Introduction

In accordance with the guidelines established by the World Health Organization (WHO), breast milk is regarded as the best nourishment for infants. It possesses attributes of safety and hygiene and is rich in antibodies that contribute to safeguarding against a multitude of prevalent childhood illnesses (WHO, 2009).¹ Breast milk is a multifaceted constantly-evolving biological fluid that contains both nutritive substances (such as amino acids such as glutamic acid, leucine and proline, proteins, fatty acids such as lactate and succinate, lipids, minerals, carbohydrates, vitamins, etc.) and non-nutritive bioactive substances (such as chemokines, cells, cytokines,  immunoglobulins, hormones, growth factors, glycans, mucins, etc.) (Ballard et al., 2013).² It offers robust defense mechanisms against infections and inflammatory responses, thereby fostering immune maturation, microbiota establishment, organ, and overall infant well-being. Human Milk Oligosaccharides (HMOs) present in breast milk play a pivotal role in bolstering infant immune defenses by promoting the growth of beneficial gut bacteria, which in turn helps prevent infections and inflammation. HMOs also contribute to immune maturation by enhancing the development of the infant’s immune system. Moreover, by fostering a healthy microbiota and supporting organ development, HMOs contribute significantly to overall infant well-being, ensuring a strong foundation for long-term health (Hemarajata & Versalovic, 2013).³ According to the guidelines provided by the WHO and Food and Agriculture Organization in 2001, probiotics are helpful living microorganisms, mainly lactic acid bacteria, that provide a host with a variety of health benefits when consumed in sufficient amounts. Dairy products contain a significant quantity of probiotic bacteria for delivery (Hemarajata & Versalovic, 2013).³ According to several studies food probiotics are a class of functional food products that are widely recognized and accepted by consumers (Al-Sahlany et al., 2023).⁴ Furthermore, it continues to meet a significant proportion of a child’s nutritional needs for 6-12 months, equivalent to approximately half or more of such needs, and up to one-third during the subsequent year of life (IYCF, 2009).⁵

Human breast milk is recognized for its established composition, characterized by approximately 87% to 88% water content and solid components present at a concentration of 124 grams per litre. These solid components comprise macronutrients, including roughly 7% (equivalent to 60–70g/L) carbohydrates, 1% (about 8–10g/L) protein, and 3.8%, indicative of other macronutrients (35–40 g/L) fat (Dror & Allen, 2018).⁶ Variations in breast milk’s composition may occur as a result of environmental influences, most notably the mother’s food intake. Colostrum, the first milk produced after childbirth, has a high percentage of protein but little fat. Additionally, the immune-protective components in colostrum are considerably enriched (Yi et al., 2021).⁷

While vitamin levels in breast milk are often sufficient to sustain normal newborn growth, there may be deficiencies in vitamins D and K, needing the infant’s supplementation (Balasubramanian, 2011).⁸ Several infant formulae closely resemble the composition of human breast milk. These formulations include breast milk-specific ingredients like Human Milk Oligosaccharides (HMOs), which boost their nutritional value (Martin et al., 2016).⁹ Human Milk Oligosaccharides (HMOs) are the second most prevalent solid and the third most abundant carbohydrate in human breast milk, with lactose ranking first in both categories. They make up about 20% of total carbohydrates in human milk, with mature milk having 12-14 g/L and colostrum having more than 20 g/L (Kim et al., 2020).¹⁰

Composition and Concentration of HMOs

Human breast milk contains approximately fifteen different structural variations of Human Milk Oligosaccharides (HMOs), each distinguished by its unique arrangement and composition of oligosaccharides. The five basic monosaccharides that makeup HMOs are galactose, glucose, sialic acid, fucose, and N-acetylglucosamine. Lactose serves as the terminal (reducing end) component in almost all HMOs. It can be expanded through the addition of Lacto-N-biose I or lactosamine. Linear structures only contain β1–3-linked N-Acetylglucosamine (GIcNAc) residue, while any β1–6-linked N-Acetylglucosamine generates branching within Human Milk Oligosaccharides (HMOs). Incorporating fucose and sialic acid monosaccharides through enzymatic reactions catalyzed by Fucosyltransferases and Sialyltransferases, respectively, might change the sequence of HMOs further by creating alpha 1-2, alpha 1-3, alpha 1-4, and alpha 2-3, alpha 2-6 links (Wiciński et al., 2020).¹¹

