Human Gastrointestinal Microbiota Modulation with Dietary Prebiotics – an Intimate Interaction Throughout Life

Human gastrointestinal microbiota modulation with dietary prebiotics – an intimate interaction throughout life Klaudyna Aneta Borewicz Thesis committee Promotor Prof. Dr H. Smidt Personal chair at the Laboratory of Microbiology Wageningen University & Research Other members Prof. Dr V. Fogliano, Wageningen University & Research Dr Elaine E. Vaughan, Sensus, Roosendaal, The Netherlands Dr R. Satokari, University of Helsinki, Finland Prof. Dr K. Venema, Maastricht University, The Netherlands This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences) Human gastrointestinal microbiota modulation with dietary prebiotics – an intimate interaction throughout life Klaudyna Aneta Borewicz Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus, Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 13 June 2018 at 11 a.m. in the Aula. Klaudyna Aneta Borewicz Human gastrointestinal microbiota modulation with dietary prebiotics – an intimate interaction throughout life, 240 pages. PhD thesis, Wageningen University, Wageningen, the Netherlands (2018) With references, with summary in English ISBN: 978-94-6343-277-1 DOI: https://doi.org/10.18174/448525 Table of contents Chapter 1. General Introduction and Outline of the Thesis....................................... 7 Chapter 2. Ecology of the human microbiome.......................................................... 21 Chapter 3. The effect of prebiotic fortified infant formulas on microbiota composition and dynamics in early life................................................... 41 Chapter 4. The association between infant faecal microbiota composition and the degradation of human milk oligosaccharides in one month old, healthy breastfed infants....................................................................................... 67 Chapter 5. The association between infant faecal microbiota composition and the degradation of human milk oligosaccharides in healthy breastfed infants at two, six and twelve weeks of age............................................. 101 Chapter 6. In vitro fermentation behaviour of isomalto/malto-polysaccharides using human faecal inoculum indicates prebiotic potential..................... 137 Chapter 7. Isomalto/malto-polysaccharides maintain normal gut functioning while promoting growth and activity of beneficial bacteria.............................. 165 Chapter 8. General Discussion and Future Perspectives........................................... 199 Summary .................................................................................................................. 227 Appendices .................................................................................................................. 231 Chapter 1 General Introduction and Outline of the Thesis 8 “everything humans need to survive is intimately coupled with the activities of microbes” 1 Caroline Harwood The world which is the most familiar to all of us is the one we can see with our eyes. However, beside this highly familiar visible realm, there exists another, very old yet obscure world, which we are now beginning to discover and understand with the help of modern technology. This is the invisible and fascinating world of microorganisms. Unlocking the secrets of the microbial world Before I can take you on a journey into the microbial world, it is necessary to mention briefly the current methods without which the research presented in this thesis would not be possible. These methods provided us with a window through which we could “see” and study microbial ecosystems [1]. They allowed us not only to identify and discover new microbial species, but also to study microbial activities and functions within the complex networks of interactions [2]. In the past, the identification and quantification of microbes was done using culture based approaches, which provided a very limited view, as the majority of microbes could not, and still cannot be grown in isolation or outside of their natural habitats [2]. Later, a broad range of molecular methods were designed to help address this limitation. These methods included, for example, the reverse transcription PCR (RT-PCR), quantitative PCR (qPCR), cloning and sequencing, Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) and targeted either the 16S ribosomal RNA (rRNA) or its encoding gene. Even though these techniques allowed us to study microbial communities without the need for culturing, they were rather slow, labour intensive and often limited in their scope of the information which they could provide [1]. A second revolution in the molecular (micro)biology started in 2005 when the so-called Next Generation Sequencing (NGS) technologies began to emerge, allowing to produce large amounts of DNA sequence data from multiple samples in parallel at a very high-throughput and depth of analysis [3]. Over the years multiple sequencing chemistries and technologies have been developed for NGS, and nowadays the Illumina system with its HiSeq and MiSeq platforms have become the most popular in the studies on microbial ecosystems [3, 4]. The NGS sequencing process allows various types of DNA templates to be sequenced and the template choice depends on the scientific questions that are being addressed. Commonly used templates include fragmented total (meta)genomic DNA extracted from environmental samples or pure or mixed cultures, PCR amplicons of a specific gene (e.g. a variable region within the 16S rRNA gene), or cDNA reverse transcribed from RNA. The sequencing process starts with the ligation of linker and/or adapter sequences to the DNA template [3]. The resulting library is then amplified, and during amplification the incorporation of each nucleotide is monitored and noted. As a result, many millions of short nucleotide reads are recorded in parallel 9 producing large amounts of data (hundreds of gigabases (GB) per run) within a period of time that is as little as a few hours to less than three days [3, 5]. When PCR amplicons are used as a template, PCR primers can be barcoded to allow mixtures of different samples to be sequenced during a single run, and then sorted out based on each barcode during data processing [6]. This approach reduces the sequencing cost while maximizing the amount of information produced during a single run, and is often used in microbiota research allowing multiple samples to be analysed simultaneously. The NGS methods are irreplaceable in modern microbiology; they enable sequencing genomes of individual organisms or entire microbial communities (metagenomics) and they can be used to collect gene expression data when mRNA is used as an initial template (metatranscriptomics - RNA-seq) [4]. Furthermore, the high-throughput approaches also provide a model that can be applied in other fields helping the development of other “omics” methods. These methods can be used to study microbial communities function and activity, for example through the large-scale studies of proteins (proteomics) and metabolic responses (metabolomics). All these methods hinge on the NGS sequencing, while also incorporating more traditional enzymatic and biochemical assays and chromatography techniques. Together, these “omics” approaches provide us with a comprehensive window to study and better understand the invisible microbial world around us. We live in a microbial world The very first living organisms on Earth were microscopic, single-celled, nucleus- lacking microbes, called prokaryotes [7]. The prokaryotes, which we can divide into two main groups, the bacteria and archaea, have evolved about four billion years ago, preceding all other microbial forms, such as fungi, viruses and protozoans, and all of the multicellular organisms, which appeared only about one million years ago [7]. Thus, the microbial world dominated our planet for much of its life, and even now, the diversity and numbers of microorganisms found on Earth largely exceed that of all other organisms found in the tree of life [7]. Microbial life occupies nearly all niches on Earth, including the most extreme environments, such as polar ice, hot springs or acidic lakes and rivers. Even though most microbes are too small to be seen with a naked eye, they are omnipresent and intimately tied with our own visible world. Microorganisms can be found everywhere around us - they are in the air we breathe, the water we drink, and in the food we eat. In fact, microbes are an inseparable part of our existence from the moment of birth, until long after we die. Microbial communities also thrive on the surface and inside the bodies of all living organisms, and all animals and plants harbour their own, highly diverse microbial ecosystems. Human bodies, too are covered with highly specialized assemblages of microbes, our microbiota, that live associated with all external surfaces (e.g., the skin, the oral cavity, the respiratory tract and the gastrointestinal tract), and also inside of our bodies (e.g., mammary glands). In fact, we could even say that we are more microbial than we are human, as it is estimated that the numbers of microbial cells in our bodies might be equal to, or possibly outnumber our own cells [8, 9]. 10 Even though historically microbes have had a bad reputation for

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