Carbohydrate Polymers 276 (2022) 118738

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Human milk oligosaccharides and infant gut microbiota: Molecular

structures, utilization strategies and immune function
Bin Zhang a, b, Long-Qing Li b, Feitong Liu c, Jian-Yong Wu b, *
a
SCUT-Zhuhai Institute of Modern Industrial Innovation, School of Food Science and Engineering, Overseas Expertise Introduction Center for Discipline Innovation of
Food Nutrition and Human Health, South China University of Technology, Guangzhou 510640, China
b
Research Institute for Future Food, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong,
China
c
H&H Group Global Research and Technology Center, Guangzhou 510700, China

A R T I C L E I N F O A B S T R A C T

Keywords: Human milk oligosaccharides (HMOs) are a unique class of non-digestible carbohydrates present in the mother
Human milk oligosaccharides milk, which play a key role in the development of infant gut microbiota, epithelial barrier and immune function.
Utilization strategies The deficiency of HMOs in the bovine milk-based infant formula has been widely recognized as a major culprit
Infant microbiota
for the much higher incidence of immune disorders of formula-fed infants. This report was to give an up-to-date
Immune functions
review on the structure characteristics of HMOs and the possible mechanisms, and strategies for their cellular
Commercial applications
uptake, and metabolism by the gut bacteria and the associated effects on the infant gut microbiome, and immune
function. Most previous studies have been carried out in animals or in vitro model systems on the utilization
strategies for HMOs in infant bacteria and their roles in infant microbiome, and gut immune function. A few
HMO molecules have been synthesized artificially and applied in infant formulas.

1. Introduction
The gold standard for commercial infant formula milk is the close
similarity to the composition of breast milk, which is achieved through
the design and optimization of the major ingredients such as proteins,
fats and functional oligosaccharides. Although bovine milk as the basal
component of formula milk can meet the basic requirements for energy
and nutrition of infant growth, it has significant differences from human
breast milk in the major ingredients and their contents. In particular,
human milk oligosaccharides (HMOs) rank the third most abundant
group of constituents (5–15 g/L) of mature breast milk, after lactose
(55–70 g/L) and lipids (16–39 g/L). The composition of HMOs is far
more diverse than the oligosaccharides in the milk of other mammals.
Up to date, more than 200 different molecular structures of HMOs have
been identified (Petschacher & Nidetzky, 2016). Most of the HMOs are
resistant to degradation by the gastric acid and digestive enzymes in the
infant gastrointestinal tract, and can reach the large intestine for mi­
crobial fermentation. HMOs provide a major source of carbohydrates
which are selectively utilized by the gut bacteria, having a profound
impact on the microbial composition of gut microbiota.
The infant gut microbiota evolves with the host since birth. The
microbial composition of infant gut microbiota undergoes significant
changes during the first three years of life, and can be affected by the
delivery mode, feeding pattern and environment. Vaginally delivered
infants on natural birth acquire bacterial communities resembling their
own mother's vaginal microbiota, dominated by Lactobacillus, Prevotella,
and Sneathia spp., whereas C-section infants harbored bacterial com­
munities which are similar to the mother's skin surface, dominated by

Abbreviations: ABC, ATP binding cassette; CAZymes, carbohydrate active enzymes; DSLNT, Disialyllacto-N-tetraose; DP, degree of polymerization; Fuc, fucose; 2′ -
FL, 2′ -fucosyllactose; 3-FL, 3-fucosyllactose; FOS, fructo-oligosaccharides; FUT2, α1-2 fucosyltransferase; FUT3, α1-3/4 fucosyltransferases; 4′ -GL, 4′ -gal­
actosyllactose; 3′ -GL, 3′ -galactosyllactose; 6′ -GL, 6′ -galactosyllactose; Gal, galactose; GlcNAc, N-acetylglucosamine; Glc, glucose; GOS, galacto-oligosaccharides;
GPCRs, G protein-coupled receptors; HDACs, histone deacetylases; HIF, hypoxia inducible factor; HMOs, human milk oligosaccharides; IBS, irritable bowel syn­
drome; LNH, lacto-N-hexaose; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; LNFP I, lacto-N-fucopentaose I; LNFP II, lacto-N-fucopentaose II; LNFP III, lacto-N-
fucopentaose III; LST, sialyl-lacto-N-tetraose; NEC, necrotizing enterocolitis; SCFAs, short-chain fatty acids; Sia, sialic acid; 3′ -SL, 3′ -sialyllactose; 6′ -SL, 6′ -
sialyllactose.
* Corresponding author.
E-mail addresses: zhangb@scut.edu.cn (B. Zhang), longqing21.li@connect.polyu.hk (L.-Q. Li), erin.liu@hh.global (F. Liu), jian-yong.wu@polyu.edu.hk (J.-Y. Wu).

https://doi.org/10.1016/j.carbpol.2021.118738
Received 8 July 2021; Received in revised form 28 September 2021; Accepted 28 September 2021
Available online 9 October 2021
0144-8617/© 2021 Elsevier Ltd. All rights reserved.
B. Zhang et al. Carbohydrate Polymers 276 (2022) 118738

Staphylococcus, Corynebacterium, and Propionibacterium spp. (Domi­
nguez-Bello et al., 2010). Bacteria present in newborn babies of a few
days old are usually aerobic bacteria and facultative anaerobic bacteria.
As oxygen in the gut is gradually consumed by the aerobic bacteria,
anaerobic bacteria such as Bifidobacterium and Bacteroides increasingly
dominate the infant microbiota (Albenberg et al., 2014). The earliest
microbiome is enriched in genes which facilitate lactate utilization with
a relatively simple structure, and mainly with degraders of simple car­
bohydrates such as HMOs. After weaning, solid foods provide gut bac­
teria with diverse fermentation substrates, resulting in stable and
mature microbiota community and function, with the ability to degrade
complex carbohydrates. Weaning is an important milestone in the
development of gut microbiome, starting the transition from infancy to
childhood. Ingestion of solid foods causes an increased abundance of
Bacteroidetes, and levels of short-chain fatty acids (SCFAs) and the
enrichment of genes associated with carbohydrate utilization and
vitamin biosynthesis (Koenig et al., 2011). After the age of three, the gut
microbiota has almost reached a stable community composition and gut
function, which are characteristic of the adult microbiome (Yang et al.,
2016).
Evidence from long-term epidemiological studies shows that
formula-fed infants are more prevalent than breast-fed infants in a
number of childhood disorders including necrotizing enterocolitis
(NEC), irritable bowel syndrome (IBS), obesity, allergies, and eczema
(He, Liu, Leone, & Newburg, 2014; Quigley, Carson, Sacker, & Kelly,
2016; Victora et al., 2016). These health problems prevalent with the
formula-fed infants are closely associated with an aberrant gut micro­
biota. HMOs are widely believed to play an important role for the
differences in the microbial composition of gut microbiota between
formula-fed and breast-fed infants (Marcobal et al., 2010).
Although much information is available on the HMO molecular
specificity, health-promoting functions (Bode, 2012; Triantis, Bode, &
van Neerven, 2018; Walsh, Lane, van Sinderen, & Hickey, 2020; Yu
et al., 2018), to the best of our knowledge, limited information has been
documented for HMO utilization strategies of typical infant bacteria
particularly Bacteroides and Lactobacillus, and the roles of HMOs in the
development of infant microbiome and gut immune function, as well as
the recent application of artificial HMOs in infant formula and regula­
tory framework. This review aims to make a critical summary on our
current knowledge and understanding of the possible mechanisms and
strategies for the cellular uptake and metabolism of HMOs by gut bac­
teria and the associated effects on the infant gut microbiome and im­
mune functions. It also provides an illustration on the major structure
characteristics of HMOs and a brief survey on the commercial applica­
tions of HMOs in infant formulas.
2. Structural characteristics of HMOs
The molecular structure of HMOs is a determining factor on their
selective utilization by the bacteria in the gut microbiota and the
consequent effects. Regardless of the structural diversity, HMOs are
generally composed of five sugar residues with a degree of polymeri­
zation (DP) ranging from 3 to 32. HMOs can be classified in three main
structural groups, fucosylated (taking up 35–50% of the total), non-
fucosylated neutral (42–55%), and sialylated HMOs (12–14%) (Totten
et al., 2012). Some terminal β1-4 chain-linked Gal can be cleaved off by