Human breast milk contains human milk oligosaccharides (HMOs), which are complex sugars. The concentrations of HMOs in mature human milk vary between 5 to 20 grams per litre (g/L), with colostrum having significantly higher levels, assuming an energy density of human milk of 64 kcal/100 mL) and 20–25 g/L of colostrum. The HMO content in the human breast milk is more abundant than the protein content, which is typically around 10 g/L (Hegar et al., 2019).¹² However, there are observable differences in women’s HMO concentrations and profiles. In addition to the influence of the genes producing galactoside 3/4-l-Fucosyltransferase and galactoside 2-l-Fucosyltransferase 2, nothing is known about the underlying causes causing this diversity. These genes influence whether α-1-2-fucosylated and α-1-3/4-fucosylated HMOs are present or absent, but additional variables affecting various HMO forms are still less explored (McGuire et al., 2017).¹³

Types of HMOs (Figure 1)

“”Fucosylated or Neutral HMOs:“These HMOs contain fucose at the end point such as: “2-Fucosyllactose (2’-FL) and lacto-N-fucopentaose.

“Non-fucosylated or Neutral N-containing HMOs: These HMOs, like Lacto-N-Tetraose, have N-acetylglucosamine at the endpoint.

(“Acidic or sialylated HMOs:”The terminal end of certain HMOs, such as 2′-sialyl lactose, contains sialic acid.

Within the initial three months postpartum, there is a decline in the overall concentration of Human Milk Oligosaccharides (HMOs). Results showed that the concentrations of individual HMOs are variable depending on the lactation period and dynamic throughout lactation. Most individual HMOs exhibited a tendency to decrease or remain stable after colostrum while 3-FL exhibited increase after colostrum (Soyyılmaz et al., 2021).¹⁴ Although there are currently more than a hundred different HMOs, it is important to realize that not all women synthesize the same kinds of oligosaccharides. This unpredictability results from the interaction of numerous elements (Ray et al., 2019).¹⁵

The aim of this review paper is to provide a comprehensive overview of the role of human milk oligosaccharides (HMOs) in human development and health. Through synthesizing existing literature and recent advancements, this paper intends to elucidate the multifaceted impacts of HMOs on various aspects of human physiology, including immune system development, gut microbiota modulation, neurological function, and long-term health outcomes. By highlighting the significance of HMOs in shaping human health from infancy through adulthood, this review aims to underscore the importance of further research and potential applications in clinical practice and nutrition science.

Figure 1: Types of Human Milk Oligosaccharides Click here to view Figure

Therapeutic Potential of Hmos

Human milk oligosaccharides (HMO) are thought to serve a variety of biological functions in addition to supplying the infant with nourishment. HMO’s hold exciting therapeutic potential due to their ability to protect against allergies. Human Milk Oligosaccharides (HMOs) are a particular kind of polysaccharide that may impact the composition of the gut microbiota, strengthen the intestinal epithelial barrier, and triggering immune system regulation. These cumulative effects result in a lower susceptibility of infants to allergies. HMO supplementation has the potential to change the gut microbiota and improve symptoms of irritable bowel syndrome (IBS), through the growth of Bifidobacterium.

HMOs and Prebiotics

Prebiotics are dietary substances that, typically in the form of oligosaccharides, regulate the diversity and activity of the microbiota in the human gut to benefit the host organism (Barile & Rastall, 2013).¹⁶ Numerous in vitro tests have demonstrated the ability of Bifidobacterium species to survive on HMOs. One of the major elements affecting newborn immune development and gut microbiota composition is this sort of commensal feeding. The gut, where most HMOs are broken down into short-chain fatty acids by certain bacteria, which produces an acidic environment.  Other helpful Bifidobacterium strains survive in the gut’s low pH environment (Chichlowski et al., 2012).¹⁷

