Chapter 4
Results
CHAPTER 4
Results
4 Proteinase A (Pr A) and beer foam stability
The quality of beer foam is of the utmost importance for the brewing industry.
Beer head volume and stability are usually the first quality characteristics observed by the
consumer, and therefore strongly contribute to his/her appreciation of the product.
Brewing companies aim at a constant and controllable foaming behaviour. The ability of
beer to produce good foam is influenced by the level of foam-active polypeptides. Specific
polypeptides with hydrophobic domains, such as Lipid Transfer Protein (LTP1), are
important components of beer foam. It is thought that Pr A plays a key role in the
degradation of these hydrophobic polypeptides responsible for the beer foam stability.
The decrease in foam stability caused by yeast Pr A in unpasteurised beer has been
reported by several researchers (Dreyer et al., 1983; Ormrod et al., 1991; Muldbjerg et al.,
1993; Yokoi et al., 1995; Kogin et al., 1995; Cooper et al., 1998b; Cooper et al., 2000; Brey et
al., 2003). It has already been extensively discussed (Chapter 3, Sections 1, 2 and 3) that Pr
A is located in the vacuoles of S. cerevisiae, but can be excreted into beer under stress
conditions or released by yeast autolysis or mechanical damage (Cooper et al., 2000). This
enzyme has a preference for cleaving peptide bonds at sites between adjacent hydrophobic
amino acid residues at pH 2–4.5. Since the substrate specificity of Pr A is not rigid, the
presence of this protease in unpasteurised beer may result in the hydrolysis of foam
proteins and a decrease in beer foam. LTP1 has been found to be partially digestible by Pr
A during fermentation (Kondo et al., 1998; Shimizu et al., 1995). The primary objective of
this research was to investigate the relationship between head retention and Pr A activity in
unpasteurised beer during storage. A second objective was to investigate changes in beer
proteins and establish the specificity between Pr A and hydrophobic polypeptides.
130
Chapter 4 – Section 1
Results
CHAPTER 4 - Section 1
Results
4.1 The influence of pasteurisation on beer foam stability
The levels of Pr A activity, hydrophobic polypeptide concentration and foam
stability were compared in beer samples of WPV and WP. WPV and WP are codes for the
two beers investigated in this study. Both beers were pils/lager type and bottled on an
industrial scale in 500 mL bottles with a crown. Both beers were produced on similar
production lines in two breweries with similar brewing protocols and the same supplier of
raw materials. Fermentations were carried out with the same lager yeast strain. WP had
0.3% less original gravity and 2-4 IBU more bitterness than WPV. The major difference in
the production regime was that in brewery 1 (producing WP) the beer was tunnel
pasteurised (15 PU) whereas brewery 2, (producing WPV) employed membrane filtration
to ensure biological shelf life and the pasteurisation step was omitted. Due to
confidentiality issues no other production parameters are described in detail.
Samples of both beers were packaged in 500 mL glass bottles at the breweries and
were immediately transported from Germany to the UK. Upon arrival the samples were
stored at both room temperature and at 4ºC and analysed for foam stability using the
Nibem method and hydrophobic polypeptide content determined, as described in the
Materials and Methods Section 2.20, over a period of 102 days.
Foam stability in WPV declined rapidly over the course of storage at room
temperature especially during the first two weeks (Figure 4.1). Over 102 days incubation at
room temperature foam stability decreased a total of 37 Nibem sec in WPV. This is
approximately equivalent to a 13% drop in foam stability. In comparison, WP maintained
head retention better over 102 days storage at room temperature with a total decrease of 8
Nibem seconds (Figure 4.1). This is equivalent to an approximately 2.5% decrease in foam
stability. WP retained the best foam stability at 4°C storage over 102 days with a loss of 5
Nibem sec which is equivalent to a total decrease of approximately 1.5%. These profound
differences in beer head retention originate most likely from the Pr A activity in the
unpasteurised, sterile filtered beer (Figure 4.3) whereas Pr A has been inactivated during the
pasteurisation step in the pasteurised, non-sterile filtered beer (Figure 4.3).