Fig. 1. HMO structures. (A) Possible linkages of HMO building blocks; (B) lactose can be fucosylated or sialylated to form HMOs; (C) lactose can be elongated
enzymatically in repeats of lacto-N-biose (type I), or N-acetyllactosamine (type II). (D) Galactosyl residues are linked to lactose through β1-3, β1-4-and β1-6 linkages
to form galactosyllactoses. (E) Elongated type I or II chains can be fucosylated or sialylated in different linkages to form a variety of structural isomers. (Abbre­
viations: 2′ -FL, 2′ -fucosyllactose; 3-FL, 3-fucosyllactose; 3′ -SL, 3′ -sialyllactose; 6′ -SL, 6′ -sialyllactose; LNnT, lacto-N-neotetraose; 3′ -GL, 3′ -galactosyllactose; 4′ -GL, 4′ -
galactosyllactose; 6′ -GL, 6′ -galactosyllactose; LNH, lacto-N-hexaose; LNFP I, lacto-N-fucopentaose I; LNFP II, lacto-N-fucopentaose II; LNFP III, lacto-N-fucopentaose
III; LST a, sialyl-lacto-N-tetraose a; LST b, sialyl-lacto-N-tetraose b; DSLNT, Disialyllacto-N-tetraose) (Cheng, Akkerman, Kong, Walvoort, & de Vos, 2021).

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B. Zhang et al.
lactase in infant small intestine, and approximately 1% of the ingested
HMOs are absorbed directly into the systemic circulation and excreted in
the urine (Dotz et al., 2014). All HMOs are built on a lactose core
(Galβ1–4Glc) at the reducing end, elongated or branched with five
monosaccharide building blocks (Fig. 1A). i.e., glucose (Glc), galactose
(Gla), N-acetylglucosaminic acid (Neu5c), fucose (Fuc) and sialic acid
(Sia). Lactose can be fucosylated either at the terminal Gal by α1-2
linkage to generate 2′ -fucosyllactose (2′ -FL) or at Glc in α1-3 linkage to
form 3-fucosyllactose (3-FL) (Fig. 1B). Alternatively, lactose can be
connected by sialic acids with α2-3 or α2-6 linkage to form 3′ -sia­
lyllactose (3′ -SL) and 6′ -sialyllactose (6′ -SL), respectively (Fig. 1B).
HMO connects galactose β-1,3-N-acetylglucosamine (Galβ1-3GlcNAc,
type 1 chain structure) on the basis of lactose to form lacto-N-tetraose
(LNT) or linked to N-acetyllactosamine (Galβ1-4GlcNAc, type 2 chain
structure) to form lacto-N-neotetraose (lacto-N-neotetraose, LNnT), as
shown in Fig. 1C. The development and application of new and
advanced analytical techniques may be helpful for profiling and struc­
ture determination of HMOs, such as matrix-assisted laser desorption/
ionization-mass spectrometry (MALDI-MS) (Huang et al., 2020) and
multi-dimensional nuclear magnetic resonance (NMR) spectroscopy (Shi
et al., 2021).
The genetic background and physiological status of women are
among the chief factors affecting breast milk composition and cause
individual differences with race, region, diet, age, and lactation stage
(Seppo et al., 2019). The type and content of HMOs in breast milk show
dramatic variations with individual women and lactation stages of the
same woman. The composition variation of fucosylated HMOs is
determined by the activities of three or more fucosyltransferases
(Andreas et al., 2015; Kunz et al., 2017), which are encoded by the
women's secretor (Se) and Lewis (Le) blood group status. The majority of
women express α1-2 fucosyltransferase (FUT2) that attach fucose resi­
dues (Fuc) to a terminal Gal through an α1-2 linkage. For example, 2′ -FL
(Fucα1-2Galβ1-4Glc) and LDFT-I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-
4Glc) are most abundant HMOs in breast milk of “secretor women”. In
contrast, non-secretor women do not express FUT2 and therefore these
HMOs are absent in the breast milk. The Le gene encodes α1-3/4 fuco­
syltransferases (FUT3), which may connect to Fuc through α1-3 linkages
such as LNFP-III (Galβ1-4Fucα1-3GlcNAcβ1-3Galβ1-4Glc), or through
α1-4 linkages such as LDFT-II (Galβ1-3Fucα1-4GlcNAcβ1-3Galβ1-4Glc)
(Moukarzel & Bode, 2017). According to the expression of FUT2 and
FUT3 (or maternal Se and Le blood type), HMO profiles in the breast
milk can be divided into four types: Se+Le+, Se− Le+, Se+Le− , and
Se− Le− . The compositions of fucosylated HMOs of each type are sum­
marized in Table 1.
Therefore, the detection of the secretion type and Lewis blood group
status can be used to predict the composition and distribution of HMOs
in breast milk. However, the composition of HMOs in breast milk does
not completely correspond to the detection of the above genotypes. The
expression of various glycosyltransferases is regulated by the Se and Le
genes, and gene deletion mutations or amino acid sequence
Table 1
Biosynthesis of fucosylated HMOs depends on Secretor and Lewis blood group
status.
Gene type Fucosyltransferase Fucosylated HMOs
HMOs
FUT2 FUT3 2′ -FL + LNFP- 3-FL + LNFP- LNFP-
I III II
α1-2 α1-3/4 α1-2 α1-3 α1-4
Secretor + +
(Se)
Lewis (Le) + + + into mono- or di-saccharides outside the bacterial cells by cell-wall
Se+Le+ + + + + + associated glycosidases, and the hydrolyzed sugars are transported
Se+Le− + − + + − into the cells for further degradation in the cytoplasm. With intracellular
Se− Le+ − + − + + digestion strategy, the HMOs are directly transported into the cells by
Se− Le−
− − − − −
3
Carbohydrate Polymers 276 (2022) 118738
modifications could lower the enzyme expression or enzyme activity
(Soejima et al., 2012). For example, breast milk from some non-secretor
women has a low concentration of 2′ -FL, due to the redundancy of
certain pathways, or enzyme activity retained by gene mutations (Totten
et al., 2012).
Although mother's genetic profile was found to have a major effect
on the HMO composition in the breast milk particularly fucosylated
HMOs, stage of lactation is a key influencing factor of the HMO quantity.
The yield of total and sialylated HMOs from the same woman is higher in
breast milk from the first few weeks postpartum and decreases over the
lactation course. In addition, preterm milk is normally associated with
higher HMO concentration compared with term milk (Gabrielli et al.,
2011).
3. Microbial utilization strategies of HMOs in Bifidobacteria,
Bacteroides and Lactobacillus
The complete degradation of HMOs with diverse molecular struc­
tures requires a series of glycoside hydrolases and/or membrane trans­
porters. These enzymes and protein transporters are available in the
infant gut microbiota and essential for the uptake, metabolism and
utilization of HMOs by the gut bacteria. Therefore, understanding of the
genes encoding the required enzymes and transporters can help reveal
the mechanisms and strategies for degradation and utilization of HMOs.
Many of these genes have been found and identified from various bifi­
dobacterial species that are among the most active species for the
metabolism of HMOs in infant gut microbiota (Walsh et al., 2020). In
addition, certain species of Bacteroides and Lactobacillus have also shown
a high HMO metabolic capacity such as Bacteroides fragilis, Bacteroides
vulgatus and Lactobacillus casei (Bidart, Rodríguez-Díaz, Monedero, &
Yebra, 2014; Marcobal et al., 2010; Yu, Chen, & Newburg, 2013).
It has been found that formula-fed and breast-fed infants have
notable differences in the microbial diversity and the keystone species of
gut microbiota. The gut microbiota of breast-fed infants usually has a
relatively low microbial diversity but a high abundance in Bifidobacte­
rium breve (B. breve) and Bifidobacterium longum subsp. longum
(B. longum), while that of formula-fed or mix-fed infants has a slightly
higher diversity and a higher abundancy in B. adolescentis,
B. catenulatum, Bacteroides, Proteus and Clostridium (Azad et al., 2013;
Van Daele, Knol, & Belzer, 2019). The microbiota of breast-fed infants
has been shown to degrade mainly the nutrients in breast milk, while
that of formula-fed infants is rich with degraders of plant-based foods
(Koenig et al., 2011).
3.1. HMO utilization strategies of Bifidobacteria
HMOs can have a significant impact on the formation and develop­
ment of infant gut microbiome, especially the establishment of Bifido­
bacterium-dominated microbial community. Certain bifidobacterial
species can selectively utilize HMOs with specific structures. Bifidobac­
teria mostly have gene clusters encoding specific HMO degrading en­
zymes, through the gene-encoded glycosidase and specific transporters.
Among the bifidobacterial species of breast-fed infants, B. longum and
B. breve are most frequently observed, whereas B. bifidum, B. longum
subsp. infantis, B. pseudocatenulatum, and B. catenulatum are less frequent
(Turroni, Van Sinderen, & Ventura, 2011).
The HMO utilization strategies employed by infant bifidobacteria are
shown schematically in Fig. 2. There are two primary degradation
strategies for Bifidobacteria, i.e., intracellular, transport-dependent
strategy and extracellular, glycosidase-dependent strategy (Katayama,
2016). With extracellular digestion strategy, the HMOs are hydrolyzed
ATP binding cassette (ABC) transporters, and then hydrolyzed into
B. Zhang et al. Carbohydrate Polymers 276 (2022) 118738

