Human Milk Oligosaccharides: Health Benefits, [622562]

nutrients
Article
Human Milk Oligosaccharides: Health Benefits,
Potential Applications in Infant Formulas,
and Pharmacology
Michał Wici ´ nski1, Ewelina Sawicka1,*, Jakub G˛ ebalski1, Karol Kubiak2
and
Bartosz Malinowski1
1Department of Pharmacology and Therapeutics, Faculty of Medicine, Collegium Medicum in Bydgoszcz,
Nicolaus Copernicus University, M. Curie 9, 85-090 Bydgoszcz, Poland; [anonimizat] (M.W.);
[anonimizat] (J.G.); [anonimizat] (B.M.)
2Department of Obsterics and Gynecology, St. Franziskus Hospital, 48145 Münster,
Germany; [anonimizat]
*Correspondence: [anonimizat]; Tel.: +48-50-123-2629
Received: 16 December 2019; Accepted: 16 January 2020; Published: 20 January 2020
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Abstract: The first months of life are a special time for the health development and protection of
infants. Breastfeeding is the natural and best way of feeding an infant, and positively influences their
development and health. Breast milk provides the ideal balance of nutrients for the infant and contains
countless bioactive ingredients such as immunoglobulins, hormones, oligosaccharides and others.
Human milk oligosaccharides (HMOs) are a very important and interesting constituent of human milk,
and are the third most abundant solid component after lactose and lipids. They are a structurally and
biologically diverse group of complex indigestible sugars. This article will discuss the mechanisms of
action of HMOs in infants, such as their anti-adhesive properties, properties modulating the immune
system, and impact on bacterial flora development. Many health benefits result from consuming
HMOs. They also may decrease the risk of infection by their interactions with viruses, bacteria or
protozoa. The commercial use of HMOs in infant formula, future directions, and research on the use
of HMOs as a therapy will be discussed.
Keywords: human milk oligosaccharides; bifidobacteria; prebitics; infections; nutrition
1. Introduction
The first months of an infant’s life are a special time, with rapid development occurring.
Breastfeeding is the natural and best way of feeding an infant, and positively influences the development
and health of the infant. Human milk is the perfect food for infants, called “living tissue” by many [ 1,2]
because it not only maintains an ideal balance of nutrients but also contains countless bioactive
ingredients such as immunoglobulins, hormones, oligosaccharides and other components [ 3,4].
One important component is human milk oligosaccharides (HMOs), which are multifunctional glycans,
naturally present in human milk. They are particularly interesting because of their quantity and
structural diversity. About 15 structure of HMO have been identified in human milk [ 5]. Figure 1 shows
represantive structures and major oligosaccharides. HMOs are made of five basic monosaccharides:
glucose (Glc), galactose (Gal), N-ethylglucosamine (GlcNAc), fucose (Fuc) and sialic acid (SA).
Almost all HMOs contain lactose (Gal-B1, 4-Glc) at the reducing end, which can be extended with
lacto-N-biose I (Gal-b1, 3GlcNAc) or lactosamine (Gal-b1, 4-GlcNAc). Branching can be linear or
branched through bonds b1-3 or b1-6. The sequence can be further modified by the addition of
Fuc and /or SA monosaccharides through alpha 1-2,3,4 and alpha 2-3,6 bonds due to the action of
fucosyltransferases and sialyltransferases [6].
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further modified by the addition of Fuc and/or SA monosaccharides through alpha 1-2,3,4 and
alpha 2-3,6 bonds due to the action of fucosyltransferases and sialyltransferases [6].

Figure 1. Representative structure of HMO and the major oligosaccharides found in breast milk. (a)
Possible linkages of HMO building blocks, ( b) type 1 (LNT) and type 2 chains (LNnT) ( c)
structures of 2 ′FL, 3′FL and 6 ′FL .
Human milk contains three major HMO types: neutral, neutral N-containing and acid.
Examples of these HMOs and the frequency of occurrence in milk are described in the Table 1. All
HMOs are based on a lactose molecule (a disacchar ide composed of a galactose molecule connected
by a β1, 4-glycosidic bond to a glucose molecule) so it is likely that HMO biosynthesis is an
extension of lactose biosynthesis. Lactose synthesis takes place in the Golgi apparatus of epithelial cells and is catalyzed by lactose synthase (LS). Th is has been well described, but oligosaccharide
synthesis needs further exploration. HMOs are resistant to low pH in the stomach and pancreatic
brush enzymes [7]. Studies on HMO metabolism have shown that 1% is absorbed into the
circulation and the rest are metabolised by gu t microbes or excreted in faeces and urine [8].