Foam stability in fresh beer was slightly higher in WPV compared to WP. This
difference of 5 Nibem sec in head stability of the fresh beer might be due to the slightly
131
Chapter 4 – Section 1
Results
higher original gravity of WPV, although the higher hopping level in WP (+2-4 EBU)
might outweigh this effect. A more likely cause of the initially diminished foam stability of
WP was the changes in protein conformation and/or precipitation due to the application
of heat in the pasteurisation step, employed in the WP brewing process. Fresh WP
contained slightly less hydrophobic polypeptides. In addition, the polymerisation of
polyphenolic substances and subsequent binding to proteins may have been facilitated in
pasteurisation thus making them unavailable for foam formation. The minor advantage in
foam stability of fresh WPV over fresh WP disappeared rapidly during aging. The rapid
decline of foam stability in unpasteurised WPV was most likely due to the relatively high
activity of Pr A present in the packaged WPV (Figure 4.3). Pr A activity in WPV was 5.5
mU/mL at “0” days and decreased to 3.5 mU/mL over 102 days storage at room
temperature. The profound effect of “normal” Pr A levels in WPV on foam stability
(Figure 4.2) can be explained by the levels of hydrophobic polypeptides in WPV with 26
mg/L in fresh beer decreasing to 14 mg/L over 102 days storage at room temperature.
WPV and WP contain approximately 80% less foam positive hydrophobic
polypeptides than many other lager type beers with approximately similar Pr A activity at
the end of conditioning (Brey, 2004). In addition, the total protein content in WPV and
WP [determined according to Bradford (1976) Coomassie Blue method] was low at
approximately 80 mg/L and 75 mg/L, respectively. Furthermore, the percentage of total
protein in WPV and WP (determined according to Bradford (1976)), which is in the
majority hydrophobic, was found to be high with 32.5% for WPV and 32% for WP. Other
mainstream lager beers contain an average of 15-20% protein measured according to
Bradford (Brey, 2004).
132
Chapter 4 – Section 1
Results
290
285
Nibem foam stability [s]
280
275
270
WPV
ohne
KZE, RT
WPV,
no pasteurisation,
RT
265
WP
mitpasteurised,
KZE, RT RT
WP,
WP,
WP
mitpasteurised,
KZE, 4C 4˚C
260
255
250
245
0
20
40
60
80
100
120
Time [days]
Figure 4.1. Foam stability over 102 days storage. RT= room temperature (approximately 20˚C).
29
Hydrophobic polypeptides [mg/L]
27
WPV
ohne
KZE, RT
WPV,
no pasteurisation,
RT
WP, pasteurised, RT
WP mit KZE, RT
WP, pasteurised, 4˚C
WP, mit KZE, 4C
25
23
21
19
17
15
13
0
20
40
60
80
100
120
Time [days]
Figure 4.2. Hydrophobic polypeptide content over 102 days storage. RT= room temperature
(approximately 20˚C).
133
Chapter 4 – Section 1
Results
6.00E-03
WPV,ohne
no pasteurisation,
WPV
KZE, RT RT
WP,mit
pasteurised,
WP
KZE, RTRT
WP
KZE, 4C4˚C
WP,mit
pasteurised,
Proteinase A Concentration [Units/mL]
5.00E-03
4.00E-03
3.00E-03
2.00E-03
1.00E-03
0.00E+00
0
20
40
60
80
100
-1.00E-03
Time [days]
Figure 4.3. Pr A activity over 102 days storage. RT= room temperature (approximately 20˚C)
134
Chapter 4 – Section 1
Discussion
CHAPTER 4 - Section 1
Discussion
4.1.1 The influence of pasteurisation on beer foam stability
A number of studies (Reichender and Narziß, 1987; Kondo et al., 1998; Cooper et
al., 2000; Brey, 2003; Brey et al., 2004) found that proteinases secreted from yeast cells into
beer during fermentation in the brewing process degraded foam-active proteins and as a
result damaged the beer foam. These findings were found to be consistent with the results
obtained during the present study. Foam stability in WPV declined rapidly over the course
of storage at room temperature especially in the first two weeks. Over 102 days incubation
at room temperature foam stability decreased 37 Nibem sec (13%) in WPV. In comparison,
WP maintained head retention better over 102 days storage at room temperature with a
loss of 8 Nibem sec (2.5%). The rapid decline of foam stability in unpasteurised WPV was
most likely due to the relatively high activity of Pr A present in WPV (Figure 4.1). Pr A
activity in WPV was approximately 5.5 mU/mL in fresh beer and decreased to 3.5 mU/mL
during 102 days storage at room temperature. This Pr A activity in WPV was not much
higher than the activities found in other all malt, 12Plato beers after conditioning (Brey,
2004). However, this activity of Pr A exerts a profound effect on the head retention of
WPV. Pr A reduces the hydrophobicity of foam positive polypeptides.