Fig. 2. Possible strategies for human milk oligosaccharide (HMO) consumption in B. bifidum, B. infantis, B. breve, and B. longum (Smilowitz, Lebrilla, Mills, German, &
Freeman, 2014). (Abbreviations: GNB, galacto-N-biose; LNB, lacto-N-biose).

monosaccharides or smaller oligosaccharides by glycosidases in the
cytoplasma.
B. infantis shows intracellular HMO degradation mechanism through
the action of many oligosaccharide transporters. Whole HMO molecules
are transported into the cytoplasm through solute binding proteins
(SBPs), and then metabolized intracellularly. Intracellular glycosyl
hydrolases (GHs) are involved in the enzymatic degradation process of
HMOs, releasing monosaccharides in cells (Sela et al., 2012). After being
degraded into monosaccharides, HMOs are assimilated to the central
metabolic pathway of B. infantis by means of the bifid shunt, yielding
acetic and lactic acids as the end products (Kim et al., 2013). A wide
variety of GHs can be secreted by B. infantis, such as α-sialidase,

Fig. 3. The role of HMOs in infant gut immune function. (a) HMOs can stimulate the growth and colonization of commensal bacteria, and (b) modulate the formation
of metabolites such as SCFAs, which can influence the gut immune system; (c) HMOs can reduce the pathogenic infections by acting as decoy receptors and (d)
stimulate epithelial cell maturation.

