Effect of hopping regime, cultivar, and yeast on terpene alcohol content in beer. Daniel C. Sharp, Andrew Molitor, Tom H. Shellhammer Oregon State University Department of Food Science and Technology Institute of Brewing and Distilling Young Scientist Symposium Chico, California April 21 st -23 rd, 2016
Basic Yeast/Hop Interactions Stripping of volatiles CO 2 production Adsorption Partitioning (e.g. foam) Solubility changes Ethanol increase Aroma masking Direct Biotransformation caryophyllene humulene Sesquiterpene loss during fermentation (King and Dickinson, 2003)
Yeast modification of hop derived compounds Carbonyls reduced to hydroxyls (Mielgard 1986) Ester hydrolysis and trans-esterification (Peacock 1981) Hop degradation products to fruity esters (Nielsen 2009) Cysteine conjugates are transformed into thiols (Nizet 2013) Monoterpene alcohols are isomerized (King 2003) Glycosidically bound aroma precursors are hydrolyzed (Kollmannsberger 2006)
Glycosides Sugar bound molecules Water soluble Non-volatile Used for storage and transport in plants Important source of aroma in wine Found in hops Linalyl Glycoside (non-volatile)
Glycoside Hydrolysis β-glucosidase Glucose Linalyl Glycoside (non-volatile) β-glucosidase optimal ph Linalool (citrus, floral) Other terpenoid aglycones Geraniol Nerol β-citronellol α-terpineol Terpin-4-ol Z-3-hexanol 1-octanol (Kanauchi and Bamforth, 2012)
Objectives 1. Determine range of β-glucosidase activity in brewing yeast 2. Monitor hydrolysis throughout fermentation 3. Determine effect of yeast β-glucosidase activity on aglycone content in beer 4. Determine effect of hopping regime on glycoside extraction
β-glucosidase Activity: Yeast Screening
Yeast β-glucosidase Activity Analysis + 4-MUG Yeast Types 35 26 4 6 8 Brett Lager Wine Other Exλ =365nm Emλ=445nm
Yeast Screening: β-glucosidase Activity Results Brett Brett Lager Lager Lager Lager Lager Brett Lager Brewing Yeasts Cell Associated Extracellular Total 0 50 100 150 200 250 Specific Activity (U L -1 O.D. 605-1 )
β-glucosidase Activity: Bench top trials
When does hydrolysis occur during fermentation? Hydrolysis Octyl-glycoside 1-Octanol Bench Top Trials 1 L wort (12P) @ 18 C, 25 ppm iso Octyl-glucopyranoside 100ppb 1-octanol Treatments Low enzyme(-) and high activity(+) ale yeast ferments Excess (>250 U/L) purified Bgase (calzyme) Control (no enzyme) Monitor 1-octanol over time via SPME-GC-MS
Percent When does hydrolysis occur during fermentation? Hydrolysis Octyl-glycoside 1-Octanol Hydrolysis of octyl-glucopyranoside 100 90 80 70 60 50 40 30 20 10 0 0 24 48 72 96 120 144 168 192 216 240 Hours ale(low) Enzyme control ale(high)
Yeast β-glucosidase activity and terpene alcohol content
Does increased yeast β-glucosidase activity increase aglycone content in beer? Hopping Simcoe Whirlpool (25 min) Treatments 12 different brewing yeasts Excess enzyme Control-no enzyme Bench Scale Ferment: 1L, 12 P wort @ 18 C SPME GC-MS Volatile Analysis (n=2) Linalool, Geraniol, Nerol, β-citronellol, α-terpineol, Terpin-4-ol, Z-3-hexanol, 1- octanol* (octyl glycoside)
ppb ppb ppb ppb Does increased yeast β-glucosidase activity increase aglycone content in beer? Mean geraniol concentration by SPME Mean linalool concentration by SPME 100 90 80 70 60 50 40 30 20 10 0 R² = 0.0456 0 20 40 60 80 100 120 350 300 250 200 150 100 50 0 R² = 0.0529 0 20 40 60 80 100 120 β-glucosidase activity β-glucosidase activity 200 180 160 140 120 100 80 60 40 20 0 Mean β-citronellol concentration by SPME R² = 0.1731 0 20 40 60 80 100 120 β-glucosidase activity 10 9 8 7 6 5 4 3 2 1 0 Mean nerol concentration by SPME R² = 0.1931 0 20 40 60 80 100 120 β-glucosidase activity
Glycoside Extraction by Different Hopping Regimes
