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oai:figshare.com:article/32641188 2026-10-01T16:25:19Z portal_1172 item_type_8 month_year_10_2026

Exploring the human milk microbiota: multiple approaches to uncover microbial diversity and interactions Ranran Li (3520220) PUREID: 640361073 Human milk microbiota donor human milk culturomics bacterial metabolism bacteriophage Human milk is a complex biological fluid, widely recognized as the gold standard for infant nutrition due to its unique composition of nutrients, immune factors, and microbiota. This study investigated the microbial composition and interactions in donor human milk (DHM) and frozen human milk using multiple approaches.<br><br>A total of 6,863 DHM samples from the Hearts Milk Bank (2017-2023) were analysed using R. Forty frozen human milk samples were analysed using traditional culture methods and an optimised culturomics approach. Approximately 70% of fresh samples had microbial load of 10³-10⁵ CFU/mL, whereas 18.3% of DHM and 50% of frozen samples showed no detectable microbial growth. Additionally, firmicutes dominated DHM samples, while frozen samples were dominated with Pseudomonadota. Interestingly, this study observed a significant (p < 0.05) negative co-occurrence of S. epidermidis with mostly opportunistic gram-negative pathogens belonging to Pseudomonadota, suggesting that the presence of S. epidermidis may suppress their growth. A culturomics protocol was optimised by this study and applied to 40 frozen human milk samples, isolating 13 unique microbial species not detected by traditional culture methods, including Bifidobacterium breve. An anaerobic transport system, comprising a W-zip gas-tight sealed pouch, a gas-generating sachet, and an anaerobic indicator, was developed and validated to maintain anaerobic conditions for at least 72 hours. Bacterial growth assays and metabolic analyses of five species raises a thought-provoking question: Is human milk composition designed to support the growth of certain bacteria, or does certain bacterial metabolism reshape its metabolic profile? Bacteriophage experiments showed that Holder pasteurization(HoP)and high-temperature short time (HTST) did not significantly affect E.coli bacteriophage viability. In contrast, UV-C significantly reduce its viability. <br>Additionally, storage at -80°C preserved bacteriophage better than -20°C.<br><br>In conclusion, this work provides a comprehensive assessment of the microbiological composition, interactions and preservation of human milk.<br><br><i>Thesis is embargoed until 31 July 2027.</i><br> 2026-10-01T16:25:19Z Text Thesis 10.17034/32641188.v1 https://figshare.com/articles/thesis/Exploring_the_human_milk_microbiota_multiple_approaches_to_uncover_microbial_diversity_and_interactions/32641188 All Rights Reserved Open Access after 2027-07-31