Chapter 6
Results
CHAPTER 6
Results
6 The importance of proteins in beer staling
The study and understanding of the mechanisms of beer oxidation is a
longstanding research priority of brewers in order to extend beer shelf life (Bamforth,
2004). The objective of this aspect of the research project was to improve beer shelf life
and flavour stability of beer produced in the 4.5 million hL/annum Foster’s brewery in
Yatala, Queensland, Australia and to shed light on the complex mechanisms involved in
beer staling, attempting to elucidate the mechanisms of ROS (Reactive Oxygen Species) in
beer staling and their interaction with thiol groups in beer proteins.
Through recent research, it has become evident that ROS play a crucial role in beer
staling and flavour deterioration (Andersen and Skibsted, 1998; Swan et al., 2003). This
chapter attempts to further elucidate the role of ROS using methods adapted from
biomedical research. Furthermore, several possible approaches for inhibiting or slowing
oxidation of the beer matrix were explored and some were successfully implemented in the
Yatala brewery. During the course of this research, it became apparent that this complex
topic can only be explored successfully if sensitive methods of analysis are available.
Methods to assess flavour stability are currently limited. In order to circumnavigate this
problem, novel methods were developed that allow near real time assessment of oxidative
stability. Furthermore, novel assays for the assessment of thiol and carbonyl groups in beer
proteins were developed. Four different lager type beers, produced on an industrial scale,
were investigated over a period of 12 weeks storage and analysed in order to monitor and
characterise the aging process. All parameters were monitored weekly in packaged lager
beer produced in the Yatala brewery. The studies reported in this chapter were carried out
as part of a collaboration between Foster’s Australia, Griffith University (Department of
Biomedical and Biomolecular Sciences), Brisbane, Australia and The International Centre
for Brewing and Distilling, Scotland. The development of novel methods for measuring
thiol and carbonyl groups in beer proteins and the Peroxide Challenge Test (PCT) were
conducted using the facilities at Griffith University. The methods of analysis were then
applied to beer produced in the Yatala brewery. Furthermore, ESR and other standard
methods of wort and beer analysis were carried out in the quality control laboratories
located in the Yatala brewery.
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Chapter 6- Section 1
Results
CHAPTER 6 – Section 1
Results
6.1 Novel procedures for detection of thiol groups in beer proteins
6.1.1 Detection of protein thiol groups using Western Blotting
A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) (MPB) was
used to detect protein SH groups on Western Blots with sensitivities in the femtomole
range. This method is widely used to detect oxidation in cancer cells (Swan et al., 1959) and
was adapted for detection of thiol groups and oxidation in beer proteins. The MPB labelled
Western Blots are depicted in Figures 6.1 and 6.2 and show that low molecular weight beer
proteins contained higher thiol concentrations. Table 6.1 gives an overview of the
characteristics of the three beers analysed for beer protein thiols. Beer C produced from a
high gravity wort (18 ºPlato) contained no detectable amounts of thiolated protein. Beer A
or B (10 and 14 ºPlato, respectively) showed distinct bands of thiolated proteins but the
molecular weight distribution varied. Beer A exhibited only one pronounced thiolated
protein band in the low molecular weight region whereas beer B showed two distinct
thiolated protein bands. Based on these results it may be concluded that the antioxidant
potential of beers are dependent on the brewing process parameters and raw materials
employed since the amount and nature of beer proteins are strongly influenced by raw
material and choices and changes in brewing parameters. Furthermore beer protein
reactivity towards MPB declined as beers A and B were aged since the free SH groups were
oxidised to disulphides which were not detected with MPB. This is evidence that the
antioxidant potential of beer declines during the course of beer shelf life and free SH
groups are increasingly oxidised to disulphides. This progressive oxidation of the beer
matrix is mainly due to oxygen intake during the beer packaging process and through
oxygen ingress into package during storage and transport of the packaged beer. It can be
assumed that in parallel to the oxidation of free thiol groups to disulphides with increasing
age of beer a multitude of other staling reactions are taking place within the complex
chemical matrix of beer leading to the undesirable stale taste and flavour that is a major
challenge for brewers. The availability of free thiol groups in beer proteins might be
beneficial for flavour stability since Reactive Oxygen Species (ROS) present in beer might
be used up in the formation of disulphides preventing the ROS from oxidising other
chemical compounds in beer to highly flavour active and thus undesirable molecules.
169
Chapter 6- Section 1
Results
Table 6.1. Characteristics of the three main beer brands (A, B and C) produced in the Yatala brewery.
PARAMETER
BRAND A
BRAND B
BRAND C
Wort gravity
10 Plato
14 Plato
18 Plato
Malt/Adjunct Ratio
Medium
Medium Plus
High
Dilution Ratio
Roasted Barley
Kettle Hopped
Wort Cooling T
Lager Yeast Strain
Fermentation T
Silica Gel / PVPP
Papain Used
Tetra hops
Reclaim allowed
Sales Alcohol (v/v)
1.25
No
No
10C
Strain A
18.5C
No
Yes
No
Yes
4.90
1.25
No
No
10C
Strain A
18.5C
Yes
No
No
No
4.90
1.90
Yes
Yes
14C
Strain A
20.0C
No
Yes
Yes
Yes
3.50
Fresh
1 months
2 months
3 months
Brand B
Fresh
1 months
2 months
3 months
Brand A
Figure 6.1. Western Blot analysis of the distribution of thiolated proteins over the course of three months
storage at 30ºC in beer B (14 ˚Plato) and beer A (10 ˚Plato). The red arrows point out the distinct
differences in thiolated protein bands between the two beers.
170
Chapter 6- Section 1
Fresh
Results
1 months
2 months
3 months
Fresh
1 months
2 months
3 months
No bands observed
Brand B
Brand C
Figure 6.2. Western Blot analysis of the distribution of thiolated proteins over the course of three months
storage at 30ºC in beer B (14 ˚Plato) and beer C (18 ˚Plato). The Western Blot for Brand B is a repeat
of the Western Blot of Brand B depicted in Figure 6.1.
.
6.1.2 Detection of protein thiol groups using an Enzyme-Linked Immunosorbent
Assay (ELISA)
Due to the large number of beer samples to be analysed for protein thiols, a high
throughput assay was developed that can be established as part of routine analysis in a
brewery. Similar to the Western Blotting technique, the beer proteins were absorbed onto a
PVDF (polyvinylidene fluoride) membrane located at the bottom of the wells of a 96-well
plate (96-well MultiScreenHTS Plates with a hydrophobic Immobilon-P PVDF membrane,
0.45 µm) as depicted in Figure 6.3 on the right. The beer samples and a standard protein
(reduced/oxidised BSA) were placed in the wells and left undisturbed for 30 min to allow
the proteins to adhere to the membrane. The 96-well plate was then positioned on a
vacuum manifold (Millipore MultiScreen™ Vacuum Manifold 96-well shown in Figure 6.3
on the left) and the beer was drawn through the membrane at the bottom of the wells. The
beer proteins absorbed onto the PVDF membrane were then derivatised with MPB. Avidin
Horseradish Peroxidase (HRP) was used as the secondary antibody and detection was
carried out with TMB (3,3’,5,5’-tetramethylbenzidine). TMB is the most sensitive
chromogenic peroxidase substrate for Western and Dot Blotting applications. Detection
171
Chapter 6- Section 1
Results
limits were decreased compared to other chromogenic membrane substrates. TMB
produced a dark blue precipitate upon reaction with HRP.
Figure 6.3. The Millipore MultiScreen™ Vacuum Manifold (left) and the Immobilon-P PVDF
membrane plates (right) suitable for use with the vacuum manifold.
Major efforts were invested into determining the optimum combination of
parameters in this complex system. The following outlines the results when assay
parameters were varied.