HMOs function as prebiotics and promote the growth of Bifidobacterium species, which is essential for the health of infants. This makes it easier for Bifidobacteria to outcompete dangerous microbes in the newborn’s digestive system, preventing infections. A study by (Okburan & Kızıler, 2023)¹⁸ reveals that some Bifidobacterium strains have unique enzymatic and genetic processes that allow them to use HMOs as growth media. Recent research, however, indicates that there is still uncertainty regarding the relationship between these bacteria’s total metabolic activity and their capacity to metabolise HMOs. Bifidobacterium longum subsp. infantis has evolved specifically to metabolize Human Milk Oligosaccharides (HMOs) in the infant gastrointestinal tract, according to genomic research.  Bifidobacterium longum subsp. infantis renders efficient utilization of both Type 1 and Type 2 Human Milk Oligosaccharides (HMOs) (Garrido et al., 2016).¹⁹ The coevolution of humans and Bifidobacterium is thought to be due to the presence of type 1 structures in human milk oligosaccharides (HMOs) and the bacterial species’ continued use of the Galacto-N-biose/Lacto-N-biose metabolic pathway (Sakanaka et al., 2019).²⁰ The consumption patterns of Human Milk Oligosaccharides (HMOs) using mass spectrometry-based glycoprofiling revealed a distinct preference for fucosylated oligosaccharides by Bacteroides vulgatus and Bifidobacterium longum subsp. infantis (Smith-Brown et al., 2016).²¹

HMOs and Infant Anthropometry

Anthropometric assessments, including measurements of head circumference, body  length (height), and, body weight  are recommended for infants and toddlers under the age of two during each well-child visit (Casadei & Kiel, 2022).²² Reported studies have investigated the relationship between human milk oligosaccharides (HMOs) and infant growth parameters. The first study observed that elevated concentrations of total HMOs were positively correlated with increased levels of infant Fat-Free Mass Index and Fat-Free Mass, while displaying negative associations with Fat Mass, Percentage of Fat Mass, Fat Mass to Fat-Free Mass ratio, and Fat Mass Index (Alderete et al., 2015).²³ Consumption of 2′-fucosyllactose (2′FL), 3-fucosyllactose (3FL), difucosyllactose (DFLac), difucosyllacto-N-hexaose (DFLNH), difucosyllacto-N-tetrose (DFLNT), and lacto-N-neohexaose (LSTb) exhibited good relationships with infant’s body composition measurements, regardless of maternal secretor status. Certain associations, however, were altered by secretor status. In infants born to secretor mothers, intake of 3′-sialyllactose (3′SL) was positively associated with body composition, but consumption of 6′-sialyllactose (6′SL) and fucodifucosyllacto-N-hexaose (FDSLNH) was adversely associated with body composition. For instance, Sialyllacto-N-tetraose a was positively correlated with weight-for-length z-score but not with weight-for-age z-score in infants. However, another study found no link between Lacto-N-Fucosylpentose I and infant growth in the first four months but suggested lower anthropometric measurements were associated with higher levels of this HMO. The ingestion of various HMOs showed associations with newborn body composition regardless of maternal secretor status, but differences based on secretor status were noted, such as the positive correlation between 3′-Sialyllactose consumption and body composition in infants born to secretor mothers (Cheema et al., 2022).²⁴

Human Milk Oligosaccharides and Immune Competence Development

The immune system of neonates differs significantly from that of adults due to its functional immaturity and its distinctive qualitative and quantitative composition (Georgountzou & Papadopoulos, 2017).²⁵ The distinctions are significant for newborns under a month old since breastfeeding slows the spread of infectious illnesses, which affects the newborns chance of survival. According to Yu et al., 2018²⁶ an newborn’s immune system matures significantly throughout the first six months of life, especially if the infant is breastfed exclusively. There is proof that type 1 diabetes, leukaemia, inflammatory bowel disease, allergies, asthma, and other conditions are less common in children who are exclusively breastfed (Liljendahl et al., 2022).²⁷ Human milk oligosaccharides (HMOs) have the potential to affect infants’ immune systems. The infant’s immune system may be indirectly impacted by the microbiome makeup and epithelial cell responses mediated by HMOs. Human milk oligosaccharides have also directly affected the immune response, (Wiciński et al., 2020).¹¹ HMOs may have local and systemic effects on lymphoid tissue cells related to the mucosa because 1% of them are absorbed and circulate throughout the body. Hence it is thought that eating HMOs can have a direct impact on the immune system (Jakaitis & Denning, 2014).²⁸

