HMO) and oligosaccharides are more effective as anti-adhesives when present
related oligosaccharides. as a mixture rather than when an individual oligosaccharide
structure is used given that more than one oligosaccharide struc-
2. Bacterial anti-adhesion ture may block more than one bacterial binding ligand.
Along with reports of less risk of diarrhoea in breastfed infants,
There have been numerous studies that have demonstrated the reports also exist on reduced risk to respiratory infection. With the
anti-adhesive effects of milk oligosaccharides and milk glyco- knowledge that fucosylated antigens are receptors for Pseudomonas
conjugates on bacterial adherence as summarised in Table 1. Many aeruginosa adhesion, and that fucosylated compounds have been
excellent reviews exist on this area of research and for more detail shown to block the adhesion of this pathogen (Scharfman et al.,
 R.M. Hickey / International Dairy Journal 22 (2012) 141e146
2001, 1999), studies were undertaken to examine the inhibition of
P. aeruginosa lectins by human milk (Lesman-Movshovich & Gilboa-
Garber, 2003; Lesman-Movshovich, Lerrer & Gilboa-Garber 2003).
The studies revealed that human milk resulted in a strong inhibi-
tion of the PA-IIL lectin but considerably lower inhibition of the PA-
IL lectin. The authors concluded that PA-IIL has excellent potential
as a rapid, reliable, and sensitive probe for fucosylated derivatives.
Interestingly, Thomas and Brooks (2004) demonstrated that 30 -
sialyllactose and 60 -sialyllactose from milk were also good inhibi-
tors of P. aeruginosa attachment. More recently, Perret et al. (2005)
investigated the structural basis for the interaction between human
milk oligosaccharides and the PA-IIL lectin. The study demon-
strated that two fucosylated epitopes, Lea (LNFP II) and Lex glucose
analogue (LNFP III) bind to the lectin. Noticeably, these residues are
present in the milk of all women irrespective of blood type and
present the highest afnity found to date for the PA-IIL lectin.
To assess if milk oligosaccharides had effects in the respiratory
tract in vivo, a pilot study was carried out by Stepans et al. (2006).
Forty-nine mothers and their infants provided milk and fecal
samples two weeks after birth. The health of the infant was
monitored at 2, 6, 12, and 24 weeks. A major human milk oligo-
saccharide, LNF-II (lacto-N-fucopentaose II), was measured to
correspond to levels of total oligosaccharides in milk and those
remaining in the faeces. The study revealed that higher milk LNF-II
levels at 2 weeks indicated a lower risk of infant respiratory illness
at 12 weeks of age and gastrointestinal illness at 6 and 12 weeks of
age. The above studies indicate that protection by human milk
oligosaccharides may extend to mucosal tissues other than the
gastrointestinal tract. Indeed, some studies indicate that human
milk oligosaccharides may protect infants from urinary tract
infection (Coppa et al. 1990; Martn-Sosa, Martn, & Hueso. 2002).
3. Fungal anti-adhesion
The number of studies investigating the anti-adhesive potential
of milk oligosaccharides against fungi is limited. Brassart, Woltz,
Golliard, and Neeser (1991) demonstrated that fucosylated human
milk oligosaccharides were capable of inhibiting attachment of
Candida spp. to epithelial cells. Indeed, the researchers used the
human milk oligosaccharide as probes, to determine the minimal
structural requirement for activity. The results demonstrated that
the anti-adhesion activity was associated with the Fuc-a1-2Galb
determinant (the H sugar sequence found on all blood group
substances of the ABO system). The authors suggest that the H
disaccharide-containing cell surface glycoconjugates may act as
host receptors for Candida albicans on the buccal mucosa as a part of
a complex adhesion mechanism probably requiring multireceptor
specicities given that total adhesion inhibitions were never ob-
tained throughout the study. Other studies have shown that ABH
non-secretors have a higher incidence of candidiasis (Burford-
Mason, Weber & Willoughby, 1988; Hurd & Domino, 2004; Lamey
et al., 1991), suggesting that fucosylated oligosaccharides may be
suitable as functional food ingredients for individuals who have
a high susceptability to oral candidiasis. The potential of milk
oligosaccharides in preventing adherence of other fungal species
remains unknown. However, given that pathogenic fungi such as
Aspergillus fumigatus interact with fucose (Mendes-Giannini et al.,
2000) and Histoplasma uses sialic acids as receptors (Lin, Huang,
Hung, Wu, & Wu-Hsieh, 2010), future anti-fungal studies should
include milk oligosaccharides as possible inhibitors of colonization.