This is
demonstrated in Figure 4.2, with the consequence of a rapid decline in foam stability
during storage as depicted in Figure 4.1. This striking effect of “normal” Pr A level in WPV
on foam stability can be explained by the levels of hydrophobic polypeptides in WPV, with
26mg/L in fresh beer decreasing to 14 mg/L over 102 days storage at room temperature
(Figure 4.2). These findings are consistent with those of Kondo et al., (1995) and Kogin et
al., (1999). They found that pasteurised and flash pasteurised beers showed no Pr A activity,
most likely due to the thermolability and denaturation during the pasteurisation step. Nonpasteurised beer contained 0.3-26.0 x 10-5 units/mL active Pr A, varying by brand
regardless of the country of origin. Muldbjerg et al. (1993) also found a correlation between
Pr A activity and foam potential and likewise Ormrod et al. (1991), who demonstrated that
proteolytic enzymes, which remain active in the final beer, damage foam stability during the
shelf-life of beer.
Dreyer et al. (1983) isolated an acid proteinase from brewers’ yeast and added it to
beer in which the original Pr A activity had been destroyed by pasteurisation. It was found
135
Chapter 4 – Section 1
Discussion
that the foam half-lives were reduced with increasing levels of enzyme activity and that
proteolytic enzymes from brewers’ yeast could digest wort proteins responsible for the beer
foam stability, leaving peptides with reduced or lacking foaming properties. Cooper et al.
(2000) compared the head retention values, measured using the Rudin method, of high (20
Plato) and low (10 Plato) gravity of pasteurised and un-pasteurised beer. It was
established, that the head retention values did not change for the high and low gravity
pasteurised beer, but the head retention of the un-pasteurised high gravity beer decreased
progressively compared with the head retention of the low gravity un-pasteurised beer
during storage.
The hydrophobic polypeptide content in both beers was low compared to the
levels found in 12 Plato wort all malt beers at the end of conditioning (Brey, 2004). WPV
and WP contained around 80% less foam positive hydrophobic polypeptides than other
lager type beers with around the same Pr A activity at the end of conditioning. This
combination makes both WPV and WP vulnerable with regard to foam stability. In
addition, the total protein content [determined according to Bradford (1976)] of WPV and
WP were low with values around 80 mg/L and 75 mg/L, respectively.
Conclusions
The present findings show that the better foam stability found in WP compared to
WPV is most likely due to low Pr A activity. It is likely that the enzyme was denatured
during the pasteurisation step since Pr A is heat sensitive and only stable up to 45°C at beer
pH. However, previous research in this Centre presented evidence that depending on the
composition of the medium, Pr A heat sensitivity can vary considerably (Brey, 2004). This
suggests that the effective denaturing temperature of Pr A will depend on beer type,
brewing procedures, raw materials etc. If Pr A is not denatured during the WP
pasteurisation step, another possible origin of the higher Pr A levels in WPV could be
different yeast management and propagation regimes. Previous research showed that
considerable amounts of active Pr A are carried over into the beer with the pitching yeast
slurry. Prolonged yeast storage under poor conditions (for example acid washing, high
storage temperature, agitation, centrifugation) promotes excretion and leakage of Pr A and
other foam negative substances from autolysing yeast cells. However, this scenario is
unlikely because even well maintained yeast excretes Pr A, which would have been detected
during the Pr A assessment.
136
Chapter 4 – Section 1
Discussion
In the case of WPV and WP beers, there is conclusive evidence that Pr A is
denatured during the pasteurisation step in the production line of WP. Consequently, low
amounts of hydrophobic polypeptides are preserved and can contribute to beer foam
stability. Introduction of a similar pasteurisation step during the WPV brewing process
would improve head retention considerably over prolonged storage or transport periods.