6.1.2.1 Assay development for protein thiol determination using ELISA: Variation of
TMB development time
In order to determine the optimum duration of TMB development, an experiment
using the reduced/oxidised BSA standards was set up as outlined above. Various mixtures
of reduced and oxidised BSA were loaded onto the Immobilon-P PVDF membrane plates
as outlined in Figure 6.4. A non coated blank was included. The protocol was conducted as
above and the TMB reaction was stopped with 1M H2SO4 after 5, 10, 15, and 20 min
development time. The results are shown in Figure 6.5. A linear response was observed in
all cases of TMB development duration, but a 100% increase in development time did not
correspond to a 100% increase in absorbance value at 450 nm. The protein loading was 1
µg/well and there was no indication that saturation of the membrane with protein was
reached. In order to ensure that this assay gave reproducible results, it was paramount that
there was not more protein loaded than the PVDF membrane could bind.
Figure 6.6 shows another display of the data in Figure 6.5. It can be seen that the
longer the TMB development time, the less linear was the response to increasing amounts
of reduced BSA, for example at 20 min development time, the absorbance value of 80%
reduced BSA was not double the absorbance of 40% reduced BSA. The shorter the TMB
172
Chapter 6- Section 1
Results
development time, the more accurate was this relationship. Thus, it was decided that for
subsequent experiments, a TMB development time of 5 or 10 min would be used.
TMB Development Time [min]
5
10
15
20
Not Coated
20% red BSA/80% ox BSA
40% red BSA/60% ox.BSA
60% red BSA/40% ox BSA
80% red BSA/20% ox BSA
100% red BSA/0% ox BSA
95% red BSA/5% ox BSA
97.5% red BSA/2.5% ox BSA
Figure 6.4. Schematic outline of the loading of the 96-well plate with BSA of various degrees of
oxidation for the determination of the optimum TMB development time for ELISA analysis of protein
thiol content (red = reduced; ox=oxidised).
1.8
R2 = 0.978
1.6
5 min TMB
1.4
10 min TMB
Absorbance 450nm
15 min TMB
1.2
R2 = 0.9273
20 min TMB
1
Linear (15 min
TMB)
Linear (10 min
TMB)
Linear (5 min
TMB)
0.8
0.6
R2 = 0.9878
0.4
R2 = 0.9904
0.2
0
0
10
20
30
40
50
60
70
80
90
% BSA Red
Figure 6.5. The influence of protein loading and TMB development time on ELISA absorbance values.
Reduced BSA was used as the standard protein and the protein loading was 1 µg/well.
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Chapter 6- Section 1
Results
2.5% BSA Red
1.8
5% BSA Red
Absorbance 450nm
1.6
10% BSA Red
1.4
20% BSA Red
1.2
40% BSA Red
R2 = 0.9519
R2 = 0.9876
60% BSA Red
1
R2 = 0.9947
80% BSA Red
0.8
0.6
0.4
0.2
0
0
Linear
Red)
Linear
Red)
Linear
Red)
Linear
Red)
Linear
Red)
Linear
Red)
Linear
Red)
(80% BSA
(60% BSA
R2 = 0.9705
(40% BSA
R2 = 0.9498
(5% BSA
R2 = 0.9565
(20% BSA
R2 = 0.9886
(10%
BSA
5
(2.5% BSA
10
15
TMB Incubation Time
20
25
Figure 6.6. The influence of TMB development time on ELISA absorbance values. Reduced BSA was
used as the standard protein and the protein loading was 1 µg/well.
6.1.2.2 Assay development for protein thiol determination using ELISA: Variation of
protein loading
An experiment was prepared where 1, 5, and 10 µg of BSA was loaded per well and
the TMB development time was selected as 5 and 10 min. The protein load of 1 µg BSA
achieved the only linear response to increasing reduced BSA concentrations, both at 5 and
10 min TMB incubation (Figure 6.7). Even with 100% (1 µg) reduced BSA per well, the
highest concentration of SH groups, the PVDF membrane did not show signs of saturation.
However, at 5 and 10 µg protein load per well it was observed that the curve flattens at
higher concentrations of reduced BSA and this is a clear sign of membrane saturation. This
effect could also be due to limitation of any of the reagents used for derivatisation (MPB),
or secondary antibody (Avidin Horseradish Peroxidase). This, however, is unlikely since
these reagents were added at many times the excess of the concentrations that should
theoretically be required for reaction with all thiol groups. These effects, seen with a 5 min
TMB incubation, were even more pronounced when a 10 min TMB incubation duration
was used. Based on these results, subsequent experiments were conducted with
approximately 1 µg protein loading per well.
174
Chapter 6- Section 1
Results
1µg protein/ well/ 10 min
10µg protein/ well/ 10 min
5µg protein/ well/ 5 min
3
5µg protein/ well/ 10 min
1µg protein/ well/ 5 min
10µg protein/ well/ 5 min
Absorbance 450nm
2.5
2
1.5
1
0.5
0
0
10
20
30
40
50
% BSA Reduced
60
70
80
90
Figure 6.7. The influence of protein loading per well and TMB development time on absorbance values.
6.1.2.3 Assay development for protein thiol determination using ELISA:
Comparison of proteins (aldolase, serum and BSA) potentially suitable as standard
proteins and the influence of protein loading
Dilutions with sodium phosphate buffer (0.02M, pH 8) of aldolase and serum
proteins to a final concentration of 40 µg/mL were prepared. These solutions were serially
diluted with sodium phosphate buffer to give protein concentrations of 20, 10, 5, 2.5, 1.25,
0.625 µg/mL. The reduced/oxidised BSA standard was prepared as described in the
Materials and Methods section 2.31. One hundred µL of the aldolase and serum dilutions
were placed onto the 96 well PVDF plate to give loadings of 4, 2, 1, 0.5, 0.25, 0.125, 0.0625
µg per well. The reduced/oxidised BSA standards were loaded as described in the previous
experiments to give a concentration of 1 µg/well. The rest of the protocol was executed as
previously described with 5 min duration of the TMB incubation step.
The purpose of this experiment was to determine if other proteins could be used as
standard proteins instead of BSA, in order to eliminate the labour intensive reduced BSA
preparation protocol. The results are depicted in Figure 6.8. Reduced BSA mixed with
175
Chapter 6- Section 1
Results
oxidised BSA and a total protein loading of 1 µg/well gave good results as expected.
Aldolase gave a linear response up to a protein loading of 0.5 µg/well; at higher protein
loadings saturation of the PVDF membrane was reached. Serum contained only very few
thiol groups and thus resulted in low absorbance values.
% BSA Red
0
10
20
30
40
50
60
70
80
90
1
0.12
Absorbance 450nm (Aldolase)
0.1
0.8
0.7
0.08
0.6
0.5
Serum
0.06
Aldolase
0.04
BSA
0.02
0.4
0.3
0.2
Absorbance 450nm (Serum, BSA)
0.9
0.1
0
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Protein loading ug/well
Figure 6.8. The influence of the type of standard protein on absorbance values. BSA loading per well is
1 µg.
176
Chapter 6- Section 1
Results
CHAPTER 6 – Section 1
Discussion
6.1.3 Novel procedures for detection of thiol groups in beer proteins
A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) biocytin
(MPB), was used to biotinylate beer proteins containing sulphhydryl groups. In
combination with appropriate avidin- or streptavidin-conjugated markers (for example
fluorescent,
enzyme-conjugated,
electron-dense),
MPB
constitutes
a
universal,
multipurpose, thiol-specific probe (Miedl et al., 2006b). The reagent was used to detect
protein SH groups on Western Blots with sensitivities in the femtomole range. This
method is widely used to detect oxidation in cancer cells (Cecil and Wake, 1962; Berlett and
Stadtmann, 1997; Laragione et al., 2003) and was adapted for detection of thiol groups and
oxidation in beer proteins. The labelling is highly specific for sulphydryl groups; proteins
lacking free SH groups are not labelled by this method. In general, cysteine and methionine
are the amino acids that are most susceptible to oxidation. Reversible oxidation/reduction
of these two amino acids may protect proteins from more damaging forms of oxidative
modifications (for example carbonyl formation).
