The contribution of ingredient glycosylation and milk glycoproteins to infant milk formulations and bacterial microbiome activity
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Date
2023-08-16Embargo Date
2025-08-18
Author
Dziembała, Iwona
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Abstract
Infant milk formula (IMF) is a food that has a specific purpose in the feeding of infants
at different stages of their development. It is modelled on human milk, which contains a
number of unique ingredients to ensure the optimal development of the child. Bovine
milk is the most widely used base in the world to create IMF. To ensure comparable
biological effects in IMF-fed infants, bovine milk is adjusted to model the composition
of human milk and the main goal of IMF manufacturers is understanding and simulating
the composition of human milk in bovine milk-based formulas.
The glycosylation of human milk contributes its complexity and bioactivities as a
selective prebiotic and antibacterial agent, ensuring optimal development of the immune
system and intestinal microbiome. Human milk oligosaccharides (HMOs) together with
oligosaccharides conjugated to proteins (glycoproteins) and lipids (glycolipids) make up
the global glycosylation of human milk. Approximately 70% of human milk proteins are
glycosylated, and these glycoproteins contribute to the unique properties of milk. Bovine
milk oligosaccharides (BMOs) are not such complex structures, but bovine milk
glycoproteins expresses complex glycosylation which is structurally altered over the
lactation period similar to HMOs. It has been suggested that glycans from bovine milk
glycoproteins may functionally replace HMOs in bovine milk. We suggest that bovine
milk glycoproteins are a potential source of components that structurally could contribute
to increasing the similarity of global glycosylation of formulas to human milk.
The infant gut microbiome and its proper development plays a key role in the acquisition
of nutrients from food, contributes to the correct development of the immune system,
and protect against pathogens and the development of infections. Feeding the intestinal
bacteria with milk glycans and glycoproteins is strongly linked to the final composition
of the infant gut microbiome, and HMOs have been particularly associated with
promotion of Bifidobacterium growth. Lactobacillus strains present in breast milk are
also present in the gut of infants, indicating the promotion of growth of this bacterial
population by components of human milk. In addition to their prebiotic effects, milk
glycans and glycoproteins show antimicrobial activity, especially those present in LF.
Many studies have reported on the activity of HMOs, but there is a gap in the literature
regarding the activity of milk glycoproteins on Lactobacillus growth and the effect on
some of the most common infant gut pathogens, Staphylococcus aureus and
enteropathogenic Escherichia coli (EPEC).
In Chapter 2 of this thesis, the contribution of successive additions of raw ingredients to
the global glycosylation of different IMF preparations was assessed and compared with
human and bovine whole milks. Incubation on lectin microarrays allowed the
determination of the global glycosylation profiles, including N- and O-linked
glycosylation. HPLC analysis allowed the analysis of free OSs in addition to N-linked
glycans. Hierarchical clustering of the resulting profiles clearly indicated that successive
additions of raw components, initially oligosaccharides such as BMOs, 2’-fucosyllactose
(2’-FL), fructo-oligosaccharides (FOS) and lacto-N-neotetraose (LNnT), altered the
global glycosylation of the IMF base. However, the greatest similarity in global
glycosylation to human milk was obtained with the addition of both BMOs and the
glycoproteins lactoferrin (LF) and osteopontin (OPN). Thus, enriching the base IMF with
these components can 'humanise' the basic formula.
Chapter 3 reports the evaluation of the potential of milk glycoproteins as selective milk
constituents in modulating the colonisation of the intestinal microbiome. The effects of
the milk glycoproteins LF, OPN, and α-lactalbumin (α-Lac) on the growth of
Lactobacilus rhamnosus, Lactobacillus paracasei and Lactobacillus brevis, and the
pathogens S. aureus (MRSA) and EPEC was assessed at different concentrations,
corresponding to their concentrations in human milk colostrum, mature human milk,
mature bovine milk and standard IMF. The milk glycoproteins at various concentrations
functioned as selective components important for long-term colonisation of intestinal
bacteria, promoting the growth of different commensals at certain concentrations and
inhibiting the growth of pathogens. In addition, the inhibitory effect of the culture
supernatant of L. rhamnosus cultured with LF against MRSA was investigated. The
greatest antagonistic effect was noted with supernatant from L. rhamnosus supplemented
with LF at the concentration corresponding to human milk colostrum. The results of the
study supports the idea of milk glycoproteins strategically modulating infant gut
colonisation at critical periods of infant microbiome development and introduces the
possibility of using products of microbial digestion of LF glycosylation by L. rhamnosus
and its growth metabolites as an anti-MRSA therapy.