IENCE C • •S T S E N C O H I N S O I V Dissertation L • O S G T 45 Y H • R G I E L S H E G A I R H C H Dietary effects on human fecal microbiota Catarina Simões VTT SCIENCE 45 Dietary effects on human fecal microbiota Catarina Simões Dissertation for the degree of Doctor of Philosophy to be presented with due permission for public examination and criticism in Lecture Hall LS2, B-building, Latokartanonkaari 9, at Faculty of Agriculture and Forestry, University of Helsinki, on the 28th November 2013 at 12 o’clock. ISBN 978-951-38-8103-0 (soft back ed.) ISBN 978-951-38-8104-7 (URL: http://www.vtt.fi/publications/index.jsp) VTT Science 45 ISSN-L 2242-119X ISSN 2242-119X (Print) ISSN 2242-1203 (Online) Copyright © VTT 2013 JULKAISIJA – UTGIVARE – PUBLISHER VTT PL 1000 (Vuorimiehentie 5, Espoo) 02044 VTT Puh. 020 722 111, faksi 020 722 4374 VTT PB 1000 (Bergsmansvägen 5, Esbo) FI-2044 VTT Tfn. +358 20 722 111, telefax +358 20 722 4374 VTT Technical Research Centre of Finland P.O. Box 1000 (Vuorimiehentie 5, Espoo) FI-02044 VTT, Finland Tel. +358 20 722 111, fax + 358 20 722 4374 Kopijyvä Oy, Kuopio 2013 Dietary effects on human fecal microbiota Catarina Simões. Espoo 2013. VTT Science 45. 86 p. + app. 49 p. Abstract The establishment of microbial populations in the gastrointestinal (GI)-tract is a complex process, involving microbial and host interactions eventually resulting in a dense and stable population. Recently, the identification of microbial species from fecal samples has become more accurate with the use of 16S RNA gene-based methods. However, although these molecular-based detection methods have apparent benefits over culture-based techniques, they involve potential pitfalls that should be taken into consideration when studying the fecal microbiota, such as the storage conditions and deoxyribonucleic acid (DNA)-extraction. Therefore, the effects of different storage conditions and DNA-extraction protocols on fecal sam- ples were evaluated in this study. Whereas the DNA-extraction protocol did not affect the numbers of Bacteroides spp., the abundance of this group showed a significant decrease after one week’s storage at -20°C. Furthermore, the numbers of predominant bacteria, Eubacterium rectale group, Clostridium leptum group, bifidobacteria and Atopobium group, were significantly higher in samples stored at -70°C after mechanical DNA-extraction than after enzymatic DNA-extraction as detected with real-time PCR (qPCR). These results indicate that rigorous mechan- ical lysis leads to the detection of higher bacterial numbers from human fecal samples than enzymatic DNA-extraction. Therefore, the use of different DNA- extraction protocols may partly explain contradictory results reported in previous studies. The composition of the human intestinal microbiota is influenced by host- specific factors such as age, genetics and physical and chemical conditions en- countered in the GI-tract. On the other hand, it is modulated by environmental factors with impact on the host during the lifespan, such as diet. The impact of diet on the gut microbiota has usually been assessed by subjecting people to the same controlled diet, and thereafter following the shifts in the microbiota. In the present study, the habitual dietary intake of monozygotic twins was associated with the fecal microbiota composition, which was analysed using qPCR and Denaturing Gradient Gel Electrophoresis (DGGE). The effect of diet on the numbers of the bacteria was described using a hierarchical linear mixed model that included the twin individuals, stratified by body mass index, and their families as random effects. The abundance and diversity of the bacterial groups studied did not differ between normal weight, overweight, and obese individuals with the techniques used. How- ever, intakes of energy, monounsaturated fat, (n-3) polyunsaturated fat, (n-6) polyunsaturated fat and soluble fibre had significant associations with the fecal bacterial numbers. In addition, co-twins with identical energy intakes had more similar numbers and DGGE-profile diversities of Bacteroides spp. than co-twins 3 with different intakes. Moreover, co-twins who ingested the same amounts of satu- rated fat had very similar DGGE-profiles of Bacteroides spp., whereas co-twins with similar consumption of fibre had very low bifidobacterial DGGE-profile similarity. Thereafter, the impact of the energy intake on the fecal microbiota of a group of 16 obese individuals was assessed during a 12 month intervention, which consisted of a 6 week very low energy diet (VLED) and thereafter a follow-up period of 5, 8 and 12 months. The diet plan was combined with exercise and lifestyle counseling. Fecal samples were analyzed using qPCR, DGGE and fluorescent in situ hybridi- zation. The effect of the energy restricted diet on the fecal bacterial numbers was described using a linear mixed model that accounted for repeated measurements in the same individual. The VLED period affected the major fecal microbial groups; in particular bifidobacteria decreased compared to the baseline numbers. Meth- anogens were detected in 56% of