The MPB labelled Western Blots (Figures 6.1 and 6.2) show that low molecular
weight beer proteins contained higher thiol concentrations. These lower molecular weight
proteins exhibited redox activity during aging. Beer SH- groups are probably able to
magnify the inhibition of the hydroxyl radical. Beer protein reactivity towards MPB
declined as beer aged since the free SH groups were oxidised to disulphides, which were
not detected with MPB. After 3 months aging of bottled or canned beer at 30ºC, the MPB
stain indicating thiols was faint compared to the fresh beer samples (Figures 6.1 and 6.2).
Furthermore, beer C produced from a high gravity wort (18 ºPlato) contained less thiolated
proteins than beer A or B (10 and 14 ºPlato, respectively). The molecular weight
distribution of the beer proteins containing thiol groups also varied between the beers.
Beer A exhibited only one pronounced thiolated protein band in the low molecular weight
region whereas beer B showed two distinct bands. Most likely this is due to the different
malt/ adjunct ratios employed and/or due to differing stabilisation regimes (see Table 6.1).
Western Blotting is a labour and time consuming method of analysis and gives only
semi-quantitative results. Due to the large number of beer samples to be analysed for
protein thiols, efforts were made to develop a high throughput assay that can also be
177
Chapter 6- Section 1
Results
established as part of routine analysis in a brewery. Major efforts were invested into
determining the optimum combination of parameters in this complex system. The
following details the results when different assay parameters were varied.
A linear absorbance response was observed in all cases of TMB development
duration but a 100% increase in development time did not correspond to a 100% increase
in absorbance value at 450 nm (Figure 6.4). The protein loading was 1 µg/well and there
was no indication that saturation of the membrane with protein was reached. In order to
ensure that this assay worked, it was paramount that there was not more protein loaded
than the PVDF membrane could bind. Furthermore, the longer the TMB development
time the less linear was the response to increasing amounts of reduced BSA, for example at
20 min development time, the absorbance value of 80% reduced BSA was not double the
absorbance of 40% reduced BSA. The shorter the TMB development time the more
accurate was this relationship. Thus, a TMB development time of 5 or 10 min was used for
subsequent experiments.
A protein loading of 1 µg BSA achieved the only linear response to increasing
reduced BSA concentrations (Figure 6.6). Even with 100% (1 µg) reduced BSA per well,
the highest concentration of SH groups the PVDF membrane did not show signs of
saturation. However, at 5 and 10 µg protein load per well it was observed that the curve
flattens at higher concentrations of reduced BSA and this is a clear sign of membrane
saturation. Based on these results, subsequent experiments were conducted with
approximately 1 µg protein loading per well. The reduced/oxidised BSA standards were
loaded as described in the previous experiments to give a concentration of 1 µg/well. The
rest of the protocol was executed as previously described with 5 min duration of the TMB
incubation step.
Aldolase and serum were also tested as possible standard proteins instead of BSA.
The results are depicted in Figure 6.8. Reduced BSA mixed with oxidised BSA and a total
protein loading of 1 µg/well gave good results as expected. Aldolase gave a linear response
up to a protein loading of 0.5 µg/well; at higher protein loadings saturation of the PVDF
membrane was reached. Serum contained very few thiol groups and thus resulted in very
low fluorescence values and was found to be unsuitable as standard protein.
Reduced/oxidised BSA appeared to be ideally suited as the standard protein for this assay
method. This assay could be useful for high throughput routine analysis of protein thiols in
beer samples. However, the method was only proven so far to work for detection of thiols
in a standard protein (BSA). Major difficulties were encountered when attempts were made
to assay thiols in beer proteins. This could be due to the heterogeneity of beer polypeptides
with regard to molecular size, hydrophobicity, charge and structure. It is likely that some
178
Chapter 6- Section 1
Results
beer polypetides compete more successfully for binding sites on the PVDF membrane than
others. Therefore, some of the beer proteins are under represented in the assay.
Furthermore, the binding of beer proteins to the PVDF membrane is very sensitive to
environmental conditions (for example pH, ionic strength) and thus minor differences
between beers will influence the results obtained. Substantial further development work
will have to be undertaken in order to optimise and adapt assay conditions in order to allow
reliable, high throughput assessment of beer protein thiol content.
The results so far indicate an important role for protein thiol groups in beer staling.
It has been found that changes in the extracellular redox state in packaged beer are
reflected in changes in the thiol levels of beer polypeptides, particularly of some of the low
molecular weight beer polypeptides.
Thiols can take part in many types of rearrangements which make them especially
interesting in terms of beer staling. ROS can be produced or destroyed at reactive sites of
proteins. Protein may be a substrate or an antagonist for ROS. Based on the findings in the
present study and on results from Swan et al. (2003), the following mechanism is proposed
to take place with beer protein thiols.
Sulphitolysis produces free thiols and sulphonates. Protein S-sulphonates can
slowly release sulphite ions in the presence of sulphydryl compounds. A series of cyclical
reactions may take place that are catalysed by oxygen. Peroxide is formed when SH-groups
are oxidised to form a disulphide. Sulphur dioxide (SO2) subsequently reduces these links
by sulphitolysis. The resulting sulphonate is labile at low pH (for example beer pH) and can
dissociate to liberate SO2. The peroxide is destroyed by the SO2. The proximity of bound
SO2 to oxidative sites allows the more effective containment of peroxide and radicals, if the
sites possess localised Fe2+or Cu or both. This will allow the SO2 to be more effective than
in free solution, to provide exaggerated protection from beer staling. Beer proteins may be
able to contribute to improved beer flavour stability by containing - 'caging' – ROS and
thus inhibit staling reactions. Figure 6.9 depicts the proposed hypothesis describing the
caging reactions on thiol groups in beer proteins when oxygen is present.
Figure 6.10 shows how the thiols of beer proteins could catalyse the destruction
of ROS in cyclical reactions within the beer matrix. This could be an effective mechanism
of slowing down oxidation reactions and thus flavour deterioration.
179
Chapter
6- Section
The
Role 1of
Beer Protein Thiols in Staling
Results
SH
O2
SH
SH
O2
O2
Beer Protein
HS
SH
SH
Caging of ROS on Beer Proteins
……SH-groups
in beer
proteins
become
oxidised
to disulphides.
…..SH-groups
in beer
proteins
become
oxidised
to disulfides.
+H2O2
O2
S
+H2O2
S
S
S
S
S
+H2O2
Beer
Protein
S
+H2O2
S
S
S
S
+H2O2
Figure 6.9. Caging of ROS on redox-active thiol groups in beer proteins (continued on next page).
180
S
Chapter 6- Section 1
Results
Caging of ROS on Beer Proteins
If SO
SO22 is
is present
present sulfitolysis
takes
place…...
If
sulphitolysis
takes
place….
SO3-S
+SO2
+SO2
SH
SH
S
+SO2
Beer Protein
SO3-S
S
Sulfonates
areare
labile
under
beerbeer
pHpH
Sulphonates
labile
under
and
and dissociate
dissociatetotoliberate
liberateSO
SO2…….
2….
SO3-S
SH
Caging of ROS on Beer Proteins
Sulphonates
labile
under
beer
Sulfonates areare
labile
under
beer
pHpH
and
to liberate
liberate SO
SO2…….
….
and dissociate
dissociate to
2
+SO2
HS
+SO2
SH
SH
S
Beer Protein
HS
S
SH
Figure 6.9. Caging of ROS on redox-active thiol groups in beer proteins.
181
+SO2
HS
Chapter 6- Section 1
Results
Caging of ROS on Beer Proteins
O2
SH
S-SO3
H2O2
O2
O2
S-SO3
H2O2
H2O2
SH
SH
O2
SH
S-SO3
H2O2
SH
SH
SO2
SO2
S - S
SH
SH
S-SO3
SO2
SO2
S - S
SH
S - S
SH
SH
H2O2
SO2
SO2
SH
SO2
S - S
SH
SH
O2
SO2
SO2
S - S
SH
S - S
SH
O2
SO2
SO2
SH
S-SO3
H2O2
Figure 6.10. Caging of ROS at thiol groups in beer proteins (Swan et al., 2003).