4. Viral anti-adhesion
A number of studies have demonstrated the anti-viral effects of
milk oligosaccharides. For instance, the HIV-1 virus crosses an infants
143
mucosal barrier when the HIV-1 envelope glycoprotein, gp120 binds
to dendritic cell-specic adhesion molecule 3-grabbing non-integrin
(DC-SIGN) on human dendritic cells (Hong, Ninonuevo, Lee, Lebrilla,
& Bode, 2009). Since some HMO structurally resemble the Lewis
blood group antigens that show high binding afnity for DC-SIGN and
some HMO carry one or multiple Lewis epitopes, HMO can compete
with gp120 for binding to DC-SIGN and reduce HIV-1 mother-to-child
transmission (Naarding et al., 2006, 2005). Interestingly, other viruses
such as Ebola virus, hepatitis C virus, Dengue virus or Cytomegalo-
virus also interact with DC-SIGN (van Kooyk & Geijtenbeek, 2003)
which raises the question do HMO have an anti-viral effect on these
organisms also?
Noroviruses (NVs) are now recognised as the principal pathogen
of non-bacterial gasteroenteritis in all age groups in both developed
and developing countries (Tan & Jiang, 2007). Recombinant virus-
like particles (VLPs) of NVs expressed in baculovirus were used as
probes to study hostepathogen interactions. The resulting studies
revealed that NVs recognise human histo-blood group antigens as
receptors (Huang et al., 2003; Marionneau et al., 2002). Moreover, it
was found that NVs are highly varied in their recognition of these
antigens and eight receptor-binding patterns have been described
(Tan & Jiang, 2007). Of major interest was the fact that non-secre-
tors who do not express the H receptor antigens are naturally
resistant to NV challenge (Lindesmith et al., 2003). Marionneau
et al. (2002) have shown that the binding of Norwalk virus to the
intestinal epithelial cells of secretors can be blocked by milk from
a secretor. To study this occurrence further, Jiang et al. (2004)
characterized the interaction of human milk samples with NV
recombinant virus-like particles (VLPs). Secretor and Lewis anti-
gens (but not A or B antigens) from human milk were responsible
for blocking NV binding to receptors. Direct evidence of human
milk protection of infants from NV infection was also obtained in
a breastfeeding cohort study by Morrow et al. (2004b). In this study,
the occurrence of calicivirus diarrhoea in infants whose mothers
milk contained high levels of the 2-linked fucosyloligosaccharide,
lacto-N-difucohexose (LDFH-I) was lower when compared with
milks with lower amounts of this oligosaccharide. Ruvon-Clouet
et al. (2006) demonstrated that certain glycoproteins in human
milk acted as inhibitors for NV capsid attachment. BSSL, MUC1 and
MUC4 all possessed tandem repeat O-glycosylated sequences
which could act as decoy receptors for the NV, depending on the
combined mother/child secretor status.
Inuenza viruses are classied into three types (species), A, B
and C, based on identity of the major internal protein antigens
(Suzuki, 2005). Using bovine colostrum oligosaccharides as probes,
Matrosovich et al. (1993) investigated the receptor-binding sites of
the H1 and H3 inuenza A and inuenza B virus hemagglutinins.
Thirty human inuenza A and B virus strains were studied in
a competitive ligand binding assay. The following bovine colostrum
oligosaccharides were shown to act as receptors for inuenza:
60 -sialyllactose, 30 -sialyllactose and 60 -sialyl-N-acetyllactosamine.
Despite these results, the design of polyvalent synthetic sialic acid-
containing inhibitors of virus attachment is seen as a more
attractive option, given that optimally designed synthetic inhibitors
revealed a higher anti-inuenza activity than known natural
inhibitors (Matrosovich & Klenk, 2003).