Human milk oligosaccharides (HMOs) help shape the infant gut microbiota and influence immune system development. They have systemic immunomodulatory effects via improving gut health and barrier function, changing gene expression, surface glycans, cytokine production, and immune cell populations. HMOs may function as immune system receptors, regulating mucosa-associated lymphoid cells and influencing dendritic cell cytokine production, so improving both innate and adaptive immunity. Specific HMOs, such as 2′-fucosyllactose (2′-FL) and 3′-sialyllactose (3′-SL), have been found to stimulate Th1 immune responses and increase protection against respiratory viral infections, including better responses to influenza vaccination. Furthermore, 2′-FL treatment improves mucosal barrier function by lowering permeability and upregulating tight junction proteins, which promotes overall immunological health (Dinleyici et al., 2023).²⁹ Plaza-Daz et al. (2018)³⁰ reported that the effects of HMOs on immune cells were studied using intestinal epithelial cells and HeLa cells as cellular models. The study’s findings included a characterization of the networks in charge of immune system differentiation, immune cell communication, and homeostasis in the intestinal mucosa (Wang et al., 2014).³¹ Furthermore, HMO use was found to reduce cytokine protein levels such as Monocyte chemoattractant protein 1/2, IL-6, IL-8, IL-1, and Monocyte chemoattractant protein-1/2. On the contrary, the amount of cytokines associated with tissue repair and homeostasis increased (Rousseaux et al., 2021).³²

HMOs and Infection Illness

HMOs stand out from other compounds because of their prebiotic effect, which promotes the growth of good microbiota over harmful microorganisms. Furthermore, these substances might prevent pathogen adherence and colonisation. The interaction of pathogen receptors and glycans on the surface of human epithelial cells is required for microbial adhesion  (Wiciński et al., 2020).¹¹ In this context, the association of HMOs and human milk glycoconjugates, such as glycoproteins and glycolipids, is critical. They work together to reduce pathogen adherence. Because they are structurally similar to surface glycans on epithelial cells, HMOs and glycoconjugates are recognized and bound by either bacterial lectin receptors or rapidly dividing epithelial cells. In both instances, this limits the colonization of pathogens (Pacheco et al ., 2015).³³

Purified HMOs have the ability to inhibit the growth of bacteria such as Acinetobacter baumannii, Streptococcus agalactiae and Staphylococcus aureus. Notably, Lacto-N-triose II (LNT-II), a critical structural component in the synthesis of various human milk oligosaccharides, exhibited the most potent antimicrobial activity against Streptococcus agalactiae (Ackerman et al., 2018).³⁴ Fucosylated and Sialylated  milk oligosaccharides decrease the frequency and intensity of gastrointestinal infections by blocking interactions between pathogens and host receptors, as evidenced by research findings for  Norovirus, Campylobacter, Escherichia coli and Rotavirus (Corona et al., 2021).³⁵

In terms of preventing respiratory tract infections, adding 2-Fucosyllactose and Lacto-N-Neotetraose to a standard formula correlates with fewer occurrences of bronchitis and medication use as reported by parents. Instead, HMOs interfere with hemagglutination, which enhances the innate immune response to influenza viruses (Cohen et al., 2016).³⁶ Only 10-15% of infants who are nursed by HIV-positive mothers get the infection, Indicating that the bioactive properties of HMOs might be a contributing factor to atypical HIV transmission through breastfeeding, higher levels of HMOs are associated with reduced postnatal HIV transmission risk in infants who are breastfed because they may prevent HIV from attaching to intercellular adhesion molecules unique to dendritic cells; specialized antigen-displaying cells that help in the fight against invasive pathogens while promoting tolerance to one’s own and safe environmental antigens (Wahl et al., 2015).³⁷

