The Science of Beer

A comparison of electron paramagnetic resonance

(EPR) spectroscopy with other staling indices to

assess the impacts of brewhouse gallotannin

addition on beer flavour stability

Jörg Maxminer1, Rod White2, Jonathan McMaster3 & David J. Cook1

1 University of Nottingham, School of Biosciences2 Molson Coors plc, 3 University of Nottingham, School of Chemistry

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Overview

• Introduction & hypothesis of research

• Trial design and analytical methods

– Pilot scale trials (16 hl)

– Large scale trials (1500 hl)

• Results

• Conclusions

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Glucose

Gallic

acid

Introduction

• Gallotannins (GT) used in

brewing

– Naturally sourced from

gallnuts

– Hydrolysable tannins

– Glucose core surrounded by

gallic acid

Glucose

Gallic

acid

o

o

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Hypothesis

• How might gallotannins (GT) influence

beer flavour stability?

– Ability to chelate metal ions (especially Fe(II))

– Radical scavenging ability

– Reduced formation of staling aldehydes and

staling aldehyde precursors

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Trial design - Pilot scale

• High Gravity Lager-style beer (16°P)

• Three different trial 16 hl brews

– Control with no addition

– Mash conversion vessel (CV) addition of 3g/hl

finished beer (FB) at mashing in

– Wort kettle addition of 2.5 g/hl FB at 10 min

before the end of boil

• Evaluate the effects of the gallotannin additions at

key points of the process and in the final product

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Trial design

Full-scale brewing trials• Used same recipe and GT dosing

regime as for pilot scale trials

• 1500 hl per brew, 1.5 or 3 brews to fill

one fermenter, respectively

• 2 controls, 2 mash CV additions, and

1 wort kettle addition runs produced

into final packaged beer

• Storage trial over 9 months at 20°C

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Analytical methods

• Staling aldehydes via solid phase

micro extraction (SPME)-GC-MS

with on fibre derivatization– Derivatization agent PFBOA

– Extraction time of 60 min at 50°C

• Thiobarbituric acid index (TBI)– Spectrophotometer reading

absorption at 448nm

• Sulphur dioxide determined by distillation method– NaOH (0.1 mmol)

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Analytical methods (2)

• Metal ion content determined by inductively

coupled plasma mass spectrometry (ICP-MS)

– Samples diluted 1/10 with nitric acid (2%)

• Sensory analysis

– Expert brewery tasting panel consisting of 9 to 11

tasters

• EPR Antioxidant potential (Area)

– PBN spin trap (50 mmol)

– Forced ageing at 60°C for 200 min

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EPR principlesPBN PBN-adduct

Radical

Detected

-4

-3

-2

-1

0

1

2

3

4

3450 3460 3470 3480 3490

EPR

sig

nal

inte

nsi

ty

Magnetic Field / G

Peak-Height

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0

0.5

1

1.5

2

2.5

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3.5

0 20 40 60 80 100 120 140 160 180 200

EPR

sig

nal

inte

nsi

ty

Forced ageing at 60°C / min

EPR metric

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0

0.5

1

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2

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3

3.5

0 20 40 60 80 100 120 140 160 180 200

EPR

sig

nal

inte

nsi

ty

Forced ageing at 60°C / min

EPR metric - Lag time

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0

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1

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0 20 40 60 80 100 120 140 160 180 200

EPR

sig

nal

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Forced ageing at 60°C / min

EPR metric - T150

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1

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0 20 40 60 80 100 120 140 160 180 200

EPR

sig

nal

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Forced ageing at 60°C / min

EPR metric - Area

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0

5

10

15

20

25

30

35

Strong wort 10 min boil End boil Cooled wort

Are

a u

nd

er

the

cu

rve

Wort Sample Points

Wort - EPR Area under curve

Control Mash CV Wort kettle

Results – Pilot scale

Mash CV add.

Wort kettle add.

