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AN ABSTRACT OF THE THESIS OF Kaylyn R. Kirkpatrick for the degree of Master of Science in Food Science and Technology presented on June 5, 2018. Title: Investigating Hop Enzymes Abstract approved: _____________________________________________________________________ Thomas H. Shellhammer According to conventional wisdom, hops are treated in the brewery as biologically inactive ingredients, added to wort or beer primarily as a flavoring agent. In the past, hops were used in relatively small quantities, with the majority of hop additions made to boiling wort. Converse to traditional hopping practices, modern brewing techniques add hops to beer during or after fermentation, a method commonly referred to as dry-hopping. The resultant flavor profiles in each case are quite distinct; generally dry-hopping is a more delicate extraction transferring highly desirable aroma compounds to beer. Brewers seeking to increase the complexity and array of hoppy flavor in beer use generous hopping rates, blends of multiple hop varieties, concentrated hop products, and dry-hopping in the presence of yeast to increase floral and fruity flavors. As a result, a variety of unique hop-forward beers have entered the market allowing brewers to distinguish their products. An unintended consequence of everchanging brewing practices is the extraction of unknown and undesirable compounds in beer during dry-hopping. In contrast to previous thought, experiments conducted in Dr. Shellhammer’s Lab indicate that enzymes in hops survive to act on finished fermented beer, posing potential quality concerns to brewers. The addition of Cascade hops to finished beer showed production of fermentable sugars glucose and maltose as a result of starch degrading enzymes in hops. Enzyme specific assays were used to detect starch degrading enzymes in Cascade pellet hops, with the highest activities reported for amylases at 0.76 U/g. Low activity levels of debranching enzymes amyloglucosidase and limit dextrinase, were additionally found in Cascade hops, the latter of which was detected at levels below the suggested assay sensitivity. Hop enzymes were further shown to alter beer carbohydrate composition over time, with high hopping rates and high temperatures showing the greatest sugar production. The extent of over- attenuation, the loss of sugars consumed by yeast, was dependent upon the length of dry-hopping time with a total loss of 1.9°P in real extract, production of 1.3% alcohol by volume and an additional theoretical 4.75% (v/v) CO2 over 40 days of contact with hops and yeast. Differences in hop enzymatic power were found across 30 hop cultivars screened for specific enzyme activity (α-amylase, β-amylase, amyloglucosidase) and non-fermentable dextrin degradation and sugar production in beer dry-hopped with different hops. Crude hop amylase activity broadly ranged from 0.04 to 0.25 U/g of α- amylase activity, and 0.14 to 0.21 U/g of β-amylase activity. The percent change in carbohydrates in finished beer dosed with hops and antimicrobial sodium azide (0.02%) was used to profile the production of simple sugars (fructose and glucose) and small malto-oligosaccharides (maltose and maltotriose); these factors when combined accounted for 90% of the variation between hop samples. Additional hierarchical cluster analysis revealed four classifications of hops with differing abilities to hydrolyze dextrins in a control beer. However, the impact of other important factors such as harvest maturity and kilning practices should be considered in future studies. The implications of this research will change the way hops are processed and valuated, and influence how brewers select and manage hops in the brewing process. Future investigations around the factors impacting hop enzymatic power will be essential in improving hops and beer quality. ©Copyright by Kaylyn R. Kirkpatrick June 5, 2018 All Rights Reserved Investigating Hop Enzymes by Kaylyn R. Kirkpatrick A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented June 5, 2018 Commencement June 2019 Master of Science thesis of Kaylyn R. Kirkpatrick presented on June 5, 2018. APPROVED: _____________________________________________________________________ Major Professor, representing Food Science and Technology _____________________________________________________________________ Head of the Department of Food Science and Technology _____________________________________________________________________ Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. _____________________________________________________________________ Kaylyn R. Kirkpatrick, Author ACKNOWLEDGEMENTS There are a number of people without whom this manuscript would not have been possible. First, to all of my former mentors and colleagues who took an interest in my education, encouraged me to continually seek truth, and inspired within me confidence in my own abilities; to all of you wonderful teachers and leaders, I am eternally grateful. My most sincere appreciation goes to my major advisor, Dr. Thomas Shellhammer, who has patiently endured the deviations from hop flavor chemistry and whole heartedly jumped into the realm of hop enzymes with me; thank you for indulging my curiosity and going down the rabbit holes. There is no doubt in my mind that your guidance and mentorship has helped me to develop as a scientist and leader in the field of brewing science. Thank you to the folks in the Shellhammer Lab; cheers to the people who tirelessly devote their passion and energy to the great cause of beer! Special thanks to Scott LaFontaine for fielding my many questions about experimental design and analytical chemistry, and Dean Hauser for his most excellent animal impressions and homemade bagels; I thank you for the reminders that we should not take ourselves all too seriously. To the graduate students in the FST Department, I couldn’t have asked for a better group of individuals to share my experience with at OSU. To the delightful faculty in the FST department, especially Joy Waite-Cusic, Elizabeth Tomasino, and Lisbeth Goddik, my sincere thanks for your words of wisdom, motivation, and compassion during this journey. And to my thesis committee members Andrew Ross, Michael Penner, and Shaun Townsend: thank you all for taking the time to participate in my education here at Oregon State University. To my dearest friend and colleague, María Del Pilar Alessandri, I cannot thank you enough for the countless home cooked meals, camping trips, words of encouragement, hugs, and for always making time for what is important in life (including reading and editing the many iterations of this manuscript). My most heartfelt thanks go to my partner in life and love, Brent Radke. Thank you for taking this grand adventure with me and making sure that I always have a smile on my face; your unwavering support and encouragement has been essential to my success and happiness here. Thank you to Zach Bodah and Jason Perkins at Allagash Brewing Company for providing me with the beginnings of a thesis and inspiring us to take on this most important project. To John Coleman, thank you for your support and generous donations of hops, and for letting me camp out on the Mt. Angel farm and roam the hills at night. To Daniel Sharp and the folks at Ninkasi who care a great deal about quality and who have kindly donated beer to this project. To all of the beer drinkers and members of the brewing industry who have given me the most interesting two years of graduate study, I owe my thanks to you. And last but not least, to the most respected and honorable Dr. Horace Brown; thank you for building the foundational work on hop diastase. TABLE OF CONTENTS Page Chapter 1 – Literature review .........................................................................................1 1. Introduction ............................................................................................. 1 1.1 Current hopping practices .........................................................................2 2. Hops ........................................................................................................ 3 2.1 Hops morphology & physiology ...............................................................5 2.2 Brewing value in hops ...............................................................................5 2.3 Comparison of modified hop products ......................................................7 3. Brewing process overview .................................................................... 10 3.1 Wort production ......................................................................................10 3.2 Hop utilization in brewing .......................................................................13 3.3 Influence of brewing process on hoppy flavor in beer ............................14 3.4 Changing beer hopping practices ............................................................16 4. Hop flavor in beer and factors that influence it ..................................... 17 4.1 Nonpolar compounds: hop acids and oils ...............................................18 4.2 Polar compounds .....................................................................................19 4.4 Interplay between hops and yeast ............................................................21

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