Conclusions
Beer protein reactivity towards MPB declined as beer was aged since the free SH
groups were oxidised to disulphides which were not detected with MPB. Furthermore, beer
produced from a high gravity wort (18 ºPlato) contained no detectable amount of thiolated
proteins. Beers produced from 10 ºPlato and 14 ºPlato wort showed distinct band(s) of
thiolated protein in the lower molecular weight ranges. The 10 ºPlato beer showed one
distinct band and the 14 ºPlato yielded two distinct bands of proteins containing free thiols.
The 10 ºPlato was brewed with 100% malt, whereas with increasing gravity increasing
amounts of sugar syrup were used. Sugar syrup does not contribute protein, whereas barley
malt contains a comparatively large amount of protein. Furthermore, high gravity brewed
beer is diluted with appropriately treated water to sales gravity; this will further decrease the
already diminished beer protein content. Thus, it can be concluded that beers brewed at
higher gravities using sugar syrup as an extract source contain less total protein compared
to beers brewed at lower gravity and this might be a contributing factor for the lower
content of protein thiols found in the beer produced from a 18 ºPlato wort. However, a
lower total protein content in high gravity beers (brewed with high amounts of sugar syrup)
could potentially decrease the amount of aging carbonyls formed and thus have a positive
effect on beer flavour stability. Increased malt content does not only contribute thiolated
182
SH
S-SO
Chapter 6- Section 1
Results
polypeptides that could have a beneficial effect with regards to flavour stability but also
contributes molecules that are pro-oxidants or molecules that can be altered by oxidation
to form undesirable stale flavours and aromas.
As demonstrated previously (Brey, 2004) protein extraction during mashing in a
high gravity process is less efficient when compared to a lower gravity process. In addition
it has been shown that the loss of certain protein fractions (especially hydrophobic
polypeptides) throughout a high gravity brewing process is increased compared to a lower
gravity process. It might be concluded that the majority of proteins with free thiols are also
hydrophobic polypeptides in nature and thus will be diminished during a high gravity
brewing process. These findings should be taken into account when considering process
intensification measures (for example increasing wort gravity) since a negative effect on
beer flavour stability could be a consequence.
Beer colloidal stabilisation treatments, decreasing the concentration of haze active
proteins, might also have a negative impact on beer flavour stability. It is not clear whether
haze active and thiol rich proteins are similar in structure and amino acid composition, but
it is unlikely that these are two distinct polypeptide groups. Further research is necessary in
order to assess the impact of malt content and wort gravity on beer flavour stability.
Western Blotting is a labour and time consuming analytical method and gives only
semi-quantitative results, thus attempts were made to develop a quantitative, high
throughput assay for measuring beer protein thiols. However, the method was only proven
to yield reliable results for the detection of thiols in a standard protein (BSA). Substantial
further development work will have to be undertaken in order to optimise and adapt assay
conditions to allow reliable, high throughput assessment of beer protein thiol content.
Beer proteins may be able to contribute to improved beer flavour stability by
containing ROS on their thiols and thus inhibiting staling reactions. A hypothesis was
proposed describing the caging reactions on thiol groups in beer proteins when oxygen is
present. The thiols of beer proteins may be able to catalyse the destruction of ROS in
cyclical reactions within the beer matrix. This could be an effective mechanism of slowing
down oxidation reactions and thus flavour deterioration.
183
Chapter 6- Section 2
Results
CHAPTER 6 – Section 2
Results
6.2 A novel assay for the detection of carbonyl groups in beer proteins
Free radical-mediated oxidation of proteins in beer results in the formation of
carbonyl groups in quantities that reflect the intensity of oxidative stress (Meilgaard et al.,
1970; Meilgaard et al., 1971; Meilgaard, 1972). Carbonyl groups are an important marker of
oxidative stress in human diseases (Meilgaard, 1972). The biomedical method of analysis
was adapted to detect carbonyl groups in beer proteins as an oxidation indicator during
aging. Further information about the importance of carbonyl groups can be found in the
Introduction Section (Chapter 1).
6.2.1 Detection of carbonyl groups using Western Blotting
Highly sensitive assays for detection of protein carbonyls involve derivatisation of
the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to the formation
of a stable 2,4-dinitrophenyl (DNP) hydrazone product. The beer was subjected to SDSPAGE analysis. This step yielded relatively pure proteins separated according to their
molecular weights. The proteins were blotted onto a PVDF membrane and the carbonyl
group specific DNPH derivatisation step of the proteins was conducted directly on the
membrane. This was followed by incubation with a DNPH specific biotinylated antibody
(Sigma D-8406, IgE). Streptavidin-biotinylated horseradish peroxidase (HRP) was used as
the secondary antibody and ECL chemiluminescence was used to detect this secondary
antibody. This novel method allows detection of individual oxidised proteins within a
mixture of proteins and only requires approximately 50 ng protein. The sensitivity is
approximately 1 pmol of protein carbonyl which equals approximately 50 ng of a 50 kDa
protein oxidised at 0.5 mol/mol. This Western Blotting technique reveals differential
susceptibility of individual proteins to oxidative modifications. Carbohydrate groups of
glycoproteins do not contribute to detected carbonyl levels but free aldehyde groups from
lipid peroxidation contribute to carbonyl levels.
This assay for carbonyl groups in beer proteins is semi-quantitative. The protein
carbonyls increased during the course of 3 months storage at 30ºC as depicted in Figure
184
Chapter 6- Section 2
Results
6.11. The 18 ºPlato beer C shows less protein carbonylation compared to beer brand A (10
ºPlato).
Fresh
1 months
2 months
3 months
Fresh
1 months
2 months
3 months
Carbonyl Group
Brand A
Brand C
Figure 6.11. Visualisation of carbonyl groups in beer proteins using Western Blotting in samples of fresh
beer and beer that was force aged for 1, 2 and 3 months at 30˚C. Brand C is a higher gravity beer (18
˚Plato) and brand A is a lower gravity beer (10 ˚Plato).
6.2.2 Detection of protein carbonyl groups with an enzyme linked immunosorbent
assay (ELISA)
Major efforts were devoted to develop Western Blotting techniques for beer
protein carbonyl groups into a rapid quantitative assay. An enzyme-linked immunosorbent
assay (ELISA) method using an anti-DNP antibody for measuring total protein carbonyl
groups that is highly sensitive, reproducible, and correlates directly with the classical
colorimetric assay was developed. The protein in the beer samples was non-specifically
adsorbed to the wells of an ELISA plate. The adsorbed protein was reacted with a
biotinylated anti-DNP antibody followed by streptavidin biotinylated horseradish
peroxidase detection (for details see Materials and Methods section 2.31). Absorbances
were related to a standard curve prepared from a mixture of oxidised and reduced bovine
serum albumin. Free DNPH and non-protein constituents were washed away and gave
185
Chapter 6- Section 2
Results
minimal interference. This potentially allows much greater sensitivity and accuracy at the
lower end of the range than for the spectrophotometric DNPH assay. In addition, the
ELISA test is easier to use, less labour-intensive, and can handle more samples per day
than the colorimetric or Western Blotting assay. The ELISA test also has the advantage
that it requires only microgram amounts (about 60 μg) of protein, Therefore, ELISA could
have wide application for measuring protein oxidation in situations where only limited
amounts of protein are available for analysis. Beer contains only 0.1-1.0 mg of total protein
per litre but only a small proportion of the total protein is susceptible for carbonyl
formation. The present set up did not yield any useable results. Further method
development work is necessary in order to get this multi-factorial system to give
reproducible results.