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

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1.0

3452.5 3462.5 3472.5 3482.5 3492.5 3502.5ESR

sig

nal

inte

nsi

ty /

x1

0 -1

Magnetic field / G

Comparison of EPR peak intensity (free radicals) of GT wort kettle addition before and after the

addition of GT

Wort kettle 10 min before end boil after 200 min

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-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

3452.5 3462.5 3472.5 3482.5 3492.5 3502.5ESR

sig

nal

inte

nsi

ty /

x1

0 -1

Magnetic field / G

Comparison of EPR peak intensity (free radicals) of GT wort kettle addition before and after the

addition of GT

Wort kettle End boil after 200 min Wort kettle 10 min before end boil after 200 min

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0

5

10

15

20

25

Strong wort 10 min boil End boil Cooled wort Bottled beerfresh

TBI

Sample point

Thiobarbituric acid index (TBI)

Control Mash CV Wort kettle

* Worts normalized to 1.048 sp. gr.

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0

20

40

60

80

100

120

140

160

Strong wort 10 min boil End boil Cooled wort Beer

Co

nce

ntr

atio

n (

pp

b)

Sample point wort sample

Fe-levels (ppb)

Control Mash CV Wort kettle

* Worts normalized to 1.048 sp. gr.

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Summary – Pilot scale

• Both Mash and Wort Kettle GT addition improved

the oxidative stability of the wort

• Mash and Wort Kettle GT additions showed

essentially the same benefits at Cooled Wort as

measured by TBI and EPR metrics

• GT addition substantially reduced iron levels in

cooled, clarified wort as validated by ICP-MS

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Summary – Pilot scale

• The benefits observed in Cooled Wort from GT

additions were negated by high Fe pick-up during

the brewing process to packaged beer

• Also the reducing power of the yeast during

fermentation could have moderated the benefits of

GT addition observed in the TBI of cooled wort

Trials repeated at full-scale using the

same addition regime

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0.0

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2.5

0 month 3 month 6 month 9 monthAre

a u

nd

er

the

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rve

/ x

10

6

Storage time at 20°C

Results – Large scale brewing trialsFinished beer EPR area

Control A Control B Mash CV A Mash CV B Wort kettle A

0

10

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90

Mn Fe Cu

Co

nce

ntr

atio

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pp

b)

Metal ions

Metal ion contents in fresh beer samples

Control A Control B Mash CV A Mash CV B Wort kettle A

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0

0.05

0.1

0.15

0.2

0.25

0.3

0 month 3 month 6 month 9 month

Co

nce

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b)

t-2-nonenal beer samples during storage at 20°C

Control A Control B Mash CV A Mash CV B Wort kettle A

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0.0

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1.0

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2.0

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5.0

Stale -Oxidised Stale -Papery

Sen

sory

sco

reSensory Analysis after 9 months storage at

20°C

Control A Control B Mash CV A Mash CV B Wort kettle A

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Summary

Full-scale brewing trials

• No significant difference between any of the trial

conditions was observed for t-2-nonenal or

sensory analysis

• No significant effect of the GT addition on Fe-

levels in finished beers

• In general very low Fe-levels, good sensory scores

and acceptable t-2-nonenal formation for all

samples

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Summary

Full-scale brewing trials

• EPR results indicated a higher radical formation

rate in the kettle addition trial, but this didn’t

correlate with sensory staling or aldehyde data

• EPR results might have been influenced by other

factors:

– Wort kettle addition showed higher Mn-level and

lower SO2-level (3 mg/l fresh beer)

– Control A had very low Fe-level and the highest

SO2-level (5 mg/l fresh beer)

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Conclusions

• Clear benefits of GT addition were observed in

the brewhouse for both addition points:

– Chelation of, and complex formation with, Fe-ions

– Scavenging of radicals

– Lower TBI

• No clear evidence of any impact of GT addition

on the flavour stability of finished beer

• Complimentary indices for flavour stability

are required to understand and predict beer

staling

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Acknowledgments

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Thank you for your

attention!

Control strong wort EPR spectrum after 200

min using a high-sensitivity cavity showing

additional unknown peaks

Degradation PBN