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

β-Glucosidase Activity:Yeast Screening

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

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

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)

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

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

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

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

Thank You

Photo credits: Lynn Ketchum and John Castle

References1. Kanauchi, M.; Bamforth, C. W. RESEARCH NOTE: beta-Glucoside Hydrolyzing Enzymes from Ale 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 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.

4. King, A. J.; Dickinson, J. R. Biotransformation of Hop Aroma Terpenoids by Ale 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.