Bifidobacterial interactions with human milk oligosaccharides and their combined potential for the advancement of infant nutrition

The primary aim of this research was to investigate the role of human milk oligosaccharides (HMOs) in shaping the infant gut microbiota and in supporting immune health. HMOs are complex carbohydrates found at high concentrations in human breast milk, where they confer various critical functions. Despite their inability to directly provide calories or nutritional value, HMOs are believed to play a crucial role in supporting infant gut health as well as in strengthening their immune system. The unique structural diversity of HMOs, including fucosylated and sialylated forms, may enhance their ability to interact with a wide range of microbial and host factors, and may influence overall host health. Given the increasing reliance on infant formula in populations where breastfeeding is not feasible, understanding the beneficial potential of HMOs to mimic the advantages of breast feeding is crucial. The research presented in this thesis aimed to investigate immunomodulatory effects of different HMO combinations, cooperative metabolism of bifidobacteria, the impact of these factors on gut microbiota composition and functionality, and the ability of HMOs to enhance bifidobacterial intestinal adhesion. Addressing these topics is of importance in order to advance infant nutrition, as the generated knowledge is expected to provide insights into how dietary components can foster a healthy gut environment, potentially reducing the risk of diseases associated with dysbiosis in early life. The central research questions guiding this thesis were: How do HMOs influence the development of the infant gut and infant gut microbiome, and what mechanisms underlie these interactions? The specific objectives included: Acquisition of a comprehensive understanding of the functional biology of HMOs, detailing their importance in infant development and gut health (Chapter I), gain insights into the characterization, production, and commercialization of HMOs, addressing the challenges of availability and synthesis that have historically limited their clinical applications (Chapter II), explore the specific adaptations of bifidobacteria to allow HMO metabolism (Chapter II), investigate the immunomodulatory effects of different HMO combinations (Chapter IV), characterize the cooperative metabolism of bifidobacteria on HMO substrates (Chapters V and VI) and assess the in vitro effects of HMOs on the adhesion of bifidobacteria to intestinal epithelial cells (Chapter VII). To achieve these objectives, a comprehensive and multidisciplinary methodology was employed, integrating both experimental and analytical approaches. The initial phase involved establishing an in vitro co-culture model using Caco-2 intestinal epithelial cells and THP-1 macrophages to simulate the intestinal environment. This model enabled the examination of immune responses under controlled conditions. The research design incorporated quantitative methodologies, including multiplex ELISA, to assess changes in inflammatory cytokines following exposure of inflamed intestinal cells to various HMO combinations. To address the research questions related to bifidobacterial interactions with HMOs, the metabolic activities of bifidobacteria were investigated through co-cultivation experiments, where different strains were grown on various HMO substrates. The research specifically delved into the characteristics of four distinct commericial bifidobacterial strains: Bifidobacterium bifidum R0071, Bifidobacterium longum subsp infantis R0033, Bifidobacterium breve M-16V, and Bifidobacterium longum subsp. infantis M-63 in the presence of HMOs. High-performance liquid chromatography (HPLC) and metabolomic analyses were employed to evaluate the consumption of HMOs and the production of beneficial metabolites, such as SCFAs, during bacterial growth. Whole-genome sequencing, in silico analysis, and label-free proteomic techniques were used to characterize strain-specific differences in HMO utilization and to identify proteins involved in these metabolic processes. Following on from this, the adherence of these probiotic strains in the presence of HMOs was evaluated using HT29-MTX human tumorigenic cell lines to mimic bifidobacterial adhesion to intestinal epithelial cells (IECs). The HT29-MTX cell line, which is capable of constitutively producing mucin, has more physiologically relevant characteristics when compared to the use of the HT29 cell line due to mucus layer formation, and has therefore been proposed as a more suitable cell line for studying host-microbe interactions. By combining these methodologies, the research aimed to provide a thorough examination of the complex interactions between HMOs, bifidobacteria, and the human host contributing to a deeper understanding of their roles in developmental health. The findings of this research yielded several significant insights into the role of HMOs in promoting gut health. One of the key discoveries was that specific combinations of HMOs, particularly 2’-fucosyllactose (2’-FL) and 6’-sialyllactose (6’-SL), were effective in modulating immune responses. These HMOs significantly reduced pro-inflammatory cytokine levels in the co-culture human cell model, indicating their potential to mitigate inflammation in the infant gut. Moreover, the investigation into the cooperative metabolism of bifidobacteria revealed that interspecies. interactions significantly enhance HMO utilization. Co-cultivation of four commercial infant-derived bifidobacteria strains demonstrated that the presence of multiple strains led to increased bifidobacterial populations and the production of beneficial metabolites, suggesting that cooperative behaviour among strains is crucial for establishing a healthy gut microbiota. Another important finding was that HMOs enhanced the adhesion of bifidobacteria to intestinal epithelial cells. The research indicated that exposure to HMOs increased the adherence of the various Bifidobacterium strains by up to 90%, underscoring the role of HMOs in promoting colonization and reinforcing gut health. HMOs may prime infant gut-associated Bifidobacterium for colonisation to intestinal epithelial cells by influencing the expression of various colonization factors. The adhesive potential of bifidobacterial strains can be substantially increased through treatment with milk oligosaccharide components. Overall, the research covered in this thesis has provided valuable insights into the role of HMOs in promoting gut health and shaping the infant gut microbiota. By addressing critical questions regarding the immunomodulatory effects of HMOs, the cooperative metabolism of bifidobacteria, and the mechanisms underlying bacterial adhesion, this research contributes to a deeper understanding of how dietary components can influence health during infancy. The findings not only support the existing knowledge of HMOs but also emphasize the potential of HMOs and commercially available bifidobacteria as key ingredients in formulating advanced infant nutrition products that mimic the health benefits of human milk, ultimately supporting the development of healthier infant gut microbiota and improved health outcomes.

Grantor dc:publisher

University College Cork

Year dc:date.issued

2025

Author dc:creator

• Walsh, Clodagh

Advisor dc:contributor.advisor

• van Sinderen, Douwe

• Breastmilk
• HMO
• Prebiotic
• Probiotic
• Infant nutrition
• Immune
• Microbiome

Statement dc:rights

• © 2025, Clodagh Walsh.

Licence dc:rights.uri

• https://creativecommons.org/licenses/by-nc-nd/4.0/

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10468/17914

OAI identifier oai:identifier

oai:cora.ucc.ie:10468/17914