Quantitative measurement of HMOs is difficult due to their biological variability and lack of standards. We currently do not have particularly sensitive and specific methods. Studies have
Figure 1. Representative structure of HMO and the major oligosaccharides found in breast milk.
(a) Possible linkages of HMO building blocks, ( b) type 1 (LNT) and type 2 chains (LNnT) ( c) structures
of 20FL, 30FL and 60FL.
Human milk contains three major HMO types: neutral, neutral N-containing and acid. Examples of
these HMOs and the frequency of occurrence in milk are described in the Table 1. All HMOs are based
on a lactose molecule (a disaccharide composed of a galactose molecule connected by a 1, 4-glycosidic
bond to a glucose molecule) so it is likely that HMO biosynthesis is an extension of lactose biosynthesis.
Lactose synthesis takes place in the Golgi apparatus of epithelial cells and is catalyzed by lactose
synthase (LS). This has been well described, but oligosaccharide synthesis needs further exploration.
HMOs are resistant to low pH in the stomach and pancreatic brush enzymes [ 7].Studies on HMO
metabolism have shown that 1% is absorbed into the circulation and the rest are metabolised by gut
microbes or excreted in faeces and urine [ 8]. Quantitative measurement of HMOs is di cult due to
their biological variability and lack of standards. We currently do not have particularly sensitive and
specific methods. Studies have shown that the amount of HMO in milk can be di erent between
women and also during di erent stages of lactation [7,9,10].
Colostrum is the first food of every newborn mammal, including humans. It is a thick, yellow liquid
secreted by the mammary gland. It begins to form in the mammary glands during pregnancy.
The highest concentration of HMO occurs in colostrum and amounts to 20–23 g /L, and then falls in

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mature milk to 12–14 g /L [11]. The milk of mothers who have given birth to premature babies has
higher HMO concentrations than the milk of mothers who gave birth at term [12].
Table 1. Human milk contains three major HMO types [13].
Neutral (Fucosylated) HMO 35% to 50% of the Total HMOe.g., 20-Fucosyllactose (20-FL) and
Lactodifucopentaose
Neutral N-containing HMO 42% to 55% of the total HMO e.g., lacto-N-tetraose
Acid (sialylated) HMO 12% to 14% of the total HMO e.g., 20-sialyllactose
Abbreviations: HMO, Human milk oligosaccharide.
Mothers can synthesize various HMOs based on their genetic background. The most important
example is related to the Lewis system and secretory status. The di erences are in the presence or
absence of fucose in the oligosaccharides.
Two fucosyltransferases—FUT2 (encoded by the secretory gene based on the 19q13.3 gene) and
FUT3 (encoded by the so-called Lewis gene based on the 19p13.3 gene)—play a key role in HMO
fucosylation. Both of these genes are expressed in the glandular epithelium [ 14]. Seventy-nine percent
of mothers have the active gene for fucosyltransferase (FUT2). Table 2 shows 4 groups of mothers with
dierent fucosylated HMO profiles.Twenty-one percent of mothers do not have the functional FUT2
enzyme and produce milk without the 1,2-fucosylated oligosaccharides 20FL and LNFP I [5,8,15].
Table 2. Groups of mothers with di erent fucosylated HMO profiles.
Lewis Positive Secretors (Se +Le+) FUT2 Active FUT3 Active
Lewis negative Secretors (Se +Le) FUT2 active FUT3 inactive
Lewis positive Nonsecretors (Se Le+) FUT2 inactive FUT3 active
Lewis negative Nonsecretors (Se Le) FUT2 inactive FUT3 inactive
Lack of FUT3 enzyme can have negative consequences. Infants of women who do not have the
FUT3 enzyme show delayed colonization of Bifidobacteria , and show more di erences in the metabolic
activity of their microflora, especially Streptococcus .
We can also distinguish four phenotypes based on the Lewis system, which are mentioned in the
Table 3.
Table 3. Phenotypes based on the Lewis system.
Le (a+b+)—Strong expression of the Lea antigen, but the Leb antigen is also synthesized with the Le and Se
allel, strong expression of the Lea antigen, but the Leb antigen is also synthesized.
(ab+)—Only Leb antigen is secreted, occurs in some people with the Le and Se alleles.
Le (a+b)—Only Lea antigen is present, occurs in people with the Le all-dominant allele who are recessive
homozygotes sese.
Le (ab)—Present in all lele homozygotes.
