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
The Science of Beer
Overview
• Introduction & hypothesis of research
• Trial design and analytical methods
– Pilot scale trials (16 hl)
– Large scale trials (1500 hl)
• Results
• Conclusions
The Science of Beer
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
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
The Science of Beer
0
0.5
1
1.5
2
2.5
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
0.5
1
1.5
2
2.5
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 - T150
The Science of Beer
0
0.5
1
1.5
2
2.5
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 - Area
The Science of Beer
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
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 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.
The Science of Beer
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
The Science of Beer
0.0
0.5
1.0
1.5
2.0
2.5
0 month 3 month 6 month 9 monthAre
a u
nd
er
the
cu
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
20
30
40
50
60
70
80
90
Mn Fe Cu
Co
nce
ntr
atio
n (
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
ntr
atio
n (
pp
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
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
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
The Science of Beer
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