Figure 2: Potential impacts of HMOs Click here to view Figure

HMOs and Cognitive Development

The initial half-year of an infant’s life is characterized by swift cerebral expansion, necessitating an elevated requirement for particular nutrients to support neurocognitive maturation. The benefits of exclusive breastfeeding have been amply documented throughout this time, which also falls within the WHO guidelines (Brown, 2017).³⁸ In fact, it has been discovered that breastfed neonates do better on “intelligence test scores” throughout infancy, with consequences for maturity as well. Hence, there exists a critical period during early developmental phases where the promotion of proper brain maturation can align with a diet known for its profound influence.

Considering that neurons’ cell membranes predominantly comprise sialic acid, recent findings have underscored the significance of sialylated oligosaccharides, specifically sialylated HMOs, in neurological development. Glycosphingolipids, which have a hydrophilic head made of sialylated oligosaccharides, are essential parts of gangliosides, these compounds participate in various neurodevelopmental processes, including synaptic transmission, memory formation, cognitive function, and the acquisition of learning (Corona et al., 2021).³⁵ The concentration of gangliosides in the brain triples from 1 trimester of pregnancy to 5 years of age of a child, providing additional evidence for the importance of this molecule during the early developmental stages of life (Palmano et al., 2015).³⁹

HMOs and Allergies

The intricate interconnected pathways that HMOs provide alter an individual’s susceptibility to allergies through their prebiotic properties and immune-modulating effects. The normal microbiota and microbiome contain short-chain fatty acids such as butyrate and propionate, that possess properties that can combat allergies and inflammation. These qualities contribute significantly to immune enhancement by reducing the prevalence of allergic disorders (Rey-Mariño & Francino, 2022).⁴⁰ These substances reduce the chance of developing asthma and food allergies.  In mice exposed to household dust, reduced gut microbial short-chain fatty acid synthesis is correlated with greater immunoglobulin E (IgE) levels, goblet cell hyperplasia, elevated mucus within the respiratory passages. Asthma and allergic dermatitis appear to be prevented by species like Bifidobacterium longum (Corona et al., 2021).³⁵

Impact of Maternal Health on HMOs Component

Women with a BMI of 14 kg/m² to 18kg/m² had considerably lower HMO concentrations compared to women with a BMI of 24kg/m² to 28kg/m². Individual HMO levels may be influenced by pre-pregnancy BMI and prenatal weight gain, as per findings from the ATLAS cohort study involving mothers from Europe. The impact of these on the HMO concentrations, however, were minimal. Pregnancy-related gestational diabetes did not alter the volume or make up of all HMOs (Biddulph et al., 2021).⁴¹

Safety 

Oligosaccharides in human milk have been scientifically known to stimulate the growth of Bifidobacterium since the 1930s. By 1954, the most common oligosaccharides in breast milk had been identified. In recent years, a small amount of Human Milk Oligosaccharides (HMOs) has been successfully produced on an industrial scale. The molecular composition of industrially produced Lacto-N-Neotetraose and 2′-Fucosyllactose is very similar to that of oligosaccharides found naturally in human breast milk. Notably, unlike probiotics, HMOs support freeze-drying and pasteurization (Hegar et al., 2019).¹² Two intervention trials were carried out to assess the safety of an infant formula containing approximately 0.2 g/L 2′-fucosyllactose. The addition of 2 g/L fucosyllactose in combination with galacto-oligosaccharides was well tolerated, had no effect on growth parameters, and had no effect on stool frequency or consistency (Alliet et al., 2022).⁴²

Significant findings included a decrease in lower respiratory tract infections up to the 12-month mark, as well as a decrease in the occurrence of bronchitis at 4, 6, and 12 months, a decreased reliance on antipyretics for the first 4 months, a decrease in antibiotic use at the 6- and 12-month intervals, and the persistence of protective effects beyond the 6-month post-intervention period among infants who received supplements containing 2-fucosyllactose and La (Fiocchi et al., 2021).⁴³