There is a relationship between the presence of specific phenotypes in the Lewis system and the
tendency for complications after exposure to pathogens. For example, Leb antigens are receptors for
Helicobacter pylori . This is especially important for people with blood type 0 because they have a higher
presence of Leb antigens [ 16]. This is important because it has been shown that people with the Le
(a+b) and Le (ab) phenotype are particularly at risk of infection with cholerae-type O1. People with
the Le (ab+) phenotype have a much lower probability of infection, and if it does occur, hospitalization
time is reduced and they have better outcomes. In contrast, people with the Le (ab) phenotype have
a reduced number of IgA antibodies to this virus (also compared to Le (a+b)).This causes diarrhea,
which resolves slowly [17].

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2. Infant Dietary Products
HMOs are absent in infant formulas, and so babies on these formulas do not receive the health
benefits of consuming HMOs. Mixtures of galactooligosaccharides (GOSs) and fructooligosaccharides
(FOSs) or inulin, known as bifidogenic, were therefore developed. These compounds may mimic the
prebiotic from mother’s milk and enter the composition of the intestinal microflora, making them
similar to breast milk [ 18,19]. HMOs are complex glycans composed of five di erent monosaccharides,
while FOSs and GOSs are much simpler structures. FOSs are linear polymers of fructose and GOSs
contain lactose at the reducing end, which is usually extended to six Gal residues that may contain
dierent branches ([Gal ( 1–3/4/6)] 1–6 Gal ( 1–4) Glc). FOSs can be variable, depending on the
method used. The first method uses the inverse fructanase and sucrase reaction or enzyme hydrolysis
of inulin [ 19], which gives short chain FOSs. They have no reducing end and contain one Glc residue,
and two or more fructose residues. The second method produces long chain FOSs. It consists of the
hydrolysis of inulin, causes free anomeric carbons and contains one fructose. GOSs are mainly produced
by enzymatic treatment of lactose -galactosidase from fungi, yeasts or bacteria [ 20].A mixture of
oligosacharides with di erent oligomer chain lengths is obtained from this process. Studies in mice
also found that mixtures supplemented with GOSs and FOSs can modulate the immune system [ 21],
reducing the incidence of infections and atopic dermatitis. GOSs and FOSs are di erent structurally
from HMOs found in human milk. Adding GOS and FOS or inulin is a reasonable and inexpensive
way to add oligosacharides to formula, improving the quality of baby formulas. Much research is still
needed to clarify the specific e ects of GOSs and FOSs. Many mechanisms of natural HMO action still
require explanation.
Two HMOs, 20-fucosyllactose (20FL) and lacto-N-neotetraose (LNnT), have recently been added
to infant formula. Indeed, a study showed that the composition of the microflora of infants fed a 20FL
(1 g/L) and LNnT (0.5 g /L) supplemented formula was di erent from that of infants fed without milk,
where the former group had flora more similar to those breastfed at 3 months of age; Bifidobacterium was
more abundant, while Escherichia and Peptostreptococcaceae were less abundant. Fecal concentrations
of several important metabolites such as propionate, butyrate and lactate in infants fed the HMO
supplement, were more similar to those in breastfed infants [20,22].
3. HMO Mechanism of Action in Building Resistance
3.1. Prevention of Pathogen Adhesion
Evidence for an anti-adhesive e ect of specific HMO comes from in vitro and ex vivo studies.
HMOs serve as soluble ligand analogs and block pathogen adhesion. Many viruses or bacteria must
attach to epithelial cell surfaces to proliferate and cause disease. Usually, the first attachment is to
epithelial sugars on the cell surface (glycans), also called the glycocalyx. HMOs resemble some glycan
structures and serve as soluble luring receptors that then block the pathogen binding to epithelial cells.
Unbound pathogens are unable to attach to the cell surface and are excreted without causing disease.
HMOs seem to have glycomic modifying e ects by changing glycan expression on the surface
where many pathogens and commensal bacteria attach. Caco-2 cells have been shown to change their
surface glycan profile after exposure to the 30SL component of HMOs. Therefore, it is possible that this
particular HMO modifies the glycan content on the surface of epithelial cells and receptor sites for
some pathogens [7,8].
Pathogen adhesion is often initiated by lectin–glycan interactions, which have been described for
many viruses such as noroviruses or rotaviruses. Similarly, Escherichia coli with type 1 fimbriae (fimbria
has the property of binding to the host protein) bind to mannose-containing glycans, whereas E. Coli
and S. fimbriae , as well as Helicobacter pylori , bind to sialylated glycans. Studies in a mouse model
have revealed that 20FL attenuates C. jejuni invasion by 80% and inhibits the release of mucosal
pro-inflammatory signals. The beneficial e ect of 20FL is thought to include a reduction in the
number of diarrhea episodes associated with C. jejuni . LNnT has been shown to reduce the number

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ofStreptococcus pneumonia cells in the lungs of an animal model [ 7]. There is a unique antibacterial
role for HMOs against the leading neonatal pathogen Streptococcus B. HMOs may act as a substrate
to modify growth of these bacteria [ 23]. The anti-adhesive properties of HMOs also apply to some
parasitic protozoa, e.g., Entamoeba histolytica , which cause amoebic dysentery or amoebic liver abscess.