186
Chapter 6- Section 2
Results
CHAPTER 6 – Section 2
Discussion
6.2.3 Novel methods for detection of carbonyl groups in beer proteins
Immunochemical techniques have been previously applied to the detection of
carbonyl groups in proteins separated by polyacrylamide gel electrophoresis (Keller et al.,
1993; Schacter et al., 1994). The potential sensitivity and specificity afforded by the
immunochemical approach prompted the investigation of its application to the
quantification of carbonyl groups in beer proteins. The measurement of protein carbonyls
following their covalent reaction with DNPH was pioneered by Levine et al. (1990) and has
become the most widely utilised measure of protein oxidation in several human diseases
(Figure 6.12). Highly sensitive assays for detection of protein carbonyls involve
derivatisation of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads
to the formation of a stable 2,4-dinitrophenyl (DNP) hydrazone product.The
spectrophotometric DNPH method (Figure 6.12) was comparatively easy to conduct but
could not be utilised for the detection of carbonyl groups in beer proteins because this
assay also detects the multitude of other carbonyls present in beer. It also has the
disadvantage of requiring relatively large (up to a milligram) amounts of protein.
Furthermore, the limited solubility of many DNP-derivatised proteins and difficulties in
eliminating free DNPH from the derivatised proteins (Cao and Cutler, 1995) makes this
method prone to interference. Other methods for carbonyl detection include HPLC
techniques based on the spectrophotometric detection of DNP-carbonyl derivatives after
separation of the proteins by gel permeation or reverse phase chromatography (Levine et
al., 1994). HPLC analysis is useful for investigating purified proteins, but is less useful in
crude mixtures (beer proteins are crude mixtures) where problems with resolution make it
especially difficult to analyse low and medium molecular weight proteins (Agarwal and
Sohal, 1995). Carbonyl groups can also be detected by labelling with tritiated borohydride
(Levine et al., 1990). This technique is highly sensitive and specific but as tritiated
borohydride also reacts with Schiff’s bases this can complicate its application to the
complex chemical matrix of beer. Immunochemical techniques have been previously
applied in medical sciences to the detection of carbonyl groups in proteins separated by
polyacrylamide gel electrophoresis. The potential sensitivity and specificity afforded by the
immunochemical approach prompted an investigation of its application to the
quantification of carbonyl groups in beer proteins. A sensitive and quantitative solid-phase
immunoassay for the determination of protein carbonyls was developed. The method is
187
Chapter 6- Section 2
Results
based on a combination of DNPH derivatisation, the preparation of blanks by treatment
with sodium borohydride, and immunological detection (Figure 6.13).
O
protein
H2O2/Fe
DNP
DNPH
oxidised
protein
DNPprotein
Absorbance
at 370 nm
Figure 6.12. Principle of the detection of total carbonyl content of beer employing a spectrophotometric
DNPH assay.
O
oxidised
protein
DNP
DNPH
DNPprotein
DNP
Anti-DNP
antibody
DNPprotein
Immunological
detection
Figure 6.13. Principle of the measurement of carbonyl groups in beer proteins employing DNP.
The Western Blot immunoassay has the advantage of avoiding complications such
as incomplete removal of the free DNPH before measurement, as it detects only DNP
groups conjugated to proteins. Small amounts of free DNPH, which may remain in the
PVDF membrane, do not react with the anti-DNP antibody even if they bind to the
transfer membrane. This assay for carbonyl groups in beer proteins is semi-quantitative.
The protein carbonyls increased during the course of 3 months storage at 30ºC as depicted
in Figure 6.11. The 18 ºPlato beer C shows less protein carbonylation compared to beer
brand A (10 ºPlato) indicating less oxidation in the high gravity beer. However, as
established in Chapter 6, Section 1 high gravity brewed beer contained less protein than
beer brewed at lower gravity. This could explain the lower levels of protein carbonylation
found in the 18 ºPlato beer.
188
Chapter 6- Section 2
Results
Conclusions
The Western Blot assay for detection of carbonyl groups in beer proteins is semiquantitative. The protein carbonyls increased over 3 months storage at 30ºC. The 18 ºPlato
beer C showed less protein carbonylation compared to beer brand A (10 ºPlato) indicating
less oxidative damage to beer constituents in the high gravity beer. However, as established
in Chapter 6, Section 1 high gravity brewed beer contained less protein than beer brewed at
lower gravity. This could explain the lower levels of protein carbonylation found in the 18
ºPlato beer. In order to eliminate bias in future research into this study the differing total
protein levels in beer have to be taken into account when using the Western Blotting
technique. Sample loading onto the PVDF membrane should not be based on equal
volumes of beer, but on equal total protein loading.
189
Chapter 6- Section 3
Results
CHAPTER 6 – Section 3
Results
6.3 The Peroxide Challenge Test (PCT): A novel method for holistic, near real time
measurement of beer flavour stability
A holistic approach is paramount in order to reliably measure beer staling. As a
consequence, the Peroxide Challenge Test (PCT) was developed as an alternative to
Electron Spin Resonance (ESR). The principle of the PCT is to mimic oxidation by
titrating beer with hydrogen peroxide. The more hydrogen peroxide a beer can quench, the
more resistant it is against oxidation and flavour deterioration. The PCT allows cost
effective, near real-time, high throughput assessment of beer flavour stability. The PCT is
convenient and inexpensive to perform and the results correlate with ESR measurements
and the perceived aged character of beer. In the present study, the production of lager
beers produced in an industrial scale process was investigated over a period of 12 weeks
storage with regard to flavour stability. One of the established methods of analysis applied
was Electron Spin Resonance (ESR) (Andersen and Skibsted, 1998; Andersen et al., 2000;
Miedl et al., 2007). However, the capital and operating costs of the ESR are significant. As
an alternative method to the ESR, the Peroxide Challenge Test (PCT) was developed. In
order to verify the PCT as a novel method of analysis that correlates with the perceived
aged character found in tastings, ESR and PCT analyses were conducted in parallel to
flavour evaluation by an experienced panel. One of the most promising holistic analysis
techniques for measurement of flavour stability is ESR. This recent development in
analytical methods utilising spin trapping reagents (Kaneda et al., 1989; Kaneda et al., 1991;
Uchida and Ono, 1996; Andersen and Skibsted, 1998) made it possible to unravel the initial
oxygen-dependent staling reactions in beer. ESR is a spectroscopic technique which detects
species that have unpaired electrons. Only ESR detects unpaired electrons unambiguously.
Other techniques, such as fluorescence, may provide indirect evidence of free radicals, but
ESR alone yields incontrovertible evidence of their presence. This technique has been vital
in the biomedical field for elucidating the role of free radicals in many pathologies and
toxicities and is now widely used to detect Reactive Oxygen Species (ROS) in the context
of beer aging (Kaneda et al., 1989; Kaneda et al., 1991; Uchida and Ono, 1996; Andersen
and Skibsted, 1998). Beer contains a complex array of chemical compounds which can
inhibit oxidative reactions within the beer matrix. Depending on the brewing process, raw
190
Chapter 6- Section 3
Results
materials, the presence of oxygen during beer production and in the final package and
other process parameters, a beer will possess a certain “anti-oxidant potential”. This ability
to withstand oxidation processes is directly related to a beer’s ability to withstand flavour
deterioration. A measure for the anti-oxidant potential of beer is the ESR lag time. The
longer the lag time, the better is a beer’s capability to suppress free radical formation when
oxygen is present. Thus, it can be expected that a beer with a longer ESR lag time will be
more flavour stable than a beer with a shorter ESR lag time. Furthermore, the intensity of
the ESR signal after 150 min of forced aging at 60˚C was reported to give an indication of
the intensity of the aged character after exhaustion of the anti-oxidant potential and the
onset of beer staling (Kaneda et al., 1989; Kaneda et al., 1991; Uchida and Ono, 1996;
Foster et al., 2002). An example of a typical ESR graph for a “standard” rated beer and for
a “reject” rated beer is shown in Figure 6.14. Each sample was analysed multiple times in
order show the reproducibility of the method. The x-axis of the graph is the duration of
the experiment (samples are held at 60˚C) in minutes and on the y-axis is the number of
free radicals detected in counts per second. The more flavour stable beer (labelled
“Standard”) repressed radical formation for approximately 50 min (ESR lag time) whereas
an aged beer (three months storage at 30˚C, labelled “Reject”) did not have any significant
ability to suppress the formation of radicals and free radicals were detected immediately
after the start of forcing at 60˚C. Furthermore after 150 min the amount of radicals created
was significantly higher in the “Reject” beer compared to the “Standard” beer,
T150: the lower the better the predicted flavour stability
Free radical formation [counts per second]
[ * 10^ 3]
Figure
250
225
35
4 67
“REJECT”
200
175
345
67
2
2
357
46
2
357
46
2
150
125
3 567
24
100
7
56
34
2
50
114
189
151617
10
89111123
75
25
3
4567
2
121314
15
189
11
110
89
16
17
189
1145
117
1231 6
110
11
89
13
12
16
89
14
15
189
110
17
11
114
15
17
16
189
1 01 3
89111
2
1145
17
116
189
1101
89
1123
1145
116
17
189
1
0
89111
123
“STANDARD”
0
0
20
40
60
80
100
[ m n
i ]
120
140
160
180
Legend:
2 : G ui nness 800m L bot t l e3 bb27.
