TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Brau- und Getränketechnologie The impact of amino acid availability and gene transcription on aroma compound profiling in Saccharomyces yeast Susanne Procopio Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. H.-Chr. Langowski Prüfer der Dissertation: 1. Univ.-Prof. Dr. Th. Becker 2. Univ.-Prof. Dr. Th. Hofmann Die Dissertation wurde am 24.02.2016 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 20.07.2016 angenommen. Crazy little thing called yeast! IMPACT OF AMINO ACIDS ON BEER AROMA Acknowledgements ACKNOWLEDGEMENTS I am very grateful to Prof. Dr. Thomas Becker for supervising and accompanying this thesis, his generous support and scientific freedom, and to have offered me the opportunity to work in a motivating and stimulating atmosphere. Further, I would like to express my thanks to - Prof. Dr. Horst-Christian Langowski for acting as chief examiner. - Prof. Dr. Thomas Hofmann who act as the second examiner, for his time and effort in reviewing my thesis. - Dr. Britta Spanier for all her help in microarray technology and for being my mentor. - the DFG - Deutsche Forschungsgemeinschaft for funding. - all the members of the Institute of Brewing and Beverage Technology at the Technische Universität München in Freising as well as the colleagues in other departments. - especially the members of my Workgroup Beverage Biotechnology for all our collaborative work and scientific dialogues. - all my students in Weihenstephan. - all my good friends. Lastly, I would like to thank my family for all their love and encouragement, especially my parents who raised me with a love of science and supported me in all my pursuits. And most of all for my loving, supportive, encouraging, and patient husband Andrea to whom I dedicate this thesis. Thank you! 2 IMPACT OF AMINO ACIDS ON BEER AROMA Publications PUBLICATIONS Peer reviewed publications The following peer reviewed publications (shown in chronological order) were generated in the period of this work (publications which are part of the thesis are indicated in bold). 1. Procopio, S., Qian, F., Becker, T.: Function and regulation of yeast genes involved in higher alcohol and ester metabolism during beverage fermentation. European Food Research and Technology 233 (2011), 721-729. 2. Procopio, S., Krause, D., Hofmann, T., Becker, T.: Significant amino acids in aroma compound profiling during yeast fermentation analyzed by PLS regression. Food Science and Technology 51 (2013), 423-432. 3. Fischer, S., Procopio, S., Becker, T.: Self cloning brewing yeast: a new dimension in beverage production. European Food Research and Technology 237 (2013), 851–863. 4. Procopio, S., Brunner, M., Becker, T.: Differential transcribed yeast genes involved in flavour formation and its associated amino acid metabolism during brewery fermentation. European Food Research and Technology 239 (2014), 421-439. 5. Procopio, S., Sprung, P., Becker, T.: Effect of amino acid supply on the transcription of flavour-related genes and aroma compound production during lager yeast fermentation. Food Science and Technology 63 (2015), 289-297. 6. Steiner, J., Procopio, S., Becker, T.: Brewer’s spent grain: source of value-added polysaccharides for the food industry in reference to the health claims. European Food Research and Technology (2015), DOI 10.1007/s00217-015-2461-7. 7. Kupetz, M; Procopio, S.; Sacher, B. and Becker, T.: Critical Review of the Methods of beta-Glucan Analysis and Its Significance in the Beer Filtration Process. European Food Research and Technology (2015). DOI 10.1007/s00217-015-2498-7. 8. Zhao X.; Procopio, S., Becker, T.: Flavor impacts of glycerol in the processing of yeast fermented beverages: a review. Journal of Food Science and Technology (2015). DOI 10.1007/s13197-015-1977-y. 9. Fischer, S., Engstle, C., Procopio, S., Becker, T.: EGFP-based evaluation of temperature inducible native promoters of industrial ale yeast by using a high throughput system. Food Science and Technology (2015). DOI 10.1016/j.lwt.2015.12.020. 