This protozoa destroys the epithelium of the large intestine. In addition to the intestinal wall, it
can also occur in the liver, lungs or spleen. Around 50 million people worldwide are infected with
E. histolytica , resulting in around 100,000 deaths annually. This is the third leading cause of death
from a parasitic disease [ 24].Entamoeba histolytica infection requires attachment to the host’s colon
mucosa. Parasites that cannot attach are excreted in faeces and do not cause disease. HMOs are only
minimally digested and absorbed in the small intestine and therefore reach the colon at the same site
asE. histolytica infection. Some HMOs significantly reduce the binding and cytotoxicity of E. histolytica
during in vivo assays [ 25]. This may explain why breastfed infants are less likely to be infected with E.
histolytica than formula-fed infants.
3.2. E ects of HMOs on Microbiota Composition
The development of the intestinal microflora of infants is a sequential process. The beginning of
this process is considered to be fetal life and the end is at about 3 years old. At this time, the digestive
tract is colonized by bacteria, mainly from the Enterobacteriacae family, especially from the Escherischa
coli,Klebsiella ,Enterobacter, Bacteroides , and Clostridia groups. In infants who are breastfed, bifidobacteria
predominate. There are fewer bacteria of the genera Clostridium and Enterococcus ,while the least
areKlebsiella and Enterobacter . In those infants that are fed artificially, the microflora resembles the
digestive tract of adults and hence its composition is more complex than those infants who are
breastfed. The dominance of bifidobacteria is the result of the presence of a bifidogenic agent in the
food consumed. Oligosaccharides are such a factor. In the large intestine, they are fermented by
bifidobacteria. The main product of this fermentation is acetic acid, which reduces the pH in the
intestine. It is bacteriostatic, inhibiting the growth of pathogenic bacteria. In addition to acetic acid,
fermentation products include butyric and propionic acid. They also have an important function.
Butyric acid is an important source of energy for colonocytes. HMOs can indirectly increase short
chain fatty acid (SCFA) production, and these elevated levels are mediated by bifidobacterial species.
SCFAs are an important source of energy for enterocytes and are key molecules for maintaining
intestinal health. Acetate, butyrate and propionate are dominant. Lactate and succinate, which are
intermediate metabolites in SCFA production, are also present but are less studied. In adults, most
SCFAs are rapidly absorbed or used by colonocytes as an energy source [ 26]. There is increasing
evidence that SCFAs have a wider systemic e ect because they act as signaling molecules and are
involved in the regulation of gene expression [ 27]. It is believed that SCFAs are associated with appetite
suppression by activating free fatty acid receptors in the intestine and increasing circulating intestinal
anorectic hormones [ 28]. SCFAs have also been shown to play an important role in the activation and
dierentiation of immune cells and are associated with inflammatory and allergic diseases [ 24,25,28,29].
The preferred composition of the intestinal microflora is one in which bifidobacteria predominate.
This composition can be seen in breastfed infants. Attempts are being made to increase the amount in
the large intestine by using probiotics. Prebiotics are defined as “a selectively fermented ingredient
that allows specific changes in both composition and /or activity in the gastrointestinal microflora,
which provides benefits to the welfare and health of the host” [ 30,31]. Prebiotics must be resistant
to stomach acidity, hydrolysis by host enzymes and gastrointestinal absorption. HMOs meet all
three criteria. The vast majority of HMOs reach the distal small intestine and colon intact and in
high concentrations [ 7]. Currently, prebiotic characteristics have been confirmed for indigestible
oligosaccharides—fructose derivatives (FOS) and galactose (GOS). Their current application and
properties will be discussed later in the article.

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3.3. Antiviral Activity
Viral infections pose a serious threat to human health. Vaccines exist for influenza or rotavirus,
but other viruses are still a problem. The main obstacle to developing e ective vaccination is a virus
evolution that generates new antigen variants. There are very few anti-viral drugs, and therefore,
most viral infections cannot be cured. Long-term treatment of viral infections can cause undesirable
eects or induce resistance strains [32–34].