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B. Zhang et al.
α-fucosidase, β-galactosidases and β-N-acetylhexosaminidases, and the
enzyme affinity covers almost all HMOs in breast milk, including 3′ -SL,
6′ -SL, 2′ -FL, 3-FL, LNnT, and LacNAc (Garrido, Ruiz-Moyano, & Mills,
2012; Sela et al., 2011; Sela et al., 2012). For example, B. infantis ATCC
15697 is a typical strain with strong HMO utilization capacity (Ward,
Niñonuevo, Mills, Lebrilla, & German, 2007), due to the certain genes of
this bacteria encoded Family 1 SBPs and a large number of glycosyl
hydrolases (GHs) (Garrido, Dallas, & Mills, 2013). Family 1 SBPs have
extremely high abundance in these microbial cells (Garrido, Kim,
German, Raybould, & Mills, 2011), part of which contributed by the
expression of various SBPs-related genes induced by HMOs (Garrido
et al., 2015). SBPs are members of the ABC transporter superfamily for
oligosaccharides, showing affinity for many different HMOs (Garrido
et al., 2011) (Fig. 3).
B. bifidum consumes HMOs through an extracellular glycosidase-
dependent molecular mechanism, utilizing various extracellular GHs
to achieve the initial degradation of HMOs outside the cells (Kitaoka,
2012). Extracellular GHs of B. bifidum have strong HMO degradation
capacity, similar to the endogenous GHs of B. infantis. The intermediate
products (e.g., monosaccharides, disaccharides) bind to SBPs and enter
the cells. Two unique extracellular GHs have been found in B. bifidum, i.
e., lacto-N-biosidase and endo-N-acetylgalactosaminidase. For example,
the lacto-N-biosidase cleaves LNT to produce lacto-N-biose (LNB) and
lactose, then LNB combines with SBPs and enters B. bifidum cells (Wada
et al., 2008). The related genes that control this process are located on
the LNB/GNB gene cluster, which is present in most bifidobacteria and
related to the LNT degradation, and its expression is induced by LNT or
other HMOs (Garrido et al., 2015; Kitaoka, Tian, & Nishimoto, 2005;
Nishimoto & Kitaoka, 2007). In addition, the intermediate products of
HMO degradation such as some disaccharides enter the cells first and
may then be excreted from the cells as monosaccharides after being
hydrolyzed by intracellular enzymes. B. bifidum SC555 was cultured
under the conditions of 2′ -FL, 3-FL or 6′ -SL, but could not digest fucose
and sialic acid when lactose was the sole carbon source (Garrido et al.,
2015). This may have been attributed to the lack of metabolic pathways
for consuming fucose and sialic acid. During its growth, fucose and sialic
acid may be released out of the cells and used by other bacteria or even
pathogens in the gut, causing adverse effects to the host (Egan, Moth­
erway, Ventura, & van Sinderen, 2014; Egan, Motherway, Ventura, &
van Sinderen, 2014).
B. breve is one of the dominant species in infant gut. Various B. breve
strains have different degradation capabilities for HMOs, depending on
the type of GHs encoded by the related genes. Table 2 summarizes the
HMO degradation profiles by selected strains. Although B. breve encodes
GH95 α-fucosidase, only strains encoding GH29 α-fucosidase can utilize
2′ -FL, LNFP I, LNFP III and other fucosylated HMOs (Ruiz-Moyano,
Totten, Garrido, Smilowitz, & Mills, 2013). Certain B. breve strains show
a high degradation capability for sialylated HMOs, such as sialyl-LNT.
Compared to neutral HMOs, B. breve SC95 prefers to utilize sialylated
HMOs (Thomson, Medina, & Garrido, 2018). B. breve UCC2003 gene
clusters encode sialic acid-ABC transporters and intracellular α-siali­
dases and other enzymes for utilization of sialic acids from HMOs or the
ones other bifidobacteria excreted (Egan, Motherway, Ventura, & van
Sinderen, 2014; Egan, Motherway, Kilcoyne, et al., 2014). As for the
neutral HMOs such as LNT and LNnT, B. breve can utilize these through
the above two degradation strategies.
Most B. longum strains consume both fucosylated HMOs (e.g., 2′ -FL,
3-FL, LDFT) but also sialylated HMOs (e.g., 3′ -SL, 6′ -SL) as summarized
in Table 2. However, the degradation strategy differs among different
strains (Yu, Chen, & Newburg, 2013). The HMO degradation strategy of
B. longum SC596 is similar to that of B. infantis, and the new gene cluster
of B. longum SC596 can encode intracellular GHs, related ABC trans­
porters, and SBPs with a unique capability for binding and degrading
LNT, SL, FL and other acidic and neutral HMOs (Garrido et al., 2016). In
addition, some B. longum strains isolated from the intestinal tract of
infants can only consume 2′ -FL, 3′ -FL and LNnT (Bunesova, Lacroix, &
5
Carbohydrate Polymers 276 (2022) 118738
Schwab, 2016).
The HMO degradation strategies and metabolic pathways are highly
dependent on the specific Bifidobacteria species or strains in view of the
obvious differences in the gene clusters related to the HMO digestion.
Likewise, a specific species or strain of Bifidobacterium can have different
utilization capabilities for specific types of HMOs (Lawson et al., 2019).
The digestion of certain HMO structures often involves multiple gene
clusters, and small differences between highly similar homologous se­
quences can also lead to differential expression of related enzymes and
transporters. It has been reported that Bifidobacteria strains lacking ABC
transporters may express extracellular glycosidases to utilize HMOs
(Matsuki et al., 2016; Zabel et al., 2019).
3.2. HMO utilization strategies of Bacteroides and Lactobacillus
Bacteroides can ferment or catabolize not only a variety of plant-
based dietary fibers, but also host-derived glycoconjugates from the
mucus layers of gut (Bjursell, Martens, & Gordon, 2006; Salyers, Ver­
cellotti, West, & Wilkins, 1977). Carbohydrates that can be degraded
and consumed by Bacteroides are similar in the sugar units to HMOs,
including N-acetylglucosamine, galactose, fucose, sialic acid, and N-
acetylgalactosamine, suggesting the possibility of HMOs being degraded
by Bacteroides.
For example, Bacteroides fragilis has been found having the same
genes that are related to mucus glycoconjugate metabolism in the HMO
degradation process (Marcobal et al., 2011). Bacteroides have a variety
of strategies for degrading HMOs to various degrees, and the mechanism
of HMO utilization as the sole carbon source is connected to the in­
duction by HMOs of specific gene expressions. Through the analysis of
the annotated genes of related carbohydrate active enzymes (CAZymes)
in the genomes of Bacteroides fragilis ATCC25285 and Bacteroides vul­
gatus ATCC8482, Marcobal et al. (2010) attained multiple CAZyme in­
formation regarding the glycoside hydrolase families that may be
involved in the HMO degradation, including GH2 (α-galactosidase),
GH3 (β-N-acetylgalactosaminidase), GH20 (β-hexosaminidase), GH27
(α-N-acetylgalactosaminidase), GH29 (α-L-fucosidase), GH33 (siali­
dase), GH42 (β-galactosidase) and GH95 (α-1,2-fucosidase). The type
and number of GHF genes are different in the Bacteroides fragilis
ATCC25285 and Bacteroides vulgatus ATCC8482, which further explains
the differences in the HMO degradation capacity and metabolic strategy
among different species/strains of Bacteroides. For example, Bacteroides
fragilis ATCC25285 showed a higher degradation capacity for non-
fucosylated HMOs and long-chain oligosaccharides compared with
Bacteroides vulgatus ATCC8482 (Xu et al., 2007). It appears that each
bacterium has adapted during evolution to retain a unique metabolic
niche, which can provide growth advantages to compete the mixed HMO
substrates (Sela & Mills, 2010).
These is very limited information about the molecular utilization
strategies of Lactobacillus species for HMOs. It has been reported that
L. casei could degrade HMOs with different transport systems and
metabolic pathways compared to Bifidobacterium (Bidart, Rodríguez-
Díaz, Monedero, & Yebra, 2014). L. casei contains a special gene cluster
gnbREFGBCDA, which participates in the metabolism of GNB, LNB and
N-acetylgalactosamine (Bidart et al., 2014). However, during the
metabolism process, the N-acetylgalactosamine parts of LNB and LGB
enter different pathways, GnbG (phospho-β-galactosidase) participates
in the hydrolysis of LNB and GNB, but GnbF (N-acetylgalactosamine-6P
deacetylase) and GnbE (galactosamine-6P isomerase/deaminase) are
only related to GNB degradation, while the utilization of LNB by L. casei
depends on nagA (N-acetylglucosamine-6P deacetylase). The galactose
degradation of LNB and LGB is accomplished through the tagatose-6P
pathway (Bidart et al., 2014).
3.3. Cross-feeding effect between HMO degraders
In the infant colon, HMOs are partially or completely degraded by
B. Zhang et al. Carbohydrate Polymers 276 (2022) 118738