137
Chapter 4 – Section 2
Discussion
CHAPTER 4 – Section 2
Results
4.2 The impact of bottle-conditioning of beer employing revitalised yeast on
extracellular Pr A levels and the consequences for foam stability
It has been discussed already that yeast excretes intracellular proteases into the
fermenting wort during fermentation. Furthermore, Pr A leaks from yeast cells undergoing
autolysis. Bottle-conditioned beer contains yeast from maturation and thus release of Pr A
continues until the beer is consumed. Pr A concentration increases, as yeast viability
declines and yeast cells autolyse (Thorn, 1971) due to the prolonged exposure to a substrate
depleted of nutrients, high ethanol and CO2 content. As previously discussed (Chapter 4,
Section 1), Pr A has been shown to degrade foam active hydrophobic polypeptide and thus
exerts a negative impact on beer foam stability (Dreyer et al., 1983; Ormrod et al., 1991;
Muldbjerg et al., 1993; Yokoi et al., 1995; Kogin et al., 1995; Cooper et al., 1998b; Cooper et
al., 2000; Brey et al., 2003).
Bottle-conditioned beer (Coopers Pale Ale) was produced in an industrial scale
process in the Coopers brewery in Adelaide, Australia. The ale yeast was removed after
maturation by centrifugation and subsequently added back to give a specified cell count in
package (no further details can be disclosed due to confidentiality). Another identical batch
of Coopers Pale Ale was produced by removing the yeast after maturation but in this case
“revitalised” yeast with high viability and vitality was added to the bottle before packaging.
“Revitalised” yeast was produced by aerating the yeast slurry and adding a small amount of
wort in order to obtain yeast in the exponential growth phase. The majority of the yeast
cells harvested from maturation were in the quiescent (G0) phase of the cell cycle to
minimise use of nutrients and increase the chance for survival. The availability of nutrients
and oxygen during revitalisation initiated another cell cycle and the majority of cells were
dividing. This yeast was then added to the ale prior to bottling. After bottling the beer was
immediately air freighted from Australia to Scotland and after arrival both batches of beer
were stored at 4ºC.
The aim of this part of the project was to evaluate how revitalisation of yeast in
bottle conditioned beer impacts on beer foam stability. Coopers expected a significant
improvement in beer foam stability by employing revitalised yeast, due to decreased yeast
138
Chapter 4 – Section 2
Discussion
stress and autolysis. Coopers Pale Ale, produced with standard (non-revitalised) yeast, was
the control and Coopers Pale Ale, produced with revitalised yeast, was the trial.
4.2.1 Viability of yeast in bottle-conditioned ale
The viability of yeast populations in fresh control and trial beers was similar (93 and
94%, respectively) but diverged after 1 week of storage at 4ºC (Figure 4.4). After 13 weeks
storage the re-vitalised yeast population (trial) showed 10% higher viability than the control
yeast. However, the viability in both populations decreased dramatically during prolonged
exposure to a substrate (beer) depleted of nutrients including oxygen, with high ethanol
and CO2 concentrations (Figure 4.4). After 146 days storage at 4ºC and 7 days storage at
40ºC, the yeast viability in the control and trial beers decreased to 0.9 and 0.3%,
respectively. The storage temperature was increased from 4ºC to 40 ºC in order to
investigate the impact of such a high temperature, that could easily be reached during
transport and warehousing in hot climes such as in Australia, the main market for Cooper’s
beers.
4.2.2 Pr A activity in bottle-conditioned beer
Figure 4.5 shows that Pr A activity increased in the beer samples with prolonged
storage and the revitalised yeast excreted less Pr A than the control yeast. Pr A activity in
both the control and trial was comparatively high, similar to the activities found in 20
ºPlato lager fermentations (Brey, 2004). However, the differences in Pr A excretion
between control and trial, and also the increase in Pr A activity during storage were small
(approximately 0.5x10-3 U/mL). Previous research (Cooper et al., 2000; Brey, 2004) suggests
that the majority of Pr A was excreted from viable yeast cells due to miss-sorting in the late
Golgi apparatus under stress conditions and not from autolysed, leaking yeast cells. Since
the yeast cells in the control beer are in their quiescent (G0) cell cycle phase, their
metabolism is reduced. Subsequently, protein synthesis is reduced to the minimum required
for survival of the yeast cell and less Pr A is produced.
