The composition of breast milk is highly complex, as it contains numerous biomolecules. Human milk oligosaccharides (HMOs) are the third-most abundant component of breast milk, after lactose and lipids. Among the synthetised HMOs, 2’-fucosyllactose (2’-FL) and lacto-N-neotetraose (LNnT) are widely studied, and are considered safe for infant nutrition. Several studies have reported the health benefits of HMOs, which include modulation of the intestinal microbiota, an anti-adhesive effect against pathogens, modulation of the intestinal epithelial cell response and development of the immune system.

The amount and diversity of HMOs are determined by the genetic background of the mothers (HMO secretors or non-secretors). The non-secretor mothers secrete lower HMOs than secretor mothers. The breastfed infants of secretor mothers gain more health benefits than those of non-secretor mothers. So, the supplementation of infant formula with 2’-FL and LNnT is a promising innovation for infant nutrition.

Composition of human breast milk

The World Health Organisation (WHO) recommends that infants must be exclusively breastfed during the first six months of life. Human breast milk provides more than half of the child’s nutritional needs during the second year of life. Infants who are formula-fed are more prone to infectious diseases such as gastroenteritis and acute otitis media, and immunemediated diseases such as allergies, when compared with the infants who are exclusively breastfed.

Human breast milk has two types of carbohydrates: lactose and oligosaccharides. Lactose, a major component of human breast milk, has a high nutrition value. HMOs are the third-most abundant component of breast milk, after lactose and lipids. HMOs in human breast milk are a complex mixture of more than 200 non-digestible and non-nutritional carbohydrates. Among the various compositional differences between human breast milk and cow milk, one of the major ones is the presence of HMOs in human milk, which are virtually absent in cow milk and infant formula.

Of course, the amount and composition of HMOs vary among women, and also during the lactation period. Generally, the total HMO concentration is higher during the early stages of lactation and decreases within the first three months. The HMO content of breast milk after term delivery is higher than that after preterm delivery. The HMO fraction is the third-most abundant component in human milk after lactose and lipids, excluding water. The HMO content usually varies between 10–15g/L of mature milk and 20–25g/L of colostrum. The HMO content in human breast milk is more abundant than the protein content, which is typically around 10g/L or 1.5g/100kcal.

Human breast milk also contains three major HMO types: fucosylated HMOs, sialylated HMOs and nonfucosylated neutral HMOs. Fucosylated HMOs include 2’-FL, while non-fucosylated neutral HMOs include LNnT. The neutral HMOs account for more than 75% of the total HMOs in human breast milk. The most abundant HMO is the 2’-FL, which constitutes nearly 30% of the total HMOs among secretor mothers. The factors that account for the variability in the secretion of HMOs include the geographical origin and genetic background of the mothers, as the HMO content in the breast milk varies among women. Globally, it is estimated that about 20% of mothers secrete lower amounts of HMOs (non-secretor mothers) than the secretor mothers.

One of the factors that affect the composition of primary gut microbiome in infants is feeding. The intestine of a neonate is already colonised at birth. A healthy gut microbiome protects the host against pathogens by various mechanisms, such as enhancing the immune development, and stimulating the digestive and metabolic functions. Other factors that affect the colonisation of the gut include the gestation period, mode of delivery, environment and medication. Dysbiosis during early life is a risk factor for immunemediated diseases such as allergy and asthma, and intestinal and metabolic diseases.

The gut microbiome of infants born through caesarean section (C-section) is different from that of infants born through vaginal birth in that the number of bifidobacteria in the intestinal microbiome of infants born through C-section may take up to six months to reach the number of bifidobacteria observed in the intestinal microbiome of infants born through vaginal delivery. As a result, infants born through C-section have a higher risk of developing allergic disease, obesity and type 1 diabetes. Additionally, infant feeding can influence the microbiome composition. The difference in the microbiome composition between the breastfed infants and formula-fed infants is mainly due to the absence of HMOs in cow milk. The consumption of infant formula not supplemented with HMOs will result in a microbiome composition that is poor in bifidobacteria, which can affect the immune development.

Also, the exposure to antibiotics during the first year of life can also affect the gut microbiome composition, and the administration of antibiotics earlier in life and the frequency of antibiotic administration affect the gut microbiome composition, since infants treated with antibiotics are at a higher risk to develop cow’s milk protein allergy, obesity, asthma, otitis, inflammatory bowel disease and diabetes than infants not treated with antibiotics. Antibiotics kill not only the pathogen but also the beneficial bacteria, which compromises immune homeostasis and deregulates metabolism. Additionally, antibiotic administration is associated with the development of antibiotic resistance.

Health benefits of human milk oligosaccharides

Several studies have reported the beneficial effects of HMOs, which include modification of the intestinal microbiota, an anti-adhesive effect against pathogens, modulation of the intestinal epithelial cell response and development of the immune system. HMOs are intrinsic components that affect the gut microbiota by providing an energy source for the beneficial intestinal bacteria.