HMOs have a high potential to provide protection against several viral pathogens [ 35–37].They can
act as an antiviral through a number of mechanisms, which were described earlier. They promote the
maturation of the immune system and create a more balanced Th1 /Th2 cytokine response. They may
stimulate the immune response and maturation of epithelial cells to protect the host against virus
infection. They a ect the diversity and concentration of microbiomes and stimulate the growth of
commensal bacteria. Another antiviral activity of HMOs is based on the fact that organism’s structure
resembles the structure of various cell surface carbohydrates. Because of this, HMOs catch viruses
that do not stick to cells. This mechanism is based on the structural similarity of HMOs to the sugar
chains of glycoconjugates present on the surface of epithelial cells. HMOs mimic the surface glycans of
epithelial cells. Soluble fucosylated and sialylated human milk oligosaccharides can be recognized and
bound by lectin receptors of fucose and /or sialyl-dependent bacteria, and /or by lectin receptors present
on the surface of host epithelial cells. In both cases, fucosylated and /or ovalized HMOs catch viruses
and participate in blocking lectin receptors. Virus lectin receptors blocked by HMOs cannot participate
in the recognition of glycotopes present on the surface of host cells, which prevents their adhesion
and colonization.
A mixture of sialylated HMOs reduces selectin-mediated neutrophil rolling and adhesion in vitro ,
as well as PNC formation and neutrophil activation ex vivo [38,39].
The discovery of HMO protection against rotavirus and norovirus infections can provide
an alternative to current therapies. HMO action is directed towards more conserved capsid
proteins. Hemaglutinin could potentially be used to treat influenza as an HMO-based medicine.
Mono-sialylated HMO (30SL and 60SL) has been shown to cause reduced influenza infection in cell
culture assays. Nevertheless, more data is still needed for the acquired knowledge to be used for
therapeutic purposes. Recent reports that HMO may reduce HIV-1 mother-to-child transmission in
breastfed infants are very interesting [40,41].
3.4. Norovirus
Noroviruses belong to the calicivirus family. This group includes viruses formerly defined as
Norwalk, Norwalk-like, Sapporo, and Sapporo-like viruses. Now, at least four of their genotypes,
GI, GII, GIII and G IV , are known. Caliciviruses also belong to the food-borne hepatitis E virus.
Noroviruses are recognized as one of the most important causes of infection of unknown viral etiology,
especially in adults. Debilitated and elderly people can become infected with these viruses, leading to
deaths caused directly by dehydration [42].
Over 10 years ago, scientists identified putative human norovirus cell receptors called histone
blood group antigens (HBGAs). HBGAs are complex terminal carbohydrates present in cells and
secreted into body fluids [ 43]. The interplay between HBGAs, HMOs, and norovirus capsid proteins
has been extensively studied in recent years. It has been shown that the addition of HMOs to foods
can help fight noraviruses. HMOs work by preventing its binding to epithelial surfaces. HMOs mimic
the structure of blood-active mucin-type O-glycans. They are an ideal source of potential competitors
for viral glycan receptors; however, knowledge of HMOs is still limited. 2-fucosyllactose (20FL)
trisaccharide is one of the smaller but more common HMOs. It drew the attention of scientists because
it is able to quite e ectively block norovirus binding [ 44,45]. 20FL is registered as a safe food additive.
It is not known, however, whether other more complex glycans in the high mass HMO fraction may
contain components with greater competitive activity in binding norovirus, and whether these glycans
can give hints as to the fine structural requirements for binding norovirus to the host epithelium [46].

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3.4.1. Rotavirus
Rotaviruses belongs to the Reoviridae family and contain a double RNA chain, enclosed in
a three-layered , non-enveloped, 20-walled capsid. Of the seven main rotavirus groups (A to G),
human disease is primarily caused by group A rotaviruses, and much less frequently by groups B
and C. Two surface proteins VP7 (G protein) and VP4 (P protein) determine the specificity of the
serotype basis for rotavirus classification (types G and P) [ 47]. Rotavirus infection is the main cause of
gastroenteritis and diarrhea in infants and young children and accounts for 5% of all deaths in children
<5 years old. Breastfed infants have a lower incidence of acute rotavirus-induced gastroenteritis than
infants fed formula-modified milk [48,49].
Like human noroviruses, a person’s blood group status has now been correlated with susceptibility
to some rotavirus genotypes. Some individuals are generally more susceptible to rotavirus infection
than others [ 44,50,51]. For example, the high incidence of Lewis positive phenotype in the population
may explain the lower occurrence of rotavirus P [ 16]. Rotaviruses interact with HMOs in a similarly
selective manner. Direct evidence for the association of rotaviruses with HMOs has been shown
in animal studies. Newborn pigs were fed with HMOs (20FL, lacto-N-neo tetraose, 60-sialyllactose,
30-sialyllactose and free sialic acid) or a mixture containing short-chain galacto-oligosaccharides (GOSs)
and long-chain fructo-oligosaccharides (FOSs). They were then inoculated with a pig rotavirus strain
on day 10. The duration of rotavirus-induced diarrhea was also shortened in piglets fed a mixture of
GOS and FOS. Pigs that received HMO treatment had almost twice as many NK cells and five times as
many basophils as mixed-fed pigs [45].