Table 2
Degradation of HMOs by selected strains of gut bacteria.
Species Strain number Glc/Lac 2′ -FL 3′ -FL 3′ -SL 6′ -SL LNnT LDFT Reference

B. infantis
BSM28-1 + + + + + Bunesova et al. (2016)
TPY6-2 + + + Bunesova et al. (2016)
DSM20082 + + + + Bunesova et al. (2016)
ATCC25962 + + + + + Garrido et al. (2015)
ATCC17930 + + + + + Garrido et al. (2015)
ATCC15697 Ward et al. (2007)
B. adolescenlis ATCC15703 + − − − − − Thongaram, Hoeflinger, Chow, and Miller (2017)

B. animalis
ATCC25527 + − − − − − Thongaram et al. (2017)
Bb-12 + − − − − − Thongaram et al. (2017)
Bf-6 + − − − − − Thongaram et al. (2017)
DSM10140 + − − − − − Thongaram et al. (2017)
JCM10602 + − − − − Garrido et al. (2015)

B. breve
DSM20213 − − − − − Bunesova et al. (2016)
TPY10-1 − − + Bunesova et al. (2016)
ATCC15700 + − − − − + Thongaram et al. (2017)
M-16V + − − − − − Thongaram et al. (2017)

B. bifidum
ATCC29521 + − − − − − Thongaram et al. (2017)
JCM1254 + + + − + Garrido et al. (2015)
JCM1255 + − − − − Garrido et al. (2015)
JCM1209 − + + − + Garrido et al. (2015)
JCM7002 + + + + + Garrido et al. (2015)

B. kashiwanohense
DSM20213 + + − Bunesova et al. (2016)
TPY11-1 + + − − − Bunesova et al. (2016)

B. longum
DSM20088 + + − − + Bunesova et al. (2016)
BRS8-2 + + + − + Bunesova et al. (2016)
TPY12-1 + + + − + Bunesova et al. (2016)
DSM20219 − − − − − Bunesova et al. (2016)
BSM11-5 + + − − − Bunesova et al. (2016)
BB536 + − − − − − Thongaram et al. (2017)
M-63 + + + + + + Thongaram et al. (2017)
ATCC15697 + + + + + + + Thongaram et al. (2017); Yu et al. (2013)
ATCC15708 + + + + + Yu, Chen, Kling, et al. (2013)
JCM7010 + + + + + Yu, Chen, Kling, et al. (2013)
JCM11347 + + + + + Yu, Chen, Kling, et al. (2013)

B. pseudolongum
DSM20092 − − − − − Bunesova et al. (2016)
BSM8-1 − − − − − Bunesova et al. (2016)
PV8-2 − − − − − Bunesova et al. (2016)

L. acidophilus
La-5 + − − − − + Thongaram et al. (2017)
NCFM + − − − − + Thongaram et al. (2017)
L. delbrueckii ATCC7830 + + − + − Yu, Chen, Kling, et al. (2013)
L. fermentum CECT5716 + − − − − − Thongaram et al. (2017)
L. gasseri ATCC33323 + − − − − − Thongaram et al. (2017)
L. jensenii ATCC25258 + − − − − − Thongaram et al. (2017)

L. johnsonii
ATCC11506 + − − − − − Thongaram et al. (2017)
ACD-1 + − − − − − Thongaram et al. (2017)
L. paracasei LCV-1 + − − − − − Thongaram et al. (2017)
L. plantarum LP-66 + − − − − + Thongaram et al. (2017)
L. reuteri DSM17938 + − − − − + Thongaram et al. (2017)

L. rhamnosus
DR20 + − − − − − Thongaram et al. (2017)
ATCC53103 + − − − − − − Thongaram et al. (2017); Yu, Chen, Kling, et al. (2013)
B. vulgatus ATCC8482 + + + + + Yu, Chen, Kling, et al. (2013)
B. fragilis ATCC25285 + + + + + Yu, Chen, Kling, et al. (2013)
B. thetaiotaomicron ATCC29148 + + + + − Yu, Chen, Kling, et al. (2013)
Clostridium perfringens ATCC13124 − − − − − Yu, Chen, Kling, et al. (2013)
Clostridium leptum ATCC29065 − − − − − Yu, Chen, Kling, et al. (2013)

Plus (+) indicating degradation of HMO and minus (− ) no degradation.