10. Kerpes, R., Knorr, V., Procopio, S., Koehler, P., Becker, T.: Gluten-specific peptidase activity of barley as affected by germination and its impact on gluten degradation. Journal of Cereal Science (2016). DOI 10.1016/j.jcs.2016.01.004. 3 IMPACT OF AMINO ACIDS ON BEER AROMA Publications Non peer reviewed publications 1. Procopio, S., Brunner, M., Becker, T.: Sequentielle Aminosäure-Aufnahme von Saccharomyces Hefen. Der Weihenstephaner 82. Jahrgang 4 (2014), 146-151. 2. Fischer, S. Procopio, S., Becker, T.: Gentechnisch modifizierte Hefen: Fluch oder Segen? Brauwelt 24-25 (2014), 735-737. CONFERENCE CONTRIBUTIONS Oral presentations 1. Procopio, S., Qian, F., Becker, T.: Einfluss der Stickstoffassimilation und Hefegenexpression auf die Synthese sensorisch aktiver Aminosäuremetabolite. 44. Technologisches Seminar. Freising, Germany, 2011. 2. Procopio, S., Qian, F., Becker, T.: Interaction of nitrogen composition on aroma-active metabolites and flavor profiling. ASBC Annual Meeting. Ft. Myers, Florida, USA, 2011. 3. Procopio, S., Becker, T.: Einfluss der Würzezusammensetzung auf das Aromaprofil. 45. Technologisches Seminar. Freising, Germany, 2012. 4. Procopio, S., Becker, T.: Impact of nutrient availability on aroma-profiling during yeast fermentation. 13th International Congress on Yeasts. Madison, Wisconsin, USA, 2012. 5. Procopio, S., Becker, T.: Fermentative Glycerinerhöhung in Bier. 46. Technologisches Seminar. Freising, Germany, 2013. 6. Procopio, S., Becker, T.: Einfluss signifikanter Aminosäuren auf die Bildung von höheren Alkoholen und Estern im Bier. 47. Technologisches Seminar. Freising, Germany, 2014. 7. Procopio, S., Becker, T.: Einfluss der Aminosäureassimilation auf das Aromaprofil von Saccharomyces Hefen. GDL Kongress – Lebensmitteltechnologie. Rodgau, Germany, 2014. 8. Procopio, S., Becker, T.: Herausforderungen an den Fermentationsprozess malzbasierter Vinegar Drinks. 47. Technologisches Seminar. Freising, Germany, 2015. Poster presentations 1. Procopio, S., Qian, F., Becker, T.: Impact of amino acid optimization on aroma-active metabolites. 33rd EBC Congress. Glasgow, Scotland, 2011. 4 IMPACT OF AMINO ACIDS ON BEER AROMA Contents CONTENTS ABBREVIATIONS 6 SUMMARY 7 ZUSAMMENFASSUNG 8 1. INTRODUCTION 9 1.1 Consumer preferences for specific beer flavour characteristics 9 1.2 Transition from brewer´s wort to the final product: fermentation 12 1.2.1 Catabolic route vs. anabolic route: the two routes of higher alcohol biosynthesis 16 1.2.2 Esters: contributing to the fruity character of fermented beverages 18 1.3 Regulatory mechanism of the production of sensory metabolites 19 1.3.1 Gene expression 20 1.3.2 Effects on enzyme activity 22 1.4 Motivation 27 2. RESULTS (THESIS PUBLICATIONS) 29 2.1 Summary of results 29 2.2 Function and regulation of yeast genes involved in higher alcohol and ester metabolism during beverage fermentation 33 2.3 Significant amino acids in aroma compound profiling during yeast fermentation analyzed by PLS regression 42 2.4 Differential transcribed yeast genes involved in flavour formation and its associated amino acid metabolism during brewery fermentation 52 2.5 Effect of amino acid supply on the transcription of flavour-related genes and aroma compound production during lager yeast fermentation 71 3. DISCUSSION 80 4. REFERENCES 94 5 IMPACT OF AMINO ACIDS ON BEER AROMA Abbreviations ABBREVIATIONS AATase alcohol acetyltransferase AEATase acyl-CoA:ethanol O-acyltransferase B regression coefficient S BCAA branched chain amino acid BCAATases branched chain amino acid transferase GATA guanine-adenine-thymine-adenine MCFA medium chain fatty acids NCR nitrogen catabolite repression mRNA messenger ribonucleic acid PLS partial least square PLSR partial least square regression qPCR quantitative polymerase chain reaction RSM response surface methodology UFAs unsaturated fatty acids VIP variable importance in the projection 6 IMPACT OF AMINO ACIDS ON BEER AROMA Summary SUMMARY Aroma is one of the most important quality characters of yeast fermented beverages. The production of beers with various aroma profiles is observed as a current trend in the brewing industry to offer a variety of beers to a new developing market. Saccharomyces yeast is responsible for fermentative activity and strongly