5. Potential of milk oligosaccharides for use as food
ingredients
Few clinical studies have been reported in the literature, which
help strengthen the link between the in vitro anti-adhesive properties
of dened oligosaccharides and in vivo prevention. Espinosa, Tamz,
and Prieto (2007) have summarised the published data which does
exist to support the biological roles of HMO in vivo. One clinical study
144 R.M. Hickey / International Dairy Journal 22 (2012) 141e146
describes the use of a pure HMO to supplement infant formula (Prieto,
2005). The oligosaccharide (lacto-N-neotetraose; LNnT) was present
in the formula at the average concentration in which it is found in
breast milk (200 mg L
1). Although the data suggests that LNnT has
a prebiotic effect, the oligosaccharide does not reduce Streptococcus
pneumoniae oropharyngeal colonization of infants fed a formula
containing LNnT. Therefore, further clinical trials are necessary in this
eld, which will require the large scale synthesis of milk oligosac-
charide structures. However, the biochemical composition of human
milk oligosaccharides appears unique and synthesis of such mole-
cules is likely to become more difcult as structural complexity
increases. Additionally, the cost of synthesizing large amounts of
these molecules for use in clinical trials may be extremely high.
Therefore, much focus has been given to the production of other
carbohydrates, e.g., oligosaccharides from lactose, that while struc-
turally different, have similar biological function. In addition to
commercial prebiotics such as GOS and FOS, researchers are also
focusing their attention on milk oligosaccharides from domestic
animals, as a source for novel functional ingredients. One alternative
may be to use bovine colostrum given its higher content of oligo-
saccharides when compared with mature bovine milk (Davis, Holt, &
Christie, 1983; Mehra & Kelly; 2006; Tao, DePeters, German, Grimm, &
Lebrilla, 2009). Infant formulae designed for nutrition during the rst
weeks after birth, are currently produced from different bovine milk
derivatives. Therefore, isolation, fractionation or enrichment of frac-
tions containing bovine milk oligosaccharides may have potential
applications if used in infant formulae. Moreover, a number of studies
have already shown that bovine oligosaccharides also possess anti-
adhesive effects against certain pathogens (Hakkarainen et al.,
2005; Matrosovich et al., 1993; Wang, Hirmo, Willen, & Wadstrom,
2001). Interestingly, these studies demonstrate that sialylated oligo-
saccharides, such as 30 -sialyllactose and 60 -sialyllactose are potent
inhibitors of pathogen adhesion and these structures are present in
both human and bovine milk (Gopal & Gill, 2000). Despite these
examples, it should be noted that bovine oligosaccharides in general,
are much less complex than those found in human milk and are
present at much lower concentrations (Tao et al., 2009). In the neutral
fraction of bovine milk oligosaccharides, linkages to fucose are rela-
tively rare, whereas linkages of galactose or N-acetylglucosamine are
dominant. Sialic acid is the most important structural element in the
acidic fraction of bovine milk oligosaccharides (Boehm & Stahl, 2007).
Although commercial prebiotics (e.g., GOS and FOS) and milk oligo-
saccharides belong to the same chemical class of oligosaccharides,
only milk oligosaccharides contain sialic acid (Kunz & Rudloff, 2006).
However, this is not to say that commercial oligosaccharides have no
effect on pathogen adhesion. Shoaf, Mulvey, Armstrong, and Hutkins
(2006) demonstrated that puried GOS reduced the adherence of
EPEC to HEp-2 and Caco-2 cells, by 65 and 70%, respectively. More-
over, certain GOS fractions were also found to inhibit V. cholerae toxin
binding to its GM1 receptor (Sinclair de Slegte, Gibson, & Rastall,
2009). However, the rationale for using sialylated bovine milk oligo-
saccharides as functional food ingredients over commercial oligo-
saccharides such as GOS is further strengthened by the many other
benecial properties which are known to be associated with sialic
acid in the diet, e.g., optimal brain development (Sampathkumar, Li, &
Yarema, 2006; Wang et al., 2007).
6. Perspectives and future directions
Milk oligosaccharides which can be associated with clearly
dened anti-adhesive effects against relevant pathogens have
enormous potential as functional food ingredients. By under-
standing milk oligosaccharide structure and functions, it is
tempting to suggest that in the future many novel foods and
beverages, having undergone regulatory approval, will be marketed
based on prevention of infection in high risk consumers. Before this
scenario can be realized, economical and rapid enrichment and
purication procedures must be put in place. Moreover, there
should be an emphasis on improving industrial processes that
convert lactose to GOS, FOS, heterooligosaccharides and other
similar compounds with superior physiological functionality that
could provide the same or similar effects like HMO. Upon the
identication of such molecules, suitable animal studies and well
controlled human clinical trials will be required to validate the anti-
infective nature of these molecules.
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