Does hopping regime influence glycoside extraction? Hopping Kettle boil (60 min) Whirlpool (25 min) Dry hop (72 hours @ 18C) Cultivar Simcoe, CTZ, HHA, Centennial Enzyme β-glucosidase 72 hours Control-No Enzyme Benchtop boils: 2L, 12 P wort (n=3, N=72) SPME GC-MS Volatile Analysis (n=2, N=144) Linalool, Geraniol, Nerol, β-citronellol, α-terpineol, Terpin-4-ol, Z-3-hexanol, 1- octanol* (octyl glycoside)
ppb Does hopping regime influence glycoside extraction? Hopping Kettle boil (60 min) Whirlpool (25 min) Dry hop (72 hours @ 18C) Cultivar Simcoe, CTZ, HHA, Centennial Enzyme β-glucosidase 72 hours Control-No Enzyme 100 90 80 70 60 50 40 30 20 10 0 1-Octanol Bgase(+) Bgase(-) DH WH KH Hopping Addition SPME GC-MS Volatile Analysis (n=2) Linalool, Geraniol, Nerol, β-citronellol, α-terpineol, Terpin-4-ol, Z-3-hexanol, 1- octanol* (octyl glycoside)
CEN CTZ HHA SIM CEN CTZ HHA SIM control CEN CTZ HHA SIM ppb Does hopping regime influence glycoside extraction? 250 Concentration Linalool by SPME E NE 200 150 100 50 0 DH KH UH WH No significant difference between enzyme (E) treatments and no enzyme (NE) treatments.
Summary Brewing yeast exhibit wide range of glycosidase hydrolysis activity. Maximum hydrolysis occurs within 3 days of primary fermentation. Aglycone content did not increase in enzyme treated beers. No strong relationship between activity and increased aglycone content.
[ug/50 g spent hops] Glycoside content varies by cultivar 350 300 250 200 150 100 50 0 Total [terpene alcohol aglycone] from spent hop extracts Cultivar Vollmer and Shellhammer
Isomerization of terpenoids during fermentation Geraniol Citronellol Nerol Linalool Total α-terpineol (King and Dickinson, 2003)
Thank You Photo credits: Lynn Ketchum and John Castle
References 1. Kanauchi, M.; Bamforth, C. W. RESEARCH NOTE: beta-glucoside Hydrolyzing Enzymes from and Lager Strains of Brewing Yeast. Journal of the American Society of Brewing Chemists 2012, 70 (4), 303 307. 2. Fia, G.; Giovani, G.; Rosi, I. Study of Beta-Glucosidase Production by Wine-Related Yeasts during Alcoholic Fermentation. A New Rapid Fluorimetric Method to Determine Enzymatic Activity. Journal of Applied Microbiology 2005, 99 (3), 509 517. 3. Kollmannsberger, H.; Biendl, M.; Nitz, S. Occurrence of Glycosidically Bound Flavour Compounds in Hops, Hop Products and Beer. Monatsschr. Brauwissenschaft 2006, 59, 83 89. 4. King, A. J.; Dickinson, J. R. Biotransformation of Hop Aroma Terpenoids by and Lager Yeasts. FEMS Yeast Research 2003, 3 (1), 53 62. 5. Meilgaard, M.C., Peppard, T.L., 1986. The flavor of beer. In: Morton, I.D., Macleod, A.J. (Eds.), Food Flavors Part B: The Flavor of Beverages. Elsevier, Amsterdam, pp. 99 170. 6. Peacock, V.E., Deinzer, M.L., 1981. Chemistry of hop aroma in beer. J. Am. Soc. Brew. Chem. 34 (4), 139 141. 7. Nielsen, T.P., 2009. Character impact hop aroma compounds in ale. In: Shellhammer, T.H. (Ed.), Hop Flavor and Aroma- Proceedings of the 1st International Brewers Symposium. Master Brewers Association of the Americas, St. Paul, Minnesota, USA, pp. 59 77. 8. Nizet, S.; Gros, J.; Peeters, F.; Chaumont, S.; Robiette, R.; Collin, S. First Evidence of the Production of Odorant Polyfunctional Thiols by Bottle Refermentation. J. Am. Soc. Brew. Chem. 2013, 71 (1), 15 22. 9. IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). XML on-line corrected version: http://goldbook.iupac.org (2006-) created by M. Nic, J. Jirat, B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8. doi:10.1351/goldbook. 10. Goldstein, H.; Ting, P.; Navarro, A.; Ryder, D. Water-Soluble Hop Flavor Precursors and Their Role in Beer Flavor. Proceedings of the European Brewery Convention 1999, 53 62.