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Growth, Metabolism and Beer Brewing with Kveik

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Growth, Metabolism and Beer Brewing with Kveik Master’s Thesis 2020 60 ECTS Faculty of Chemistry, Biotechnology and Food Science Growth, metabolism and beer brewing with kveik Nadia Marlen Aasen Food Science Foreword This master thesis was written as a finishing part of a master’s degree in Food Science, and it was delivered at the Faculty of Chemistry, Biotechnology and Food Science at the Norwegian University of Life Sciences in June 2020. The research that was performed took place in the time period September 2019 to April 2020. The thesis consists of 60 ECTs. I chose an assignment with the topic of brewing and fermentation as these processes are very interesting and complex. I have been very lucky to be able to use a broad specter of methods and analyses, which has made the assignment fun, varied and extremely interesting. The work has been time-demanding, but the experience and knowledge that has come as a result of the work has made it more than worth it in the end. I would like to thank everyone who made this assignment possible. My supervisors Trude Wicklund and Hilde Marit Østlie have been truly inspirational, and it has been an honor and a pleasure to work with them on this project. A special thanks goes out to May Aalberg, Ahmed Abdelghani, Kari Olsen and Davide Porcellato, who were a great help and inspiration throughout the entire process, and to the wonderful people at the Writing Centre at NMBU. My deepest thanks to everyone who shared their kveik, their knowledge and their expertise with me at the 2019 Norwegian Farmhouse Ale Festival. A sincere thank you to Lars Marius Garshol, who shared generously of his vast knowledge on kveik and traditional Norwegian brewing. To my family, friends and classmates; thank you for your support and encouragement. I could not have done this without you. Norwegian University of Life Sciences Faculty of Chemistry, Biotechnology and Food Science Ås, June 1st, 2020 ____________________________________________________________________ Nadia Marlen Aasen Sammendrag I denne oppgaven ble den mikrobiologiske sammensetningen av ti ulike typer kveik studert. Stammer av bakterier og gjær fra de fire kveikene Ørjasæter, Gausemel, Otterdal og Gamlegrua er blitt isolert, sekvensert og identifisert. Eukaryote og prokaryote mikrobiota analyser ble utført på disse, i tillegg til kveikene Espe, Tormodgarden, Midtbust, Ebbegarden, Sigmund og Stalljen. De antibakterielle egenskapene hos einer ble studert mot melkesyrebakteriene (MSB) Lactobacillus plantarum, Lactobacillus buchneri og Lactobacillus brevis. Kveik typene Ørjasæter, Gausemel og Gamlegrua er blitt studert i vekst- og metabolisme studier, ved temperaturene 22 °C, 30 °C og 37 °C. Vekst- og metabolisme studier ble utført for å analysere kveikene ved flere inkubasjonstemperaturer og parametre enn det som var mulig å utføre i pilot-skala bryggingen. Målinger av pH, cellevekst, sukkerinnhold og aroma komponenter ble tatt ved 0, 2 og 7 dagers inkubering. Disse kveik typene ble deretter brukt i pilot-skala brygging. Hensikten med bryggingen var å studere forskjeller i metabolitter og sensoriske karakteristikker mellom de ulike kulturene når fermenteringen ble utført ved ulike temperaturer. Triplikat (n = 3) av 9 øltyper, kvadruplikat (n = 4) av 1 øltype og duplikat (n = 2) av 1 øltype, der de tre ulike fermenteringstemperaturene 22 °C, 30 °C og 37 °C ble benyttet, ble analysert ved hjelp av «headspace» gasskromatografi (HSGC). Gjæren Saccharomyces cerevisiae ble isolert og identifisert ved Sangersekvensering i Ørjasæter, Gausemel, Otterdal og Gamlegrua. Bakteriene Lb. plantarum og Acetobacter malorum ble isolert og identifisert ved Sangersekvensering i Gausemel og Gamlegrua. Det kunne observeres sekvensvarianter av gjæren S. cerevisiae i kveikene fra eukaryot mikrobiota analyse. Den prokaryote mikrobiota analysen viste at alle ti kveiker i denne studien inneholdt bakterier. Antibakterielle egenskaper av einerekstrakt ble observert mot MSB. Forskjeller i innhold av flyktige komponenter og totalinnhold av fenoler mellom de ferdige ølene ble observert, både som resultat av ulik fermenteringstemperatur og kultur benyttet til å utføre fermenteringen. Konklusjonen ved studien er at einerlog laget av kvist og nåler fra einer har en antibakteriell effekt mot MSB. Noe effekt ble observert av umodne bær. Alle ti kveik typene som er inkludert i denne studien består av sekvensvariasjoner av S. cerevisiae og sekvensvariasjoner av flere bakterier, derav Acetobacter spp., Lb. brevis, Lb. backii og Lactococcus lactis. Lavere fermenteringstemperaturer resulterer i høyere innhold av fenoler og estere i øl fermentert med kveik. Bruk av einerlog i ølbryggingen fører til et høyere totalinnhold av fenoler og et lavere innhold av diacetyl og