: G
9. ui
06nness 800m L bot t l e4 bb27.
: G
9. ui
06nness 375m L bot t l e bb2
Lag time: the longer the better the predicted beer flavour
5 : G ui nness 375m L bot t l e6 bb27.
: G
9. ui
06nness 375m L bot t l e7 bb08.
: G
8. ui
06nness
HR 800m L bot t l e bb0
stability
8 : VB bot t l e bb24. 4. 06 Yat9al a: 2mVB
ontbot
h tRlT,e bb24.
Y/ A com
4. 06
p Yat10
al a: 2mVB
ontbot
h tRlT,e bb24.
Y/ A com
4. 06
p Yat al a
11 : VB bot t l e bb24. 4. 06 Yat12
al a: 2mVB
ontbot
h tRlT,e bb24.
Y/ A com
4. 06
p Yat13
al a: 2mVB
ontbot
h tRlT,e bb24.
Y/ A com
4. 06
p Yat al a
14 : VB bot t l e bb22. 4. 06 Abby
15 2: m VB
ont bot
h RtT,l eY/bb22.
A com
4. 06
p Abby
16 2m
: ont
VBhbot
RT,t l e
Y/ bb22.
A com4.p06 Abby 2m
6.14. Example
of a typical ESR analysis
of a ont
“standard”
rated4.and
a 19
“reject”
ratedRT,beer.
The
17 : VB bot t l e bb22. 4. 06 Abby
18 2: m VB
bot
h RtT,l eY/bb22.
A com
06
p Abby
2m
: ont
VBhbot
tl e
Y/ bb22.
A com4.p06 Abby 2m
samples were run multiple times in order to demonstrate the reproducibility of the method.
191
Chapter 6- Section 3
Results
As an alternative method to the ESR, the Peroxide Challenge Test (PCT) was
developed. The basic principle of this method is simply to mimic rapid oxidation by
titrating the beer or process sample with increasing amounts of hydrogen peroxide.
Depending on the ability of the redox active beer constituents to quench or neutralise
hydrogen peroxide, the breakthrough point of peroxide gives an indication of the
antioxidant potential of beer. The more peroxide a beer can quench, the more resistant it is
against oxidation and hence flavour deterioration. The assay can be conducted in 96-well
plates and therefore allows high throughput and near real-time assessment.
Figures 6.15, 6.16 and 6.17 show the results of the PCT analysis of the freshly
packaged beers A, B and C. Similar to the ESR lag time these fresh beers exhibit a “PCT
lag time” that is actually not a time, but a hydrogen peroxide concentration that the beer is
able to neutralise. The ability to quench peroxide is directly related to a beer’s antioxidant
potential and thus flavour stability. After the addition of a specific amount of peroxide, the
antioxidant potential of a beer is exhausted (“Peroxide Breakthrough Point”) and any
further addition of peroxide will result in an accumulation of peroxide as seen in Figures
6.15, 6.16 and 6.17, through an increase of the measured peroxide concentration. Another
important observation was the substantial batch-to-batch variation found in the
investigated beer brands. This finding indicates that different batches of one brand are by
Peroxide Challenge Test (PCT)
no means uniform with regard to flavour stability. Further research is necessary to establish
Beer Brand A - fresh
the origin of this significant bandwidth within one brand.
Beer A
Beer A
measured Peroxide
Concentration
[uM]
Measured
Peroxide
Concentration
[μM]
100
PCT Lag time
90
Peroxide Breakthrough Point
80
70
Substantial batch-to-batch
variation within one beer
brand
60
50
40
30
20
10
0
0
50
100
150
200
250
300
350
400
450
-10
-20
Peroxide Concentration [uM]
Addedadded
Peroxide
Concentration [μM]
Figure 6.15. Peroxide Challenge Test analysis of fresh beer A. Similar to the ESR lag time these fresh
beers exhibit a “PCT lag time” that is actually not a time but a hydrogen peroxide concentration that the
beer is able to neutralise.
192
Chapter 6- Section 3
Results
Beer B fresh
100
Measured Peroxide Concentration [μM]
90
80
70
60
50
40
30
20
10
0
0
50
100
150
200
250
300
350
400
450
-10
-20
Added Peroxide Concentration [μM]
Figure 6.16. Peroxide Challenge Test analysis of fresh beer B. Similar to the ESR lag time these fresh
beers exhibit a “PCT lag time” that is actually not a time but a hydrogen peroxide concentration that the
beer is able to neutralise.
Beer C
100
Measured Peroxide Concentration [μM]
90
80
70
60
50
40
30
20
10
0
0
50
100
150
200
250
300
350
400
450
-10
-20
Added Peroxide Concentration [μM]
Figure 6.17. Peroxide Challenge Test analysis of fresh beer C. Similar to the ESR lag time these fresh
beers exhibit a “PCT lag time” that is actually not a time but a hydrogen peroxide concentration that the
beer is able to neutralise.
193
Chapter 6- Section 3
Results
A comparison of the peroxide breakthrough points (PBP) of the fresh beers A, B
and C is given in Figures 6.18 and 6.19. Beers B and C quenched more peroxide than beer
A, indicating diminished resistance to flavour deterioration in beer A. The beer samples
were aged for one and two months at 30˚C and the PCT analysis repeated. After one
month aging, the PBP differences between the beers decreased but beer B exhibited a
slightly higher antioxidant potential than beers A and C (Figure 6.20). After two months
aging at 30˚C, all three beers have deteriorated further, however, beer B showed the highest
antioxidant potential followed by beer A and beer C as depicted in Figure 6.21.
Figure 6.22 depicts the summarised PCT results of beer C analysed fresh, after one,
two and three months aging at 30˚C. Fresh beer C was able to quench approximately 70
μM of hydrogen peroxide; one month aged beer neutralised approximately 40 μM of
peroxide; two months aged beer 27 μM and three months aged beer only around 14 μM of
peroxide. This clearly shows the decreasing resistance of beer to suppression of oxidation
reactions with increasing age. Similar results were obtained with beers A and B (data not
shown).
PCT on beers A, B and C - fresh
100
Beer A
90
Beer B
measured Peroxide Concentration [uM]
80
Beer C
70
60
50
40
30
20
10
0
0
50
100
150
200
250
300
350
400
450
-10
added Peroxide Concentration [uM]
-20
Figure 6.18. Peroxide Challenge Test analysis of fresh beers A, B and C. The red, orange and green
symbols represent the average of a triplicate analysis. All data points are shown in black and white in order
to demonstrate reproducibility.
