Groundbreaking analytical techniques give new insights into human milk

Within a single drop of human milk, an extraordinary range of functional ingredients can be found – each serving its own purpose, but all working together for the baby’s benefit. Among the three most abundant structures in human milk are human milk oligosaccharides (HMOs), a group of complex carbohydrates essential for an infant’s digestion and healthy development. HMOs promote gut health by acting as prebiotics that selectively feed beneficial bacteria, while simultaneously helping modulate the immune system. 

Over the past few decades, extensive research on HMOs has driven important  advances in infant nutrition, including improvements in infant formulas. But given the complexity and diversity of HMO isomeric structures (molecules that share the same molecular formula but differ in structural arrangement and biological properties), much remains to be uncovered. As a result, innovations in analytical methods remain a top priority for researchers seeking to better  understand how human milk supports infant health.

This is where Danone R&I’s Human Milk Research team and their academic  partners are helping to fill in the gaps. In a series of papers published in peer-reviewed journals in 2025, they describe both novel techniques and enhanced applications of established methods to characterize HMOs. 

“Collectively, our latest publications highlight how advances in sensitivity and  diversity of analytical methods are key in reshaping this field of study,” says Bernd Stahl, Danone R&I’s Director of Human Milk Research & Analytical Science and Associate Professor at Utrecht University. 

Bernd was part of a team of experts spanning multiple academic centers that  combined ion mobility spectrometry with mass spectrometry (IM-MS) to sequence and identify more than 300 unique glycan structures in human milk without relying solely on existing databases. Their research, recently published in Nature Communications, contributes to a more comprehensive understanding of the functional components present in human milk. ¹

The second in this trio of important papers published in 2025 appeared in the journal Analytical Chemistry and presented findings from the application of the ’AIF LC-ESI-IM-MS approach’.² “Our team developed this method to indentify a wide range of HMOs at scale without sacrificing structural resolution. This may help to further elucidate the relationships between HMO structures and their function in infants’ healthy development,” explains Kelly Dingess, Senior Scientist and Program Leader in the Human Milk Research & Analytical Science team.

Finally, research published in the journal Analytical and Bioanalytical Chemistry showed how a team of scientists – including Bernd Stahl, Sibel Goeraler, Marko Mank and John Gonsalves from Danone R&I – applied innovative analytical techniques to identify four new isomers in human milk.³ According to Bernd: “These could not have been synthesized purely through the mammary gland, suggesting the existence of biosynthetic routes that remain to be discovered and opening further avenues for research”.

For more than 50 years, Danone R&I has been at the forefront of exploring the complexity of human milk, translating scientific insights into safe, effective nutritional solutions to support infant health—especially for those who cannot be breastfed. 

The Human Milk Research team’s latest analytical innovations strengthen this mission by directly linking molecular discovery with nutritional insight. Reflecting on the broader implications of these advances, Marko Mank, Senior Team Leader of Advanced Analytical Discovery at Danone R&I notes: “We are building on a long history of research. When we uncover new compounds and structural features of molecules, we can connect them to existing knowledge. Ultimately, this enables us to translate robust science into meaningful improvements in, for example, infant formulas – bringing us closer to nutrition that is ever more inspired by human milk.” 

1. Javier Sastre Toraño, Julia C. C. Vreugdenhil, Gaël M. Vos, Kevin C. Hooijschuur, Shannon Vogelaar, Christian Klein, John Fjeldsted, Bernd Stahl & Geert-Jan Boons | De novo sequencing of glycans by ion mobility-mass spectrometry using a self-expanding database
2. Gonsalves, J., J. Bauza-Martinez, B. Stahl, K. A. Dingess and M. Mank (2025). "Robust and High-Resolution All-Ion Fragmentation LC-ESI-IM-MS Analysis for In-Depth Characterization or Profiling of Up to 200 Human Milk Oligosaccharides." Anal Chem 97(10): 5563-5574.
3. Abikhodr, A. H., S. Warnke, A. Ben Faleh, T. R. Rizzo, S. Goeraler, J. Gonsalves, B. Stahl and M. Mank (2025). "Characterization of rare trifucosylated human milk oligosaccharides by cryogenic infrared ion spectroscopy (CIRIS)." Anal Bioanal Chem.