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Mapping probiotic interactions with human milk oligosaccharides
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Date
2025
Authors
Kiely, Leonie Jane
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Publisher
University College Cork
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Abstract
Human milk is the ‘gold standard’ nutrition for infants, providing essential nutrients for growth and development, however, breastfeeding is not always feasible. Therefore, research into supplementing infant formula with breast milk bioactives is of critical importance to ensure adequate nutrition, while mimicking the functions of human breast milk where possible. One approach to achieve this is the addition of human milk oligosaccharides (HMOs) to infant formula. HMOs are resistant to human digestive enzymes allowing them to reach the large intestine intact. In the colon, HMOs may be metabolised by resident bacteria, in particular certain Bifidobacterium species. Health benefits associated with HMOs include prebiotic effects leading to increased levels of bifidobacteria with concomitant production of perceived beneficial metabolites, such as short-chain fatty acids (SCFAs). The aim of this thesis was to investigate the interactions of 23 strains of Bifidobacterium, Lacticaseibacillus, Lactiplantibacillus, Lactobacillus and Pediococcus, the majority of which are commercially available, with HMOs. Specific objectives included: to perform a comprehensive evaluation of the mechanisms of HMO degradation utilised by a range of commensal gut microbes when metabolising HMO structures (Chapter I), characterise potential probiotic traits in the 23 strains, many of which are commercially available (Chapter II), examine growth and metabolism of these strains on a specific blend of eight HMO structures (8-HMO) (Chapter III), investigate the adhesive capacity of these strains to human intestinal cells and the impact of pre-exposing the bacteria to 8-HMO blend plus a combination of 6’-sialyllactose and 3’-sialyllactose (6’-SL and 3’-SL) on this adhesion (Chapter IV), and, finally, to examine the microbiome modulating effects of 2’-fucosyllactose (2’-FL) and an 8-HMO blend on their own, or in combination with Bifidobacterium bifidum R0071 through a faecal fermentation study (Chapter V).
Chapter II aimed to characterise specific probiotic traits using in silico genomic analysis combined with phenotypic evaluation in 23 strains belonging to the genera Bifidobacterium, Lacticaseibacillus, Lactiplantibacillus, Lactobacillus and Pediococcus. Probiotic traits investigated included evaluation of carbohydrate utilisation capabilities, colonisation and survival genotypes, genome mining for bacteriocin-associated gene clusters with phenotypic antimicrobial activity evaluation, exopolysaccharide (EPS) gene cluster prediction and production analysis, bile salt hydrolase (BSH) genotype analysis and activity characterisation. Comparative analyses of genomic predictions and phenotypic production of these traits provided key insights into how well genotype corresponds with phenotype in these particular strains. Probiotic properties varied significantly between strains, re-enforcing the need for strain-level selection in probiotic development.
To assess bacterial interactions with HMOs, Chapter III investigated growth, HMO utilisation and metabolite production following bacterial cultivation on a blend of eight predominant HMOs (8-HMO) including: (2’-FL, 3-fucosyllactose (3-FL), Lacto-N-tetraose (LNT), Lacto-N-neo-tetraose (LNnT), 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), Difucosyllactose (DFL) and Lacto-N-fucopentaose I (LNFP-I)). Firstly, we identified the presence of specific glycoside hydrolase-encoding genes associated with HMO utilisation in all investigated genomes. The 8-HMO blend was shown to support growth of several strains, with superior growth observed for four infant-associated Bifidobacterium species (B. longum subsp. infantis, B. bifidum, B. breve and B. longum subsp. longum). High-performance anion-exchange chromatography (HPAEC) was used to evaluate the consumption of specific HMOs by individual bacteria. B. bifidum R0071 was identified as the most efficient utiliser of HMOs present in the 8-HMO blend (97 % at 24 h). Metabolomics analysis using high-performance liquid chromatography (HPLC) was employed to evaluate the production of metabolites, such as SCFAs, following bacterial growth. The main fermentation end-products generated were identified as acetate and lactate. This work not only characterised HMO metabolism in various bifidobacterial species using a unique 8-HMO blend, but also highlighted preferential utilisation of specific structures by particular species as well as strain-specific preferential utilisation, emphasising the importance of strain selection in HMO and probiotic containing products.
Chapter IV investigated the adhesion of various commercially available strains to human intestinal epithelial cell lines (HT29 and HT29-MTX) and the impact of pre-exposure of such bacteria to HMOs on this adhesion. Previous research suggests that HMOs prime infant-gut associated species such as Bifidobacterium for colonisation by upregulating colonisation related genes. Bacterial genomes were assessed for genes involved in bacterial adhesion. Strains were examined for adhesion to cell lines following pre-exposure of bacteria to either 8-HMO, No HMO or a combination of 6’-SL/3’-SL. Pre-exposure to the 8-HMO blend increased adhesion of a number of bacteria to human intestinal epithelial cells, particularly B. bifidum R0071 (67 %). This result reinforces the positive role of HMOs in promoting Bifidobacterium adhesion. Quantitative proteomic analysis characterised differences in protein abundances following exposure of bacteria to human intestinal cells and exposure of bacteria to HMOs. This work demonstrates that HMOs enhance the adhesion ability of commensal bacteria to intestinal epithelial cells, a property which may result in improved gut barrier function and protection from pathogen adhesion. In chapter V, we examined the impact of two prebiotic preparations (2’-FL and 8-HMO blend), B. bifidum R0071 and their synbiotic combinations on the faecal microbiota obtained from six exclusively breastfed infants, using an advanced faecal fermentation model. Fermentation parameters (pH, gas production, and SCFA production) were analysed alongside gut microbiome composition. To evaluate the effects of these specific test products on gastrointestinal (GI) health and in turn the effect of the associated metabolite production, gut permeability and tight junction gene expression were assessed. This was performed by exposing the human intestinal epithelial cell line, Caco-2 to the faecal supernatant and subsequently investigating gene expression. Both 2’-FL and the 8-HMO blend boosted metabolic activity, as evidenced by a decrease in pH due to acetate and lactate production. Addition of B. bifidum R0071 to 2’-FL or the 8-HMO mix evoked the most pronounced effects (compared to 2’-FL or 8-HMO in isolation), reflecting apparent infant-specific synbiotic effects, including enhanced acetate production for two infants, while 8-HMO plus B. bifidum R0071 further enhanced lactate production for one infant. The findings presented here highlight the role of HMOs and synbiotic combinations with particular bacteria in shaping the infant gut microbiota and promoting infant gut health, through lowering pH and increasing production of purported beneficial metabolites.
The research described in this thesis provides insights into the complex interactions which occur between HMOs, and a wide range of health-promoting bacterial species and the human host, contributing to a deeper understanding of their roles in infant health and development. This work expands the existing knowledge on the health-promoting benefits of HMOs and the potential of specific HMO blends, plus key bacterial species as functional ingredients in infant formula and infant nutrition products, for infants who cannot receive breast milk. Our findings support the development of and should aid in the selection of novel HMObiotics™, which are defined as the combination of specific bacteria matched with specific HMOs with benefits for infant health.
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Keywords
Human milk oligosaccharides (HMOs) , Probiotics , Prebiotics , Infant gut microbiota , Bifidobacteria
Citation
Kiely, L. J. 2025. Mapping probiotic interactions with human milk oligosaccharides. PhD Thesis, University College Cork.