Amount of complex oligosaccharides in 1L of human breast milk.

Additionally, HMOs affect the health of the host by serving as a decoy receptor for the opportunistic pathogens in the mucosal surface. When infants are fed with a formula supplemented with 2’-FL and LNnT, they develop a distinctive stool bacterial profile that is more similar to that of the breastfed infants compared with the infants that are fed with a formula not supplemented with prebiotics. The bacterial diversity of infants at the age of three months exhibited increased colonisation with beneficial bifidobacteria and decreased colonisation with pathogenic bacteria.

Non-digestible and non-nutritional carbohydrates form HMOs in breast milk.

HMOs improve the host defence mechanism by strengthening the gut barrier function, as well as reduce preterm mortality and morbidity by modulating the gut microbiome to protect against necrotising enterocolitis, candidiasis and several immune-related diseases. HMOs are also able to directly affect the intestinal cell response by reducing the cell growth, and by inducing differentiation and apoptosis. Intestinal health and barrier function are considered to be the first line of defence in innate immunity, and HMOs can reputedly increase the intestinal cell maturation.

Immunomodulation properties

One of the important properties of HMOs is the immunomodulation. HMOs directly modulate the gene expression of intestinal cells, leading to changes in the expression of cell surface glycans and other cell responses. HMOs modulate lymphocyte cytokine production and enable a more balanced TH1/TH2 response. An increasing number of in vitro studies suggest that HMOs exert microbiota-independent effects by directly modulating the immune response, and by regulating the immune cell population and cytokine secretion. HMOs may either act locally on the mucosa-associated lymphoid tissue or at a systemic level. The plasma concentration of inflammatory cytokines in the breastfed infants and infants fed with experimental formula supplemented with 2’-FL was markedly lower than that in the infants fed with control formula supplemented with galactooligosaccharides. This data indicates that infants fed with a formula supplemented with 2’-FL exhibit lower plasma inflammatory cytokine profiles, which is similar to those of a breastfed reference group. HMOs were more effective than non-human prebiotic oligosaccharides in modulating the systemic and gastrointestinal immune cell responses in pigs. These altered immune cell populations may mediate the rotavirus infection susceptibility.

HMOs and their metabolic products, such as sialic acid, have a role in brain development, neuronal transmission and synaptogenesis as they are a source of sialic acid, an essential nutrient for optimal brain development and cognition. L-fucose and 2’-FL stimulate brain development, and dietary 2’-FL affects cognitive domains, and improves learning and memory in rodents. The HMOs 3’-sialyllactose and 6’-sialyllactose support normal microbial communities and behavioural responses during stress by modulating the gut-brain axis.

Oligosaccharides were identified in the 1930s as the bifidogenic factor in human milk. The most abundant oligosaccharides in human breast milk were discovered and characterised in 1954. However, the industrial production of some of the HMOs was recently achieved. The molecular structure of industrially produced 2’-FL and LNnT is identical to that of the oligosaccharides present in the human breast milk. Unlike probiotics, HMOs are resistant to pasteurisation and freeze-drying.

The US FDA categorised three HMOs as generally regarded as safe: 2’-O-fucosyllactose, 2’-FL, and LNnT. In the EU, HMOs are considered as novel foods and the oligosaccharides, 2’-FL and LNnT, and their combination passed the safety assessment. Among the HMOs, 2’-FL and LNnT are widely studied and have a chemically simple structure. These two HMOs are more abundant in human breast milk compared to other HMOs. Additionally, these two HMOs can be produced on an industrial scale. Hence, these HMOs can be used as supplements to infant formula.

The right formula

HMOs can serve as soluble decoy receptors that block the attachment of viral, bacterial or protozoan parasitic pathogens to the epithelial cell surface receptors, which may aid in preventing infectious diseases. HMOs are also antimicrobials that act as bacteriostatic or bactericidal agents. Additionally, HMOs enhance host epithelial and immune cell responses in the neonate.

Although the functions of HMOs were known previously, a strategy for industrial production was not available. Hence, non-human milk oligosaccharides – mainly FOS and GOS – were used as an alternative supplement for infant formula. Currently, 2’-FL is added to infant formula as the industrial production capacity has increased. The industrial production of other HMOs such as LNnT is still limited. Hence, LNnT is not routinely used as a supplement to infant formula.

The gastrointestinal microbiome of infants fed with a formula supplemented with 2’-FL is similar to that of the infants who are exclusively breastfed. To date, there have been no adverse effects reported for 2’-FL. Clinical studies have demonstrated that infants fed on a formula supplemented with 2’-FL exhibit a normal growth pattern and normal defecation. Therefore, it can be concluded that 2’-FL is a safe supplementation for infant formula.