estere i det ferdige ølet. Øl fermentert med kveik inneholder høyere nivå av estere, og lavere nivå av høyere alkoholer og diacetyl, sammenlignet med øl fermentert med US-05. Abstract In this thesis, the microbiological diversity of ten different types of kveik were studied. Strains of bacteria and yeast from the four kveiks Ørjasæter, Gausemel, Otterdal and Gamlegrua were isolated, sequenced and identified. Eukaryotic and prokaryotic microbiota assays were performed on these kveiks, in addition to the kveiks Espe, Tormodgarden, Midtbust, Ebbegarden, Sigmund and Stalljen. The antibacterial effects of juniper were studied against the lactic acid bacteria (LAB) Lactobacillus plantarum, Lb. buchneri and Lb. brevis. The kveiks Ørjasæter, Gausemel and Gamlegrua were studied in growth and metabolism studies, at the temperatures 22 °C, 30 °C and 37 °C. The purpose of the growth and metabolism studies were to test the kveiks using additional temperatures and parameters than what was possible to do in the pilot-scale brewing. Measurements of pH, cell count, sugar content and aroma components were done after 0, 2 and 7 days of incubation. These kveiks were also used in pilot-scale brewing. The purpose of the brewing was to determine differences in both metabolites and sensory characteristics between the cultures when the fermentation was done at different temperatures. Triplicate (n = 3) of 9 beers, a quadruplicate (n = 4) of one beer and a duplicate (n=2) of one beer, using the three fermentation temperatures 22 °C, 30 °C and 37 °C, were analyzed using Headspace gas chromatography (HSGC). The yeast Saccharomyces cerevisiae was isolated and identified by Sanger sequencing in Ørjasæter, Gausemel, Otterdal and Gamlegrua. The bacteria Lb. plantarum and Acetobacter malorum were isolated and identified by Sanger sequencing in Gausemel and Gamlegrua. It was observed sequence variants of the yeast S. cerevisiae in the kveiks from the eukaryotic microbiota assay. The prokaryotic microbiota assay showed that all ten kveiks analyzed in this study contained bacteria. There were observed antibacterial effects of juniper extracts against LAB. Differences in content of volatile components and total phenol content between the finished beers were observed, both as results of differences in fermentation temperature and culture used to perform the fermentation. The conclusion to this study is that einerlog prepared from juniper twigs and needles have an antibacterial effect on LAB. Some effect was observed by unripe berries. All ten kveiks included in this study consists of sequence variants of S. cerevisiae and sequence variants of several bacteria, such as Acetobacter spp., Lb. brevis, Lb. backii and Lactococcus lactis. Lower fermentation temperatures result in higher total phenolic content and ester content in beer fermented with kveik. The addition of einerlog in the brewing process resulted in a higher total phenol content and a lower content of diacetyl and esters in the finished beer. Beers fermented with kveik contains higher levels of esters and lower levels of higher alcohols and diacetyl than beers fermented with US-05. Table of Contents 1 Introduction ............................................................................................................................. 1 2 Theory ...................................................................................................................................... 3 2.1 History and characteristics of the kveiks used in this study ............................................. 3 2.1.1 Geiranger................................................................................................................... 3 2.1.2 Hornindal .................................................................................................................. 4 2.1.3 Voss........................................................................................................................... 5 2.1.4 Stordal ....................................................................................................................... 5 2.1.5 Sykkylven ................................................................................................................. 5 2.2 The modern brewing process ........................................................................................... 5 2.2.1 Malting ...................................................................................................................... 5 2.2.2 Mashing..................................................................................................................... 6 2.2.3 Hop addition and wort boiling .................................................................................. 8 2.2.4 Fermentation ............................................................................................................
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