This may be an explanation for the small increase in Pr A activity during storage at
4ºC. After the beer was exposed to 40ºC for 7 days, following storage at 4ºC, Pr A activity
increased in both beers due to autolysis of dead cells (Figure 4.4 and 4.5). However, the
beer with revitalised yeast showed a more pronounced increase in Pr A activity.
4.2.3 Hydrophobic polypeptides
An important factor affecting the loss of wort hydrophobic polypeptides during
fermentation and storage is Pr A activity. Due to the long contact time between Pr A and
139
Chapter 4 – Section 2
Discussion
the foam positive polypeptides during the storage period in the un-pasteurised Pale Ale, the
loss of hydrophobic polypeptides was high. This effect appeared to be reinforced by the
continuous excretion of Pr A from the yeast cells during storage. In addition, the high
percentage of autolysed yeast cells will exacerbate the leakage of Pr A into beer.
As shown in Figure 4.6, there was no difference in hydrophobic polypeptide content
observed between trial and control during the first 146 days storage at 4ºC. In both beers
the concentration of hydrophobic polypeptides decreased by approximately 25mg/L
during this time period. After subjecting the beer to 7 days storage at 40°C the
hydrophobic polypeptide content in the control beer declined rapidly (a further 22 mg/L)
whereas the decrease in hydrophobic polypeptide concentration in the trial beer was only
11 mg/L in the same time period. This correlates with the decrease of foam stability and
the increase in Pr A activity. Overall, the concentration of hydrophobic polypeptides in
Coopers Original Pale Ale was high, compared to values reported by Brey (2004).
The exceptionally high content of hydrophobic polypeptides explains the excellent
foam stability of Coopers Pale Ale, despite the comparatively high Pr A activity in this beer.
Due to confidentiality issues no detailed description of the production parameters of
Coopers Pale Ale are disclosed but the high levels of foam positive hydrophobic
polypeptides could for example, be due to the use of 100% malt, mashing parameters that
facilitate maximum extraction of these polypeptides and suppression of fermenter foaming
to retain hydrophobic polypeptides in solution.
4.2.4 Foam stability of bottle conditioned beer
Overall, the foam stability of this ale was excellent. During the course of 13 weeks
storage at 4˚C, the head retention decreased from approximately 320 to 285 Nibem sec but,
as depicted in Figure 4.7, no large difference in head retention was observed between the
control and trial beer samples.
Assessment of foam stability after the beer was subjected to 40ºC for 7 days,
following storage at 4ºC, showed a dramatic deterioration of the head retention values in
the trial from 276 to 228 Nibem sec. The foam stability in the beer without revitalised yeast
(control) also decreased, but not as drastically as in the beer with revitalised yeast. The high
levels of Pr A activity released by autolysed yeast cells as well as other foam-negative cell
contents leaking into the beer are likely to be the cause of the rapid decline in foam stability
during the 40ºC storage period.
140
Chapter 4 – Section 2
Discussion
Viability 0,1,5,9,13 weeks
100
90
Viable Cells [%]
80
70
60
50
40
30
318-03
Control
20
Trial
318-03V
10
0
0
50
100
150
200
Storage [days]
Figure 4.4. Yeast viability during storage of Coopers Pale Ale at 4ºC, followed by 7 days storage at
40ºC. The arrow indicates when the beer was moved from 4ºC to 40ºC.
Proteinase A Activity
Proteinase A Activity [10E-3 units/ml]
6
5.5
5
4.5
4
Control
318-03
Trial
318-03V
3.5
3
0
50
100
150
200
Storage [days]
Figure 4.5. Pr A activity during storage of Coopers Pale Ale at 4ºC, followed by 7 days storage at 40ºC.
The arrow indicates when the beer samples were moved from 4ºC to 40ºC.
141
Chapter 4 – Section 2
Discussion
Hydrophobic Polypeptides
Concentration hydrophobic
Polypeptides [mg/l]
230
220
210
200
190
180
318-03
Control
170
Trial
318-03V
160
150
140
0
50
100
150
200
Storage [days]
Figure 4.6. Concentration of hydrophobic polypeptides during storage of Coopers Pale Ale at 4ºC,
followed by a 7 day storage period at 40ºC. The arrow indicates when the beer samples were moved from
4ºC to 40ºC.