6
B. Zhang et al.
certain bacteria. The incompletely degraded HMO products can be
further degraded and utilized as the carbon and energy sources by other
bacteria. The cross-feeding effect of infant gut bacteria is particularly
prominent among bifidobacterial species/strains. The HMO utilization
by certain bifidobacterial strains can affect the growth of other strains by
sharing the degradation products. Cooperation with other bacteria
(including both HMO consumers and non-HMO consumers) to promote
the degradation and metabolism of HMOs to the greatest extent can
contribute to increasing the diversity and attaining a dominant position
of Bifidobacteria (Lawson et al., 2019). A study has found a cross-feeding
mechanism for HMO degradation among four bifidobacteria strains with
complete HMO degradation gene clusters, which can theoretically
metabolize all structural types of HMOs, but some HMO degradation
products were not further degraded in the in vitro culture (Gotoh et al.,
2018). When these strains were added to the infant fecal inocula, these
HMO degradation products were further degraded by other bifido­
bacterial species/strains, which significantly stimulated the growth of
non-HMO consumers, and promoted the overall advantage of bifido­
bacteria in the microbial community. The cross-feeding effect of bifi­
dobacteria is very dependent on the extracellular degradation by
ectoenzymes. Only when the cells are lysed or the metabolites are
secreted by the cells, can the HMO degradation products be utilized and
metabolized through the cross-feeding effect (Mee, Collins, Church, &
Wang, 2014; Nishiyama et al., 2018; Nishiyama et al., 2017). In addi­
tion, other non-bifidobacterial species can also benefit from the cross-
feeding mechanism.
4. Effects of HMOs on infant microbiome and immune functions
4.1. The role of HMOs in the development of infant microbiome
HMOs can play an important role in the development and maturation
of infant immune function through their strong influence on the infant
gut microbiota and the gut epithelial barrier. HMOs can contribute to
the development infant microbiome ecology from the colonization of
aerobic, and facultative anaerobic bacteria (e.g., Escherichia coli,
Enterococcus, Streptococcus, Staphylococcus, etc.) and anaerobic bacteria
(e.g., Bifidobacterium, Bacteroides, Clostridium, etc.). The infant intestinal
mucosal immune system gradually matures in the process of continuous
contact, colonization and stimulation of the mucosal epithelial surface
by these gut bacteria. Certain beneficial or commensal bacteria specif­
ically bind to the surface of infant mucosal epithelial cells to inhibit
pathogens through space-occupying effect, nutritional competition, or
secretion of antimicrobial metabolites such as bacteriocins, or directly
stimulate the immune cells in the lamina propria of intestinal mucosa to
induce the maturation of the immune system (Bode & Jantscher-Krenn,
2012). For example, Bifidobacterium, a common dominant bacterium in
the intestinal tract of breast-fed infants, can stimulate immune cells to
secrete more IL-1 and IL-6, while IL-1 can promote Helper T cells to
secrete IL-2 and B lymphocytes to secrete antibodies (Maslowski &
Mackay, 2011). In the meantime, IL-6 promotes the differentiation and
maturation of B lymphocytes, making them secrete antibodies, and can
also directly induce the proliferation of T lymphocytes (Maslowski &
Mackay, 2011).
The HMO molecular utilization strategy of bifidobacteria and the
microbial cross-feeding mechanism provide strong support for the
establishment and balancing of infant microbiome, and HMOs may also
use their specific structures to influence immune functions (Bode &
Jantscher-Krenn, 2012). While increasing the diversity of the gut mi­
crobial population is generally considered beneficial for adults, it is not
the case for infants, as bifidobacteria are mainly beneficial to infant
nutrition absorption, disease prevention, and growth promotion (Bel­
kaid & Hand, 2014; Huda et al., 2014). HMOs promote the establish­
ment of gut barrier function and the development of the intestinal
immune system in infants by affecting the microbiota composition and
their metabolites. HMOs can induce genes encoding HMOs-type SBPs in
7
Carbohydrate Polymers 276 (2022) 118738
Bifidobacteria. Because of the structural similarity of HMOs to intestinal
epithelial glycoconjugates, some SBPs can bind to the intestinal
epithelium and the epithelial-associative SBPs enhance the binding of
Bifidobacteria to intestinal epithelial cells. In addition, Bifidobacteria
metabolism of HMOs can increase the expression of anti-inflammatory
cytokines and tight junction proteins (Chichlowski, De Lartigue,
German, Raybould, & Mills, 2012).
4.2. The impact of HMOs on infant microbiota changes
The intestinal microbiota has been co-evolved with the host since
birth. The composition of infant microbiota undergoes significant
changes during the first three years of life, and then gradually stabilizes
to form an adult-like shape. Infants begin to contact with a variety of
microorganisms from mother's body and environment after being
delivered, but not all microorganisms can colonize the infant gut. Only
those microorganisms that adapt to the baby's intestinal environment
can survive. Aerobic and facultative anaerobic bacteria usually exist in
newborn babies such as Streptococcus and Enterobacteria, and the species
are closely related to the delivery mode and environmental factors.
Naturally delivered infants have similar gut bacterial varieties to the
mother's vagina, such as Lactobacilli, while newborn babies with C-sec­
tion have more bacteria that are similar to those from the mother's skin
and external environment, such as Staphylococcus, Streptococcus and
Propionibacterium (Dominguez-Bello et al., 2010). With growing age,
oxygen in the infant intestine is gradually consumed by aerobic bacteria,
and the decrease in oxygen content allows the growth of anaerobic
bacteria. For example, Bifidobacterium and Bacteroides gradually domi­
nate the infant microbiota (Albenberg et al., 2014). Low diversity and
high variability are the major characteristics of the infant microbiota,
and the differences in the shape and composition of microbiota among
individual infants could be even higher than the adults (Chu et al.,
2017).
During the first six months of life, the infant microbiota has a single
structure and a small number of species, mainly degraders of simple
carbohydrates. Bifidobacterium comprises 90% of the gut microbiota in
breast-fed infants, whereas less Bifidobacterium and more Bacteroides are
typical of formula-fed infants (Underwood et al., 2015). Breast-fed in­
fants normally have a lower microbial diversity in the gut compared
with the formula-fed infants (Azad et al., 2013; Van Daele et al., 2019).
Bifidobacterium dominates the breast-fed infant microbiota, due to the
high capability of HMO utilization. HMOs are the preferred substrate for
B. infantis and other bifidobacteria, which can stimulate the growth of
beneficial bacteria and prevent harmful bacteria from HMO utilization.
Bacterial fermentation of HMOs produces SCFAs, resulting in a low-pH
environment in the colon, which is conducive to the growth of benefi­
cial bacteria and inhibits potential pathogens (Yu, Chen, & Newburg,
2013). Bifidobacterium has an advantage in competing with pathogens
for limited nutrients, and its colonization helps reduce the proportion of
pathogens and other harmful bacteria.
A recent clinical study has shown that the secretor status of women is
closely associated with the abundance of Bifidobacterium species in the
infant gut, whereas non-secretor breast milk delays the colonization of
bifidobacteria, with promotion of Clostridium and Enterobacteria (Lewis
et al., 2015). The bifidobacteria isolated from feces of secretor breast
milk-fed infants were able to utilize 2′ -FL as the sole carbon source,
indicating a more pronounced bifidobacterial metabolic activity tar­
geting fucosylated HMOs (Lewis et al., 2015).
Numerous studies have shown that HMOs can selectively stimulate
the growth of beneficial strains such as B. infantis, B. breve, B. bifidum and
B. longum (Asakuma et al., 2011; Bunesova et al., 2016; Thomson et al.,
2018; Xiao et al., 2010). For example, B. longum and B. breve are more
active to digest LNT, whereas B. infantis is prone to utilize LNnT, and
B. infantis and B. bifidum have shown a preference to utilize fucosylated
HMOs (Suzuki et al., 2008; Wada et al., 2008). In addition, the molec­