influences the quality and aroma characters of the final product. Several studies have been performed to investigate the influence of different fermentation parameters, including osmotic pressure, oxygen content in wort and the nitrogen source and its availability on final sensory active metabolites. Amino acid assimilation by yeast during wort fermentation is linked to the aroma profile. However, many aspects of the genetic and metabolic processes that occur during alcoholic fermentation and the accompanying aroma compound production remain poorly understood. Accordingly, in the first part of this thesis, the significance of wort amino acids in the production of important higher alcohols and esters during fermentation is discussed. The focus of the investigations was on amino acid variation in synthetic media, calculated by an experimental design that reduced primary insignificant parameter variation. Aroma-active higher alcohols and esters produced in each fermentation were quantified, and their correlation with the most significant amino acids for two different Saccharomyces yeast strains was determined. Multivariate data analyses were used to establish the relationship between amino acids and the resulting concentrations of aroma compounds by means of pattern recognition. Thus, a fingerprint of amino acid importance in the detected aroma compound spectrum was created. In the second part of the study, DNA microarray analyses were performed. The results provide a comprehensive overview of the transcriptomic changes during model beer fermentation, with focus on the transcription of genes associated with amino acid assimilation and its derived aroma-active compounds and will serve as a reference for further studies. In subsequent experiments, the effect of amino acid supply on the transcription level of genes involved in the biosynthesis of aroma-relevant higher alcohols and esters was investigated. Amino acids were chosen according to the previous multivariate data analysis. These results show that the supply of single amino acids can determine the final concentrations of some important aroma-contributing metabolites. This provides evidence that the differential expression of genes participating in the biosynthesis of aroma-active compounds is affected by the concentrations of some amino acids. This information may help to improve understanding of the importance of specific aroma biosynthesis genes in the production of defined concentrations of aroma-active higher alcohols and esters during brewery fermentation. 7 IMPACT OF AMINO ACIDS ON BEER AROMA Zusammenfassung ZUSAMMENFASSUNG Aroma wird als eines der wichtigsten Merkmale für die Qualität hefefermentierter Getränke diskutiert. Ein derzeitiger Trend in der Brauindustrie besteht darin, möglichst viele Biere mit unterschiedlichen Aromaausprägungen zu produzieren, um den Verbrauchern eine variierende Auswahl an Bieren am sich stetig wandelnden Markt anbieten zu können. Hierbei haben die für die Fermentation verantwortlichen Saccharomyces Hefen entscheidenden Einfluss auf die Geruchs- und Geschmacksausprägung des finalen Produktes. Mehrere Studien haben sich bereits mit der Auswirkung der Fermentationsparameter, wie osmotischer Druck, Sauerstoffgehalt als auch der Stickstoffquelle und deren Verfügbarkeit in der Würze, auf die Synthese sensorisch aktiver Metaboliten beschäftigt. Gerade die Aminosäureassimilation der Hefe während der Fermentation scheint stark mit dem daraus resultierenden Aromaprofil verknüpft zu sein. Dennoch sind nicht alle genetischen und metabolischen Vorgänge während der Hefefermentation und der damit einhergehenden Synthese der Aromastoffe vollständig aufgeklärt. In dem ersten Teil der vorliegenden Arbeit wurde zunächst die Signifikanz der in der Würze vorliegenden Aminosäuren für die Synthese der bedeutenden höheren Alkohole und Ester ermittelt. Der Schwerpunkt der Untersuchungen lag auf Aminosäurevariationen in einem synthetischen Medium. Die synthetischen Würzen wurden jeweils mit zwei verschiedenen Saccharomyces Hefestämmen fermentiert, die charakteristischen aroma-aktiven höheren Alkohole und Ester quantifiziert und deren Korrelation zu den