Studies have also been performed on a monkey epithelial cell line (MA-104) that was mixed with
HMOs consisting of 30SL, 60SL, 20FL and galactooligosaccharides (GOSs). This study showed that
both sialylated and fucosylated oligosaccharides can reduce the infectivity of human rotaviruses. It is
thought that many human rotavirus strains, including G1P [ 9], use internal sialic acid residues on
cell surfaces as receptors. However, HBGA is increasingly being considered as an important human
binding agent for rotaviruses. Specifically, fucosylated glycans, including H-type 1 and Lewis b HBGA,
are currently recognized as potential binding partners for both G1P [ 9] and G2P [ 4,52,53]. This study
showed that all oligosaccharides tested in this study can reduce the infectivity of human rotavirus
strains G1P [9] and G2P [54].
3.4.2. Respiratory Viruses
Influenza viruses belong to the Orthomyxoviridae family. Types A, B and C are distinguished
among them. Of all its representatives, only type A viruses have the potential to infect a wide spectrum
of hosts: both animals and humans. Di erent strains are characterized in this group due to the di erent
antigenic properties of the two surface, spiny glycoproteins: hemagglutinin (HA) and neuraminidase
(NA). The genes encoding HA and NA are very variable, resulting in only 30% of the amino acid
sequence being conserved within all their subtypes. Currently, 16 di erent HA and 9 NA subtypes
have been identified [55].
When consumed, HMO bathes the laryngopharyngeal region. It was postulated that it may reduce
pathogen adhesion at the entrance to epithelial cells of the respiratory mucosa [ 15]. This also applies
to locally residing or transient-immune cells (lymphocytes, dendritic cells, monocytes, macrophages,
NK cells and M cells) in the palate and lingual tonsils. HMO, with the participation of neutrophils,
has been shown to suppress platelets and the inflammatory response. It is absorbed into the circulation
with potential access through the tonsils or intestinal mucosa [56–59].
HMO interaction studies were performed with respiratory epithelial cells or peripheral blood
mononuclear cells. The e ect of HMOs on the course of viral infection and cytokine expression was
observed. 20FL has been shown to reduce RSV viral load, while LNnT and 60SL reduce the influenza
viral load. It has been concluded that HMO at or below the level found in breast milk increases
innate immunity to respiratory viruses in vitro and can a ect innate immunity. As a result of blocking
hemagglutination of influenza viruses, immobilized 30SL and 60SL attachments prevent infectivity of

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influenza viruses [ 60,61]. A number of external HMO connections have been identified that bind to
influenza virus [ 60,61]. A number of additional sialic acids containing HMO that bind to influenza
virus have also been identified. One interesting study in vivo showed that 20FL enhanced responses
to vaccination in mice. Administration of 20FL in mice improved the humoral and cellular immune
response to vaccination. It is thought that this is a direct 20FL eect on the di erentiation of immune
cells [62].
3.4.3. Human Immunodeficiency Virus
HIV is a RNA virus in the Lentivirus genus of the Retroviridae family. Any HIV infection leads to the
acquired immunodeficiency syndrome (AIDS). The virus targets CD4 +Th lymphocytes, macrophages,
monocytes and dendritic cells [ 54]. Infection occurs through sexual or perinatal transmission, or as
a consequence of exposure to secretions or tissues containing the virus. The infectious material is
blood, semen, pre-ejaculate, vaginal secretion, rectal secretion, milk, and unfixed tissues. HIV-1 virus
can be transmitted from the mother during breastfeeding. However, only about 10–15% of infants fall
ill from a mother infected with HIV through breastfeeding when breastfeeding exclusively [ 63]. The
HIV virus binds to the dendritic cell-specific intercellular adhesion molecule (DC-SIGN) on human
dendritic cells that carries the virus across the mucosal barrier. The most likely mechanism of HMO
action on disruption of HIV infection is that HMOs interfere with the binding of HIV to DC-SIGN.
DC-SIGN also has a strong a nity for Lewis blood group antigens, which form part of the HMOs in
the milk of some women [40].
3.5. Immunity System Development
The newborn’s immune system di ers from the adult’s immune system because it is functionally
naive and has a di erent quantitative and qualitative composition. The di erences are important
for newborns under 1 month of age because breastfeeding reduces the incidence and alleviates the
course of infectious diseases, which a ects the survival of the newborn. Breastfeeding for the first
six months of an infant’s life has been shown to a ect the maturity of the immune system. There
is evidence that exclusive breastfeeding reduces the incidence of many diseases such as asthma,
allergies, inflammatory bowel disease, type 1 diabetes, celiac disease and leukemia.