ular structure of HMOs probably also affects the colonic fermentation
B. Zhang et al.
rate and SCFA production. Some studies have shown that fermentation
of SL and 2′ -FL increased the concentration of SCFAs and lactic acid,
though different HMOs had distinct SCFA profiles during the fermen­
tation. For example, Perdijk et al. (2019) established an in vitro
fermentation model and found that SL can greatly increase the con­
centration of propionic acid compared with GOS. Salli et al. (2019)
found that 2′ -FL can increase the concentration of lactic acid in the in
vitro fermentation model compared with GOS. However, there have
been very few comparative studies to reveal the structure-microbial
fermentation outcomes of various HMOs.
Jing, Chen, Yu, He, and Newburg (2016) have carried out in vitro
experiments to evaluate the 2′ -FL utilization by single strains (B. longum,
L. acidophilus, etc.), and found that 2′ -FL, as the sole substrate for
anaerobic fermentation of certain strains, was readily consumed, pro­
ducing high concentrations of total SCFAs. Yu, Chen, Kling, et al. (2013)
found that Bifidobacterium effectively consumed 2′ -FL and 3-FL during
the in vitro anaerobic fermentation, and the increase of the lactic acid
and SCFA concentration significantly inhibited the growth of E. coli and
Clostridium perfringens that can hardly use fucosylated HMOs. In a ran­
domized double-blind controlled multicenter clinical trial (Berger et al.,
2020), it was found that after three-month intervention, infant formula
containing 2′ -FL and LNnT significantly increased the abundance of
Bifidobacterium and Streptococcus, and turned the microbiota of
caesarean section infant group of 0–6-month-old to the vaginal delivery
infants.
Short-chain fatty acids play an important role in connecting micro­
biome with the host immune system. Acetic, propionic, and butyric acids
are commonly found in the colon with relatively high concentrations,
and are absorbed by intestinal epithelial cells. SCFAs can modify cell
gene expression, differentiation, proliferation and apoptosis and other
processes to change cell functions, interact with the activation of G
protein-coupled receptors (GPCRs), and inhibit histone deacetylases
(HDACs) activity and other signal transduction pathways related to the
stabilization of hypoxia inducible factor (HIF). Microbiota and the im­
mune system are connected through regulating GPCRs and the activity
of enzymes and transcription factors to further modulate the develop­
ment, survival and function of IECS and leukocytes. Each SCFA has its
special functions to the infant health. Both acetic and propionic acid
have been suggested to reduce the risk of infant asthma (Fukuda et al.,
2011). Butyric acid can stimulate intestinal epithelial metabolism,
consume intracellular oxygen, stabilize HIF, and enhance epithelial
barrier function (Kelly et al., 2015). In addition, butyric acid has been
shown to enhance regulatory T cells (Tregs) activity and proliferation,
and inhibit the pro-inflammatory activity of a variety of other immune
cells, and reduce the risk of diarrhea by improving the gut barrier
function (Thorburn et al., 2015). In addition to SCFAs, other metabolites
derived from gut microbiota such as bacteriocins, amino acids and vi­
tamins, play a vital role in activating the intestinal immune response,
thereby resisting external pathogens (Li et al., 2018).
4.3. The impact of HMOs on the gut immune function of infants
The gut immune function in newborn infants is immature, with
characteristics of weak barrier function and low microbiota diversity as
well as immature immune response (Yu et al., 2018). The HMO mole­
cules may modulate neonatal immunity indirectly, by altering host gut
microbiota composition and the metabolites as mentioned in the pre­
vious section, and also directly, by affecting host epithelial and immune
cell responses, or systemic immune function in the intestine and other
sites. A recent study has suggested that some neutral HMO fractions
produced significant immunomodulation activities in RAW264.7
macrophage cells via the upregulated expression of nitric oxide synthase
and cy-clooxygenase 2 (Zhang et al., 2019). HMOs have structural
similarity to the surface glycans of intestinal epithelial cells, and could
prevent infections by binding pathogens to intestinal epithelial cells
(Morozov, Hansman, Hanisch, Schroten, & Kunz, 2018). HMOs can also
8
Carbohydrate Polymers 276 (2022) 118738
directly act on intestinal epithelial cells. By regulating the gene
expression of epithelial cells, the glycans on the cell surface are changed,
thereby preventing pathogenic microorganisms from contacting intes­
tinal epithelial cells (Kunz, Rudloff, Baier, Klein, & Strobel, 2000).
It has been suggested that HMOs modulate the immune functions and
regulate the responses of epithelial cells and immune cells by altering
cell proliferation, differentiation, and apoptosis processes, as well as cell
signaling pathways and cell surface glycosylation. The intestinal
epithelium lining in the small intestine and colon is a physical barrier
between the intestinal lumen and the circulatory system and the first
line of defense for innate immunity. The permeability of intestinal
epithelium mainly depends on the structure and expression of tight
junction proteins. Nutrients can be absorbed by epithelial cells or be­
tween tight junctions. Perturbation of the intestinal barrier integrity can
quickly lead to a series of immune responses, including gastrointestinal
infection, inflammation, and allergies (Vancamelbeke & Vermeire,
2017). HMOs with different structures can have a direct effect on
different cells of the intestinal epithelial barrier. For example, Cheng,
Kong, Walvoort, Faas, and de Vos (2020) used the LS174 T goblet cell
line to test the effects of 2′ -FL and 3-FL on the intestinal mucus barrier
function and found that 3-FL regulates IL-13 and TNF-α to improve
MUC2 gene expression. Similarly, HMO intervention increased the
expression of MUC2 and decreased the permeability of the intestinal
epithelium in a neonatal mice model (Wu et al., 2019).
The glycocalyx acts as the backbone of proteoglycans, which pro­
vides binding sites for microorganisms and shows intestinal barrier
function. Some in vitro studies have shown that HMOs can enhance the
intestinal barrier function by changing the expression of glycocalyx.
Kong et al. (2019) proved that fucosylated HMO (2′ -FL and 3-FL)
significantly increased the thickness of absorbed albumin, and 3-FL
increased the area coverage of albumin, heparan sulfate and hyaluron­
ic acid in the glycocalyx of Caco-2 cells, with enhanced stability of the
glycocalyx and reduced adhesion of pathogenic bacteria. The immature
glycocalyx layer is associated with changes in the expression of heparan
sulfate and hyaluronic acid and may lead to gastrointestinal diseases
such as inflammatory bowel disease (IBD) (Kong et al., 2019).
HMOs can directly interact with infant intestinal epithelial cells,
affecting their gene expression, cell cycle, and cell surface glycosylation
and regulating their growth, differentiation and apoptosis (Kuntz,
Rudloff, & Kunz, 2008). He et al. (2014)) assessed the effect of HMOs on
gene expression in fetal immature intestinal mucosa, and identified
several immune-related pathways, such as immune cell communication,
homeostasis, and immune differentiation. Sialyllactose can promote the
differentiation and growth of intestinal epithelial cells by up-regulating
the expression of alkaline phosphatase, indicating that sialyllactose can
be recognized by the receptors of the epithelial cells and beneficial to
promote intestine barrier function (Kuntz et al., 2008). It has been re­
ported that 3′ -SL had anti-inflammation effect by reducing the IL-12 and
IL-8 expression in Caco-2 cells mediated by the NF-κB pathway and the
anti-inflammatory nuclear receptor PPARg (Zenhom et al., 2011).
Apart from regulating the mucus and glycocalyx, HMOs can stimu­
late the maturation of epithelial cells. Holscher, Davis, and Tappenden
(2014)) used HT-29 and Caco-2Be mixed cells to model different dif­
ferentiation patterns along the crypt-villi axis, and evaluated the effects
of LNnT, 2′ -FL and 6′ -SL on the differentiation, apoptosis and prolifer­
ation of epithelial cells. It was found that LNnT and 6′ -SL lead to a
decrease in cell proliferation, and LNnT can reduce the permeability of
epithelial cells (Holscher et al., 2014). In a later report from the same
