Microbial quality of barrel aged beers in the craft brewing industry Word count: 16 264 Klaas Vanderpoorten Student number: 01309542 Supervisors: Prof. dr. Franck Carbonero, Prof. dr. ir. Tom Van de Wiele Tutors: Dr. Daya Kshitiz Marasini, Msc. Jana De Bodt A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Bioscience Engineering: Food Science and Nutrition Academic year: 2017 – 2018 The author and the promotors give the permission to use this thesis for consultation and to copy parts of it for personal use. Every other use is subject to the copyright laws, more specifically the source must be extensively specified when using results from this thesis. De auteur en de promotoren geven de toelating deze scriptie voor consultatie beschikbaar te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting de bron te vermelden bij het aanhalen van resultaten uit deze scriptie. Ghent, June 8th, 2018 The promotors, Prof. dr. ir. Franck Carbonero Prof. dr. ir. Tom Van de Wiele The tutors, Dr. Daya Kshitiz Marasini Msc. Jana De Bodt The author, Klaas Vanderpoorten 2 Table of Contents List of abbreviations ........................................................................................................................................ 1 Abstract ........................................................................................................................................................... 3 Summary in Dutch ........................................................................................................................................... 4 A. Introduction .......................................................................................................................................... 5 B. Literature review .................................................................................................................................. 6 1. Historical perspective on craft brewing .................................................................................................... 6 2. Brewing process from microbiological point of view ................................................................................ 7 2.1. Raw materials .................................................................................................................................. 7 2.1.1. Grain ........................................................................................................................................ 7 2.1.2. Hops ........................................................................................................................................ 9 2.1.3. Yeast ........................................................................................................................................ 9 2.2. Production process ........................................................................................................................ 11 2.2.2. Lautering ................................................................................................................................ 11 2.2.3. Boiling .................................................................................................................................... 12 2.2.4. Fermentation ......................................................................................................................... 12 2.3. Influence of brewery environment ................................................................................................. 12 3. Description of the most common barrel aged beers ............................................................................. 13 3.1. Influence of oak barrels on beer .................................................................................................... 13 3.1.1. Important oak-related chemicals in aged beers .................................................................... 13 3.2. Lambic and American coolship ales .............................................................................................. 14 3.2.1. Lambic ................................................................................................................................... 15 3.2.2. American coolship ales .......................................................................................................... 17 3.3. Red-brown acidic ales ................................................................................................................... 18 3.4. Other barrel aged beers ................................................................................................................ 19 C. Survey ............................................................................................................................................... 20 1. Introduction ............................................................................................................................................. 20 2. Materials and methods ........................................................................................................................... 20 3. Results and discussion .......................................................................................................................... 20 4. Conclusion .............................................................................................................................................. 26 D. Investigation of microbiological profile of barrel aged beers ............................................................. 27 1. Materials and methods ........................................................................................................................... 27 1.1. Sample collection .......................................................................................................................... 27 1.2. Microbial DNA extraction ............................................................................................................... 27 1.3. Quality check of DNA extraction .................................................................................................... 28 1.4. Sequencing .................................................................................................................................... 28 3 2. Results ................................................................................................................................................... 29 2.1. General overview of microbiota profile .......................................................................................... 30 2.2. Microbiota profiles per brewery ..................................................................................................... 33 2.2.1. Brewery 1............................................................................................................................... 33 2.2.2. Brewery 2............................................................................................................................... 37 2.2.3. Brewery 3............................................................................................................................... 40 3. Discussion .............................................................................................................................................. 46 4. Conclusions ............................................................................................................................................ 50 E. Suggestions for future research ........................................................................................................ 51 F. References ............................................................................................................................................. 52 G. Annexes ............................................................................................................................................. 57 1. Annex 1: Explanatory email accompanying survey ............................................................................... 57 2. Annex 2: Survey ..................................................................................................................................... 59 4 List of abbreviations Table 1: The meaning of the abbreviations used throughout this thesis. Abbreviation Meaning AAB Acetic acid bacteria ACA American coolship ale AOB American Oud bruin BOB Belgian Oud bruin LAB Lactic acid bacteria 1 Acknowledgements First and foremost, I would like to thank Dr. Franck Carbonero for his guidance, expertise and the opportunity to go to the United States to work on this project and experience life abroad. Next, I would like to thank professor Van de Wiele for the support and the opportunity to work at CMET. I am also very grateful to Jana De Bodt and Daya Marasini for their help and expertise in the lab. Without their guidance, the experiments would have never be completed successfully. I would also like to thank my dad and my friends Stephen and Nicolas who went to breweries for me to collect additional samples and my brother Jeroen for collecting empty tubes at the lab. Next, I would like to thank the breweries to welcome my dad, my friends and me in their facilities, to let us take samples and to tell us so passionately about their products. I am also very grateful to my roommates and friends in Arkansas who supported