HMOs are a very important component of breast milk as they can a ect the infant’s immune system
through various mechanisms. The composition of the infant microbiome and the HMO-mediated
epithelial cell responses may indirectly a ect the infant’s immune system. However, many in vitro
studies have suggested that HMOs can also directly modulate the immune response. HMOs may act
locally on lymphoid tissue cells associated with the mucosa, and at the systemic level, as 1% of the
HMOs is absorbed and reaches the systemic circulation. HMOs are detected in the plasma of infants
fed human milk in concentrations of 1–133 mg /L, therefore it is believed that HMOs in the diet can
directly a ect the immune system [64,65].
Many immunoreceptors recognize the oligosaccharide structures of their glycoprotein ligands.
HMOs are structurally similar to selectin ligands, so it is believed that they may bind directly to
immune cells [ 66,67]. Such binding can cause signaling that leads to changes in the populations and
functions of immune cells. During inflammatory processes, E and P selectin, present on the surface
of endothelial cells, recognize and participate in interactions with sialyl-Lewis X glycotopes (sLeX).
sLeX is a member of the glycoconjugates, on the surface of leukocytes, which are one of the elements
involved in the process of leukocyte extravasation and mucosal infiltration [ 11]. HMO enzymatic
modifications such as fucosylation and sialylation allow binding to selectins [68].
Umbilical cord blood T cells exposed to sialylated HMO cause an increase in the number
of CD3 +/CD4+and CD3 +/CD8+cells producing
interferon, and CD3 +/CD8+cells producing
interleukin-13 (IL-13). This is important because sialylated HMO is thought to a ect lymphocyte
maturation and promote the shift of T-cell responses towards more balanced Th1 /Th2 cytokine
production and low immunity. Some sialylated HMOs have been shown to contribute to the prevention

Nutrients 2020 ,12, 266 9 of 14
of allergies, such that sialylated HMOs reduced IL-4 production in the lymphocyte subgroup from
adult patients with a peanut allergy [56,66,67,69].
The e ect of HMOs from colostrum on fetal human intestinal mucosa cells has been investigated.
A cell model with intestinal epithelial cells (T84 /HCT8 /FHs74) and HeLa cells was used for this purpose.
The result of the research was characterization of the networks controlling the communication of
immune cells, di erentiation of the intestinal mucosa immune system and homeostasis. It has also
been found that the use of HMOs reduced the levels of cytokine proteins such as IL-8, IL-6, monocyte
chemoattractant protein 1 /2 and IL-1 . In contrast, the level of cytokines involved in tissue repair and
homeostasis increased.
4. NEC
Necrotising enterocolitis (NEC) leads to severe and often fatal destruction of the infant’s intestine.
It aects 5–10% of premature babies with low birth weight (less than 1500 g). More than 25% of infants
with NEC die. Infants who survive, often have long-term neurological complications. Breastfed infants
have a 6 to 10 times lower risk of developing NEC than formula-fed infants [ 70,71]. The etiology and
pathogenesis of NEC remain poorly understood. Treatment is limited and based on cessation of enteral
nutrition, antibiotic therapy, and, in severe cases, surgical removal of necrotic intestine, which can be
accompanied by long-term complications. Although infant milk formulas have improved in the last
10–15 years, the di erence in NEC risk between formula-fed and breast-fed infants remain unchanged.
It has been concluded that HMOs contribute to the protective e ects of human milk against NEC.
Studies conducted on a rat model have shown that HMOs do protect against NEC. If these results
translate to NEC in humans, disialyllacto-N-tetraose (DSLNT) could be used to prevent or treat NEC in
formula-fed infants, and its concentration in the mother’s milk could serve as a biomarker to identify
breastfed infants at risk of developing this disorder. It may also be useful for developing innovative
therapies against NEC. Additional research and DSLNT e ects in NEC extension should be performed.
There are currently no data in infants confirming the e ect seen in animal models [72,73].
5. New Possibilities of HMO Applications
Considering the action and therapeutic potential of HMOs, other applications have been sought.
A study was carried out to check whether a GOS /FOS mix had e ects on bone health. It was found that
it could help maintain bone health by reducing bone resorption, and increasing bone mineralization,
density and structure with a simultaneous increase in absorption of Ca, Pi and Mg. The results of these
studies are very promising, although studies are still needed that would confirm the e ectiveness of
the GOS /FOS mix. If the results are confirmed, the GOS /FOS mixture may be used in the future in
combination with traditional pharmacological treatments [74].