signifikanten Aminosäuren ermittelt. Mittels multivariater Datenanalyse wurde die Beziehung zwischen den Aminosäuren und den daraus resultierenden Konzentrationen der einzelnen Aromastoffe evaluiert. In einem zweiten Teil der Thesis wurden DNA-Microarrays angefertigt, deren Daten einen umfassenden Überblick über die Änderungen des Hefetranskriptoms während der Fermentation, mit Fokus auf die Expression von Genen des Aminosäuremetabolismus und der Aromastoffbiosynthese, bieten. In weiterführenden Experimenten wurde die Wirkung der zuvor als signifikant ermittelten Aminosäuren auf den Transkriptionslevel aroma-relevanter Gene untersucht. Hierbei konnte einigen Aminosäuren eine induzierende Wirkung hinsichtlich der Genexpression zugeschrieben werden. Auch die Endkonzentrationen der am Bieraroma maßgeblich beteiligten höheren Alkohole und Ester korrelieren mit den Aminosäuredotierungen. Diese Ergebnisse deuten darauf hin, dass eine gezielte Aminosäurezusammensetzung die Expression aroma-relevanter Gene beeinflusst und dazu beitragen kann, definierte Mengen an aroma-aktiven Metaboliten in fermentierten Getränken biosynthetisch zu generieren, um somit spezifische sensorische Eindrücke im Bier einzustellen. 8 IMPACT OF AMINO ACIDS ON BEER AROMA Introduction 1. INTRODUCTION 1.1 Consumer preferences for specific beer flavour characteristics Odour and taste are two of the most important quality attributes of yeast fermented beverages. The current trend in the brewing industry consists of producing beers with different aroma profiles to offer a variety of beers to a developing market to meet specific consumer preferences. Beer aroma is the result of a plurality of interactions between chemical compounds that can exert synergistic or antagonistic effects. The chemical profile of beer is derived from the raw materials: barley and hops, the fermenting yeast and the ageing and storage conditions. The composition of beer has been intensively studied, and compounds have been identified that are responsible for certain individual aroma profiles of various beer types (Meilgaard, 1975a, 1975b; Saerens, Delvaux, Verstrepen, & Thevelein, 2010; Verstrepen, Derdelinckx, Dufour, Winderickx, Thevelein, et al., 2003a). The various flavours of the many styles of beer generally fall into two classes: lager and ale. Together, these two classes of beer collectively account for thousands of varieties of beer, such as India Pale Ale, Stout, Pilsner, Export and Wheat beers. In general, the global beer market is around US$500 billion each year, and around 5% of this sum is captured by the fast-growing craft and specialty brewing sector. There is a global demand for new beers with novel flavours. It is predicted to grow further over the next few years as more breweries respond to this consumer demand. Several thousand unique varieties are created from different combinations of ingredients and added flavours. Fruit, vegetables and spices are added to both lagers and ales to create beverages with flavours such as cherry wheat and pumpkin spice. Spirit-flavoured brews such as rum, bourbon and tequila beers have become popular, particularly in the UK beer market. Nowadays, hops are of great interest and used mainly to produce special citric, mandarin- and litchi-like aromas (Lutz, Kneidl, Kammhuber, & Seigner, 2013). Thus, brewers focus on identifying new varieties of hops that can deliver these desired flavour and aroma characteristics. Hop flavours and aromas come mainly from essential oils that are easily dissolved into hot wort and tend to fall into several categories: floral, fruity, citrus, herbal, earthy, piney and spicy. More specific descriptors include geranium, berry, grapefruit, orange, minty, grassy, woody, resinous, liquorice and pepper. The high volatility of these hop oils has led to the dry hopping technique. The basic dry hop technique is to add hops post- fermentation to capture the aroma oils in the beverage (De Keukeleire, 2000). However, when beers are divided into different categories, it turns out that lager is the most favoured beer in the world (see Table 1 - 1). Approximately 94% of the beer market is accounted for by lagers, 9