194
Chapter 6- Section 3
Results
PCT on beers A, B and C - fresh
Continued...
measured Peroxide Concentration [uM]
Beer A
Beer B
10
Beer C
0
0
20
40
60
80
100
added Peroxide Concentration [uM]
-10
Figure 6.19. Peroxide Challenge Test analysis of fresh beers A, B and C. Detail from Figure 6.18. The
red, orange and green symbols represent the average of a triplicate analysis. All data points are shown in
black and white in order to demonstrate reproducibility.
15
measured Peroxide Concentration [uM]
Beer A
Beer B
10
Beer C
5
0
0
20
40
60
80
100
-5
-10
added Peroxide Concentration [uM]
.
Figure 6.20. Peroxide Challenge Test analysis of beers A, B and C after 1 month aging at 30˚C. The
red, orange and green symbols represent the average of a triplicate analysis. All data points are shown in
black and white in order to demonstrate reproducibility.
195
Chapter 6- Section 3
Results
15
Beer A
Beer B
measured Peroxide Concentration [uM]
10
Beer C
5
0
-10
10
30
50
70
90
110
-5
-10
added Peroxide Concentration [uM]
Figure 6.21. Peroxide Challenge Test analysis of beers A, B and C after 2 months aging at 30˚C. The
red, orange and green symbols represent the average of a triplicate analysis. All data points are shown in
black
andon
whitebeer
in orderCto–demonstrate
reproducibility.
PCT
fresh, 1 month,
2 months and 3 months aging at 30˚C
Measured Peroxide Concentration [μM]
20
CMS
fresh Average
Fresh
15
1 1month
30˚C
CMS
month Average
CMS2
month Average
2 months
30˚C
10
CMS
mointh Average
3 3months
30˚C
5
0
0
10
20
30
40
50
60
70
80
-5
-10
Added Peroxide Concentration [μM]
Figure 6.22. Peroxide Challenge Test analysis of beer C, fresh, and after 1, 2 and 3 months forced aging
at 30˚C. The red, orange and green symbols represent the average of a triplicate analysis. All data points
are shown in black and white in order to demonstrate reproducibility.
196
Chapter 6- Section 3
Results
A summary of PCT-PBP’s of beers A, B and C, fresh, and after one, two and three
months aging at 30˚C is shown in Figure 6.23. The degree of beer flavour stability
(excellent to very stale) was determined based on the differences in PCT-PBP’s and graded
according to the scale shown on the top right of Figure 6.23. It was found that with
increasing age, all three beers exhibited diminished antioxidant potential. However, this was
more pronounced in beer C (BPB at approximately 17 μM peroxide) compared to beers A
and B, which were still able to neutralise approximately 32 μM of hydrogen peroxide after
three months aging at 30˚C. These results suggest that beer C is more susceptible to
oxidation and hence flavour deterioration in the trade.
In order to establish whether the PCT results have a reliable correlation with actual
beer flavour deterioration, an established method of analysis (ESR) and taste evaluation
with an experienced panel were applied to fresh and aged beers A, B and C (Figures 6.24
and 6.25). Figure 6.24 depicts the decrease in ESR lag time with aging. Again, all three
beers showed a decrease in ESR lag time, but this was most pronounced in beer C (ESR lag
time of approximately 23 min after three months aging at 30˚C) followed by beer A (ESR
lag time of 28 min). Beer B stood out again as the most resistant against oxidation and it
can thus be assumed that beer B will exhibit the highest resistance against flavour
deterioration. These ESR results confirm the PCT findings. Figure 6.25 summarises the
taste evaluation results. Again there was no large difference in flavour ratings for the fresh
beers. However, in the course of aging differences in the flavour score appeared. As already
observed with the PCT and ESR analyses beer C deteriorated faster than beer A, and beer
B performed best with regard to flavour stability. Figures 6.26, 6.27 and 6.28 illustrate the
correlations between PCT and ESR analysis and taste evaluation with more clarity. Both,
PCT and ESR analysis appear to mimic actual perceived flavour deterioration sufficiently.
However, neither of these analytical methods was able to predict beer flavour deterioration
from analysis of a fresh beer sample. Beer C (which performed worst during aging) was
actually rated as the best beer when analysed fresh with the PCT, and rated on the same
level with beer B (which performed best during aging at 30˚C) when analysed fresh with
the ESR method. Based on these findings it can be concluded that the ESR and the PCT
do reliably correlate with perceived flavour deterioration, but do not allow a reliable
prediction of the speed of flavour deterioration in a given beer based on the analysis of the
fresh beer. This statement is only valid for the three beers evaluated during this study but
predictive measurement might be possible for other beer types or brands.
Table 6.2 is a summary and comparison of all results derived from the PCT and ESR
analysis and from the taste evaluation. All ESR lag times and PCT- PBP’s were graded
according to rating systems that were developed over the duration of this project. It is
197
Chapter 6- Section 3
Results
important to note that the grading schematics have to be adjusted and verified for other
beer brands and types. As already discussed, beer C deteriorated faster than beer A. Beer B
showed the highest resistance against oxidation and staling and these findings were
confirmed using PCT and ESR analyses as well as taste panel evaluation.
Summary of PCT Results
Staling Resistance
Beer A
80
>80
Beer B
Very good
70-80
Good
60-70
Beer C
Average
50-60
Fair
40-50
Poor
30-40
Stale
20-30
Very stale
<20
70
PCT Break Through Point [uM]
PCT Lag Time [min]
Excellent
60
50
40
30
20
10
fresh
2 months 30˚C
1 months 30˚C
3 months 30˚C
Figure 6.23. Summary of Peroxide Challenge Test lag time results of beers A, B and C, fresh, and after
1, 2 and 3 months aging at 30˚C. The degree of beer flavour stability (excellent to very stale) was
determined based on the differences in PCT lag times.
198
Chapter 6- Section 3
Results
Predicted Staling Resistance
100
Beer A
Beer B
Beer C
90
ESR Lag Time [min]
80
70
ESR Lag Time [min]
Excellent
>90
Very good
80-90
Good
70-80
Average
60-70
Fair
50-60
Poor
40-50
Stale
30-40
Very stale
<30
60
50
40
30
20
10
0
1 months 30˚C
fresh
2 months 30˚C
3 months 30˚C
Figure 6.24. Electron Spin Resonance lag time results of beers A, B and C, fresh and after 1, 2 and 3
months of aging at 30˚C. Displayed are the averages across all assessed production batches of each beer
brand.
4.5
Staling intensity (Scale from 1-4)
4
1-2
Standard
2-3
Minor defects
3-4
Major defects
4
Reject
3.5
3
Beer A
Beer B
Beer C
2.5
2
1.5
1
fresh
1 months 30˚C
2 months 30˚C
3 months 30˚C
Figure 6.25. Taste panel results of staling intensity of beers A, B and C, fresh and after 1, 2 and 3
months of aging at 30˚C. Displayed are the averages across all assessed production batches of each beer
brand.
199
Chapter 6- Section 3
Results
3
Beer A (PCT)
80
Beer A (ESR)
Beer A (Taste)
70
60
2
50
40
30
1
Staling Intensity [4-Score)
PCT Breakthrough Point [uM PO]; ESR Lag Time [min]
90
20
10
0
0
fresh
1 months 30˚C
2 months 30˚C
3 months 30˚C
Figure 6.26. Correlation of Peroxide Challenge Test lag time, Electron Spin Resonance lag time and
taste results for beer brand A; fresh, and after 1, 2 and 3 months aging at 30˚C.
90
3
Beer B (ESR)
Beer B (Taste)
80
70
60
2
50
40
30
1
Staling Intensity (4-Score)
PCT Breakthrough Point [uM PO]; ESR Lag Time [min]
Beer B (PCT)
20
10
0
0
fresh
1 months 30˚C
2 months 30˚C
3 months 30˚C
Figure 6.27. Correlation of Peroxide Challenge Test lag time, Electron Spin Resonance lag time and
taste results for beer brand B; fresh, and after 1, 2 and 3 months aging at 30˚C.