the participants at every sampling time point, regardless of the change in dietary intake. Furthermore, the change in numbers of the fecal bacterial groups studied followed the dietary intake and not the weight changes during the 12 months. These findings confirm that the diet and energetic intake play an important role in modulation of the fecal microbiota. Finally, the potential of utilising the information on expression levels of selected stress genes in assessing the quality of probiotic products was evaluated. For this purpose, reverse transcription-qPCR methods were developed to study the expres- sion of clpL1 and clpL2 stress genes in Lactobacillus rhamnosus VTT E-97800 cells after exposure to processing-related stress conditions or to freeze-drying. Heat treatments were performed with L. rhamnosus VTT E-97800 in laboratory scale, whereas acid treatments were performed both in laboratory and fermenter scale. RNA was extracted from fresh cells and freeze-dried powders. clpL1 and clpL2 transcripts were analysed by qPCR using SYBR Green I. clpL1 was induced in L. rhamnosus VTT E-97800 cells exposed to 50°C and to a much lesser extent in cells exposed to 47°C. No induction was observed for clpL2 during either acid or heat treatment in any of the conditions applied. RNA isolation from freeze-dried powders was unsuccessful, although several attempts were made with high quality products. These results suggest that developing quality indicators for probiotic products based on differences in the expression of stress genes will be a challeng- ing task, since rather harsh conditions are apparently needed to detect differences in the gene expression. In addition, the unsuccessful RNA isolation from freeze- dried powders hampers the applicability of this technique in the quality control of probiotic products. Keywords human fecal microbiota, DNA-extraction, diet, very low energy diet, qPCR, stress response 4 Preface The research work presented in this dissertation was carried out at VTT Technical Research Centre of Finland in Espoo during the years 2008 to 2012, and at the Rowett Institute for Nutrition and Health, University of Aberdeen, Scotland, during the period of 18th April to 20th May 2011. This study was financially supported by the Portuguese Foundation for Science and Technology (4 year doctoral studentship SFRH/BD/40920/2007), the Europe- an Science Foundation/ European Network for Gastrointestinal Health Research (Exchange visit grant to Scotland), the Finnish Funding Agency for Technology and Innovation [PREBTEKN (40076/06) and FIBREFECTS (40136/09)] and the European Commission [TORNADO (FP7-KBBE-222720) and ETHERPATHS (FP7-KBBE-222639)], which are gratefully acknowledge. I thank the University of Helsinki for giving me the opportunity to perform my PhD studies in a high quality education environment and for supporting the writing of this dissertation. I deeply thank Prof. Tapani Alatossava for all the interest while coordinating my studies. I am grateful to VTT Biotechnology, in particular to Dr. Maria Saarela for accepting me as a PhD Student in her research group, for the excellent working conditions and for guiding me during these years. I deeply thank Dr. Johanna Maukonen for being my mentor and for introducing me to the fabu- lous world of the intestinal microbiota. I sincerely thank Dr. Hanna-Leena Alakomi for supervising part of my work at VTT. I also thank Dr. Karen P. Scott for hosting and supervising my work at the Rowett Institute. The technical staff, in particular Maija-Liisa Saalovara, Merja Aarnio and Jenny Martin, is greatly acknowledged for the technical knowledge shared with me. I sincerely thank my co-authors for their contributions: Hanna-Leena Alakomi, Jo- hanna Maukonen, Maria Saarela, Jaakko Kaprio, Aila Rissanen, Kirsi Virtanen, Karen P. Scott and Kirsi Pietiläinen. I also thank Christian Ritz for the statistical assistance. The pre-examiners of this dissertation, Prof. Paul O’ Toole and Prof. Riitta Korpela, are greatly acknowledged for the revision of the manuscript. I also thank Michael Bailey for the fast and efficient English language revision of this dissertation. I warmly thank all the colleagues and friends from VTT that in different ways became part of my life during the years in Finland. Finally, I heartily thank my family for all the support, patience and love. Catarina Simões 5 Academic dissertation Custos Professor Tapani Alatossava Department of Food and Environmental Sciences University of Helsinki Helsinki, Finland Supervisor Docent Maria Saarela VTT Technical Research Centre of Finland Espoo, Finland Reviewers Professor Paul O’Toole Department of Microbiology and Alimentary Pharmabiotic Centre University College Cork National University of Ireland Cork, Ireland Professor Riitta Korpela Institute of Biomedicine University of Helsinki Helsinki, Finland Opponent Professor Atte von Wright Institute of Public Health and Clinical Nutrition University of Eastern Finland Kuopio, Finland 6 List of publications This thesis is based on the following original publications, which are referred to in the text as I–IV.