Head Retention [Nibem seconds]
Head Retention 0,1,5,9,13 weeks
340
320
300
280
260
318-03
Control
240
Trial
318-03V
220
200
0
50
100
150
200
Storage [days]
Figure 4.7. Head retention during storage of Coopers Pale Ale at 4ºC, followed by a 7 day storage
period at 40ºC. The arrow indicates when the beer samples were moved from 4ºC to 40ºC.
142
Chapter 4 – Section 2
Discussion
CHAPTER 4- Section 2
Discussion
4.2.5 The impact of bottle-conditioning of beer employing revitalised yeast on
extracellular Pr A levels and the consequences for foam stability
The process of bottle-conditioning is a traditional practice in brewing and bottleconditioned beers have seen a revival internationally over the last few years. The objective
of bottle-conditioning is to produce CO2 from the extract remaining after the main
fermentation and thus yeast is added to the packaged product for this purpose. In addition,
bottle-conditioning increases the biological stability and flavour shelf life of beer. The
viable yeast cells in contact with the beer will remove oxygen from the package and this
protects the beer from oxidative staling reactions. Furthermore, viable yeast cells in the
beer limit the growth of beer spoiling organisms and thus biological shelf life is increased.
The prolonged contact of the yeast cells with the beer also results in a multitude of
continued flavour developments in the package which gives bottle conditioned beers their
distinct flavour profile.
The exceptionally high content of hydrophobic polypeptides explains the excellent
foam stability of Coopers Pale Ale, despite the comparatively high Pr A activity in this beer.
During the course of 13 weeks storage, the head retention decreased from approximately
320 to 285 Nibem sec, but as depicted in Figure 4.7, no large difference in head retention
was observed between Coopers Original Pale Ale with revitalised and the control yeast.
One would have expected less head retention with the control yeast due to the lower
yeast viability and higher Pr A levels in this beer. Why this did not occur might be
explained by an unusual observation made during viability determinations with the light
microscope. The yeast sample with revitalised yeast contained an increased amount of small,
dead yeast cells, which appeared to be fallen-off yeast buds. These cells were much smaller
than a normal yeast cell and were not taken into consideration for the viability counts. The
viability of the revitalised yeast population would have been considerably lower if these
dead cells had been taken into account. Presumably these immature buds found in the
revitalised yeast population could not tolerate the stressful environmental and nutritional
conditions in the beer during storage. This may have triggered apoptosis in the young buds,
143
Chapter 4 – Section 2
Discussion
whereas fully grown cells were able to withstand these conditions to a greater extent. In
comparison, the control yeast sample contained only a few budding cells and the majority
of the cells were fully grown. Since there are many small, dead cells in the revitalised yeast
population, more foam negative cell contents, such as lipids and proteinases, leak into the
surrounding beer compared to the population with the control yeast. This effect seems to
outweigh the slightly higher Pr A levels in the beer with the control yeast, so that in the end,
the head retention in both beers is approximately the same.
Conclusions
This study suggests that employing a yeast population in the logarithmic growth
phase (revitalised) for bottle conditioning does not achieve an improvement in head
retention after prolonged storage at 4ºC compared to control yeast. This may be due
mainly to autolysis of a large number of dead, immature yeast buds which might release
foam negative substances (lipids?) into the beer. Furthermore, when trial and control beers
were subjected to extreme storage conditions (40ºC for 7 days) it showed that the head
retention in the beer containing revitalised yeast deteriorated to a greater extent than the
control beer.
Based on these results, it can be suggested that employing a healthy yeast population
in stationary growth phase (G1) for bottle conditioning results in decreased Pr A secretion,
increased levels of hydrophobic polypeptides and consequently improved beer foam
stability. In this scenario, the yeast cells use the remaining nutrients in the beer for
maintaining survival, but not for entering cell replication (budding) and logarithmic growth
phase. After depletion of nutrients and with the high ethanol and CO2 concentrations
present in beer, the yeast enters the quiescent growth phase, with reduced metabolism and
protein synthesis. Low concentrations of Pr A and decreased leakage of other foamnegative substances through autolysing cells (mainly buds) and improved foam stability are
the consequence. Since beer is not always stored under optimum conditions (4ºC), it would
be interesting for future work to investigate the effect of storage at room temperature on
head retention and foam stability. A major effect would be expected, since the metabolism
of the yeast cells contained in the beer will be enhanced by higher temperatures and more
Pr A and other foam negative substances will be released into the beer.
144