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 21st-23rd, 2016
• Stripping of volatiles
• CO2 production
• Adsorption
• Partitioning (e.g. foam)
• Solubility changes• Ethanol increase
• Aroma masking
• Direct Biotransformation
Basic Yeast/Hop Interactions
Sesquiterpene loss during fermentation (King and Dickinson, 2003)
caryophyllene humulene
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)
• Sugar bound molecules
• Water soluble
• Non-volatile
• Used for storage and transport in plants
Glycosides
• Important source of aroma in wine
• Found in hops
Linalyl Glycoside (non-volatile)
Linalyl Glycoside (non-volatile) Linalool (citrus, floral)
Glucose
Glycoside Hydrolysis
β-Glucosidase optimal pH
(Kanauchi and Bamforth, 2012)
Other terpenoid aglycones• Geraniol• Nerol• β-citronellol• α-terpineol• Terpin-4-ol• Z-3-hexanol• 1-octanol
β-Glucosidase
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
4 6
35
26
8
Brett Lager Wine Ale Other
Yeast Types
Yeast β-Glucosidase Activity Analysis
+
4-MUG
Exλ =365nm Emλ=445nm
Yeast Screening: β-Glucosidase Activity Results
AleLagerBrett
AleAle
LagerAle
LagerAleAleAleAleAleAleAleAleAleAleAleAleAleAleAleAle
LagerAleAle
LagerAleAleAleAle
LagerBrettBrett
0 50 100 150 200 250
Specific Activity (U L-1 O.D.605-1)
Brewing Yeasts
Cell Associated
Extracellular
Total
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
When does hydrolysis occur during fermentation?
Hydrolysis
Octyl-glycoside 1-Octanol
0102030405060708090
100
0 24 48 72 96 120 144 168 192 216 240
Pe
rce
nt
Hours
Hydrolysis of octyl-glucopyranoside
ale(low) Enzyme control ale(high)
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
SPME GC-MS Volatile Analysis (n=2)• Linalool, Geraniol, Nerol, β-citronellol, α-terpineol, Terpin-4-ol, Z-3-hexanol, 1-
octanol* (octyl glycoside)
Bench Scale Ferment: 1L, 12°P wort @ 18°C
Does increased yeast β-glucosidase activity increase aglycone content in beer?
R² = 0.1931
0
1
2
3
4
5
6
7
8
9
10
0 20 40 60 80 100 120
pp
b
β-glucosidase activity
Mean nerol concentration by SPME
R² = 0.1731
0
20
40
60
80
100
120
140
160
180
200
0 20 40 60 80 100 120
pp
b
β-glucosidase activity
Mean β-citronellol concentration by SPME
R² = 0.0456
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120
pp
b
β-glucosidase activity
Mean geraniol concentration by SPME
R² = 0.0529
0
50
100
150
200
250
300
350
0 20 40 60 80 100 120
pp
b
β-glucosidase activity
Mean linalool concentration by SPME
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
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)
Benchtop boils: 2L, 12°P wort (n=3, N=72)
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
SPME GC-MS Volatile Analysis (n=2)• Linalool, Geraniol, Nerol, β-citronellol, α-terpineol, Terpin-4-ol, Z-3-hexanol, 1-
octanol* (octyl glycoside)
0
10
20
30
40
50
60
70
80
90
100
DH WH KH
pp
b
Hopping Addition
1-OctanolBgase(+) Bgase(-)
Does hopping regime influence glycoside extraction?
0
50
100
150
200
250C
EN CTZ
HH
A
SIM
CEN CTZ
HH
A
SIM
con
tro
l
CEN CTZ
HH
A
SIM
DH KH UH WH
pp
b
Concentration Linalool by SPME
E NE
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 aglyconecontent.
Glycoside content varies by cultivar
0
50
100
150
200
250
300
350
[ug
/50
g s
pe
nt
ho
ps]
Cultivar
Total [terpene alcohol aglycone] from spent hop extracts
Vollmer and Shellhammer
Isomerization of terpenoids during fermentation
Geraniol Citronellol
Nerol Linalool
α-terpineol
(King and Dickinson, 2003)
Total
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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 Microbiology2005, 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.
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6. Peacock, V.E., Deinzer, M.L., 1981. Chemistry of hop aroma in beer. J. Am. Soc. Brew. Chem. 34 (4), 139–141.
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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.