200
Chapter 6- Section 3
Results
90
3
Beer C (ESR)
Beer C (Taste)
80
70
60
2
50
40
30
1
Staling Intensity (4-Score)
PCT Breakthrough Point [uM PO]; ESR Lag Time
[min]
Beer C (PCT)
20
10
0
0
fresh
1 months 30˚C
2 months 30˚C
3 months 30˚C
Figure 6.28. Correlation of Peroxide Challenge Test lag time, Electron Spin Resonance lag time and
taste results for beer brand C; fresh, and after one, two and three months aging at 30˚C.
Table 6.2. Summary of the flavour stability ratings for beers A, B and C, fresh, and after 1, 2 and 3
months aging at 30˚C derived from taste panel Electron Spin Resonance and Peroxide Challenge Test lag
Beer A
Beer B
PCT
Good
Good
Very good
Fresh
ESR
Good
Very good
Good
Taste
Standard
Standard
Standard
1 month
PCT
Poor
Poor
Fair
ESR
Poor
Fair
Stale
Taste
Minor defects
Minor defects
Minor defects
2 months
PCT
Poor
Poor
Stale
ESR
Stale
Fair
Very stale
Taste
Major defects
Minor defects
Major defects
3 months
time data.
PCT
Poor
Poor
Very stale
ESR
Very stale
Poor
Very stale
Taste
Major defects
Major defects
Reject
201
Beer C
Chapter 6 – Section 3
Discussion
CHAPTER 6 – Section 3
Discussion
Beer flavour stability is a complex issue (Engan 1969; Drost et al., 1971; Graveland
et al., 1972; Hashimoto 1972; Bernstein and Laufer, 1977; Doderer et al., 1981; Baxter, 1982;
Barker et al., 1983; Baxter, 1984; Bohman, 1985a; Bohman, 1985b; Barker et al., 1989;
Kobayashi et al., 1993; Harayama et al., 1994; Kobayashi et al., 1994; Bamforth, 1999a,
Bamforth, 1999b; Angelino et al., 1999; Bamforth 2000b; Kobayashi et al., 2000; Foster et
al., 2001; Araki et al., 2002; Garbe et al., 2003; Bamforth, 2004). The assessment of the
mechanisms involved in beer staling is a longstanding research priority of brewers in order
to extend beer shelf life. No single chemical reaction is responsible for any given flavour
change. Furthermore, the sorts of chemical species which are thought to take part in these
reactions are those which are an integral part of any beer as we know it. They include the
bitter compounds from hops, the amino acids from malt, the higher alcohol products of
yeast metabolism and the melanoidin colouring materials and many other compounds
(Bamforth, 2004). As each of these categories of substance have their place in beer
production and quality, it is not a practical option to eliminate them from beer in
anticipation of preventing flavour change. The extent to which these changes occur will
differ from beer to beer and the solution to the problem will similarly vary.
Three lager beers were analysed over 12 weeks storage in order to predict their
antioxidant potential with the PCT and ESR methods. Sensory analysis by an experienced
panel demonstrated that the PCT and ESR results correlated with the rate of flavour
deterioration. The PCT, applied to fresh and aged beers, showed a similar pattern to the
ESR measurement. After an initial period of constant peroxide resistance, where the
antioxidants in beer were able to neutralise the peroxide, the breakthrough of peroxide
could be clearly detected by a sharp increase in luminescence. The PCT appears to be a
useful tool to predict the endogenous antioxidant activity of beer, which determines the
beer flavour stability towards oxidation.
It was demonstrated that aged beer can be distinguished from fresh beer. Even
differences between production batches can be identified and benchmarking values for
each brand and different breweries defined. Both, PCT and ESR analyses mimic actual
perceived flavour deterioration sufficiently. However, neither of these analytical methods
was able to predict beer flavour deterioration from analysis of a fresh beer sample. Beer C
(which performed worst during aging) was actually rated as the best beer when analysed
202
Chapter 6 – Section 3
Discussion
fresh with the PCT, and rated on the same level with beer B (which performed best during
aging at 30˚C) when analysed fresh with the ESR method. Based on these findings, it can
be concluded that the ESR and the PCT correlated with perceived flavour deterioration but
did not allow a reliable prediction of the speed of flavour deterioration in a given beer
based on the analysis of the fresh beer.
A summary of the process parameters and characteristics of the three beer brands
discussed in this chapter is shown in Table 6.1. A variety of process parameters such as raw
material type and amount of hop addition, boiling system, duration and evaporation rate,
wort gravity, yeast strain, fermentation duration and temperature, cold conditioning
duration and temperature, stabilisation agents and regime, packaging equipment, material
and design could be responsible in part for the differences in flavour stability observed
between beers A, B and C. The major differences that set beer C (worst performing) apart
from beer A and B is the very high gravity process with sugar syrup addition, 18 ˚Plato
wort gravity, a high fermentation temperature of 20˚C and a large dilution ratio to a sales
alcohol content of 3.50 % (v/v). Previous research by Miedl et al., 2005d has found that
beer proteins, especially the heavily thiolated low molecular weight fraction play a
fundamental role in preventing oxidation and flavour deterioration. The thiol groups in
these beer proteins have the ability to “cage” reactive oxygen species and thus prevent
oxidation. The use of large amounts of adjunct, the high gravity process and the significant
dilution to sales alcohol content will most likely decrease the concentration of the lower
molecular weight protein fraction that has been shown beneficial for beer flavour stability.
This will negatively impact on beer flavour stability of beer C. Beers A and B contained a
higher proportion of malt as extract source and the wort gravity was 10 ˚Plato and 14
˚Plato, respectively. Furthermore, the dilution ratios of beers A and B were lower
compared to beer C. The higher sales alcohol content in beers A and B might also have
contributed to their superior flavour stability. Papain, an enzyme with proteolytic activity,
was used as a stabilising agent in beers A and C and might have contributed to increased
proteolysis which could have, in turn, diminished the content of beer proteins protecting
flavour stability. Papain was not used during production of beer B.
203
Chapter 6 – Section 3
Discussion
Conclusions
ESR is considered “a cutting edge” and holistic method of analysis to determine
beer flavour stability (Andersen and Skibsted, 1988; Kaneda et al., 1991; Kaneda et al., 1989;
Uchida and Ono, 1996). Extensive research work has shown that ESR measurement does
correlate with the onset of beer staling and perceived flavour deterioration over time.
However, there are a number of arguments that diminish the viability of ESR as a flavour
stability tool. Firstly, ESR analysis involves forcing the beer at 60˚C for the duration of
measurement to speed up the formation of free radicals. Such temperatures are far
removed from the usual environmental conditions encountered by beer during the
distribution chain in most countries. Chemical reactions, including oxidation, and
consequently beer staling are highly temperature dependent and this should be taken into
account when attempting to measure flavour stability. Secondly, ESR analysis requires
significant capital expenditure for the purchase of the ESR equipment- hardware and
software. Furthermore, ESR running expenses are significant due to the cost of the spin
trapping reagent and the manpower required to operate the machine.
The PCT is, similar to the ESR, also a holistic and near real time method of flavour
stability analysis and as shown by this research, PCT results do correlate reliably with the
onset of staling and flavour deterioration. A considerable advantage of the PCT over the
ESR is the high sample throughput (96-well assay) and the lower running costs (chemicals
and manpower). There is some capital expenditure necessary for the purchase of the
luminometer required for PCT analysis. However, the costs for a luminometer are lower
than
for
a
basic
ESR
instrument.
Furthermore,
if
a
combined
luminometer/spectrophotometer is purchased many routine assays used in quality control
in the brewery could be adapted to a 96-well format, making substantial savings in time,
manpower and chemicals realistic. The PCT allows cost effective, near real time, high
throughput assessment of beer flavour stability. The PCT is convenient and inexpensive to
perform and the results correlate with ESR measurements and the perceived aged character
of beer.
204