research group, Holscher, Bode, and Tappenden (2017)) found that 2′ -
FL, 3-FL and 6′ -SL alone or in combination can reduce cell proliferation,
increase the differentiation of intestinal epithelial HT-29 and Caco-2Be
cells, and promote the maturation of intestinal epithelium.
B. Zhang et al.
5. Application of artificial HMOs in infant formula and
regulatory framework
For many years, it has been difficult to isolate or synthesize human
milk oligosaccharides with defined molecular structures and to apply
these oligosaccharides in infant formula. Instead, alternative prebiotic
oligosaccharides such as galacto-oligosaccharides (GOS) and fructo-
oligosaccharides (FOS) have been supplemented to infant formula to
mimic HMOs. However, several studies have shown that these oligo­
saccharides cannot mimic the prebiotic effects or biological function of
HMOs. For example, 2′ -FL has the advantages over FOS in reducing
intestinal pH and SCFA production, and is preferentially utilized by
Bifidobacterium and Lactobacilli to colonize and form dominant bacteria
in infant gut (Yu, Chen, Kling, et al., 2013). It has been reported that
GOS can be utilized non-selectively by various bacteria, including
pathogenic bacteria (Yu, Chen, Kling, et al., 2013). It is unlikely that
GOS, FOS or other non-human oligosaccharides with different structures
from HMOs can mimic the structure-specific effects of HMOs (Bode &
Jantscher-Krenn, 2012).
The increasing awareness of the significance of HMOs in infant
health and development has stimulated both research and commercial
interests world-wide in the synthesis and manufacture of HMOs or their
active analogues as supplements to infant formula. In recent years,
major technology breakthroughs have been made for production of
HMO molecules such as 2′ -FL, 3′ -SL, 6′ -SL, LNT, LNnT and DFL through
cell engineering, chemoenzymatic and chemical synthetic processes.
This has brought revolutionary changes to the infant nutraceutical in­
dustry so that several companies have acquired the capability for
manufacturing these HMOs at large scale such as Glycom A/S, Abbott
Laboratories, Glycosyn LLC, Inbiose, Jennewein, and GeneChem etc. In
2015, chemically synthesized 2′ -FL and LNnT from Glycom A/S and
biotechnologically synthetized 2′ -FL from Jennewein received the first
regulatory approvals as GRAS from US Food and Drug Administration
(FDA) (Salminen, 2017). The supplementation of synthetic or artificial
HMO compounds to infant foods has already been approved by US, EU,
Australia and other regulatory agencies (Bych et al., 2019). The Euro­
pean Food Safety Authority approved the use of 2′ -FL and LNnT in infant
formula and other infant food products in 2015, and indicated that the
addition of 2′ -FL and LNnT at a ratio of 2:1 (w/w) to infant foods is safe
for babies under 1 year old, with the maximum dosage of 2′ -FL (1.2 g/L)
Fig. 4. Overview over the commercial application in the last three decades of HMOs in infant formula products by some major food companies.
9
Carbohydrate Polymers 276 (2022) 118738
and LNnT (0.6 g/L). The addition of 2′ -FL alone can also be applied to
food products for babies over 1 year old, or added with LNnT at a ratio of
2:1, with the maximum dosage of 2′ -FL (1.2 g/L) and LNnT (0.6 g/L). In
2016, biotechnologically synthetized 2′ -FL and LNnT from Glycom A/S
received Novel Food status in EU and GRAS notification in US. Since
2018, Glycosyn and Inbiose have manufactured 2′ -FL with receiving
GRAS notification from US FDA. In 2018, LNT and DFL from Glycom A/S
and 3′ -SL from Genechem received the GRAS notification from the US
FDA (Bych et al., 2019). In 2019, US FDA stipulated that the maximum
dosage of 2′ -FL in infant formula for 0 to 12 months old infants is 2.4 g/
L, and the maximum dosage of LNnT in infant formula is 0.6 g/L. In the
same year, Food Standards Australia and New Zealand (FSANZ) rec­
ommended that the maximum dosage of 2′ -FL and LNnT in infant for­
mula is 96 mg/100 kJ if only 2′ -FL is added, or 24 mg/100 kJ for LNnT
and 96 mg/100 kJ for the sum of 2′ -FL and LNnT if both are added. The
establishment of valid and official standards by regulatory and profes­
sional authorities has safeguarded the safety and nutrition quality of
infant formula and other infant food products but also paved the way for
their commercial applications.
Among a variety of HMOs, 2′ -FL, 3′ -SL, 6′ -SL, 3′ -GL, LNnT and DFL
have been most widely used as ingredients of infant formula, dietary
supplements, and health foods (Fig. 4). The first infant formula products
appeared in 2016 and more products have continued to emerge. For
example, Abbott has applied 2′ -FL to the infant formula (Similac® Pro-
Advance) with positive health benefits for brain development, reduction
of infection, inflammation and NEC through microbiome modulation. In
addition, Aptamil applied 2′ -FL and 3′ -GL with the combination of FOS
and GOS to the infant formula to mimic HMO formula in breast milk.
Wyeth also uses HMO in a variety of infant formula products. For
example, 0.25 g/L 2′ -FL is added to S-26® infant formula stage 1–4; in
the Illuma series, 2′ -FL and LNnT are added to 1–4 stage of infant for­
mula. In Biostime HMO infant formula, every 800 g of milk powder
contains 2′ -FL in a dose as high as 3.12 g. In addition, Mead Johnson,
Nestle, etc. have also applied HMOs to their infant formula products. In
Nestle's NAN formula milk, the addition amount of 2′ -FL is 0.26 g/L, and
the addition amount of LNnT is 0.13 g/L; in Nestle's BEBA formulas, 2′ -
FL is added up to 1 g/L and LNnT is added up to 0.5 g/L. With the
gradual expansion of HMOs in the field of infant formula, applications
have also expanded to dietary supplements and medical foods. For
example, Dupont added 2′ -FL and 3-FL into UHT milk and yogurt
B. Zhang et al.
products (Christensen, Skov, Lendal, & Hornshoj, 2020). Biostime used
freeze-drying technology to combine 2′ -FL with a variety of highly
active probiotics, and has launched a new product for children aged 3 to
12 and newborns from 1 to 6 months.
6. Concluding remarks and future directions
There is accumulating evidence that HMOs play a key role in the
development of the gut microbiome, and the immune system in early life
and are also beneficial to infant health and wellbeing. The beneficial
effects of HMOs on the infant health are strongly dependent on their
cellular uptake and metabolism by the gut bacteria and the associated
effects on the infant gut microbiome and immune function. The uptake
of HMOs is structure-dependent and selective by the gut bacterial spe­
cies. This review has summarized the HMO utilization strategies of
major infant bacteria such as Bifidobacterium, Bacteroides and Lactoba­
cillus, and their functions in the development of infant microbiome and
gut immune function. Currently only a few HMO molecules have been
synthesized artificially and found commercial application in infant and
baby foods.
Although significant research progress and technology break­
throughs have been achieved on the function and application of HMOs
in infant nutrition and health, much more effort is needed for the
research and development in several important areas as listed below.
1. Only a few HMO molecules are available from commercial suppliers
for research and even fewer for infant formula applications.
2. Systematic studies at the molecular level through various -omics
approaches, e.g. genomic, metabolomic, proteomic, and glycomic,
are most helpful to reveal the molecular strategies and mechanisms
for the cellular uptake and metabolism by different microbes and the
immune function of HMOs.
3. The current knowledge and understanding of HMOs in infant health
has been mainly based on findings from animal or in vitro experi­
ments. Further studies on human subjects or humanized animal
models are needed with consideration of the gene background and
physiological status of mothers to ascertain the functions of the
mixed HMO formulation for the different individuals.
4. More rigorous and placebo-controlled interventions with the HMOs
in human subjects are needed to justify the epidemiological evidence
and the significance for infant nutrition and immune function.
Declaration of competing interest
All authors declare there is no conflict of interest.
Acknowledgements
This work was financially supported by the Hong Kong Scholar
Program (XJ2019049), The Hong Kong Polytechnic University, and the
111 Project (B17018).
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