HMOs are found in the plasma and urine of breastfed infants. Although the “normal” concentration
has not yet been determined, HMOs have been found in the urine of pregnant women at the end of the
first trimester. Test results confirm the postulate that HMOs result in fewer cases of GBS infections
in breastfed infants during pregnancy due to the anti-bacterial properties of HMOs. Studies have
also shown synergy of HMOs with some antibiotics, so they may be useful in the treatment of GBS
infections. Future research can help identify the relationships of individual HMOs, such as LNT,
with the risk of GBS infection. The development of new anti-infective strategies based on the natural
standard of human milk seems likely [23].
Researchers studied the e ects of oligosaccharides on constipation in mice. The aim of this
study was to assess the e ects of three di erent types of oligosaccharide—FOS, GOS and IMO on
loperamide-induced constipation. Oligosaccharides were administered intragastrically to healthy mice
once a day for 17 days. The indicators of constipation, changes in intestinal microflora and metabolic
activities were analyzed, and oligosaccharides were shown to treat constipation. They increase the
water content in the feces, reduce the time of intestinal transit, modulate the composition of the
intestinal microflora and increase the concentration of short chain fatty acids in the feces of mice

Nutrients 2020 ,12, 266 10 of 14
with constipation. After oligosaccharide treatment, Lactobacillus and Bifidobacterium dominated in
intestinal microbiota and decreased levels of Odoribacter, Alistipes and Bacteroides were found [75].
HMO supplementation studies were conducted in 100 healthy adults consuming
20-O-fucosyllactose (20FL) and /or lacto-N-neotetraose (LNnT) at various daily doses and mixtures,
or placebo , for 2 weeks. It was shown that 20FL and LNnT supplementation in daily doses up to 20 g are
safe and well tolerated. Analysis showed supplementation modified the microflora of adult intestines.
The treatment changed the relative abundance of Actinobacteria and Bifidobacterium, as well as the
reduction of Firmicut and Proteobacteria . This study is the first to show the safety, good tolerance and
eects of HMOs on the intestinal microflora of adults. Thanks to the results of these studies, we can
conclude that HMO diet supplementation may be a valuable opportunity to shape the human intestinal
microflora, and especially to promote the growth of beneficial Bifidobacteria .
It has been suggested that HMOs may have therapeutic potential in allergic diseases. The e ect of
two HMOs, 20FL and 60SL, on anaphylactic e ects was investigated by studying the symptoms caused
by oral ovalbumin in an ovalbumin-sensitized mouse model of food allergy. The results of these studies
suggest that 20FL and 60SL reduce symptoms of food allergy by induction of IL-10 ( +) regulatory cells
and indirect mast cell stabilization. Another study showed that the HMOs 20FL and 60SL modulate
human epithelial cell responses. In particular, 60SL may have additional antiallergic benefits because it
inhibits the release of chemokines induced by antigen–antibody complexes and other inflammatory
signals. This leads to inhibition of the influx of inflammatory cells into the intestines. These studies
encourage further exploration of the therapeutic potential of HMOs in food allergies [76].
6. Conclusions
In conclusion, it can certainly be said that HMOs are a very important component of breast milk.
They contribute to the development of the infant’s microflora and immune system. By acting via various
mechanisms, they protect against many infections and alleviate their course. They have been shown to
have anti-bacterial, anti-viral and anti-inflammatory e ects. Exclusive breastfeeding up to 6 months is
very important because it protects the health and life of infants. Many diseases, including diarrhea,
respiratory and urinary tract infections, otitis media, bacteraemia and necrotizing enterocolitis are less
common in breastfed children. Breastfeeding also has an impact on the course of other immune-related
diseases such as celiac disease, asthma, allergy, type 1 diabetes, acute lymphoblastic leukemia and
acute myeloid leukemia. It also reduces the incidence of these diseases.
Research on the possibility of giving infants ingredients that are functionally similar to HMOs,
which are supposed to bring the same health benefits, is very important. Prebiotic oligosaccharides,
which are a mixture of fructooligosaccharides (FOSs) and galactooligosaccharides (GOSs), are already
used in modified milk. The potential of HMOs is larger than this and research in many areas is
ongoing. However, faithful reproduction of breast milk composition is not possible at this stage
of knowledge and a lot of research is still needed. Interdisciplinary teams consisting of chemists,
pediatricians, nutritionists, microbiologists, glycobiologists and many others are needed.
Author Contributions: M.W., supervision, funding acquisition; E.S., formal analysis, resources,
writing manuscript ; J.G., writing manuscript and editing; K.K. participated in data collection and critical
revision of the article; B.M., critical revision of the article and final approval. All authors approved the final
manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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