What Are HMOs in Formula, and Why Do They Matter for Infants?

Human milk oligosaccharides (HMOs) are a key component of breast milk that support gut microbiota development, strengthen the immune system, and may contribute to cognitive development. Advances in science have made it possible to include certain HMOs in infant formula, aiming to replicate some of the benefits of human milk.

As research highlights their significance, understanding how HMOs are incorporated into formula and what sets them apart from other ingredients is essential for parents making feeding decisions.

Composition And Classification Of HMOs

HMOs are structurally diverse carbohydrates that are the third most abundant solid component of human milk, following lactose and lipids. These complex sugars are indigestible by infants but serve as prebiotics, promoting the growth of beneficial gut bacteria. More than 200 distinct HMOs have been identified, each varying in structure based on their monosaccharide composition and specific linkages. Their complexity arises from different combinations of five basic building blocks: glucose, galactose, N-acetylglucosamine, fucose, and sialic acid. The presence of fucose or sialic acid further classifies HMOs into neutral or acidic subtypes, influencing their function.

The most abundant and well-studied HMO is 2′-fucosyllactose (2′-FL), a neutral fucosylated oligosaccharide found in high concentrations in the milk of secretor mothers—those who express the FUT2 gene. Other major neutral HMOs include lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), which serve as foundational structures for more complex oligosaccharides. Acidic HMOs, such as 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL), contain sialic acid, influencing their interactions with gut microbes and host cells. The relative abundance of these HMOs varies due to genetic factors, maternal diet, and lactation stage.

HMOs are synthesized in the mammary gland through enzymatic modifications of lactose, with glycosyltransferases adding specific sugar residues. The enzymatic activity responsible for fucosylation and sialylation is genetically determined, leading to variations in HMO composition among mothers. Secretor milk contains higher concentrations of 2′-FL and related fucosylated structures, while non-secretor milk is enriched in alternative oligosaccharides. These differences may influence infant development.

Methods Of Production For Formula

Producing HMO-enriched infant formula requires biotechnology, fermentation, and purification techniques to ensure both safety and efficacy. Since HMOs are not naturally present in cow’s milk—the primary base for most formulas—scientists have developed methods to synthesize them while maintaining consistency in large-scale manufacturing.

Microbial fermentation is a widely used approach, employing genetically engineered bacteria or yeast strains to synthesize specific HMOs. Escherichia coli and Saccharomyces cerevisiae have been studied for their ability to produce oligosaccharides like 2′-FL and LNnT through controlled enzymatic reactions. These microorganisms are modified to express human glycosyltransferase enzymes, facilitating the stepwise addition of sugar residues. Once synthesis is complete, HMOs are purified to remove microbial byproducts and residual genetic material, ensuring compliance with safety standards set by regulatory agencies like the FDA and EFSA.

Enzymatic synthesis is another method, using glycosidases and glycosyltransferases to construct HMOs from simpler sugar precursors. This approach allows greater control over structure and reduces purification steps. However, scalability remains a challenge due to production costs.

Chemical synthesis has been explored for producing highly purified HMOs with defined structures. This method relies on sequential chemical reactions to build oligosaccharides from monosaccharide building blocks. While it provides precise structural fidelity, it is labor-intensive and costly, limiting its use in large-scale production. It is primarily used for research or producing small quantities of rare HMOs.

How HMOs Are Added And Labeled

Integrating HMOs into infant formula requires precision to ensure stability, bioavailability, and regulatory compliance. Since HMOs are water-soluble and structurally delicate, they must remain intact during processing, storage, and digestion. Manufacturers typically introduce HMOs during the liquid phase of formula production, dissolving them in a controlled environment before heat treatment and spray drying. This ensures even distribution and preserves their molecular structure.

Unlike traditional prebiotics such as galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS), which are added in larger quantities, HMOs are included in concentrations that align with their natural levels in human milk—typically 0.2 to 2 g/L for 2′-FL, the most commonly added HMO.

Labeling follows strict guidelines from regulatory agencies like the FDA and EFSA, requiring clear identification of specific HMOs in the ingredients list (e.g., “2′-fucosyllactose” or “lacto-N-neotetraose”). Some manufacturers also specify HMO concentrations per 100 mL of prepared formula. Unlike broad prebiotic claims, HMO labeling must distinguish these bioactive compounds from other oligosaccharides.

Marketing claims about HMOs must be evidence-based, with health benefits substantiated by clinical research. For example, if a formula brand claims its HMOs “support gut health,” it must provide scientific data demonstrating that the included oligosaccharides foster beneficial bacteria growth. This ensures transparency for parents and healthcare providers.

Differences From Other Oligosaccharides

HMOs differ from other oligosaccharides in their structural complexity and specific roles in infant nutrition. Unlike GOS and FOS, which serve as general prebiotics, HMOs have intricate molecular architectures with branching patterns and specialized linkages that influence their biological activity. While over 200 HMOs have been identified in human milk, GOS and FOS are simpler, consisting of linear chains of repeating sugar units.

Another key distinction is their resistance to enzymatic digestion in the small intestine. While GOS and FOS are partially broken down, HMOs pass through the upper gastrointestinal tract intact, reaching the colon where they serve as substrates for specific bacterial species, particularly Bifidobacterium infantis. This selective utilization helps shape the gut microbiome more precisely than non-specific prebiotics. Studies show that infants fed formula with HMOs develop gut microbiota compositions more similar to breastfed infants compared to those receiving only GOS or FOS.

Analytical Approaches To Confirm HMO Presence

Ensuring that HMOs are present in infant formula at intended concentrations requires advanced analytical techniques. These methods verify ingredient composition and purity, distinguishing HMOs from other oligosaccharides and maintaining consistency across batches.

High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) is widely used to separate and identify individual HMOs based on molecular weight and structure. Tandem mass spectrometry (MS/MS) provides a detailed profile, distinguishing closely related oligosaccharides. Capillary electrophoresis (CE) separates molecules based on charge-to-mass ratio, offering a complementary approach to HPLC-MS. Nuclear magnetic resonance (NMR) spectroscopy has also been explored for structural confirmation, though its high cost and complexity limit routine use.

These analytical tools ensure that formulas containing HMOs meet stringent quality standards, providing caregivers with confidence in ingredient accuracy.