Shotgun metagenomics-based exploration of kefir microbiomes

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Walsh, Liam
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University College Cork
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Fermentation is among the world’s oldest forms of food processing, having been applied for millennia to preserve or enhance foods and still accounts for a significant component in the human diet. One such fermented food is milk kefir, which is regularly consumed in eastern European countries and is becoming increasingly popular in western society as a functional food, with numerous scientific studies and reviews associating health benefits with its consumption. Water kefir represents another fermented food of considerable interest, which is increasingly being perceived as a non-dairy alternative to milk kefir. In this thesis, we demonstrate that metagenomic analysis is a valuable tool to (i) expand our, and the general public’s understanding on the microbiology of kefir fermentations, (ii) determine the potential functionality of specific microbes therein and (iii) investigate the impact of milk kefir on the host gut microbiome. In chapter 1, we critically analyse the tools and pipelines that have been used, or that could be applied, to the analysis of metagenomic and metatranscriptomic data relating to fermented foods. In addition, we critically analyse a number of studies of fermented foods in which these tools have previously been applied to highlight the insights that these approaches can provide. Chapter 2 is focused on the shotgun metagenomics-based analysis of 256 kefir milk samples produced from milk kefir grains sourced from various parts of the world. This chapter provides considerable insight into the heterogeneity of these populations, while also uncovering conserved features such as the presence of Lactococcus lactis and Lactococcus cremoris, which may help to define the minimal components required for a fermented milk product to be considered a milk kefir. In chapter 3, we show that shotgun metagenomics, when used alongside metabolomics, can provide evidence that milk kefir modulates the gut microbiome. We show that daily consumption of kefir in a healthy cohort has a subtle impact on the urinal metabolome and gut microbiome. The principle change to the gut microbiome was the detection of Lactococcus raffinolactis post kefir consumption. The detection of Lactococcus raffinolactis is particularly notable given its low relative abundance across kefir metagenomes generated in chapter 2. In chapter 4, we describe Kefir4All, a citizen science project designed to provide the general public with an opportunity to expand their awareness, knowledge and practical skills relating to microbiology, introduced from the perspective of producing a fermented food, i.e., milk kefir or water kefir. In chapter 5, we highlight how research relating to the milk kefir and water kefir microbiome was greatly extended through the efforts of the Kefir4All citizen scientists through the investigation of compositional, functional and evolutionary change in milk and water kefir microbial communities over 21 weeks of repeat regular fermentation by citizen scientists. Overall, this thesis highlights that bioinformatic analysis of high throughput sequencing datasets can expand our knowledge of microbial communities associated with fermented foods and in the host following consumption, while also highlighting the merits of employing fermented food-related studies to raise awareness, knowledge and interest in microbiology and fermentation.
Milk kefir , Metagenomics , Lactic acid bacteria , Microbiology , Identification , High-throughput DNA sequencing , Bioinformatics , Citizen science , Public engagement
Walsh, L. 2023. Shotgun metagenomics-based exploration of kefir microbiomes. PhD Thesis, University College Cork.
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