Table 1: HMOs functions reported in literature

Future Prospects and Constraints

The future prospects for utilising the benefits of human milk oligosaccharides (HMOs) in newborn development are promising with current research emphasizing their critical role in forming gut microbiota and boosting immune function. HMOs have the potential to improve infant health outcomes by lowering the risk of infection and encouraging healthy growth and development. However, obstacles like as the complexity of HMO structures, difficulties in large-scale manufacture, and regulatory concerns must be overcome. Efforts to overcome these barriers, together with ongoing advances in biotechnology and formulation science, could open the way for the broad introduction of HMOs into infant feeding products, ultimately improving the health and well-being of infants globally.

Further research into the unique health benefits of human milk oligosaccharides (HMOs) holds promise for the development of novel infant formulae supplemented with HMOs that closely resemble the composition of human breast milk. HMOs may have implications beyond infant nutrition, such as functional foods and dietary supplements that improve gut health and immunological function in both infants and adults. HMOs (Human Milk Oligosaccharides) and oligosaccharides isolated from animal milk, such as bovine milk, exhibit similarities in their prebiotic effects and potential immunomodulatory properties. However, they differ significantly in their structural composition, source, and specific functional benefits due to their tailored roles in human and animal nutrition respectively. HMOs have inherent therapeutic potential, including the ability to alter gut microbiota and immunological responses, and may present innovative paths for treating a variety of health issues, including gastrointestinal illnesses and immune-related diseases. Commercial infant formulas now include added HMOs to better resemble breast milk. Marketed as “HMO-added” or “HMO-enriched,” these formulas aim to mimic breast milk’s nutritional benefits, though they may not match its composition entirely. Ongoing research means formulations may evolve. For the latest, consult manufacturers or recent infant nutrition literature. Biotechnology advancements may make it possible to produce HMOs at a lower cost by microbial fermentation or enzymatic synthesis, increasing availability to these beneficial substances. However, the structural complexity of HMOs complicates their isolation, characterisation, and synthesis, necessitating the development of efficient large-scale manufacturing and purification techniques. Regulatory clearance processes for novel food components and therapeutic treatments, including HMOs, are stringent and time-consuming, emphasising the significance of establishing safety and efficacy in order to acquire commercial acceptability.

Conclusions

HMOs occupy a distinct position in disease prevention and treatment, thereby upholding the well-being of both pediatric and adult populations. Findings  on HMO’s are the newest development in neonatal nutrition and fulfil a substantial function in the immunological protection that breast milk offers to vulnerable neonates. The advancement of research and applications in infant feeding and therapeutics depends on resolving critical challenges with the isolation of Human Milk Oligosaccharides (HMOs). Human milk oligosaccharides (HMOs) are difficult to isolate because of their complex structures, low breast milk quantities, and high purity requirements. The main challenges include addressing ethical issues, scaling up production while keeping cost efficiency, and achieving specificity without adding contaminants. Clinical research has advanced to the point that it now views HMOs as a favourable option for infants unable to receive breastfeeding. Even though research has contributed to advanced knowledge, substantial issues with isolating human milk oligosaccharides still need to be solved. These problems include increasing purity while lowering contamination, developing scalable and economical processes, and advancing structural characterization to achieve the full potential of these molecules in infant nutrition and therapeutics.

Acknowledgements

The authors are grateful to former Vice Chancellor Late. Prof Aditya Shastri, Banasthali Vidyapith, Rajasthan, India.

Funding Sources

There is no funding sources

Conflict of Interest

There are no conflict of interest.

Authors’ Contribution

Neelam Chaturvedi: detailed conceptualization and writing of original draft, formulation of methodology, reviewing, editing and supervision; Bhargavi Gupta: writing-original draft and reviewing; Neha Sahrawat: conceptualized the original draft, formulated methodology, reviewed and edited the manuscript; Saloni Dua: gathered sources, reviewed and edited the manuscript; Chandra Kumari: edited the original draft and Srishti Mittal: writing-original draft.

Data Availability Statement

Not applicable

Ethics Approval Statement

Not applicable

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